WO1989010786A2

WO1989010786A2 - Process for forming and using microdroplets

Info

Publication number: WO1989010786A2
Application number: PCT/US1989/001716
Authority: WO
Inventors: James C. Weaver; Gregory B. Williams
Original assignee: Microdrop, Inc.
Priority date: 1988-04-22
Filing date: 1989-04-21
Publication date: 1989-11-16
Also published as: AU4062189A; WO1989010786A3

Abstract

Gel microdroplets, and means for formation therefor, are disclosed, wherein such gel microdroplets have two or more distinguishable gel or liquid regions. Composite gel microdroplets provide means for segregating first biological entities from second biological entities within one gel microdroplet, and for similarly segregating biological entities, marker entities such as fluorescent beads, and force coupling entities such as magnetic particles, from other entities within one gel microdroplet. This invention further relates to general means for providing physical, chemical or biological external influence on biological entities, wherein biological entities contained within microdroplets are changed in position so as to reversibly move microdroplets to a location near a source of external influence, maintain microdroplets near the source, and to move microdroplets into a measurement apparatus. A process for determining the number of analyte molecules in a sample by a homogeneous specific binding assay is also disclosed.

Description

PRQCESS FOR FORMING AND USING MICRODROPLETS

Description

Background of the Invention

5 Determination of cell growth, cell activity and the active behavior of other biological entities such as enzmes and viruses is important to biological research, and has many applications in fields such as clinical microbiology, cancer diag¬ nosis and treatment, environmental science, food safety, toxicology, and research and development in basic and applied biology. Although a significant number of

10. assays and tests are directed towards viruses, nucleic acids and antibody-antigen complexes, many features, particularly disadvantages, of the most well established methods can be described by considering the prior methods used to determine growth of cells. For this reason, the main features of prior methods relating to biological measurements, tests, assays and isolation of biological entities are 5 presented with an emphasis on prior methods for determining cell growth.

Present cell analysis methods involve two major classes of assays. The first class rapidly detects and identifies specific cells directly from a primary sample, but does not determine cell viability. The most widely used in this class are specific ligand binding assays, e.g. immunoassays and genetic probes. However, D they require many cells, and do not distinguish between dead and viable cells.

This restricts their use to samples in which sufficient numbers of cells are present, and to determinations in which direct assessment of the physiological state of the cell is irrelevant. The second class of assays is used for viable cell determina¬ tions either directly using the primary sample, or using a subculture of the pri- 5 mary sample. The most traditional and widely used method is the plate count, which allows determination of single cell viability, based on growth, under many test conditions (see, for example, Hattori The Viable Count: Quantitative and Environmental Aspects, Brock/Springer, Madison, 1988). An important attribute of viable plate enumeration is that time required to obtain a determination is 5*- independent of the concentration of the cell in the sample, because formation of each colony proceeds from an initial single cell. The major disadvantage is its slowness, as typical determinations require one-half to several days, and are also labors and materials-intensive.

The disadvantages of viable plating can better be appreciated by drawing lO^'Z attention to its basic attributes. Viable plating is a well established, important method for qualitatively determining the growth of cells, particularly the presence or absence of growth for given conditions, and is often based on the growth of initial cells into distinct colonies. Viable plating typically involves the spreading of a suspension of cells onto the surface of a gel-containing petri dish, with or

15^' without the pouring of a gel layer over the first gel surface. The gels are pro¬ vided with nutrients, such that following an incubation period at a suitable tem¬ perature, many generations of growth occur, which leads to formation of visible colonies. For many microorganisms formation of visible colonies requires growth for 22 to 30 generations and therefore produces colonies containing 10⁷ to 10⁹

20 cells. (See Sharpe, in Mechanizing Microbiology, A. N. Sharpe and D. S. Clark

(Eds.) Charles C. Thomas, Springfield, 19 - 40, 1978). Although conventional viable plating leads to formation of colonies, and thereby provides a basis for counting viable cells by counting colonies, the presence or absence of colonies only allows inference that the conditions present in the gel support or do not sup-

25 port growth. For this reason, conventional viable plating is not well suited to quantitative determinations such as cell growth rate and lag time, because viable plating based on visual inspection counts the number of colonies formed, but does not determine how the cellular material or amount of cellular constituents in the colonies varies with time. An additional complication arises because the nutrient

30 and metabolite concentrations within a colony comprise a microenvironment, which generally changes with time in an unknown way as microcolonies increase to form larger colonies with many cells in close proximity. The microenviron¬ ment within a large colony can also have significant heterogeneity of chemical composition within the microcolony, so that different cells within a large colony

35 experience different growth conditions. Further, although some methods are based on a straightforward extension and application of scanning optical methods for determination of optical properties of colonies on or in gel slabs, such methods suffer from relatively large cost and size, and, because of the relatively large gel slab size, do not allow incubation conditions to be changed rapidly at the site of the cells within the gel. (See Glaser in New Approaches to the Identification of Microorganisms Proceedings of a Symposium on Rapid Methods and Automation in Microbiology, C.-G. Hedέn and T. Illέni (Eds.), Wiley, New York, 3 - 12, 1975).

Instrumented methods for rapidly determining cell or culture growth and/or metabolic activity have been developed which only partially address the limita- tions of the viable plate assay. These include optical techniques for growth deter¬ mination such as those which measure the change in light scattering due to many cells in a liquid suspended culture (See, for example, Edberg and Berger, in Rapid Methods and Automation in Microbiology and Immunology, K. O. Haber- mehl, Ed., Springer- Verlag, Berlin, 215 - 221, 1985), and a variety of metabolic activity based techniques which measure changes due to many cells in an analyzed sample. Examples of these include the measurement of changes in extracellular pH (See, for example, Cowan and Steel's Manual for the Identification of Medical Bacteria, Cambridge University Press, Cambridge, 1974; Manual of Methods for General Bacteriology, P. Gerhardt, (Ed), American Society for Microbiology, Washington, 1981), carbon dioxide release (see, for example, Courcol et al., J. Clin. Microbiol., 25: 26 - 29, 1986; Manca et al., J. Clin. Microbiol., 23: 401 - 403, 1986), electrical impedance (see, for example, Stewart, J. Exp. Med., 4: 235 - 245, 1899; Eden and Eden Impedance Microbiol¬ ogy, Research Studies Press, Letchworth, 1984; Hadley and Yajko, in Instrumen- tal Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim,

193 - 209, 1985; Bishop and White, J. Food Protect., 49: 739 - 753, 1986), chem- iluminescence (see, for example, Neufeld et al., in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 51 - 65, 1985; Rapid Methods and Automation in Microbiology and Immunology, Habermehl (Ed.), Springer- Verlag, Berlin, 1985) or fluorescence (see, for example, Rossi and

Warner in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 1 - 50, 1985; Rapid Methods and Automation in Micro¬ biology and Immunology, Habermehl (Ed.), Springer- Verlag, Berlin, 1985). A disadvantage of all such metabolic activity methods is that they are based on combined effects of a large number of cells. Therefore, this generally require an initial process, based on plating, to obtain initial colonies for purposes of inoculation of the analyzed sample, such that the determinations based on many cells at least are based on a monopopulation, i.e. a population comprised nomi¬ nally of the same type of cells. For this reason, although a total population cell determination may itself be rapid, it is generally preceeded by a viable plating method, or its equivalent, which is slow. Thus, the total analysis time, counted from receipt of a primary or non-plated sample to a cell growth determination, is the sum of both, and therefore still long.

Further, because such determinations are based on the combined effect of a large, but unkown number, of cells, such total population determinations do not actually yield a count. In contrast, determinations based on many individual measurements, each associated with an initial single cell, can yield a count.

Finally, because these total population methods are based on the combined effects of many cells, the time required for a determination becomes significantly longer as the number of cells decreases, i.e. as the sample's cell concentration ^,_ decreases.

Similarly, prior use of flow cytometry for cell growth measurements (see, for example, Hadley et al. in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 67 - 89, 1985) is limited, because con¬ ventional use of flow cytometry performs measurements on individual cells, or clumps of cells which naturally adhere, in an aqueous liquid suspension, and therefore does not have the capability to measure colony formation. For this rea¬ son, prior use of flow cytometry can only measure total numbers of cells in a volume in order to determine average growth, must also, therefore, involve a careful volume measurement, and is dependent on the signal-to-noise ratio of sin- ^" gle cell measurements. This signal-to-noise ratio is less than satisfactory for many measurements (see, for example, Shapiro Practical Flow Cytometry, R. Liss, New York, 1985; Hadley et al. in Instrumental Methods for Rapid Micro¬ biological Analysis, Nelson (Ed.), VCH, Weinheim, 67 - 89, 1985).

Likewise, quantitative microscopy and image analysis combined with con- ventional gel preparations, such as gel slabs, petri dishes and the like, although capable of determining colony formation, is tedious in manual versions, and con¬ ventional gel slabs, petri dishes and the like cannot provide physical manipulabil- ity or a sufficiently fast characteristic diffusion time within the gel, so that cells cannot be rapidly and conveniently exposed to different growth conditions, such ^" as rapid changes in concentrations of nutrients, drugs, hormones, enzymes, antibo¬ dies and other chemicals. In addition, conventional gel slabs, petri dishes and the like cannot be readily manipulated physically because of their size, and therefore cannot be readily used for exposure of gel-entrapped cells to in vivo conditions.

For example, agarose slabs can be used to protect fragile biological entities such as plant protoplasts in cases wherein both free cells and cells entrapped in 5 agarose beads are found to be damaged (see Pasz and Lurquin, BioTechniques 5:

716 - 718, 1987), but such gel slabs have relatively long characteristic diffusion times; and cannot be readily inserted into animals in order to provide biological influence.

In another example, guiding human cancer chemotherapy by determining

100 human cancer cell growth in immunodeficient mice, which are exposed to trial chemotherapy conditions, can be approached by insertion of a small tissue piece into a mouse (see Bogden, Annal. Chirurgiae Gynaecol., Vol. 74 Suppl. 199, 12 - 27, 1985; Favre et al., Eu. J. Can. Che. Oncol., 22: 1171 - 1178, 1986), but the insertion of conventional gel slabs or petri dishes into the mice would be simi-

15.^" larly cumbersome, requiring considerable manual manipulation. For example, slices of clot about 1 to 1.4 mm on a side containing about 1 x 10⁵ to 2 x 10⁵ human cancer cells can be prepared and inserted into a mouse, and subsequent combined growth due to this large number of cells can be determined by meas¬ urement of the resulting tumor dimensions. Colony formation is not determined,

20. however, so that such procedures both require manual manipulations and do not result- in cell preparations which are readily analyzed quantitatively for cell growth, such as average cell growth rate, or more importantly, the quantitative growth of individual cells (see Fingert, Cancer Res., 47: 3824 - 3829, 1987). Further, in the absence of applied physical forces, transport of molecules within

25 gel materials occurs by diffusion, for which the characteristic diffusion times are dependent on the square of the thickness of the gel slabs or petri dish gel material, which thicknesses are typically 10^-1 to 3 x lO^-1 cm or larger. For this rea¬ son characteristic diffusion times for even small molecules are long, as can be estimated by calculating -_di_ffusi_on ~ x²/D, wherein x is a characteristic dimension or 0 thickness of the gel. The diffusion constant, D, is about 10^"S cm²/sec for small molecules, and is signficantly smaller, often about 10^-7 cm²/sec for large molecules such as antibodies and enzymes, so that typically ^s^.. ~ 17 to 50 minutes for small molecules, and about 2.8 to 8.3 hours for large molecules. It is generally well known that times of about St^^_a, to 5t_diffusion are required to obtain changes 5 which are 95 to 99% complete for diffusion controlled processes. Thus, the times corresponding to 3tdj_f.u-.o_n for a 95% complete change are about 50 minutes to about 2 1/2 hours for small molecules, and about 8 1/2 hours to about 1 day for large molecules, and still longer for a 99% complete change, so that it is generally impossible to provide rapid, completed changes of chemical concentrations at the site of cells contained in conventional gel slabs, Petri dishes or large gel particles 55 by the general means of changing external chemical concentrations (see, for example, Nilsson et al., Nature, 302: 629 - 630, 1983; Nilsson et al., Eur. J. Appl. McroBioL Biotechnol., 17: 319 - 326, 1983).

Similarly, prior methods for determining growth of viruses involve the use of cell culture, such that sample viruses infect one or more of provided cells, and 103 grow within the infected cells, often resulting in cell lysis and releasing a large number of virus. Prior methods for determining growth of chlamydia also involve growth within host cells.

Further, prior methods for determining growth of nucleic acids involve a cyclic incubation which results in a doubling of specific nucleic acids with each 15^"; cycle (see Mullis et al., U.S. Patent 4,683,195). Finally, prior methods for deter¬ mining growth of immunological complexes such as antibody-antigen complexes are carried out in a homogeneous phase wherein the reactants are in solution, or using a solid phase, wherein one or more reactants are attached to a solid surface.

The optical path length associated with optical measurements on cells in 20 conventional gel slabs or Petri dishes can often be equal to a significant fraction of the thickness, or the entire thickness, of the gel slabs or petri dish material. For this reason, the size of conventional, macroscopic gel preparations also places limitations on optical measurement methods which can be used with the gel preparations, as light absorbtion, light scattering and auto fluorescence can be 25. significant, and result in difficulty in making accurate measurements on individual or small numbers of cells within such macroscopic gels.

In view of these limitations of prior methods for determining growth of bio¬ logical entities, particularly cells, significantly more rapid and automated methods for biological growth determination allow rapid changes in growth conditions to 30 be made, based on a small number of biological entities, preferably individual biological entities, and therefore would not require prior incubation. Additionally, these methods should be capable of determining biological growth under a variety of conditions, using samples which contain small numbers of biological entities.

Furthermore, many assays and tests are based on exposure of cells to com- 35 pounds and agents wherein the compounds and agents are chemically modified by first ex osing the compounds and a ents to activatin entities. These entities include whole animal metabolism, isolated organ preparations, tissue extracts, cell preparations and biochemical preparations such as enzymes. The modification of compounds and agents by such biological and biochemical processes is generally termed activation. Following activation, cells are exposed to the modified com- pounds and incubated to allow biological processes such as cellular growth, meta¬ bolism, genetic transformation and biomolecule secretion to occur.

In order to carry out tests and assays based on such biological and biochemi¬ cal interactions, one or more properties or behaviors of the cells is then deter¬ mined. For example, within a period during and/or after such activation the amount of cell growth is determined, so that the cell killing effects, cell inhibitory effects and cell growth stimulating effects, and the like, can be directly deter¬ mined. Such assays and tests are widely used, and have important applications in fields such as clincial microbiology, cancer diagnosis and treatment, environmen¬ tal science, food safety, toxicology, and research and development in basic and applied biology.

Present methods for activation of cells have significant disadvantages. For example, it is generally believed that the most realistic condition for providing activation involves the use of whole animals, wherein the complex interaction of activating pathways in the cells of one or more tissues and/or organs is utilized. Thus, for example, in testing for possible genetic toxicologial effects of chemicals it is often believed that the tested compound should be administered to an animal, such that complex organ systems such as the gastrointestinal tract and the liver can activate the tested chemical. While such methodology is desirable because it represents the complex activating biological and biochemical pathways, there are signficant problems associated with the use of animal tests to estimate the effects of the tested chemical on humans. Even whole animal test results can be misleading and inaccurate when applied to humans because typical animals such as rats, dogs, goats and primates are significantly different than humans.

One consequence of obtaining different results from different animal species is the perceived need to use, in many cases, more than one type of animal for each test of a chemical, as it is hoped that it is more likely to obtain results which will be useful in estimating human toxicity.

Furthermore, in many cases a problems exists with respect to reproducibility between different animals, such that no one individual animal is respresentative. Such animal to animal variability often requires that several animals be used for each test, which significantly increases the cost of carrying out animal tests. Further, present methods for in vitro activation have disadvantages which are particulalry relavant to assays and tests using microdroplets (MDs). Specifically, present in vitro activation methods involve co-culture, wherein testor cells are grown in close proximtry to activating cells by using soft agarose or similar materials, such that both testor and activating cells reside within the same piece of gel, and therefore cannot be readily removed from one another. Additionally, existing methods using co-culture in soft agarose do not allow the cell density to be controlled once the co-culture has begun, and it is well known that cell density is often critically important to cell culture. Similarly, guiding human cancer chemotherapy by determining human cancer cell growth in immunodeficient mice, which are exposed to trial chemoth¬ erapy conditions, can be approached by insertion of a small tumor tissue piece into a mouse (see Bogden, Annal. Chirurgiae Gynaecol., Vol. 74 Suppl. 199, 12 - 27, 1985; Favre et al., Eu. J. Can. Che. Oncol., 22: 1171 - 1178, 1986), but the insertion of conventional gel slabs or petri dishes into the mice would be simi¬ larly cumbersome, requiring considerable manual manipulation. For example, slices of clot about 1 to 1.4 mm on a side containing about 1 x 10^s to 2 x 10^s human cancer cells can be prepared and inserted into a mouse, and subsequent combined growth due to this large number of cells can be determined by meas- urement of the resulting tumor dimensions. Colony formation is not determined, however, so that such procedures both require manual manipulations and do not result in cell preparations which are readily analyzed quantitatively for cell growth, such as average cell growth rate, or more importantly, the quantitative growth of individual cells (see Fingert, Cancer Res., 47: 3824 - 3829, 1987). In contrast, by using GMDs to deteπnine individual growth of a large number of cells taken from a sample of a patient's cancer cells, a faster more meaningful test can be performed. GMDs can provide growth measurements within one, two or a few division times, which for human cancer cells corresponds to times of about one, two or a few days. Further, it is well known that cancer cells often differentiate to some degree, so that, for example, the cancer cells from an individual patient show varying subpopulations with different resistance to chemotherapeutic chemicals. As a consequence, a candidate chemi¬ cal can kill or inhibit the growth of, for example, 95% of the cells in a sample, but the remaining 5% can be resistant, and can survive a treatment, grow, and lead to the death of the patient. In a situation such as this, measurment of the total cell growth or inhibition, as by measuring the growth of a tissue piece, can miss the small growth due to a subpopulation, and thereby give misleading test results. It is, therefore, highly desirable to have a test which can measure a large number of individual cells for the effectiveness of a candidate chemotherepeutic chemical, and which can be carried out rapidly, but while still providing the important biological influence of metabolic activation and processing of the chemotherapeutic chemicals.

Further, in the absence of applied physical forces, transport of molecules within gel materials occurs by diffusion, for which the characteristic diffusion times are dependent on the square of the thickness of the gel slabs or petri dish gel material, which thicknesses are typically 10"¹ to 3 x 10"¹ cm or larger. For this reason characteristic diffusion times for even small molecules are long, as can be estimated by calculating t_d^_a, ~ x²/D, where x is a characteristic dimension or thickness of the gel. The diffusion constant, D, is about 10^"5 cm²/sec for small molecules, and is signficantly smaller, often about 10^~7 cm²/sec for large molecules such as antibodies and enzymes, so that typically .-m^_^ - 17 to 50 minutes for small molecules, and about 2.8 to 8.3 hours for large molecules. It is generally well known that times of about 31,^^ to St^_a,,-, are required to obtain changes which are 95 to 99% complete for diffusion controlled processes. Thus, the times corresponding to 3 i_ffusi_on for a 95% complete change are about 50 minutes to about 2 1/2 hours for small molecules, and about 8 1/2 hours to about l day for large molecules, and still longer for a 99% complete change, so that it is generally impossible to provide rapid, completed changes of chemical concentrations at the site of cells contained in conventional gel slabs, Petri dishes or large gel particles by the general means of changing external chemical concentrations (see, for example, Nilsson et al., Nature, 302: 629 - 630, 1983; Nilsson et al., Eur. J. Appl.

Microbiol. Biotechnol., 17: 319 - 326, 1983).

Overall, the ability to provide biological influence such as metabolic activa¬ tion using intact in vivo conditions provided by whole animals, or by using in vitro conditions provided by using feeder cells, tissue extracts, viable cell preparations, and the like, would be important in providing realistic tests. The additional ability to make individual cell measurements, and/or population distri¬ bution measurements, and to make such measurements rapidly, would provide an extremely general, flexible and rapid basis for the effect of agents and compounds on cell growth, wherein such agents and compounds are not only used directly, but also in modified forms as the result of activation and biochemical processing. Furthermore, determination of the amounts of specific molecules and other analyte entities contained in a sample is widely recognized as important, with applications in fields such as biological research, biotechnology, cancer testing, veterinary medicine, home health care, infectious disease, and food safety. Specific binding assays are recognized as valuable because of the specificity asso¬ ciated, with reagents such as specific binding molecules including antibodies, antigensi.nucieic: acids, avidin-biotin, enzyme inhibitors and Iectins. Although prior specific binding assay methods relating to avidin-biotin, enzyme inhibitors and Iectins are of interest, basic issues relating to specific binding assays can be described in terms of specific binding nucleic acid assays, and in terms of immu- noassays based on the specific binding of antibodies and antigens.

Specific nucleic acid assays are well known to be extremely important, and the recent development of amplification of nucleic acid has lead to assays which can amplify initial nucleic acid molecules by very large factors, so that the much larger quantity of nucleic acid can then be specifically measured (see Mullis et al,

US Patent 4,683,195, 1987; SaiM et al, Science 239: 487 - 491, 1988). Although extremely powerful, such amplification methods are not used to directly count the initial individual nucleic acid molecules present in a sample, as such counting, even in a small sample, would require that the amplification process be carried out separately in a large number of parallel assays on separate portions of the sample.

Immunoassays are also well known to be extremely important, and are gen¬ erally recognized as a general means of providing both highly specific and sensi¬ tive determinations, that is, of providing assays with a low detection limit for specific molecules. Thus, immunoassays are generally regarded as having the ability to detect very small amounts of specific molecules in the presence of a great variety of other molecules. However, the detection limits of prior immu¬ noassays are not always adequate. Therefore, better detection limits, that is meas¬ urement of smaller analyte entity concentration, is desirable. Finally, the funda- mental limit of detecting individual analyte entities such as individual antigenic maαomoleeules has not been described in the prior art.

Both: solid phase immunoassays and homogeneous immunoassays have been developed, and have features which represent the general aspects of specific bind¬ ing assays. Solid phase or heterogeneous assays are characterized by the use of one or more solid surfaces to which specific binding sites have been attached.

The use of heterogeneous assays generally requires wash steps which are often manual or difficult to automate, and detection limits are limited by non-specific binding to solid surfaces. In constrast, homogeneous assays do not involve solid surfaces, and thereby avoid washing steps and most of the problems associated with non-specific binding. However, prior homogeneous assays have generally exhibited detection limits inferior to those of inhomogeneous assays.

Since the introduction of the radioimmunoassay, a great amount of research and development has been directed towards immunoassays generally. Such efforts have sought to improve features of immunoassays such as specificity, speed, cost and detection limit. Additionally, efforts have sought to eliminate the use of radioactive material. As part of these efforts, a large number of different types of homogeneous immunoassays have been devised, including homogeneous assays based on fluorescence quenching, fluorescence enhancement, fluorescent substrates, fluorescence excitation energy transfer, fluorescence polarization, chemiluminesence quenching, chemiluminesence enhancement, chemiluminesence energy transfer, change in electron spin resonance, nephelometry, liposome lysis inhibition and zymogen activation (see, for example, Campbell et al in Alternative Immunoassays, Collins (Ed.), Wiley, pp. 153 - 183, 1985).

Enzyme based homogeneous assays are generally based on alteration of enzyme activity, or enzyme inhibition, of an enzyme molecule coupled to an antigen, wherein the antigen is usually a small molecule, and the sensitivity or detection limit is poorer than that of inhomogeneous immunoassays (see, for example, Voller and Bidwell in Alternative Immunoassays, Collins (Ed.), Wiley, pp. 77 - 86, 1985). Other versions of the enzyme activity alteration homogeneous assay can be used to measure larger molecules such as protein. Still other homo- geneous immunoassays suitable for large molecule assays are based on fluores¬ cence principles, and result in homogeneous immunoassays based on fluorescence excitation transfer or fluorescence protection (see, for example, Rubenstein in Immunoassays for the 80s, Voller et al (Eds), University Park Press, Baltimore pp. 127 - 132, 1981). Solid phase immunoassays generally have better detection limits than homo¬ geneous immunoassays, and are limited primarily by non-specific binding, that is the false non-specific signal resulting from non-specific binding of immunocom- pounds. However, solid phase immunoassays require one or more washing steps wherein non-specifically bound reagents are to be removed, and these additional steps are undersirable. Overall, the importance of obtaining highly specific assays with low detection limits has resulted in continued research and development of immunoassays, and additional improvement is desired (see, for example, Alterna¬ tive Immunoassays, Collins (Ed.), Wiley, 1985). It is particularly relevant that no method to date has yet achieved a detection limit wherein individual antigenic entities such as macromolecules without large numbers of repeated antigens can ; be generally assayed. The detection limit is thus a fundamental limit.

In another field of investigation, that of enzyme activity and enzyme dena- turation,. an entirely manual, difficult and tedious method for measuring the fluorescent product molecules of one, two or more individual enzyme molecules of the enzyme β-galactosidase was demonstrated many years ago by Rotman (PNAS 47: 1981 - 1991, 1961). There has been no repetition of this study, and no subsequent utilization of the measurement of individual enzyme molecule activity in assays. Therefore no application to the counting of individual enzyme molecules which could be used as the basis of a specific binding assay has been shown. Further, although enzyme channeling, wherein the product of a first enzyme catalyzed reaction is the substrate for a second, has been described for use with some established enzyme immunoassays (see, for example, Voller and Bidwell in Alternative Immunoassays, Collins (Ed.), Wiley, pp. 77 - 86, 1985), there has been no application of enzyme channeling to the measurement of enzyme activity within small volumes such as those provided by the microdroplets employed by

Rotman.

Likewise, although enzyme cycling, wherein the product of a first enzyme catalyzed reaction is the substrate for a second enzyme catalyzed reaction, one of whose products is a substrate for the first enzyme, thereby forming a cycle, has ^' been described for use in enzyme assays (see, for example, Siddle in Alternative

Immunoassays, Collins (Ed.), Wiley, pp. 13 - 37, 1985), there has been no appli¬ cation of enzyme cycling to the measurement of enzyme activity within microdro¬ plets.

Additionally, although flow-through-microfluorimetry has been used with 65 to 100 μ. gel particles to investigate the kinetics of immobilized β-galactosidase with the fluorogenic substrate resorufin-β-D-galactopyranoside, very large numbers of enzyme molecules were bound to the covalently gel particles because that investigation was directed towards understanding continuously stirred tank reactors. No attempt was made to measure very small amounts of enzyme within a gel particle (see Hofmann and Sernetz, Anal. Chim. Acta 163: 67 - 72, 1984). Presently, in spite of the recognized great need for improved specific binding assays, particularly immunoassays, there are many problems associated with detection limits, particularly of the homogeneous immunoassays, which have the highly desirable attribute of not requiring a solid phase or surface, and therefore not requiring washing steps. No prior specific binding assay has been based on the counting of individual labeled analyte entities such as proteins or other macro- molecules.

Summary of the Invention

The present invention consists of composite gel microdroplets (GMDs), and processes for forming composite gel microdroplets (GMDs). Such processes involve forming first GMDs, and subsequently processes for forming GMDs wherein the first GMDs are incorporated within second GMDs. This yields GMDs with two or more distinguishable regions, and can be extended to the for¬ mation of composite GMDs with a plurality of distinguishable gel regions. The various gel regions can be provided with different properties, or used to entrap biological entities at different relative locations within GMDs, or used to provide or entrap assay-assisting entities at different locations within GMDs. Further, one or more gel regions can be subsequently liquified, such that composite GMDs with one or more interior liquid regions are formed. This invention also pertains to a process for providing external influence on biological entities, wherein biological entities such as cells are contained within gel microdroplets such that the gel microdroplets are physically manipulated to be introduced into the proximity of a source, maintained in the proximity of a source, or removed from the proximity of a source of influence such as feeder ^" cells, a perfused organ or a site within living animal. Following the provision of such influence, further processes relating to measurement and isolation of the gel microdroplets, and the biological entities contained therein, can be carried out. This invention provides general means for exposing cells and other biological entities to complex sources of influence under approximate in vivo conditions, including metabolic activation, cancer radiation therapy, cancer hyperthermia therapy and cancer chemotherapy.

Finally, the invention also consists of a specific binding assay process whereby the fundamental limit of detection of individual analyte-specific binding molecule complexes molecules is the basis of a homogeneous immunoassay, such that, for example, individual antibody-antigen complexes are measured and counted. A sample is first reacted with at least two labeled specific binding molecules, and the sample used to form microdroplets. The frequency-of- occurrence of the amount of label in individual microdroplets is measured for size ranges of microdroplets, and compared to that expected from purely random occurrence. The difference in frequency-of-occurrence is then used with statisti¬ cal-analysis to compute the number of analyte entities present in the sample. Individual complex measurement and counting is best accomplished by providing th^'e-ability to detect the activity of individual enzyme molecules used as labels for specific binding molecules, such that a homogeneous enzyme linked immunoassay is achieved.

Brief Description of the Figure

The figure illustrates composite GMDs. Part (a) indicates a composite GMD (10) in which the first gel region (12) is completely surrounded by the second gel region (14). Part (b) indicates a composite GMD (20) in which the first gel region (22) is partially surrounded by the second gel region (24). Part (c) indi¬ cates a composite GMD (30) containing a four-cell microcolony (32) within the first gel region (34), which is completely surrounded by the second gel region (36). Part (d) indicates a composite GMD (40) containing a four-cell micro¬ colony (42) within the first gel region (44), which is partially surrounded by the second gel region (46). Part (e) indicates a composite GMD (50) in which the first gel region (52) is completely surrounded by the second gel region (54), which in turn is completely surrounded by the third gel region (56).

Detailed Description of the Invention

Microdroplets This invention relates to microdroplets, which are very small volume entities comprised of liquid or gel material, and which can contain zero, one or multiple biological entities. More specifically, the term microdroplet (MD) includes both the¹ gel microdroplet (GMD), the liquid microdroplet (LMD), with or without con¬ tained biological entities. Thus, unless restricted by specific use of the term "gel microdroplet" or "liquid microdroplet", the term "microdroplet" refers to both gel and liquid microdroplets. Liquid microdroplets (LMDs) are very small volumes of predominantly liquid material, which can contain solutions, typically aqueous solutions with inorganic and/or organic chemical compounds, and which can additionally contain biological entities. LMDs have volumes which are defined by a boundary comprised of another liquid, such as a non-aqueous fluid, or by a permeability barrier such as a membrane, such that the membrane is capable of retaining bio¬ logical entities of interest within a LMD, and also capable of passing other bio¬ logical entities such as molecules.

Although LMDs can be of any shape, LMDs are often approximately spheri- cal because of the tendency of interfacial forces associated with the boundaries of

LMDs to round up the deformable LMDs. Other forces, for example hydro- dynamic shear associated with stirring a LMD suspension, adhesion to a surface, or gravity, tend to cause departure from a spherical shape. Further, LMDs which contain or occupied by entities whose volume is a large fraction of the LMD volume can result in LMDs which are non-spherical. Thus, for example, a cell surrounded by a thin coating of an aqueous solution, which in turn is surrounded by a non-aqueous fluid, is a LMD. Similarly, a non-biological particle sur¬ rounded by a thin coating of an aqueous solution, which in turn is surrounded by a non-aqueous fluid, is also a LMD. If spherical, LMDs have diameters between about 0.2μ to about l,000μ, preferably between about 5μ and about 500μ. Gen¬ erally LMD volumes are between about 8 x 10^~15 to about l x 10^~3 ml, preferably between about 1 x 10^~10 to about l x 10"⁴ ml.

Liquid microdroplets can be formed by a variety of methods, which are gen¬ erally well known, and include methods based on breakup of a liquid jet, a spray- ing process, and by dispersion. For example, an aqueous liquid jet issuing into air can be forced to breakup into liquid microdroplets of nearly uniform volume (see, for example, Kachel and Menke in Flow Cytometry and Sorting, Melamed et al (Eds), Wiley, New York, pp. 41 - 59, 1979), or by spraying an aqueous liquid (see, for example, Rotman, PNAS 47: 1981 - 1991, 1961). Likewise, the use of dispersion to create emulsions consisting of a non-continuous aqueous phase of aqueous liquid microdroplets is well established (see, for example, Weaver et al., Ann. N.Y. Acad. Sci., 434: 363 - 372, 1984).

GMDs are very small volume entities which comprise at least one gel region, and which provide a mechanical matrix capable of entraping or surround- ing, without necessarily contacting, biological entities such as small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules. GMDs can consisit entirely of gel, in which case containment of biological entities can occur by entrapment of the bio¬ logical entities by the gel matrix. The general ability of gel matricies to entrap or immobilize biological entities is well known, having been established for a variety of macroscopic gel preparations such as Petri dishes, gel slabs and gel beads (see, for example, Immobilized Cells and Organelles, Vols. I and II, Mat- tiasson (Ed), CRC, Boca Raton, 1983).

Alternatively, GMDs can consist of a shell of gel matrix material which sur- rounds at least one aqueous liquid region, in which case containment of biological entites can occur by entrapment of the biological entities by the gel matrix material, or can occur by surrounding biological entities with a shell of gel matrix material, with or without contacting the biological entities.

Further, GMDs can consist of a plurality of regions comprised of gel material and liquid material. Representative configurations of GMDs with a plu¬ rality of gel regions include a first gel region entirely surrounded by a second gel region, wherein the second gel region can be comprised of a gel material different from the gel material of the first gel region. Alternatively, a second gel region can be comprise of essentially the same gel material as a first gel region, but the second gel can contain different entities such as entrapped beads and macro- molecules, or the second gel can have distinquishable molecules such as fluores¬ cent molecules attached to a constituent of the second gel matrix.

Similarly, GMDs can contain liquid regions which are surrounded by at least one gel region. Representative GMDs with such liquid regions include GMDs which consist of a shell of gel material which surrounds at least one liquid region, such as an aqueous liquid core surrounded by gel. Such GMDs provide a general means for entrapping biological entities without necessarily contacting the biological entities with a gel matrix, as it is only necessary that the gel matrix be impermeable to the surrounded biological entities, and that the gel matrix be sufficiently mechanically strong that such GMDs remain intact during any desired physical manipulation process of GMDs.

Liquid regions and gel regions of GMDs which contain no biological entities are termed non-biological regions of a GMD.

Although GMDs can be of any shape, GMDs are often approximately spher- ical, with diameters between about 0.2μ to about l,000μ, preferably between "¹⁵ to about 1 x 10^"3 ml, preferably between about 1 x 10^"10 to about 1 x 10^"4 ml.

The term gel refers to a porous matrix with a high water content. Structures have the ability to entrap biological entities while allowing transport of many molecules within the aqueous medium of the gel matrix. The gel matrix can also contain a chemical solution, typically an aqueous solution with inorganic and/or organic chemical compounds. For example, the gel matrix can contain a phy¬ siologic solution or cell growth medium, which is comprised of inorganic ions and molecules and/or organic ions and molecules. Representative natural gel material for creation of GMDs includes kappa-carrageenan, iota-carrageenan, sodium alginate, furcelaran, zein, succinylated zein, succinlylated cellulose, agarose, collagan, fibrin, proteoglycans, elastin, hyaluronic acid and glycoproteins such as fibronectin and laminin, and other naturally occuring extracellular matri¬ cies, or the like. Representative synthetic gelable material synthetic water soluble polymers include those formed from vinyl pyrolidone, 2-methyl-5-vinyl pyrridine-methyl acrylate-methacrylic acid copolymer, vinyl alcohol, vinyl pyrri- dine, vinyl pyrridine-styrene copolymer or the like.

GMDs can be created by a variety of methods, including the subdivision of a macroscopic gel volume, but preferably GMDs are formed or created by con¬ verting an aqueous suspension into liquid microdroplets, followed by formation of a gel state from the liquid state of the liquid microdroplets. Liquid microdroplets

(LMDs) are very small, deformable volumes of liquid which are surrounded by another distinct fluid, either liquid or gas, or are coated by a membrane material. A general process for creating GMDs involves first creating LMDs, wherein the LMDs are created from a liquid which contains gelable material, such that upon subsequent exposure to gelation conditions, the LMDs are transformed into

GMDs. Formation of the gel state can be caused by a variety of well known gelation processes, including temperature changes, ion concentration changes, chemical concentrations, enzyme catalysis, and photopolymerization.

The associated gelation processes may be reversible without hysteresis, reversible with hysteresis or irreversible. In the case of reversible gelation without hysteresis, LMDs can be converted into GMDs, and GMDs can be con¬ verted into LMDs, by simply reversing the conditions, for example returning to a temperature which first caused gelation. In the case of reversible gelation with hysteresis, LMDs can be converted into GMDs, and GMDs can be converted into LMDs, by reversing the conditions beyond the conditions needed to cause gela¬ tion, for example returning to and then passing a temperature which first caused gelation. In the case of irreversible gelation, the conditions for reversing the gelation process cannot be achieved without creating conditions which are harm¬ ful to the biological entities contained in the LMDs or GMDs. One example of irreversible gelation is the formation of GMDs created by photopolymerization. In one general procedure the liquid suspension is forced through a nozzle or vibrating, orifice to form a liquid stream which breaks up, either because of the surface-tension of a capillary jet, or by application of a shearing force, to form liquid^'micrσdroplets. Subsequently the liquid microdroplets are gelled by expos¬ ing the liquid microdroplets to conditions such as a temperature change, or by directing the liquid microdroplets into a solution containing ions which cause - gelation. One attribute of the nozzle or vibrating orifice GMD creation method is that most GMDs are about the same size.

Another method for creating GMDs involves the first creation of a macros¬ copic gel volume, followed by subsequent fragmentation, cutting, disruption, or subdivision of the macroscopic gel volume such that a plurality of very small volume gel fragments or gel particles are created. This general method emphasizes that GMDs need not be spherical, nor even approximately spherical. Instead, it is only necessary that GMDs consist of very small volumes of gel material, with volumes between about 8 x 10^~15 to about 1 x 10^~3 ml, preferably between about 1 _c 10^~10 to about 1 x 10"⁴ ml.

It is generally preferred to use a dispersion method for creating GMDs from a liquid suspension, as dispersion methods are simpler, less expensive and gen¬ erally freeer from clogging problems than are fluid jet methods. The dispersion methods consist of dispersing the liquid suspension into an immiscible liquid such as a heavy alcohol, mineral oil or silicone fluid, by means such as stirring or vor- texing the liquid suspension and immiscible liquid together, thereby creating liquid microdroplets surrounded by the immiscible liquid. The liquid microdro¬ plets are gelled during or after the dispersion process by any of a variety of well known gelation processes such as ion exchange or temperature change. Disper- sion.methods generally create GMDs with a relatively wide range of sizes, for examples diameters of about 5μ to 500μ.

GMDs can also be formed by the process of fragmenting, cutting, disrupting or otherwise converting a macroscopic volume of gel into very small volume gel particles, such that said GMDs can have irregular shapes. For example, a macroscopic gel slab can be formed in which biological entities such as cells are entra ed at random ositions, the el slab can be cooled to a low tem erature, and the gel slab then mechanically impacted so as to fragment the macroscopic gel into pieces, many of which have a very small volume and thereby constitute GMDs.

GMDs can also be formed by processes which cause a gel coating to form around one or more entities, such that the gel entirely surrounds, or essentially surrounds, the entities. For example, by contacting cold cells with a warmer solution of material which contains gel material which gels upon cooling, a coat¬ ing of gel can be formed around the cells, such that the cells are thereby incor¬ porated into GMDs. In this case the GMDs can be markedly non-spherical, as the gel coating often forms with the shape of the cells. Similarly, non-biological entities such as cell culture microcaπier beads, soil particles and food particles can be incorporated into GMDs by gel-coating processes, such that the resulting GMDs often have shapes which approximate the incorporated non-biological enti¬ ties. In those cases wherein GMDs are formed within a non-aqueous fluid, it is often desireable to transfer the GMDs into an aqueous medium, in order to expose biological entities to a variety of conditions relating to growth, metabol¬ ism, secretion, transmembrane potential development, membrane integrity and enzyme activity, and also to conditions which favor measurement and isolation of GMDs. An exemplary method for such transfer is gentle agitation if the GMD aqueous interiors contain suitable surfactant agents, including naturally occuring surfactants such as those present in serum.

Composite Gel Microdroplets

Composite GMDs are GMDs which contain more than one distinguishable region of gel material or of liquid material, which gel or liquid materials are non-biological regions which may entrap or surround biological entities, and can be formed by several methods. Thus, a composite GMD is characterized by a plurality of non-biological regions which are further characterized by having at least one non-biological region having a first property surrounding substantially, or entirely, all of at least one non-biological region having a second property

Composite GMDs can contain both gel and liquid regions, wherein Uquid regions are surrounded by one or more gel regions, so that such composite GMDs must contain at least one gel region However, in order to be useful for making meas¬ urements on biological entities, and for isolating biological entities, at least some composite GMDs contain biological material In one general method the aqueous suspending medium is provided with any cells, microbeads or other marker entities or force-coupling entities which are desired to be incorporated or entrapped in a first gel region. First GMDs are then formed from a first gellable material, using any of processes described elsewhere in this disclosure. The first GMDs are then suspended in a medium containing a second gellable material, which second gellable material may be of the same or difSaren composition as the first gellable material. Additionally, the second gel¬ able^ material can be comprised, partially or entirely, of material which has optical properties such as light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence, so as to .dis¬ tinguish the second gel region from the first gel region. In this way, by using any combination of one or more of such methods, the second gel can provide compo¬ site GMDs, in this case of two distinguishable gel regions, termed GMD/GMDs, with desirable optical properties. For example, an optical signal for GMD diame- ter determination can be obtained from the second gel region while avoiding, with high probability, the contacting of first GMD entrapped cells with the second gel region.

More generally, it is useful to form composite GMDs wherein at least one region contains a first material with a first optical property selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence, and a second region contains a second, optically distinguishable material with optical properties selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chem- iluminescence.

Alternatively, marker entities including beads, non-biological particles, cry¬ stals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts, vesicles, stains and dyes can be incorporated into the first gel region or first GMDs, and biological entities such as small multicellular organ- isms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules can be incorporated into the second gel region in order to provide means for enhanced measurement of composite GMDs. In this case, composite GMDs with at least one gel region containing marker entities selected from the group consisting of beads, non-biological particles, crystals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts, vesicles, stains and dyes are provided Such composite GMDs are par¬ ticularly useful in the case that the optical properties of the marker entities are selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chem- 5 iluminescence. Composite GMDs can also be characterized by a plurality of non-biological regions which are further characterized by having at least one non-biological region having marker entities surrounding substantially, or entirely, all of at least one non-biological region having a second property.

In order to form composite GMDs, several general processes can be used.

10 One method for producing GMDs having a plurality of non-biological regions, with at least one non-biological region having different properties than at least one other non-biological region, consists of the following general steps: (a) form¬ ing GMDs of a first gel using any of the processes described elsewhere in this disclosure, (b) suspending the gel microdroplets in a material capable of forming

15 a second gel, and (c) incorporating gel microdroplets of the first gel into gel microdroplets of the second gel, thereby forming gel microdroplets with distinct non-biological gel regions.

Another general method for producing GMDs having a plurality of non- biological regions, wherein at least one non-biological region has different proper- 20 ties than at least one other non-biological region, consists of the following general steps: (a) forming GMDs, (b) suspending said GMDs in a material capable of forming LMDs, and (c) incorporating GMDs of the first gel into LMDs, thereby forming composite GMDs with distinct non-biological regions, in this case com¬ posite GMDs with one or more liquid regions.

25 Still another general method for producing GMDs having a plurality of non- biological regions, wherein at least one non-biological region has different proper¬ ties than at least one other non-biological region, involves the following general steps: (a) forming GMDs of a first gel capable of liquification; (b) suspending said GMDs in a material capable of forming a second gel, (c) incorporating

30. GMDs of said first gel into GMDs of said second gel, and (d) liquifying the first gel, thereby forming GMDs with distinct non-biological liquid regions, in this case also with at least one liquid region.

In order to use the gel microdroplets of this invention in processes involving measurement and/or isolation of biological entities, the composite GMDs should 35 be formed from a suspension or solution which contains the appropriate biological entities, and therefore which contains biological material The composite GMDs of this invention are useful in cases wherein the biological material is composition of biological entities such as small multicellular organisms, groups of cells, indi¬ vidual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of 5 molecules , and is particularly useful in cases wherein the cells are selected from the group consisting of animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells,

or are selected from the group consisting of normal human cells, human cancer cells, pathogenic bacteria, pathogenic yeast, mycoplasms, parasites, and patho_^ 0 genie viruses.

Force coupling entities such as beads, non-biological particles, bubbles, and non-aqueous fluid inclusions with force coupling properties selected from the group consisting of ferromagnetic properties, diamagnetic properties, paramag¬ netic properties, dielectric properties, electrical charge properties, electrophoresis 5 properties, mass density properties, and optical pressure properties can be also incorporated into one or more gel regions, or liquid regions, in order to provide means for physical manipulation of composite GMDs. Thus, it is useful to pro¬ vide composite GMDs which contain one or more regions with contain force- coupling entities such as beads, non-biological particles, bubbles, and non- 0 aqueous fluid inclusions with force coupling properties Such provision of force- coupling entities allows composite GMDs to be manipulated by applying forces such as

The invention also includes extension of the basic process to the creation of composite GMDs comprising more than two distinguishable gel regions. For 5 example, composite GMDs which are GMD/GMDs can be used in a GMD for¬ mation process to form GMD/GMD/GMDs, that is, composite GMDs with three distinguishable gel regions.

GMDs containing more than one non-biological gel region can be formed, such that composite GMDs are thereby formed, wherein said composite GMDs f ' are comprised of regions of different gel material, and/or of the same gel material but with different entrapped or bound entities. Thus, for example, a GMD which is a composite GMD made from two different gel materials can contain a first inner region which is comprised of a soft, low density gel such as 0.5% agarose, and a second outer region which is comprised of a harder, higher density gel such nuin can support the growth of cells with less compressive force on the cells, while the second outer region can better confine the growing cells.

GMDs containing at least one liquid region can also be formed. An exem¬ plary process for formation of GMDs wherein a gel material region surrounds a liquid region is as follows. A first step comprises using a process to form GMDs from a gel material capable of subsequent liquification, a second step comprises formation of GMDs which consist of a second gel region completely surrounding the first GMDs, such that the second gel material is different from the first gel material, and is capable of remaining a gel under conditions which liquify the ' ^■ first gel, and a third step comprises liquification of the first gel material, with the result that GMDs with a liquid region surrounded by a gel region are thereby formed. A more specific illustration of this process is as follows. The first step comprises forming liquid microdroplets which contain sodium alginate by forcing a suspension of biological entities with sodium aliginate through a vibrating ■ orifice, thereby breaking up the resulting liquid jet which contains both biological entities and sodium alginate, allowing the resulting liquid microdroplets to enter an aqueous medium containing calcium ions, thereby forming calcium alginate GMDs. The second step comprises concentrating the calcium alginate GMDs by means such as filtration and centrifugation, adding molten agarose at about 37°C, following which the calcium alginate GMD suspension is dispersed into mineral oil, and cooling the dispersion, thereby forming agarose GMDs which contain alginate GMDs, The third step comprises exposing said composite GMDs to an aqueous solution containing sodium chloride and essentially zero calcium, such that sodium and calcium ions can be exchanged, and thereby liquifying the cal- _^' cium alginate within the composite GMDs.

For convenience it is useful to term GMDs comprised of more than one gel and liquid region as composite GMDs, and to use notation such that GMD/LMDs refers to GMDs formed with a first formed region which is liquid and a second formed region which is gel, and GMD/GMD refers to GMDs formed with a first region which is gel and a second formed region which is gel. Thus, in the preceeding more specific illustration, upon completion of the second step GMDs comprising GMD(agarose)/GMDs(calcium alginate) are formed, and upon com¬ pletion of the third step GMDs comprising GMD(agarose)/LMDs(sodium alginate) are formed. Measurements of Microdroplets

The term measurment refers to the process of quantifying the amount of a parameter, and includes the term detection, as detection is a coarse measurement which determines whether or not a parameter is greater than or equal to a thres- -^• Hold condition, or is less than a threshold condition. Thus, for example, optical measuremeπt-of a microdroplet involves quantifying at least one optical signal associated wi^'tifcai microdroplet. The term quantifying refers to assigning a value to the signal, such that said quantifying has a resolution which allows the parame¬ ter to be assigned one of two more than two different values. Li contrast, detec- ^' tion refers to measurement wherein the resolution allows the parameter to be assigned to only one of two values, one value which corresponds to sub-threshold and therefore non-detection, and the other which corresponds to threshold or supra-threshold and therefore to detection.

Although many useful processes relating to measurement and manipulation of microdroplets, and of any biological entities contained therein, can be carried out without explicit measurements of microdroplet parameters, enhanced measure¬ ment and manipulation is often achieved if microdroplet parameters such as microdroplet volume, V_MD, microdroplet diameter, D_UD, (if approximately spheri¬ cal), microdroplet mass and microdroplet mobility are determined. The volume, V_MD , of microdroplets can be measured by measuring signals associated with the interface between two fluids which define a MD, or by signals associated.with the difference in physical properties of the fluid within a MD and the fluid external to a MD. The physical basis for V_MD measurement can be selected from the group consisting of optical, weighing, sedimentation, field flow sedimentation fractionation, acoustic, magnetic, electrical and thermal measure¬ ment.

A variety of measurements can be based on a mass density difference between the fluid within a MD and the fluid external to a MD, and include weighing measurement on a microbalance such as a submerged piezoelectric sen- sor, sedimentation measurement based on an acceleration field such as gravity, rotationa acceleration such as centripetal acceleration which is the basis of centri- fugation, and/or non-rotational acceleration, which is used to separate MDs on the basis of size and field flow sedimentation fractionation measurement wherein MDs are gently separated according to size (see, for example, Levy and Fox, Biotech. Lab. 6:14 - 21, 1988). Other methods which are partially based on include measurements based on acoustic measurement wherein variation in acous¬ tic properties of MDs relative to the surrounding fluid are utilized (see, for exam¬ ple Quate, Physics Today, August 1985, pp. 34 - 42), magnetic measurement wherein differences in magnetic properties, particularly paramagnetic, diamagnetic and ferromagnetic properties, are utilized, and thermal measurement wherein differences in thermal properties relative to the surrounding fluid are utilized, par¬ ticularly differences in thermal conductivity, thermal diffusivity and specific heat (see, for example, Bowman et al, Ann. Rev. Biophys. Bioengr. 4: 43 - 80, 1975).

The generally preferred method of measuring V_m involves optical measure- ments selected from the group consisting of light scattering, light absorbance, fluorescence, phosphoresence and chemiluminescence, as optical measurements are flexible, rapid and non-contacting measurements. Exemplary optical measure¬ ments using light scattering can be based on differences in the index of refraction between the aqueous fluid within MDs and a non-aqueous fluid external to a MD, or can be based on differences in light absorbance or colorimetry, phosphoresence or chemiluminescence between the aqueous fluid within MDs and the non- aqueous fluid.

More specifically still, it is preferred to utilize differences in fluorescence of the MD relative to the surrounding fluid, as fluorescence is particularly sensitive. Although both the fluid within a MD and external to MDs can be fluorescent, it is preferred to make measurements wherein either the fluid within the MD, or the non-aqueous fluid external to the MD, have significant fluorescence. Thus, for example, at least one fluorescent molecule type can be incorporated in MDs, such that when surrounded by a non-fluorescent, non-aqueous fluid the volume of a MD can be determined by the total fluorescence intensity associated with the fluorescent molecule, and subject to the further condition that said fluorescent molecule not significantly partition into the surrounding non-aqueous fluid. Still more specifically, a fluorescent molecule such as FTTC-dextran can be incor¬ porated into the aqueous medium which comprises the fluid within a MD, and the total fluorescence emission intensity of FITC-dextran measured. Likewise, at least one fluorescent molecule type can be incorporated into the non-aqueous fluid which surrounds MDs, such that the decrease in fluorescence associated with the presence of a non-fluorescent MD provides the basis of V_MD measurement (see, for example, Gray et al, Cytometry 3: 428 - 434, 1983). In addition to the measurement methods which can be used with microdro¬ plets generally, GMDs containing one or more marker entities can also be measured by measuring signals associated with at least one marker entity which is incorporated into a gel matrix of at least one GMD, wherein said marker entity is capable of measurement In the case of GMDs which exist prior to measurement, marker entities can be incorporated into the pre-existing GMDs prior to measure- ment. Alternatively, marker entities can be incorporated into GMDs by supplying marker entities in the aqueous medium from which GMDs are formed, thereby incorporating marker entities into GMDs during the formation of GMDs

The volume, V_CMD, of a GMD can be determined by first measuring the marker entities contained within a GMD, followed by analysis of the amount of marker entities in at least one gel microdroplet so as to determine the size or volume of said gel microdroplet. Statistical analysis can be applied to one or more types of measurements relating to GMD properties, and/or to one or more types of measurements relating to biological enities. It is preferred to combine measurement of biological entities with measurement of V_GMD , so that statistical analysis relating to the number of biological entities in GMDs can be used, and for such combined measurements biological entities are incorporated into GMDs prior to measurement of the GMDs based on marker entities.

Exemplary types of marker entities include beads, non-biological particles, crystals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts, vesicles, stains and dyes. Non-biological particles include particles comprised of inorganic material such as silica, of organic material such as charcoal or carbon, and of combinations of inorganic and organic material wherein the organic material can be of biological or non-biological origin. Such marker entities allow a variety of measurement to be made, including optical, weighing, sedimentation, field flow sedimentation fractionation, acoustic, mag¬ netic, electrical and thermal measurements However, it is preferred to measure GMDs optically by using marker entities which can be measured by using light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluores¬ cence, phosphorescence and chemiluminescence. In cases wherein the presence of marker entities does not adversely affect biological entities contained within GMDs, or the ability of gel material to form a gel matrix, gellable material can be pretreated so as to attach at least one type of marker entity to at least one gellable material prior to formation of GMDs, thereby resulting in formation of GMDs having enhanced measurement properties. Thus, for example, pretreatment of gelable material by chemically attaching marker entities com risin fluorescent molecules renders the GMDs b measurable by fluorescence. Alternatively, GMDs can be first formed, and marker entities subsequently introduced. For example, macromolecules such as dextrans can be labeled with a fluorescein derivative, agarose GMDs exposed to said macromolecules, whereupon the fluorescent dextran can diffuse into the agrose, and then be subsequently precipitated or complexed, such that the dextran is essentially trapped within the gel, and the GMDs are thereby provided with marker entities in the form of fluorescent labeled dextran

In some preparations of GMDs, a significant number of biological entities, such as cells, can either remain free in suspension, or can escape from some types of GMDs during vigorous physical manipulation. This results in suspensions which contain both cells trapped within GMDs and free cells, wherein the term free cells refers to cells free in suspension and not contained within MDs. This occurrance is usually undesirable. Thus it is highly desirable to provide measure¬ ment means for distinguishing free cells from cells contained within GMDs. For example, the formation of microcolonies within GMDs provides a general method for determining growth of biological entities, particularly cells, and allows direct determination of plating efficiency following an incubation by quantitatively com¬ paring the number of microcolonies to single cells. However, if significant free cells can sometimes occur in suspension, and free cells usually cannot be dis- tinguished from cells contained within GMDs, so that significant error can result.

In such cases it is preferred to measure at least one type of marker entity con¬ tained within GMDs, such that it is possible, by measurement of both marker entities and biological entities, to determine that there is a high probability that the biological entity is associated with a gel microdroplet. In other cases the measurement of marker entities can comprise detection, wherein the detection of one or more GMDs allows measurements of biological entities to be associated, with high probability, with the containment of said biological entities within GMDs. For example, non-growing individual cells within GMDs can be dis¬ tinguished from individual cells which are free in suspension Often it is also desirable, in order to enhance subsequent statistical analysis, to determine V_GMD for a GMD associated with a measured biological entity. In this case the measured parameter used for a marker entity type is selected to be distinguishable from measured parameters which relate to measurements of bio¬ logical entities. For example, biological entities which contain double stranded nucleic acids can be measured by using well known staining protocols utilizing propidium iodide (PI), and measuring the Red Fluorescence associated with PI, while marker entities such as entrapped microbeads, or covalently attached fluorescein, with Green Fluorescence provide the basis for measurement of V_Giω, as the magnitude of the Green Fluorescence signal is proportional to V_GUD . The combined measurements of V_GMD and biological entities further provide the basis for determining the frequency-of-occupation of GMDs by biological entities, and thereby, enhance statistical analysis methods such as those provided by using Pois- so statistic-Sϊor modified Poisson statistics.

Marker-entities can be selected with a variety of physical properties which result in enhancement of GMD measurement upon incorporation of said marker " entities into GMDs. Useful physical properties which provide the basis for meas¬ urement of marker entities include optical properties, mass density properties, acoustic properties, magnetic properties, electrical properties and thermal proper¬ ties. Because of their speed, specificity, non-perturbing nature and non-contacting nature, it is preferred to use optical measurement means, including flow cytometry ^" apparatus, flow-through-microfluorimetry apparatus, optical particle analyzer apparatus, fluorescence microscopy apparatus, light microscopy apparatus,image analysis apparatus and video recording apparatus to measure marker entities. More specifically, in the case of flow cytometry, it is useful to measure optical pulses such as maximum pulse magnitude, pulse time integral and pulse duration, all of which are well known (see, for example, Shapiro, Practical Flow

Cytometry, A. R. Liss, New York, 1985).

The marker entities can also be measured using well known electrical meas¬ urements, particularly electrical resistance measurements, electrical measurements which- provide the basis of particle analysis, such as the electrical resistance based particle- measurements (see, for example, Kachel in Flow Cytometry and Sorting,

Melamed et al (Eds), Wiley, New York, pp. 61 - 104), and dielectric property measurement (see, for example, Harris et al, Enzyme Microb. Technol. 9: 181 - 186, 1987). These electrical measurements are well known and generally desir¬ able because of the relative ease and relative low cost of making such electrical ^". measurements.

Statistical Analysis of Measurements

The use of microdroplets generally provides means for making a large number of individual measurements relating to a biological sample. This is in constrast to most established measurement methods, as most established measure- ment methods are responsive to the total effect on measured parameters by biological entities contained in a sample. Thus, although useful measurements can be made using small numbers of microdroplets, in general, the use of large numbers of individual microdroplet measurements provides the basis for making significantly improved measurements on biological entities of a sample. Although significant measurement information can be obtained without explicitly carrying out statistical analysis of large numbers of microdroplet measurements, significant improvement in measurements is achieved by applying statistical analysis to microdroplet measurements.

Measurements on microdroplets are often made wherein more than one parameter is measured. For example, it is generally preferred to use optical measurements, particularly fluorescence measurements, in which case simultane¬ ous measurements such as Green Fluorescence measurement and Red Fluores¬ cence measurement are often made. In the exemplary case of measurements relating to a mixed biological population, a Green Fluorescence labeled antibody can be used to measure the amount of biological material associated with a first type of biological entity, and a Red Fluorescence labeled antibody can be used to measure the amount of biological material associated with a second type of bio¬ logical entity. Following an incubation the magnitude of the Green Fluorescence and Red Fluorescence signals can be used to determine the amount of growth of each type of biological entity, such that the frequency-of-occurrence distribution of the Green and Red Fluorescence signals can be obtained, and then statistically analyzed to determine the variation in growth, and the variation in lag time, for both types of biological entities. In this exemplary case, however, it is not neces¬ sary to use statistical analysis in combination with microdroplet volume measure- ment, but only with the Green and Red Fluorescence measurements. Thus, although statistical analysis of microdroplet measurements is generally useful, sta¬ tistical analysis does not necessarily involve the use of microdroplet volume measurements, nor does statistical analysis necessarily relate to occupation of microdroplets. Instead, statistical analysis can relate to the frequency-of- occurrence of measurements relating to the biological entities themselves.

Individual and Multiple Microdroplet Occupation

In many cases it is useful to determine, at least approximately, the statistical distribution of occupation of microdroplets by biological entities. As used herein, the term "occupation" refers to the presence of initial biological entities, that is, those biological entities present shortly after formation of microdroplets, and before any incubation is used. Thus, for example, in the representative case wherein biological entities are cells, microdroplets can have a high probability of zero occupation, individual occupation, or of multiple occupation. As used herein, zero occupation or unoccupied refers to the case wherein a microdroplet contains zero initial cell, individual occupation refers to the case wherein a micro¬ droplet contains one initial cell, and multiple occupation refers to the case wherein a microdroplet contains at least two initial cells. Following an incuba¬ tion, growth may occur and result in increases in size and number of cells, such that an individually occupied microdroplet subsequently contains progeny cells of the initial single cell, and is nevertheless termed an individually occupied MD, and a multiply occupied microdroplet subsequently contains progeny cells of the initial multiple cells, and is nevertheless termed a multiply occupied MD.

In the general case wherein a microdroplet contains at least two types of biological entities, the term occupation can be used separately with each type of biological entity. Thus, for example, in the case of a mixed biological population comprised of two types of cells, type A and type B, a microdroplet can initially contain, prior to any incubation, one A cell and several B cells. In this case the microdroplet is termed individually occupied by type A cells and multiply occu¬ pied by type B cells, and the same designation is also used subsequent to any incubation which results in growth. That is, continuing this example, if incuba¬ tion subsequently leads to A cell progeny, the microdrop is still deemed individu¬ ally occupied by type A cells. This terminology is straightforwardly extended to all types of biological entities.

Analysis Using Poisson Statistical Methods It is often preferred to make measurements on microdroplets that are indivi¬ dually occupied, so that the measurements can be interpreted as measurements relating to one biological entity. In order to form microdroplets which have a ^• significant fraction of microdroplets with individual occupation, it is generally useful to estimate the distribution of occupation for different size microdroplets. Many methods for forming microdroplets, in which biological entities are incor¬ porated into microdroplets, are random, or well approximated by randomness, such that statistical analysis involving one or more MD parameters, such as diam¬ eter or volume, is useful for determining the probability of occupation of different size microdroplets. As a result, it is generally useful to determine the size of microdroplets that have a high probability of having zero, individual or multiple occupation, so that the probability of having less than two initial biological entities, and of having at least two initial biological entities, in microdroplets of different size or volume 5 ranges can be estimated. If the concentration of the suspended biological entities is known approximately, or can be estimated, then the suspension can be diluted so as to provide an average, known number of biological entities in liquid micro¬ droplets of a particular size or volume being made. A mathematical formula or equation which describes the relation between the average number of biological

10 entities and liquid microdroplet volume is the Poisson probability distribution,

P .n fi), (see, for example, Gosset, Biometrika, 5: 351 - 360, 1907; Weaver et al., Ann. N.Y. Acad. Sci., 434: 363 - 372, 1984; Weaver, Biotech, and Bioengr. Symp. 17, 185 - 195, 1986; Williams et al., Ann. N. Y. Acad. Sci., 501: 350 - 353, 1987), which in its application to microdroplets gives the probability, P(njϊ),

15 of finding a particular number, n , of initial biological entities in microdroplet volume V_MD if the average number of initial biological. entities found in the volume V_MD is n . More specifically

A mathematical relation governing n is n = ρV_MD , where p is the concentration of 20. the biological entities in the suspension which was converted into liquid micro¬ droplets, and the term "average occupation" is defined to be n, and refers to the average or mean number of initial biological entities present before any incuba- . tion. For a sample with randomly distributed biological entities, such as a well- stirred sample, the probability of finding particular numbers of biological entities 25 is described by the Poisson formula. Thus, the probability of having zero initial biological entities or being unoccupied is P (0,7f), the probability of having one biological entity or being individually occupied is (l,n), the probability of hav¬ ing two biological entities is PQ-n), and so on. Of particular interest to some applications of GMDs is the situation wherein a LMD is initially occupied by 0^; more than one entitiy, that is, multiply occupied. The probability of multiple occupation is P(>ljϊ), and, because the sum of all possible probabilities equals one, the probability of initial multiple occupation is as follows: Pι>lmn) ^~- l - P(0JΪ) - P(l r) (2)

Generally, the transition from LMDs to GMDs does not involve significant changes in volume, i.e. V_UD = v_mD = V_GMD in most cases. In such cases the Poisson probability can be used interchangably with either LMDs or GMDs. If the.- volume change is significant, a volume scaling factor, /_vo , is used according to V_GMLr = f_wiVi_MD , such that there is a common relation between the volumes of all GMDs and the LMDs from which they were created. This scaling factor, /_Ϊ0/J is generally a well known macroscopic property of gel materials.

In the process of creating MDs, a sample volume, V_s, is mixed with a volume of additional material, V^a. In the case of GMD formation, the volume

Vj_u usually contains gellable material which forms the gel matrix of the subse¬ quent GMDs. The corresponding dilution by a factor f_D is as follows:

fo = V_s + V_Add (3)

and is straightforwardly computed following measurement of both V_s and V_G. The diluted biological entity concentration, p, is therefore related to the sample biological entity concentration, p_s by the following:

where p is the biological entity concentration used in the Poisson equation.

In the case of biological entities which naturally aggregate, in the Poisson equation, the mathematical parameters n, n and ρ_s refer to the particular number of aggregates, the average number of aggregates and the concentration of aggre¬ gates, respectively. Thus, for biological entities which naturally aggregate as colony forming units (CFU), in the Poisson equation n, n and p_s refer to the par¬ ticular number of CFUs, the average number of CFUs and the concentration of CFUs, respectively.

In some cases it is desirable to make measurements on more than one MD at a time, and thereby to make measurements on clusters or groups of MDs which are associated with individual MDs. In this case the Poisson statistics formula is applied with the change that V_m is replaced by V._roup, wherein V._roup is the sum of the individual MD volumes in the group.

In some cases, such as those wherein the volume of n biological entities, nV_BE, within a MD is a significant fraction of the volume of a MD, V_MD , it is use¬ ful to employ a modified form of the Poisson formula. An example of a modified Poisson function is given in equation (5).

P(n,n) ^~- $-- -- with n ~~ p(V_UD - nV_BE) (5)

in which the "available volume" = V_UD - nV_BE replaces V_MD , which provides a better description of the frequency of occupation for cases wherein V_BE is a significant fraction of V_MD . This approximate equation uses the definition that the volume of a MD containing n cells is defined to be the volume of the gel material plus nV_BE, and is consistent with the inability of two or more nominally idential biological entities initially occupying a MD of volume less than 2V_BE. In the case that non-identical biological entities are initially present, the quantity nV_BE is replaced by V._0U.ι s_E, the total volume of the biological entities.

Another approximate, modified version of the Poisson function is is most readily presented in terms of a separate mathematical expression for each of the probabilities, _°(0,n), P( Zn). P(2,W), etc. The first two expressions are

P(0 i) = 1 if V_MD < V_BE and _e ⁽-" ^{+ p}^ if V_MD ≥ V_BE (6a)

P (1,7T) - 0 if V_m < V_BD ; (1 - e ^{n + p β£)}) if V_BE < V_MD ≤ SV_BE (6b)

These modified Poisson forumulae or equations agree with equation (1) in the mathematical limit that V_BE approaches zero, but provide better descriptions results in cases wherein the volume of the initial biological entities within a MD is a significant fraction of V_MD .

Further, other extensions or modifications of statistical analysis can be used to analyze cases wherein the analyzed MDs have a significant range of volumes. The basic concept underlying such analysis is that each size range of MDs separately obeys the same probability equation, specifically Poisson statistics or modified Poisson statistics.

The preferred embodiment of the invention initially uses conventional Pois- 5 son statistics for all cases, but then uses either the version given by equation (5) ox by equations (6a), (6b), (6c) and extensions thereof, if occupations of small volume MDs differ significantly from those obtained from large volume MDs in the same preparation made from the same sample. It is also possible to utilize any other extension of Poisson statistics which provides descriptions of the proba- 0^"= bility of occupation in the case that V_BE is a significant fraction of V_MD. Depend¬ ing on the relative size, shape and means for forming MDs, other version of modified Poisson statistics formulae may be utilized.

The most conceptually simple measurements relate to measurements which simultaneously measure one MD. Such measurements can be made using a 5 variety of measurement apparatus based on optical, weighing, sedimentation, field flow sedimentation fractionation, acoustic, magnetic, electrical and thermal means, but preferably optical means in the form of flow cytometry apparatus, flow- through-microfluorimetry apparatus, optical particle analyzer apparatus, fluores¬ cence microscopy apparatus, light microscopy apparatus,image analysis apparatus 0 and video recording apparatus. Electrical means can include dielectric property measurement apparatus or a particle analyzer based on electrical resistance meas¬ urement As before, microdroplet measurements are made in such measurement apparatus by operating the apparatus in a mode wherein there is a high probabil¬ ity that less than two MDs are simultaneously within the measurement volume of 5^' the apparatus. Thus, in the exemplary case of flow cytometry, the instrument is operated in a mode wherein there is a high probability that less than two MDs are simultaneously in the focal volume of the optical illumination region. Further, in the exemplary case of microscopy, the instrument is operated in a mode wherein there is a high probability that less than two MDs are simultaneously in the field D^' view used in a measurement. A general advantage relating to making measure¬ ments on less than two MDs simultaneously relates to simplicity of interpretation, as measurements can be interpreted in terms of measurements on individual bio¬ logical entities or their progeny.

Somewhat more conceptually complex measurements relate to measurements 5 which simultaneously measure at least two MDs, wherein a plurality of MDs is termed herein as a rou or cluster of MDs. As described elsewhere in the present disclosure, such measurements can be made using a variety of measure¬ ment apparatus based on optical, weighing, sedimentation, field flow sedimenta¬ tion fractionation, acoustic, magnetic, electrical and thermal means, but preferably optical means in the form of flow cytometry apparatus, fiow-through- microfluorimetry apparatus, optical particle analyzer apparatus, fluorescence microscopy apparatus, light microscopy apparatus,image analysis apparatus and video recording apparatus, or electrical means in the form of dielectric property measurement apparatus or a particle analyzer based on electrical resistance meas¬ urement Microdroplet measurements are made in such measurement apparatus by operating the apparatus in a mode wherein there is a high probability that at least two MDs are simultaneously within the measurement volume of the apparatus. Thus, in the exemplary case of flow cytometry, the instrument is operated in a mode wherein there is a high probability that less than two MDs are simultane¬ ously in the focal volume of the optical illumination region. Further, in the exemplary case of microscopy, the instrument is operated in a mode wherein there is a high probability that less than two MDs are simultaneously in the field view used in a measurement. Advantages relating to making measurements on at least two MDs simultaneously include larger measurement throughput rates of MDs, for example in cases wherein many MDs are unoccupied, and reduced technical complexity of measurement apparatus because of generally larger meas¬ urement region volume.

The most conceptually simple use of microdroplets involves MDs with indi¬ vidual occupation, as measurements of individually occupied MDs can be readily related to measurements of biological material associated with an initial individual biological entity. For example, measurement of an individually occupied MD provides the basis for analysis and interpretation of growth of an initial biological entity.

A more conceptually complex use of microdroplets involves microdroplets with multiple occupation. Multiple occupation may be selected for a variety of reasons, such as allowing more biological entities to be measured for the same number of MDs, or to provide biological entities initially at a higher concentra¬ tion within MDs. The multiply-occupied MD measurement itself, however, is essentially equivalent to the measurement of multiple MDs, that is, measurement of groups or clusters of two or more MDs. As used herein, measurement of mul- tiple MDs consists of either the making or combining of measurement of two or more MDs. For example, the mutliple measurement of five MDs consisits either of making a simultaneous measurement on five MDs, or on making measurements of any subgroup of the five MDs followed by summing the subgroup measure¬ ments to obtain the multiple MD measurement.

Even though measurement results cannot be as readily interpreted in terms 5 of individual biological entities, advantages of making measurements on multiply occupied MDs include making measurements on biological entities at a higher rate in comparision to individually occupied MDs, and to lower costs associated with requiring less material and time to make measurements. In addition, impor¬ tant information can often be obtained which cannot be readily obtained from

10 non-microdroplet measurement methods. Specifically, measurement of multiply occupied MDs provides the basis for analysis and interpretation of growth of ini¬ tial biological entities, wherein said growth is the average growth associated with the occupation of the MD. Similarly, secretion by one hyperactive biological entity, or its progeny, can be measured in the presence of other, poorly secreting

IS^"* biological entities. For example, in the case that MDs with average occupation by three cells is measured, the measurement can still reflect any significant varia¬ bility which is present in the cells, as the measurement of average property of three cells can be significantly affected by one unusual cell, thereby determining that an unusual cell is present. Measurements can be useful with multiply occu-

20 pied MDs containing 2 to about lO³ initial biological entities, but are preferably used with multiply occupied MDs containing 2 to about 10 initial biological enti¬ ties.

For example, consider the illustrative case wherein according to Poisson statistics the mean or average occupation is three, so that W = 3, the mathematical 255 Poisson probability formula predicts the distribution of particular occupation values, n, given in the table below.

n P(njT=3) n P(n,π=3)

0 0.050 5 0.101

1 0.149 6 0.050 3 ^'^ 2 0.224 7 0.020

3 0.224 8 0.008

4 0.168 In this illustration, there is a low probability (0.05) of having unoccupied MDs, a high probability (0.95) of having occupied MDs. The probability of individual occupation is 0.15 so the probability of multiple occupation is 0.80. Thus, 80% of MDs initially contain at least two biological entities. However, even in this ^" example for which the mean occupation is high, the multiply occupied MDs have a peaked distribution of occupation, such that there are equal, maximum probabil¬ ities of 0.224 of having either 2 or 3 initial biological entities, and rapidly decreasing probabilities of having 4 or more initial biological entities. As a result, the probability, P (>7,Λ) of having more than seven (7) initial biological 0 entities is

which is low, about 0.006. In summary for this illustration, the use of multiply occupied MDs with n = 3 results in multiply occupied MDs for which only n = 2 to 7 have significant probability. Thus, for example, in contrast to relatively macroscopic preparations such as test tubes, cell culture flasks and microtiter wells which are often used with thousands of biological entities such as cells, and which therefore usually result in small effect on the total population by one biological entitiy, in the present exam¬ ple it is highly probable that a single biological entity will have seven or fewer other biological entities within the same MD. For this reason, an unusual pro¬ perty of such a single biological entity, such as significantly greater growth or significantly greater secretion rate, can have a large fractional effect, and thereby be measured, although non-optimally, in the presence of the other biological enti¬ ties within the same MD. J It is also possible to straightforwardly carry out measurements on multiple

MDs, that is groups or clusters of MDs, wherein one or more of the MDs are multiply occupied. Such a process of measuring multiple multiply occupied MDs is equivalent to measuring one large MD of same total volume as the sum of the MD volumes of the MDs which comprise the group or cluster of MDs, and can have the advantage of providing still larger throughput measurement rates while still retaining other advantages of MDs, such as the generally short characteristic diffusion time, τ_D. Manipulation of Microdroplets Surrounded by Non-Aqueous Fluids

It is generally desirable to provide means for applying forces on MDs, so as to provide means for manipulating MDs, such as changing or maintaining the locations of MDs, so that external influence from physical, chemical and biologi- cal sources can be provided, for purposes such as measurement, incubation and isolation. It is useful to distinguish two classes of processes for applying forces which accomplish changing or maintaining positions of MDs. These two classes are forces which depend on MDs being surrounded by a non-aqueous fluid, and forces which depend on MD force coupling entities, or intrinsic properties of MDs, which are surrounded either by a non-aqueous fluid or an aqueous fluid.

In the case of MDs which are GMDs, following GMD creation in a non- aqueous fluid, the GMDs are left suspended, allowed to settle, or captured on a grid or filter, while surrounded by the non-aqueous fluid, such that the GMDs can now be manipulated through the use of physiochemical interactions that exploit differences in properties of the aqueous GMD interior fluid and the GMD- surrounding non-aqueous fluid.

Alternatively, if GMDs are created in an aqueous fluid, the GMDs can be transfered to a non-aqueous fluid, wherein the GMDs are manipulated by the use of physiochemical interactions that exploit differences in properties of the aque- ous GMD interior fluid and the GMD-surrounding non-aqueous fluid.

In this invention MDs can be surrounded by non-aqueous fluids in order to provide a non-aqueous environment which surrounds or suspends microdroplets Representative suitable non-aqueous fluids include liquid hydrocarbons, mineral oils, silicone fluids, ferrofluids, and heavy alcohols. This invention further involves physical manipulation of microdroplets surrounded by a non-aqueous fluid so as to change the position of such microdroplets by applying one or more physical forces, which are due to differences in properties of the MDs and the surrounding non-aqueous fluid.

More particularly, such forces can be applied by selecting forces selected from the group consisting of . While most of these forces are extremely well known in general, the term optical pressure force as applied to particles the size of MDs is more recent (see, for example, Ashkin et al, Nature 330: 769 - 771, 1987). In the process of this invention it is preferred to utilize electrical force selected from the group consisting of electrophoresis force, iontophoresis force, electrokinetic force, dielectric force and coulombic force, which are well known properties which differ from the dielectric properties of the medium surrounding the entities.

A particular process involving such electrical force is carried out using the following steps: (a) providing a non-aqueous fluid environment, (b) providing a plurality of charged electrodes within said non-aqueous fluid environment wherein at least two electrodes are of opposite polarity, (c) injecting an electically charged material capable of forming electrically charged microdroplets into the non- aqueous fluid; and (d) moving one or more charged microdroplets by means of an electrical force associated with potential differences which are applied between two or more electrodes, such that it is possible to produce microdroplets within a non-aqueous fluid, to introduce microdroplets into a non-aqueous fluid, to move microdroplets within a non-aqueous fluid, and to remove microdroplets from a non-aqueous fluid. Alternatively, MDs formed by any means and surrounded by a low electrical conductivity medium can be charged by contacting the MDs with a charged electrode.

It is thus possible to use such forces to manipulate the position of MDs with respect to measurement entities such as light emitting diodes, lasers, electrical capacitors, electrical inductors, electrical resistors, thermistors, thermocouples, fiber optics, photodiodes, phototransistors, photocells, piezoelectric sensors, specific ion electrodes, oxygen electrodes, carbon dioxide electrodes, pH elec¬ trodes and electrodes allowing dielectric property measurement. More specifically, such physical force is utilized to move MDs into proximity to such measurement entities, to maintain MDs in proximity to such measurement entities, and to remove MDs away from such measurement entities. In addition to physically manipulating MDs with respect to one or more such measurement entities, in the process of this invention physical force is also utilized to move MDs which are located within measurement apparatus such as light measuring instruments, light microscopes, fluorescence microscopes, lumi- nometers, fluorometers, photometers, time-resolved fluorometers, image analysis systems, colorimeters, spectrofluorimeters, particle counters, particle measuring systems, photoacoustic instruments, acoustic absorption instruments, acoustic microscopes, dielectric spectrometers, electrical impedance measuring instruments, calorimeters, thermal property measurement instruments and piezoelectric mass loading instruments. Similarly, the process of this invention can be used to cause MDs to come into contact with other MDs, or for contacted MDs to be separated. The former is particularly useful to enhancing collisions and coalescence of LMDs, while the latter is particularly useful for separating weakly adhering GMDs.

Likewise, the process of applying one or more forces to MDs surrounded by a non-aqueous fluid can be used to physical force is used to provide physical ^r manipulations of microdroplets selected from the group consisting of moving microdroplets. into proximity of, maintaining microdroplets in the proximity of, andiremovihg; microdroplets away from so that such MDs are exposed to external sources of physical influence, or exposed to sources of chemical influence. Such MDs surrounded by a non-aqueous fluid can be exposed to any physical source of 0 external influence, including sources of heat, electric fields, magnetic fields, elec¬ tromagnetic radiation, optical radiation, ionizing radiation, acoustic radiation and acceleration. Suitable sources of chemical influence include those which produce, release, modify or consume chemical compounds which are capable of dissolving in both the surrounding non-aqueous fluid and the aqueous interior of such MDs. ^" Finally, all of such manipulations are of particular utility in the case that at least one of the microdroplets contains at least one biological entity such as small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules. - In another embodiment of this invention it is possible to chemically manipu- late_-MDs- which are surrounded by a non-aqueous fluid. The present embodiment relates- tσ> the chemical manipulation of MD interior fluid composition while the MDs are_- maintained surrounded by a non-aqueous fluid. This invention thereby allows biological entities within non-aqueous fluid surrounded MDs to be exposed to waterrsoluble agents and compounds, and allows water soluble chemical

- reagents to be added to such MDs, thereby significantly extending the ability to carry out biological and chemical assays and tests using MDs.

More specifically, this invention consists of a process for delivering water soluble, entities and water insoluble entities, through an intervening non-aqueous fluid_into MDs, with means for subeqently determining the quantitiy of material soDd ivered. The major types of delivery processes involve:

(1) Introduction of LMDs or GMDs which contain (a) water soluble species to be delivered, and (b) water soluble optical indicator species which allow subsequent measurement of the amount water soluble material delivered to each MD, or (2) Dissolution of ampiphillic species (soluble in both aqueous and non-aqueous fluids) into the surrounding non-aqueous fluid, such that stirring and diffusion within the non-aqueous fluid provides essentially homogeneous dis- _* tribution of the ampiphillic species in the non-aqueous fluid, which in turn

5 allows partitioning of such species into the non-aquoeus surrounded MDs.

A- more complete disclosure of these processes is provided in the following sec¬ tions_^ If. not already surrounded by a non-aqueous fluid, it is first necessary to surround the microdroplets with an environment which comprises a non-aqueous fluid

10 The general process of this embodiment involves chemically manipulating

MDs, which are surrounded by a non-aqueous fluid, by altering the concentration of at least one chemical compound within the MDs by altering the composition of the surrounding non-aqueous fluid. A general means for accomplishing such chemical manipulation involves the dissolution of at least one chemical compound

15 in the non-aqueous fluid, such that the chemical can partition into the aqueous interior of the MDs, and thereby cause a change in the chemical composition of the aqueous interior of the MDs.

Alternatively, the composition of the non-aqueous fluid can be altered by adding MDs to the non-aqueous fluid, such that the MDs contain at least one 20 water soluble chemical compound. A general procedure for accomplishing such chemical manipulation comprises the steps of: (a) dissolving at least one chemical compound in a first non-aqueous fluid, (b) contacting said first non-aqueous fluid with a second non-aqueous fluid, said second non-aqueous fluid surrounding at least one microdroplet, said chemical compound being soluble in the first non- , 25^: aqueous fluid, in the second non-aqueous fluid, and in aqueous medium, and (c) allowing time for partitioning of said chemical compound from the first non- aqueous fluid into the second non-aqueous fluid, and subsequently into at least one microdroplet.

Often it is desirable to use the additional step of mixing in order to shorten 30 the time needed for partitioning of chemicals compounds into microdroplets from the non-aqueous phase, as such mixing reduces or eliminates the large concentra¬ tion gradients that arise upon dissolving a chemical compound in the non-aqueous fluid Another general process for accomplishing chemical manipulation of MDs surrounded by a non-aqueous fluid involves providing additional MDs in the non-aqueous fluid, such that the additional MDs contain the chemicals which are to be supplied to the original MDs. Such additional MDs can be provided in the form of an emulsion, by contacting the emulsion to the non-aqueous fluid, such that the,non-continuous phase of the emulsion comprises the additional MDs, and such that.the: continuous phase of the emulsion is comprised of the same, or a miscible, non-aqueous fluid as that which originally surrounds the original MDs. Often it is desirable to improve this process by mixing the emulsion and non- " aqueous fluid after the emulsion and non-aqueous fluid are contacted.

Another version of this process involves the use of an emulsion wherein the non-continuous phase of the emulsion consists of GMDs, rather than LMDs.

Although in some applications it is not necessary to know how much of a chemical compound is delivered to MDs, in some applications, for example the ^" delivery of an antimicrobial, it is important to know the amount or concentration of the chemical following the chemical manipulation of the MDs. In order to provide means for such measurement, it is useful to provide at least one tracer compound with measurable properties which is delivered to MDs by the same means as the chemical compounds , such that MDs can then be measured for the amount of tracer compound In addition, a measurable tracer can also be provided in the non-continuous phase of an emulsion contacted with the non-aqueous fluid, as this provides a measure of the total amount of the emulsion so contacted with the non-aqueous fluid Overall, the use of such tracers is important in that meas¬ urement of the amount of at least one tracer compound allows determination of - the amount of at least one other chemical compound which has been delivered to the MDs.

In order to measure such tracers, the tracer compounds are selected to have measureable properties selected from the group consisting of optical properties, mass density properties, acoustic properties, magnetic properties, electrical proper- ties and thermal, properties. Of these, it is preferred to utilize tracers with optical properties, such- as light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence A particu¬ larly useful optical property is fluorescence, which can be provided by using tracer compounds such as fluorescein, rhodamine, coumarin, lucifer yellow, phy- coerythrins and their chemical derivatives In order to enhance collisions and contact between the non-continuous phase of the emulsion and the MDs, it is possible to electrically charge the non- continuous phase of the emulsion, for example, by using means similar or identi¬ cal to those for forming MDs with electrical charge by forcing an aqueous medium from an electrically conducting needle into an agitated non-aqueous fluid while a large electrical potential difference is maintained between the needle at a large area electrode on the outside of the non-aqueous fluid container

In conducting of the process of this invention, it is useful to alter the chemi¬ cal concentration within MDs by delivering chemical compounds having proper- ties such as antiviral activity, enzyme inhibitory activity, antimicrobial activity, antifungal activity, cytotoxic activity, and chemotherapeutic activity It is further useful to deliver compounds which are reactants for one or more enzyme catalyzed reactions. , and it is particularly useful to utilize reactants such as fluorescein-di-β-D-galactopyranoside, resorufin-β-D-galactopyranoside, fluorescein diacetate, carboxyfluorescein diacetate, fluorescein isothiocyanate diacetate, fluorescein digalactoside, 4-methyl umbelliferone butyrate, 4-methyl umbelliferone phosphate, 1-napthol phosphate, 2-napthol phosphate, 3-μ-methyl-fluorescein phosphate, napthol AS phosphates, diacetyl 2-7-dichloro fluorescein, homovanillic acid, homovanillic acid + rhodamine lead,nicotinamide adenine dinucleotide (NAD) and resazurin

Microdroplet Incubation

Incubation consists of providing conditions for a time interval such that biochemical and biological reactions have an opportunity to occur. Incubation includes biochemical reactions and processes relating to replication of genetic material, synthesis of biological material, degradation of biological material, metabolism, secretion, uptake, ligand binding, aggregation based on specific bind¬ ing such as occurs in antibody-antigen reactions, the reactions which comprise the formation and/or growth of molecular complexes and aggrgates, and the complex reactions which comprise growth of virus, cells and small mutlicellular entities. Thus, during incubation biological entities have an opportunity to increase in size and/or number, and also to exhibit biochemical activity. Further, by altering the concentration of one or more compounds exhibiting properties such as antiviral activity, enzyme inhibitory activity, antimicrobial activity, antifungal activity, cytotoxic activity, and chemotherapeutic activity compounds, before, during or after an incubation, such compounds can be allowed to affect biological entities. The term biological entity refers to small biological structures which are capable of being incorporated into liquid microdroplets and/or gel microdroplets, and includes small multicellular organisms, groups of cells, individual cells, pro¬ toplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules. Small mul¬ ticellular organisms include fertilized eggs, blastomeres, embryos, small nema- todes and small helminths, which are of a size that can be incorporated into GMDs. Groups of cells include colony forming units, or CFUs, of naturally aggregating cells, and also microcolonies that result from growth of cells follow- ing one or more incubations. General types of cells of interest include animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells. Organelles include mitochondria, choroplasts, ribosomes and lysosomes. Protoplasts include those made from cells with cell walls by enzymatic digestion and other wall removing processes, or by mutants which lack cell wall synthesis ability. Virus includes those such as Herpes simplex, cytomegalo virus, Epstein-

Barr virus, adenoviruses, influenza A or B virus, parinfluenza 1, 2 or 3 virus, mumps virus, measles virus, coronavirus, poliovirus, coxsackie A and B virus, echovirus, rhinovirus and hepatitis A and B virus, and human immunodeficiency virus or HIV. Nucleic acids include both DNA and RNA. Antibody molecules include IgA, IgG, and IgM obtained from animals such as human, mouse, rat, goat, dog, pig and monkeys. Antigen molecules include large antigenic molecules with multiple distinct epitopes and/or overlapping epitopes, and small molecules such as haptens. Aggregates of molecules include immunoprecipitates, antigens which have bound one or more antibodies, with or without labels, hybri- dized nucleic acids and non-covalently bound complexes of two or more molecules. Although many of the examples of use of biological entities with microdroplets are described in terms of cells, particularly cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells, the broader group is intended. Further, as used here, biological entity also refers to any of these previously mentioned entities which have been reacted with one or more labling molecules, stains, dyes or with one or more intervening molecules.

Capturing Molecules at Binding Sites in GMDs

This invention relates to GMDs which contain one or more provided binding sites within the GMDs. Such GMDs are used to capture molecules within the GMDs at the provided binding sites. Such GMDs, and processes carried out with such GMDs, allow important measurements, manipulations and isolations to be carried out. The process of capturing molecules consists of: (a) incorporating specific sites and biological entities into GMDs, (b) allowing molecules released from biological entities in GMDs to move by diffusion, convection or drift within the GMDs, such that some molecules encounter the binding sites and are bound at such sites, thereby capturing molecules released from biological entities at binding sites within GMDs.

Molecules captured within GMDs by this process can then be measured, in order to determine properties or behavior of biological entities contained within the GMDs. Useful measurement means for measuring the captured molecules include optical, weighing, sedimentation, field flow sedimentation fractionation, acoustic, magnetic, electrical and thermal means It is preferred to use optical measurements such as measurements based on light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chem¬ iluminescence.

If the captured molecules have one or more measureable optical properties, the captured molecules can be measured by measuring a naturally occuring opti¬ cal signal associated with the captured molecules, including optical signals such as light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence.

In the practice of this invention it is often useful to provide one or more incubations, in order to allow different conditions to effect the production and/or release of molecules from the biological entities. For example, it is useful to pro- vide one or more incubations in order to allow molecules released by secretion from non-growing cells to accumulate in sufficient numbers at binding sites that measurement of the captured molecules is more readily accomplished. Likewise, it is often useful to provide one or more incubations which provide growth condi¬ tions for biological entities, particularly cells, in order that cells can increase in size and/or number, such that the total ability of the biological entities within a

GMD to produce and secrete molecules within a GMD is increased, thereby resulting in capture of more molecules at binding sites within such GMDs.

Gel microdroplets can also be physically isolated on the basis of measure¬ ment of the captured molecules, followed by release, if desired, of biological enti- ties from the isolated GMDs by processes such as dissolution of GMDs, mechani¬ cal disruption of GMDs and outgrowth by the biological entities, such that biological entities contained within the isolated GMDs are physically isolated based on the measurement of captured molecules. Examples of physical isolation include removing GMDs from a suspension and placing the GMDs in another suspension, sorting the GMDs by using a flow cytometer/cell sorter, identifying ^" GMDs by microscopy and utilizing micromanipulation to remove the GMDs, and using optical pressure to move GMDs to a known location. Following physical isolatiόπ-.of GMDJsi-bioIogical entities contained within the GMDs can be released from GMDs- by processes such as dissolution of the gel matrix, mechanical disr¬ uption of the gel matrix and outgrowth from the gel matrix by at least one biolog- * ical entity

Li some uses of this invention, a measurement of captured molecules is omitted, and instead forces which interact with one or more captured molecules are used to physically isolate GMDs, and thereby the biological entities contained therein. This can be followed by release of biological entities from the isolated GMDs by processes such as dissolution of GMDs, mechanical disruption of

GMDs and outgrowth by the biological entities, such that biological entities con¬ tained within the isolated GMDs are physically isolated based on physical forces interacting with the captured molecules.

In cases wherein it is desired to measure captured molecules, the captured molecules do not necessarily have properties which allow satisfactory measure¬ ment. In this case it is often possible to provide a subsquent step which comprises exposing GMDs to one or more labeling molecules which have measurable properties and also are capable of binding to, and thereby labeling, the captured molecules. Following the process of exposing GMDs to one or more labeling molecules, the labeling molecules are then measured, using either

_ physical or chemical means. Examples of labeling molecules include antibodies, antigens, nucleic acids, Iectins, receptors, enzyme inhibitors, and protein A, all with measurable labels. i the practice of this invention it is preferred to use optical measurements - to measure laheling. molecules which have bound to the captured molecules, includingroptical measurements of labeling molecules with labels which can be measured by light scattering, light absorbance or colorimetric, fluorescence, time- delayed fluorescence, phosphorescence and chemiluminescence. Exemplary label¬ ing molecules which are labled with such labels include antibodies, antigens, nucleic acids, Iectins, receptors, enzyme inhibitors, and protein A, all with measurable labels. Physical isolation of GMDs, and the biological entities contained within the GMDs, can be accomplished by using labeling molecules having a label capable of coupling with a physical force, such that following exposure of GMDs to labeling molecules, the GMDs with labeled captured molecules are manipulated by at least one physical force in order to physically isolate such GMDs. In this embodiment it is preferred to provide and use magnetic labels

It is often desirable to expose GMDs containing captured molecules to one or more intervening molecules before exposing, or simultaneously with exposing the GMDs to labeling molecules. This is accomplished by: (a) supplying at least one intervening molecule type and (b) supplying labeling molecules, such that intervening molecules bind to captured molecules and labeling molecules bind to intervening molecules. Examples of intervening molecules include antibodies, antigens, nucleic acids, Iectins, protein A and avidin. For use as an intervening molecule, it is preferred to use unlabeled antibodies obtained from an animal species different than that from which a labled antibody is obtained as a labeling molecule. For example, a mouse antibody to a captured molecule can be used as an intervening molecule, and a fluorescence-labeled goat anti-mouse antibody can be used as a labeling molecule for the intervening molecule, such that a first exposure to this intervening molecule, and a second or simultaneous exposure to the labeling molecule results in the ability to measure the captured molecules.

If desired, an intervening molecule can also have one or more labels, such that the binding of both intervening molecules and labeling molecules increases the amount of label associated with captured molecules, thereby enhancing the measurement of captured molecules It is useful to practice this invention using the biological entities including cells, spores, protoplasts, vesicles and small multicellular organisms. , and it is preferred to use small multicellular organisms and cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells.

In cases in which biological entities do not secrete molecules of interest, or ' secrete molecules at a less than desired rate, it is useful to cause biological enti¬ ties to release cells by providing one or more external stimuli. A general method for providing stimulus to cells, vesicles, protoplasts and small multicellular organ¬ isms involves the application of an electromagnetic stimulus which results in elec- troporation (see, for example, Sowers and Lieber, FEBS Lett. 205: 179 - 184, 1986) Determination of Biological Growth

Prior use of GMDs has been based on determination of cell activity, more particularly metabolic activity, of one or more biological entities contained within the very small volume of a cell-occupied GMD. A general feature of such

55 activity based determinations is that GMDs are provided with a permeability bar¬ rier,, in the form of a coating, or by suspending the GMDs in mineral oil (see Weaver et al., Ann. N.Y. Acad. Sci., 434: 363 - 372, 1984; Weaver, Biotech, and Bioengr. Symp. 17, 185 - 195, 1986; Williams et al., Ann. N. Y. Acad. Sci., 501: 350 - 353, 1987). Activity based determinations using GMDs are based on the lQϊ extracellular accumulation of cell products within the very small volumes of

GMDs, and the use of chemical indicators or chemical assays in combination with changes in the extracellular environment within a very small volume. The determinations are fundamentally based on a time integration of the production rate for cell products which are released into the extracellular environment, and

15. which are retained within the very small volume of a GMD. Further details con¬ cerning the production of GMDs may be found in U.S. Patents 4,399,219, 4,401,755 and 4,643,968, each of Weaver, the teachings of which are incor¬ porated herein by reference.

In such prior use of GMDs cell growth itself has not been determined, but

20 instead the accumulated effect of cell activity, which is due to both to the activity per cell and to cell number, was the basis of the determinations. Thus, for exam¬ ple, an increase in cell products within a GMD with a permeability barrier could be due either the presence of one highly active cell, such as a single yeast cell, or could be due to an initial bacterium which rapidly grows to form a microcolony

25 of several cells, which microcolony has activity several times that of an indivi¬ dual bacterium. In the first example, a single yeast cell, without the occurrence of growth, is the basis of a determination, while in the second example, the deter¬ mination occurs primarily because of the increase in cell biological material, and corresponding increased activity, due to growth. As demonstrated by this illustra-

30 tion, for different cell types the same change in extracellular chemical concentra¬ tion due to activity can occur with or without growth, depending on the relative individual cell activity and the relative cell growth rate, so that for this reason the prior use of GMDs does not provide a general means for determining cell growth. Further, although a significant advantage of the prior use of GMDs is that cell

35" growth is not necessarily the basis of determinations, generally the type of activity must be known. For example, although a great many microorganisms produce significant amounts of acids, allowing GMD determinations based on extracellular pH changes within the very small volume of a GMD with a permea¬ bility barrier, the production of significant acid is not a universal property of cells. For this reason, prior use of GMDs has required knowledge of the type of 5 biochemical activity exhibited by a cell, whereas, in contrast, the present inven¬ tion is much more general.

For the above reasons, neither the previous methods of others, nor the previ¬ ous use of GMDs to detect cells based on metabolic product retention, actually provides a rapid determination of cell growth, a fundamental process in which

10 biological entities increase in biological material and/or in number. In contrast, the present invention provides a general means for biological entity growth deter¬ mination, wherein entrapment of initial and progeny biological entities within MDs, preferably GMDs surrounded by an aqueus fluid or medium, is combined with measurement of the biological material within MDs, and which, because of

15 the very small size MDs, allows incubation conditions and analysis conditions to be changed rapidly because of the small diffusion times within MDs. In the case of MDs which are GMDs, this invention also allows optical measurements to be made with small optical path lengths within a gel matrix. This reduces optical measurement error associated with light scattering, light absorbtion or

20 autofluorescence in a gel matrix.

In the case of cells which naturally aggregrate and adhere to each other in small numbers, the present invention determines the growth of colony forming units (CFUs).

In the general practice of this invention MDs are formed by any of the pre- 25 viously described means, such that biological entities such as small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules are incorporated into MDs. The resulting MDs can contain biological entities such that MDs of different sizes have a high 30. probability of being mostly unoccupied, individually occupied or multiply occu¬ pied. Measurement of biological material is then accomplished by making meas¬ urements on the MDs, either individually or in small groups, such that measure¬ ments responsive to biological material are made. Biological material consists of the constituative molecules and structures found in biological entities such as 35 small multicellular organisms, groups of cells, individual cells, protoplasts, vesi¬ cles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules. Thus, examples of biological material include proteins, nucleic acids, phospholipids, polysaccharides, enzymes, antibodies, cell receptors and other well-known biological molecules.

Measurement of biological material can be accomplished by using measure- ments based on optical, weighing, sedimentation, field flow sedimentation fractio¬ nation, .acoustic,. magnetic, electrical and thermal means. Such measurements can be^ιbased:onιproρerties- such as optical properties, mass density properties, acous¬ tic properties, magnetic properties, electrical properties and thermal properties. It is preferred to utilize measurements based on optical properties such as are meas- ureable utilizing light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence. Thus, use¬ ful measurement apparatus includes flow cytometry apparatus, flow-through- microfluorimetry apparatus, optical particle analyzer apparatus, fluorescence microscopy apparatus, light microscopy apparatus-image analysis apparatus and video recording apparatus.

In some cases naturally occurring optical properties of the biological material of biological entities can be used to measure the biological material, as this provides a naturally occurring optical signal Examples of such naturally occurring signals are light scattering, light absorbance or colorimetric, fluores- cence, time-delayed fluorescence, phosphorescence and chemiluminescence.

However, in order to enhance the measurement of biological material in GMDs it is often useful to employ staining protocols which utilize stains such as stain indi¬ cative of biological composition, stain indicative of enzyme activity, and stain indicative of cell membrane integrity, as such stains are responsive to the amount, activity and/or state of biological material. More specifically, it is often desirable to use stains in the general catagories of fluorescent stains, light absorbance stains and light scattering stains, and more specifically stains such as nucleic acids stains, protein stains, lipid stains, cell membrane stains, cell wall stains, stains responsive to enzyme activity, stains responsive to transmembrane potentials and cell surface receptor stains. Still other useful types of stains include transmem¬ brane potential, stains, membrane exclusion stains and intracellular enzyme activity responsive stains.

It is generally preferred to use fluorescent stains such as propidium iodide, ethidium bromide, FTTC (fluorescein isothiocyante), fluorescein diacetate, carboxyfiuorescein diacetate and FITC diacetate. Many other suitable fluorescent stains for biolo ical material are well known see for exam le Hau land Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,

Junction City).

In the general practice of this invention it is preferred to make measurements

_ of MDs involving predominantly measurements of individual or single MDs 5 However, measurements can also be made simultaneously on groups of two or more MDs. Likewise, it is preferred to make measurements on MDs consisting predominantly of microdroplets with a high probability of containing less than two biological entities prior to incubation , but measurements can also be made on microdroplets specimens consisting predominantly of microdroplets with a 10 high probability of containing at least two biological entities prior to incubation.

Although it is useful to measure the amount of biological material in GMDs without requiring measurement of the volumes of individual GMDs, or of groups of GMDs, it is often useful to measure such GMD volumes. This data can be used to provide the basis for further analysis or interpretation of the biological 15 material measurements. For example, measurement of such GMD volumes pro¬ vides the basis for determining the volume of a sample which was contained in the measured GMDs, as the measured GMD volumes can be summed to yield the total volume analyzed, with correction for dilution if needed.

Additionally, it is often useful to analyze the measurement of MD volumes, 20 or volumes of groups of MDs, with statistical formulae which relate the occupa¬ tion of MDs to MD volume and the average concentration in the suspension or solution from which MDs were formed. More specifically, it is often useful to analyze MDs and measurements of the amount of biological material, _> :h that measurement of the biological material provides a basis for classifying each MD, 25 or group of MDs, as being unoccupied or occupied. It is then useful to further analyze the MDs and measurements by using Poisson statistics formulae and modified Poisson statistics formulae.

If control analysis is desired, one or more specimens of MDs are measured without incubating. Other MDs are then exposed to conditions for which growth

30) determination is sought, and incubated for one or a plurality of incubation periods, and some or all of the MDs then measured, individually or in groups, for the amount of biological material present in the individual MDs or in the groups of MDs. The results of such MD measurements are interpreted as the amount of change in biological material during the control or incubation periods, and such

35 change in biological material is attributed to the amount of growth which occured ti n ri Following such measurement of the change in the amount of biological material, one or more forces can be applied such that MDs containing biological material with a first change in the amount of biological material are isolated from

MDs not containing biological entities with the first change in the amount of bio-

5 logical material

In the preferred embodiment of the invention, MDs are created with a wide range- σ£ sizes, such that some range of sizes has a high probability of containing zero or one initial biological entities. One subpopulation of MDs is measured without incubation in order to provide a control, and one or more other subpopu- £P lations of MDs are incubated under desired conditions and then measured indivi¬ dually for the relative amount of biological material in each MD.

It is useful to separately consider the case wherein the volume, V_MD , of each MD is known or measured, and it is also known that the sample contains biologi¬ cal entities at approximately known concentration, p_s, so that following concen- 5:- tration or dilution of the sample, and after addition of gelable material, the result¬ ing concentration, p, is known, and is the suspension from which MDs are made.

A preferred embodiment of this invention involves formation of MDs which are GMDs, followed by suspending these GMDs in an aqueous or non-aqueous medium. Following one or more incubations of these GMDs the growth of indi- 0 vidual biological entities into microcolonies of two or more biological entities can occur. In the case in which GMDs are surrounded by a non-aqueous fluid, the GMDs are transfered from the non-aqueous fluid into an aqueous fluid. A stain¬ ing procedure for biological entity biological material is then used to provide the basis of one or more optical measurements, such that the magnitudes of the opti- 55 cal signals provides a determination of the biological material present in each measured GMD.

In a representative case of the general invention, wherein MDs are GMDs suspended in an aqueous medium, the biological entities are cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold D^"> cells. A fluorescent dye is used to stain nucleic acids, the fluorescence associated with the dye is measured in the GMDs, preferably in individual GMDs, and the frequencey-of-occurrence of a certain magnitude of a fluorescence signal can be displayed as a function of the magnitude of fluorescence signal. This type of plot is generally termed a histogram. Such analysis shows that incubated GMDs with 5 growing cells produce histograms with fluorescence peaks that occur at larger occurrence versus logarithm fluorescence, because cells growing in the exponen- tial phase then exhibit a linear or proportional increase in the average log fluores¬ cence as a function of time. It is found that such plots generally exhibit peaks * with increasingly larger average fluorescence for GMDs incubated for longer

5. times. The area of such peaks provides a measure of the average growth of cells, and thereby the average growth rate. It has been found that such GMD based average growth rate determinations compare favorably with conventional, total population methods. The present invention also provides a measure of the varia¬ tion in growth rate, or growth rate distribution, which is reflected in the variation 10 of fluorescence within the fluorescence peaks which is greater than the variation or instrumental error in the fluorescence measurement apparatus itself, and which is not determined by conventional methods

This invention can also be used to determine the lag time in growth of bio¬ logical entities. This lag time is the delay in achieving an exponential growth

15 rate following exposure to changed conditions. Such determinations are made by comparing the magnitude of the change in the amount of biological material in GMDs incubated under particular changed conditions to the change in the amount of biological material in other GMDs incubated under control conditions. Such lag time determinations utilize the growth rate determinations described elsewhere

20 in this disclosure.

Further, in some cases such analysis reveals a fluorescence peak at approxi¬ mately the location of the initial or non-incubated peak, whose peak area provides a measure of non-growing cells for the incubation conditions used. If such condi¬ tions correspond to conditions which ordinarily support growth of the cell type,

25 then the ratio of the number of GMDs with growing cells to the number of both growing and non-growing cells can be interpreted as the cloning or plating efficiency in GMDs for these conditions. Conventional rapid growth measure¬ ments based on the combined effects of many cells do not provide direct determi¬ nation of non-growing cells, but instead can only provide a determination based

30 on the combined effects of growing and non-growing cells, and the relative numbers of growing and non-growing cells is not known a priori

In addition to measuring the biological material consisting of the total amount of one or more types of biological material, it is often useful to supply chemical compounds in order to utilize reactions such as degrading reactions of 35 stainable biological material and blocking reactions of stainable biological material, so that enhanced measurement is achieved. For exam le, in the case of GMDs in an aqueous medium wherein it is useful to measure total stainable nucleic acid to provide a measurement of growth, it is also possible, for biologi¬ cal entities that contain DNA which is replicated, to provide a RNA degradation process such as exposing the GMDs to RNAase, which degrades the RNA while leaving the DNA, such that subsquent exposure to a nucleic acid stain allows

DNA to be measured in GMDs. Such measurement of DNA then provides the basis; for determining replication of biological entities.

The process of the_.present invention can provide determinations of biologi¬ cal entity growth which are based on measurement of biological material within MDs which initially had a high probability of having zero or one biological entity. This biological entity growth determination process is rapid, often requir¬ ing about one average generation or doubling time. However, the process can also be advantageously carried out using more generations or doublings, in order to allow possibly unstable cells to cease growing, and/or in order to utilize less sensitive and less expensive optical measurement apparatus.

Although the previous illustration describes a preferred embodiment, in which there is a high probability of some MDs, of some size or volume range, V_MD. being unoccupied or individually occupied it is also possible to make growth measurements using MDs in a size range, relative to the biological entity suspen- sion concentration, p, for which there is a moderate or high probability of multi¬ ple occupation. In this case the measurement of optical signals from individual MDs relates to the total biological material within each MD, and therefore correponds to an average growth determination, wherein the average is over the small number of biological entities initially present for each MD size. For exam- pie, if the biological entity concentration, p, just prior to MD creation results in n

= 3 biological entities in MDs of size V_MD , then the average growth determination has a high probability of corresponding to the average growth behavior of 3 bio¬ logical entities, and can reveal some aspects of growth heterogeneity or variability which cannot be determined by conventional methods which are based on the combined effects of many, typically 10⁴ or more, biological entities.

Alternatively, in a related embodiment, MDs with a high probability of inital occupation by zero or one biological entities can be measured in small groups rather than individually. This can be advantageous, for example, in flow cytome- ter measurement of GMDs suspended in an anqueous medium, wherein measure- ment conditions allow several GMDs to be present transiently in the optically measured region, and thereby allows a higher rate of GMD analysis. Likewise, this can be advantageous in measurement using microscopy, of MDs suspended in, or surrounded by, a non-aqueous fluid, wherein measurement conditions allow several MDs to be present in the measurement field of view, and thereby allows a higher rate of MD analysis. In this embodiment, measurements on groups of 5 MDs incubated for two or more incubation periods are compared, and the differences in optical signals from the groups of MDs are interpreted as resulting from. differences in biological entity material within the groups of MDs. These differences are interpreted as arising from biological entity growth within the groups of MDs. For example, measurements on groups of incubated MDs are

109 compared to measurements on groups of non-incubated MDs, such that differences in optical signals from the incubated groups of MDs and non- incubated groups of MDs are interpreted as resulting from differences in biologi¬ cal entity material. These differences are further interpreted as resulting from biological entity growth during the incubation period. This embodiment is related

15- to the previous embodiment, in that the biological material optical signal corresponding to several initial biological entities forms the basis of the measure¬ ment, and therefore provides the average growth determination as having a high probability of corresponding to the average growth behavior of several biological entities. This method can thus reveal some aspects of growth heterogeneity or

20- variability which cannot be determined by conventional methods which are based on the combined effects of many, typically 10⁴ or more, biological entities.

In the present invention, after creation, the MDs are placed in desired condi¬ tions for determining growth. Typically MDs can be suspended in aqueous or non-aqueous media which provide a wide range of different chemical composi-

25. tions, and at different pH, temperature, partial pressures of oxygen and carbon dioxide, etc.. This allows biological growth under a wide range of conditions to be determined. As an alternative to suspension, MDs can be held stationary by allowing MDs to sediment under the influence of an applied force such as gravi¬ tational or centripetal force. In the case of GMDs, the GMDs can be temporarily

30: trapped; against a porous mesh or filter by a perfusing flow, which provides a sup¬ ply of growth medium past the trapped GMDs.

The transport of chemicals within MDs is generally governed by diffusion. Thus, in the case of MDs surrounded by a non-aqueous fluid, the supply and removal of chemicals to biological entities within the MDs is governed by the 35 partioning of chemicals between the non-aqueous fluid and the interior aqueous fluid of the MDs, and is further governed by diffusion within the MDs. Because of the relatively small size of MDs, the characteristic diffusion time, ^_di_ffusi_on =χ²lD can be short, as x is the characteristic distance over which diffusive transport occurs, and r_MD ~ x. This yields τ_t_{f f} sio from about 4 min to about 5 x 10"⁶ sec for MDs with diameters of lOOOμ to 0.2μ, and from about 1 min to about 6 x lOr³ sec for MDs with diameters of 500μ to 5μ for small molecules with

D~10^~S cm² sec^-1. This value is approximately representative of the size molecule which can readily partition and diffuse between the non-aqueous fluid surrounding a MD and the interior aqueous fluid of the MD. As a result, if the kinetics of partioning and transport within the non-aqueous fluid are not limiting, the charac- - teristic diffusion time, τ_D, governs changes, and can be short. Thus, the concen¬ tration of some types of molecules, specifically those with significant solubility in both the non-aqueous fluid and the aqueous interior medium can be changed rapidly in such MDs.

Similarly, the supply and removal of chemicals to biological entities within GMDs surrounded by an aqueous medium is governed primarily by diffusion, and by partitioning between the external aqueous medium surrounding the GMDs and the aqueous medium within the GMDs, because the gel matrix effectively clamps viscous flow, that is, increases resistance to viscous flow. The partioning between the external aqueous medium and the GMD interior medium is often non- selective, because many gels used to form GMDs do not exclude, absolutely or partially, most chemicals of interest. In some cases, however, gel materials can have charge or size exclusion properties so as to partically or absolutly exclude some molecules from the interiors of GMDs.

Further, because of the relatively small size of GMDs compared to conven- ^; tional, more macroscopic gel preparations, the characteristic diffusion time,

^_di_ff io_n = x²lD . where x is a characteristic dimension such as the thickness of a macroscopic gel slab and D is the diffusion constant within the aqueous liquid within the gel, can range from shorter to much shorter than for conventional gel preparations. Depending on the type of biological entity used, and the chemicals of interest, i_&ffxuio- can have a wide range of values, as can be seen by using r_GMD

~ x, which gives τ_ώ7/l4-_ώ„ from about 4 min to about 5 x 10^"6 sec for GMDs with diameters of lOOOμ to 0.2μ, and from about l min to about 6 x 10^~3 sec for GMDs with diameters of 500μ to 5μ for small molecules with £>=10^~5 cm² sec^-1, and about a factor of 100 longer for macromolecules with £>=10^~7 cm² sec^-1. As a result, even the concentration of macromolecules can be changed rapidly in GMDs with diam¬ eters of about 200μ or less, as the corresponding diffusion time is about l hour, a value much smaller than the doubling time of typical mammalian cells. This value can be changed even more rapidly in the smaller GMDs which can be used with smaller microorganisms such as bacteria and yeast, which microorganisms have shorter doubling times. As a further example, 20μ GMDs used with rapidly growing bacteria for which the doubling time, t_, is typically about 20 min, have an appropriately short τ_&i_f _._on of about 0.1 sec for small molecules and about 10 sec for macromolecules.

After one or more incubation periods the MDs are measured, preferably by optical means. Other measurement means include methods sensitive to mass den- sity, such as weighing, sedimentation, and sedimentation field flow fractionation, and additional methods based on acoustic, magnetic, electrical and thermal pro¬ perties of MDs containing different amounts of biological material. Sedimenta¬ tion field flow fractionation force can be provided by simultaneously utilizing a hydrodynamic force and a sedimentation force (see, for example, Levy and Fox, Biotech. Lab. 6:14 - 21, 1988). Acoustic measurements utilize sound absorbtion and reflection of biological material, as is utilized in acoustic microscopy (see, for example Quate, Physics Today, August 1985, pp. 34 - 42). Magnetic measure¬ ment utilizes diamagnetic, paramagnetic and, occasionally, ferromagnetic proper¬ ties of biological material. Thermal measurement utilizes thermal conductivity, thermal diffusivity and specific heat properties of biological material (see, for example, Bowman et al, Ann. Rev. Biophys. Bioengr. 4: 43 - 80, 1975). Electri¬ cal measurement utilizes electrical resistance and dielectric properties of biologi¬ cal material, such that measurement of the dielectric properties at various frequen¬ cies can provide measurement of biological material (see, for example, Kell in Biosensors: Fundamentals and Applications, Turner et al (Eds), Oxford Univer¬ sity Press, Oxford, pp. 427 - 468; Harris et al, Enzyme Microb. Technol. 9: 181 - 186, 1987). The measurement of electrical resistance of biological entities such as cells is well known to provide a means for measuring cell size, and is the basis for particle analyzers such as the Coulter Counter (see, for example, Kachel in Flow Cytometry and Sorting, Melamed et al (Eds), Wiley, New York, pp. 61 -

104). In the present invention, the prefered electrical measurement is utilized with GMDs suspended in or surrounded by an aqueous medium, is preferably used with biological entities with bilayer membranes. These entities include small multicellular organisms, cells, vesicles and protoplasts. The electrical measurement is based first on a rapid diffusional exchange of medium within a

GMD from a defined electrical resistance medium such as physiological saline, followed by a second step of passing GMDs though a particle analyzer such as a Coulter Counter. The gel matrix of such a GMD provides negligible electrical resistance compared to such biological entities, thereby allowing measurement of the amount of biological material associated with cells contained in GMDs. It is presently preferred to utilize optical measurements in order to measure biological material contained within MDs. Well known general optical measure¬ ments: sensitive to biological material include light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chem¬ iluminescence. ^ Biological material contained within MDs can in some cases be adequately measured utilizing naturally occuring optical properties of the biological material. Thus, for example, fluorescence of the biological material, light absorbance by the biological material, and light scattering by the biological material can sometimes be used. ^"J Prior to optical measurements, however, it is often preferred to expose MDs, particularly GMDs surrounded by an aqueous medium, to one or more staining processes. These processes can be general (e.g. nucleic acid stains) or they can be specialized (e.g. fluoresence-labeled antibodies), depending on the type of bio¬ logical entity and the purpose of the biological entity growth determination. In the case of MDs surrounded by a non-aqueous fluid, stains can be introduced through the surrounding non-aqueous fluid by dissolving the stains in the non- aqueous fluid, or by supplying the stains in the non-continuous phase of an emul¬ sion which is contacted with the non-aqueous fluid. In the case of GMDs sur¬ rounded by an aqueous fluid or medium, stains can be introduced through the sur- 5 rounding aqueous fluid by dissolving the stains in the aqueous fluid.

In the general case wherein growth analysis without further use of the bio¬ logical entities is desired, any biological entity staining process, including those which kill biological entities, can be used. In the case wherein further analysis or use of viable biological entities is desired, biological entity staining which allows „- biological entity survival is used. As is well known in the art, there are biologi¬ cal, material stains for nucleic acids stains, protein stains, lipid stains, cell mem¬ brane stains, cell wall stains, stains responsive to enzyme activity, stains respon¬ sive to transmembrane potentials and cell surface receptor stains.

Following exposure of the MDs to suitable stains, the MDs are individually measured, or the MDs are measured in small groups, provided that the probability the exemplary case of biological entities stained by fluorescent compounds, opti¬ cal analysis such as digital fluorescence microscopy or flow cytometry is used to measure individual MDs, using a wavelength band sufficiently different from that used for any detection of measurement of MD properties. This method allows simultaneous, or serial, measurement of MD properties and of biological entities with said MD. The associated fluorescence signals are acquired and measured, with correction for spectral overlap if necessary, by conventional means.

The relatively small size of MDs results in the possibility of more flexible analysis. For example, conventional flow cytometers have flow biological entity channel diameters of several hundred microns, which prohibits the use of flow cytometry with conventional macroscopic gel preparations, but which readily allows the use of MDs in the size range from somewhat less than the flow biolog¬ ical entity channel size and smaller.

The magnitude of the optical signal due to the biological entity stain in each MD, or group of measured MDs, is compared to the optical signal of individual biological entities, whether or not such individual biological entities are entrapped in MDs, thereby providing a calibration. Comparison of the MD optical signal magnitude to that of individual biological entities provides the basis for determi¬ nation of growth of individual biological entities, for which the growth determina- tion can often be made within one generation time, but without a need for significant prior culture to obtain large numbers of biological entities.

By making a large number of such individual biological entity growth deter¬ minations, the distribution of growth rate, distribution of lag time, and the plating efficiency can be automatically determined by computer calculation. Manual or visual inspection and scoring of MDs can also be used, but is relatively labor intensive and therefore more prone to error, so that the preferred processes are those conducted using automated measurement means.

Determination of Effects of Compounds on Biological Entities

The present invention further provides means for determining important pro- perties of chemical compounds and agents as said properties relate to the effects of said compounds on biological entities, particularly the growth of biological entities. Alternatively, this invention also provides means for determining impor¬ tant properties of biological entities, particularly cells, relating to the susceptibility behavior of the biological entities. This is especially useful for determining the effects of compounds on the growth behavior of biological entities which can be determined by measuring the amount of biological material associated with bio¬ logical entities contained in MDs. A general process for determining the effect of 5 compounds on the growth of biological entities comprises the steps of: (a) expos¬ ing MDs to at least one compound, said compound being such that its effect on the; growth of said biological entities is to be determined, and (b) measuring bio¬ logical' material within at least one MD In some applications of this invention, MDs can be supplied which already contain biological entities, but in other appli- 10^" cations in is necessary to first incorporate biological entities into gel microdro¬ plets. The incorporation can be accomplished using any of previously described processes for the formation of MDs.

As used in this invention, the term specimen of microdroplets encompasses both specimens of LMDs and specimens of GMDs, and refers to a subset of the 15.5 MDs formed from a sample. Thus, for example, if a sample is processed so as to lead to the formation of about 10⁵ MDs, these MDs can be divided into ten approximately equal specimens of MDs wherein each specimen of MDs contains about 10⁴ MDs.

The effect of compounds or agents on biological entities is generally not 20 revealed instantaneously, but instead after a period of time has been allowed to elapse, such that at least one incubation is generally desirable in order to bring out the effect of of compounds or agents on biological entities. Further, the effect of compounds or agents on biological entities can often be advantageously determined by exposing at least two specimens of microdroplets to different con- 25^" centrations of the compounds or agents. In order to interpret changes caused by the exposure of biological entities in MDs to compounds and agents, it is often desirable that at least one specimen of MDs not be exposed to the compounds or agents, thereby providing at least one control condition.

The effects of many compounds and agents on biological entities can be 30 determined by this invention, including the effects of compounds such as antibiot¬ ics, antimicrobial compounds, antifungal compounds, chemotherapeutic com¬ pounds, toxic compounds, cytotoxins, irreversible inhibitors, reversible inhibitors, mutagenic compounds, hazardous compounds, hormones, growth factors, growth enhancers, nutrients, vitamins, food preservatives, pesticides and insecticides. 35 Further, many different types of biological entities can be used in this invention, including small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules. It is pre¬ ferred to use cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells. Additional flexibility can often be obtained by: (a) using at least two incuba¬ tion periods, and (b) using at least one change in the concentration of the com¬ pounds between incubation periods. For example, if it is desired to test the rever- sability of a growth altering compound or agent, a first incubation can be used with the compound or agent present, followed by a second incubation with the compound or agent absent.

Control conditions can be provided by: (a) exposing a single specimen of microdroplets to at least one change in concentration of at least one compound or agent, (b) using a first incubation as a control for growth, and (c) using at least one subsequent incubation with exposure to the compound or agent, thereby pro- viding a serial process in which a control is followed by exposure to compounds or agents Alternatively, a process can be carried out by: (a) exposing at least one specimen of microdroplets to at least one change in concentration of at least one compound, (b) using at least one other specimen of microdroplets without expo¬ sure to said compound, in order to provide a control for growth, and (c) using separate incubation of at least one exposed specimen and one control specimen.

More specifically, the process of this invention can be used to determine the antimicrobial susceptibility of microorganisms to various compounds. Specimens of GMDs which contain one or more microorganisms are exposed to an antimi¬ crobial at one or more concentrations, and the growth determined by using an incubation for each concentration. By comparing the amount of growth at different concentrations, the effectiveness of the compound to inhibit growth of the microorganism at these concentrations can be determined. This determination corresponds to a determination of the antimicrobial susceptibility of that microor¬ ganism for the tested compound. A related process provides determination of the sensitivity of cancer cells to compouds such as chemotherapeutic compounds, in which case it is preferred to use GMDs. Specimens of GMDs which contain one or more cancer cells are exposed to a compound at one or more concentrations, and the growth determined by using an incubation for each concentration. By comparing the amount of growth at different concentrations, the effectiveness of the compound to inhibit growth of the cancer cells at these concentrations can be determined. This determination corresponds to a determination of the chemotherapeutic susceptibil¬ ity of the cancer cells to the tested compound. An advantage of the present invention is that it is possible to make measurements on GMDs which have a high probability of being occupied by a small numbers of cells, preferably less 5 than two cells. Further, measurements can be made on large numbers of GMDs which contain cells. As a result, for example, this invention provides the ability to determine the chemotherapeutic susceptibility of large numbers of individual cancer cells, for example 1,000 cells to 10,000 cells. Thus, it is possible to deter¬ mine the distribution of amount of growth by a large number of cells, which

10. ^' growth is not necesarily identical, thereby allowing the distribution in cancer cell growth to be determined. This, in turn, provides a determination of the distribu¬ tion of susceptibility of the cancer cells to the compound. Thus, for example, if a subpopulation comprising 10% of the cancer cells is resistant to a compound, or mixture of compounds, significant growth will be found in about 10% of the

155 occupied GMDs. Such determinations can have good statistical significance because a large number of individual GMD measurements can be made, wherein there is a high probability that GMDs are occupied by less than two cells.

Continuing this example, if a resistant subpopulation comprising about 10% of the cells is found on the basis of measuring 10⁴ cells, the number of GMDs

20 occupied by resistant cells is about 10³. The corresponding statistical error in sampling, and therefore in determining the size of the resistant subpopulation, is due to the error in counting randomly occurring events. This statistical error is well known to be described by -\N_{raπdom evtΛt}, so that in this illustration the error is is Naώtot_f ceii = ftθ³ = 32, which corresponds to about 3% error, and is therefore

25^: highly accurate in determining the size of the resistant subpopulation.

Measurements of the amount of biological material in GMDs can provide the basis for measurement of growth, and other biological activity and function, by biological entities such as cells within GMDs. Suitable measurement means for measuring the biological material include optical, weighing, sedimentation, 30 field flow sedimentation fractionation, acoustic, magnetic, electrical and thermal means. It is preferred to use optical measurement means, particularly measure¬ ments based on light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence.

As described elsewhere in this disclosure, groups of microdroplets consisting 35 predominantly of single microdroplets can be simultaneously measured, or, alter¬ natively, groups of microdroplets consisting predominantly of at least two microdroplets can be simultaneously measured. As also described elsewhere, the measured microdroplets can consist predominantly of gel microdroplets with a high probability of containing less than two biological entities prior to incubation Alternatively, the measured microdroplets can consist predominantly of microdro- plets with a high probability of containing at least two biological entities prior to incubation.

Finally, this invention can be used with a step wherein measurement of growth is used to determine the effect of at least one compound on growth characteristic behavior selected from the group consisting of plating efficiency, growth rate distribution, average growth rate, growth lag time distribution and average growth lag time

Enumeration of Viable Biological Entities

This invention involves also the determination of the number of viable bio¬ logical entities per volume, which comprises enumeration of viable biological entities, a measurement which is widely used in biology and medicine. For example, it is common to determine the number of viable microorganisms per ml of a fluid sample. In this process it is important to make determinations which are rapid, based on the number of microorganisms, and based on a stringent cri¬ terion for microorganism viability. As described herein, it is often possible to extend the concept of growth to include the growth of biological entities such as small multicellular organisms, groups of cells, individual cells, protoplasts, vesi¬ cles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules. It is preferred, how¬ ever, to carry out the process of this invention with cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells, par¬ ticularly for normal human cells, human cancer cells, pathogenic bacteria, patho¬ genic yeast, mycoplasms, parasites, and pathogenic viruses. This invention pro¬ vides a general means for rapidly enumerating such biological entities using the criterior viability based on growth, and also, for some biological entities, using criteria provided by vital stains.

More specifically, this invention provides means for determining the number of viable biological entities per volume of a sample, the process comprising the steps of: (a) exposing at least one MD, which contains at least one biological entity, to conditions for which the number of viable biological entities is to be determined, (b) measuring biological material in GMDs, (c) measuring the volumes of the associated GMDs, and (d) thereby determining the number of viable biological entities per volume in the sample. In cases wherein MD volumes are not known, the additional step of measuring MD volumes is used. Prior to making this determination, it is necessary to incoporate biological entities 5 - into MDs. This can be accomplished using previously described means for form- ing-MDs. with sample material which contains biological entities

Although an indication of viability can, in some cases, particularly for cer¬ tain types-' of cells, be determined by use of vital stains such as membrane poten¬ tial responsive dyes, membrane exclusion dyes such as the fight absorbance dye

10ϋ trypan blue, and such as the fluorescent dyes propidium iodide and ethidium bromide, and intracellular enzyme/membrane integrity dyes such as fluorescein diacetate, carboxyfluorescein diacetate and fluorescein isothiocyanate diaceate, (see, for example, Shapiro, Practical Flow Cytometry, A. R. Liss, New York, 1985) it is generally desirable to use a more stringent criterion for determining

155 that a biological entity is viable. Many biological entities, particularly cells and viruses, are stringently determined to be viable only by determining that the bio¬ logical entities are capable of growth, that is, of increasing in size and/or number. Thus, the present invention can be utilized to enumerate biological entities by a process wherein at least one incubation is provided, in order to provide an oppor-

20 tunity for growth prior to measurement of the amount of biological material in

MDs.

In the practice of this invention it is preferred to measure the change in the amount of biological material in MDs subsequent to one or more incubations. More specifically, it is preferred to utilize MDs with individual occupation such

25-i that the amount of biological material associated with individual biological enti¬ ties, particularly cells, can be measured prior to at least one incubation, and also subsequent to at least one incubation, so that the change in amount of biological material is measured. Thus, it is particularly useful to carry out the proceeding process wherein the change in amount of biological material is used as an indica-

30.. tion of viability of biological entities.

In cases in which the stringent criterion of biological entitiy growth is not required as the basis of determining viability, and the biological entities consist of small multicellular organisms, groups of cells, cells, protoplasts, vesicles, spores, organelles and parasites, it is possible to use this invention with short incubation, 35 or essentialy no incubation, by using vital stains in combination with the volume measurements of MDs. Vital stains res ond to one or more im ortant biochemical or physical functions of biological entities, particularly cells, such that said functions can often be measured more rapidly than growth. Representa¬ tive types of vital stains include transmembrane potential stains, membrane exclu¬ sion stains and intracellular enzyme activity responsive stains. Specific vital stains include cyanine dyes, propidium iodide, ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate and fluorescein isothiocyanate diacetateif n LP. Thus, by exposing MDs to at least one vital stain and subsequently measur¬ ing both the vital stain and volumes of the associated MDs, an enumeration of viable biological entities can be obtained Although this invention can be used to obtain an approximate enumeration without statistical analysis applied to the MD measurements, the most accurate determinations involve statistical analysis which utilizes both biological material measurement and MD volume measurement. Such statistical analysis involves scoring each MD, or specimen of MDs, as occupied or unoccupied. Additional information can be obtained by further scoring each MD according to the amount of biological material, so that growth of biological entities is measured and used as the basis for determining viability. In the case that each MD, or specimen of MDs, is scored as occupied or unoccupied, the volume of the corresponding MD, or volume of the correpsonding specimen of MDs, is utilized, such that a statisti- cal frequency distribution of the occurrence of occupation for different ranges of

MDs volumes, or MD specimen volumes, is determined from the measurements, and this frequency distribution used to determine the average number of viable biological entities per volume of sample which was used in the formation of MDs, and which therefore comprises a viable enumeration for the sample. It is preferred to utilize Poisson statistics or modified Poisson statistics with the measured frequency-of-occurrence of occupation in MDs within different MD volume ranges. As in the conventional, standard method of viable plating of cells, random mixing and the Poisson probability distribution are used to obtain an enumeration. The use, if necessary, of iterative computations results in a self- consistent determination described by the Poisson probability function if the bio¬ logical entities were randomly distributed into MDs during the MD creation pro¬ cess. An intitial, trial value of p is used, and the initial occupation distribution for the measured V_MD distribution is computed. The initial value of p is then adjusted, according to whether the computed distribution results in more or less occupation than measured. This process is continued until there is agreement, preferably to within 10 to 30%, but depending upon the application, and then this

value of p is corrected for dilution to obtain p_s, which is the desired viable cell enumeration, expressed as the number of viable biological entities per volume of sample.

The average number of initial biological entities, n, in MDs within a range of volumes Vur, is related to the sample's cell concentration, p, through the rela¬ tion

» = P UD so that s = -^~~- . — (8)

provides a determination of p_s, the viable enumeration. Here f_D is the dilution factor defined by equation (3). The determination of n and V_MD for a statistically significant number of occupied MDs allows p_s to be determined with sufficient accuracy, typically +.10%, to +_30%, which is better or about the same as typical enumerations obtained by conventional viable plating. If desired, increased accu¬ racy in _s can be obtained by making and using measurements on a larger number of occupied and unoccupied MDs and/or groups of MDs. Computation using suitable probability distributions such as the Poission probability formula, or modified Poisson statistics formulae, is also used, self- consistently, to identify which range of sizes within a MD specimen have a high probability of being unoccupied, individually occupied or multiply occupied. Examples of suitable MD creation processes are described elsewhere in this dis- closure. An advantage of processes which produce a wide range of MD sizes is that a wide range of sample cell concentrations, p, can be used. These conditions are useful for samples having a large specimen of MDs in which some significant fraction of the MDs will have volumes which correspond to having a high proba¬ bility of being unoccupied or individually occupied. After formation, the MDs are placed in desired conditions for determining growth. Typically MDs can be suspended, or located, in a medium of a wide range of different compositions, and at different pH, temperamre, partial pressures of oxygen and carbon dioxide, etc., so that, as described elsewhere in this disclo¬ sure, growth under a wide range of growth medium conditions can be determined. After an incubation period the MDs are exposed to one or more staining processes, depending on the type of biological entity and the purpose of the growth determination. In the general case wherein growth analysis without further use of the biological entities is desired, any staining process, including both those which kill biological entities and those which do not kill entities, can be used. In the case wherein further analysis or use of viable biological entities is desired, staining which allows biological entitiy survival is used.

Following exposure of the MDs to suitable stains, MDs are individually measured, or measured in small groups, provided that the probability of finding more than one biological entity-containing MD in the group is small. In the exemplary case of cells stained by fluorescent compounds, optical analysis such as digital fluorescence microscopy or flow cytometry is used to analyze individual MDs, or groups of MDs, using a wavelength band sufficiently different from that used for any detection of measurement of MD properties. Thus, simultaneous, or serial, measurement of MD properties and of cells within said MDs is possible.

The associated fluorescence signals are acquired and analyzed, with correction for spectral overlap if necessary, by conventional means.

The magnitude of the optical signal due to the biological entity stain in each MD, or MD group, is compared to the fluorescence of individual cells, whether or not such individual biological entities are entrapped in MDs, thereby providing a calibration. Comparison of the MD, or MD group, signal magnitude to that of individual biological entities provides the basis for determination of growth of individual biological entities. For example, in the important case of cells, such comparison of signal magnitude provides the basis for determination of growth of individual cells into microcolonies of two or more biological entities, which can be made within about one generation time, but without a need for significant prior culture to obtain large numbers of cells, and provides the basis for establishing that the occupied MDs contain viable cells, as determined by the requirement of growth from one into two or more cells This invention can be used to obtain a viable enumeration of biological enti¬ ties such as small multicellular organisms, groups of cells, individual cells, proto¬ plasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, anti¬ body molecules, antigen molecules, and aggregates of molecules It is preferred to use this invention to enumerate cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells.

Representative suitable means for measuring biological material within MDs, using naturally occuring properties of biological entities, or using stains, includes physical means such as optical, weighing, sedimentation, field flow sedimentation fractionation, acoustic, magnetic, electrical and thermal means. It is preferred to use optical measurements wherein biological material and gel microdroplet volumes are measured using optical phenomenona such as light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphores¬ cence and chemiluminescence.

Optical measurements can be often enhanced by treating or exposing MDs to at least one staining process, wherein at least one stain is utilized to enhance the measurement of biological material. Representative suitable types of stains include stain indicative of biological composition, stain indicative of enzyme activity,'.and: stain indicative of cell membrane integrity Such stains are generally selected to>ha.ve readily measureable properties such as fluorescent stains, light absorbance stains and light scattering stains, and can be further selected according to the class of biological material which is stained, including, therefore, stains such as nucleic acids stains, protein stains, lipid stains, cell membrane stains, cell wall stains, stains responsive to enzyme activity, stains responsive to transmem- brane potentials and cell surface receptor stains.

It is also useful to practice this invention wherein optical measurement is made using apparatus such as flow cytometry apparatus, flow-through- microfluorimetry apparatus, optical particle analyzer apparatus, fluorescence microscopy apparatus, light microscopy apparatus,image analysis apparatus and video recording apparatus

Electrical measurements also have significant advantages, as electrical sig- nals can be coupled directly to computational means. Thus, it is useful to prac¬ tice this ^invention by employing electrical measurement means to measure biolog¬ ical material within MDs and also the volume of MDs. Electrical measurements useful with GMDs include those involving electrical resistance particle analysis apparatus and dielectric property measurement apparatus, while those useful with LMDs involves dielectric property measurement apparatus.

For example, it is well established that a resistive cell counter, often termed a Coulter Counter, can use electrical resistance measurement to determine cell volume (see, for example, Kachel in Flow Cytometry and Sorting, Melamed et al (Eds), Wiley, New York, pp. 61 - 104). In the case of GMDs, the gel matrix of GMDs generally has a high molecular weight cutoff property, such that only large molecules are- excluded, with the result that the gel matrix offers only small electrical resistance if GMDs are suspended in an aqueous medium comprising an aqueous electrolyte with small ion composition similar to that of physiological saline (about 0.9% NaCl). For this reason, GMDs without biological entities such as small multicellular organisms, cells, protoplasts, vesicles and spores have electrical resistance essentially indistinguishable from such aqueous electrolytes, and therefore are not electrically measured, while cells contained with the GMDs * are measured. Specifically, formation of microcolonies in GMDs leads to electri¬ cal resistance of the GMDs which increases with microcolony size, and thereby ^■ provides an electrical means for measuring the amount of biological material in

5 GMDs.

The volume of the corresponding GMDs, or specimens of GMDs, can be obtained by using other means, including optical means responsive to the gel matrix of GMDs, or responsive to marker entities provides within GMDs. Alter¬ natively, by providing marker entities with measureable electrical or magnetic

10 properties in GMDs, it is possible to measure the volume of the corresponding

GMDs, or specimens of GMDs, by electrical or magnetic means. For example, by providing marker entities comprising particles of a high dielectric constant such as barium titanate, it is possible to measure the total amount of such dielec¬ tric material in each GMD, or specimen of GMDs, and thereby to measure the

15 volume of said GMDs. A similar embodiment involves the use of marker entities with measureable magnetic properties, such as magnetite particles which have measureable magnetic properties which can be measured by well known means such as positioning a coil in proximity to the orifice of the resistive particle counter. In these exemplary cases the electrical resistance of GMDs can be

20 insignificantly altered by the presence of the marker entities, because even in the case that large numbers, e.g. 10⁵, of marker entities are used in a 50 μ diameter GMD, the spacing of the marker entities within the gel matrix is sufficient so as to not significantly impede the movement of the small ions which predominantly determine the electrical resistance of such GMDs

25^ Measurements on Mixed Biological Populations

Although many samples contain a single type of biological entitity, for _s example, a monoculture of microorganisms wherein all of the microorganisms are of the same type, a great many samples obtained in biology and medicine are mixed populations, in that at least two types of biological entities are present in

3.0 the sample, generally such that neither the relative numbers of the different types nor the absolute numbers is known a priori. The present invention provides gen¬ eral means for measuring biological entities in mixed population samples, while requiring minimal or no pretreatment of the sample, and can yield such measure¬ ments rapidly. The general process of this invention comprises the steps of: (a)

35/ creating microdroplets from a sample of the mixed population, and (b) making at least one measurement which is sensitive to at least one type of biological entity, and one additional measurement which is sensitive to at least one other type of biological entity.

In this process it is preferred that there is a high probability that each MD 5^" contains less than two types of biological entities, but the process can also be car¬ ried, out under conditions in which there is a high probability that each MD con¬ tains, ar.least one type of biological entity. In many cases, but not all, it is further desirable to provide the additional step of measuring MD volumes.

For example, formation of MDs which are LMDs from a sample containing Q3 two different types of microorganisms, I and II, with I having significantly greater metabolic acid production and secretion rate than It, when provided with the par¬ ticular growth medium provided. Then, with or without dilution of the sample, by forming LMDs which have a range of volumes, V_UD = V_MO . or forming LMDs which have essentially the same volume, LMDs can be formed which have a high 5 probability of being unoccupied or individually occupied, so that each occupied

LMD has a high probability of containing one initial type I, or one initial type π, microorganism. By providing optical pH indicators, either light absorbance or fluorescent, the LMDs can be incubated such that the acid production of the type I allows measurement and identification of the LMDs which contain that microor- 0 ganism, thus providing the basis for measurement of one type of biological entity in that mixed population.

In another example, if GMDs are formed from a suspension containing two different types of cells, type A and type B, comprising the mixed biological sam¬ ple, and it is further assumed that: (a) type A grows significantly faster than type 5 B for the conditions provided, and (b) type A can be labeled with a Green

Fluorescence labeled antibody to type A surface antigens, and type B can be labeled with a Red Fluorescence labeled antibody to type B surface antigen, then the growth of type A and type B can be separately and simultaneously deter¬ mined. Continuing this example, a portion of the mixed population sample is 0 converted into GMDs, thereby incorporating cells of both types into GMDs. A

.φecimen of the GMDs can be exposed, simultaneously or consecutively, to both antffiodies, such that antibodies enter the GMDs and bind to surface antigens, thereby labeling the amount of biological material, in this case amount of surface antigen, for both cell types. The amount of Green Fluorescence and Red Fluores- 5 cence in GMDs is then measured, to thereby provide a measurement of the amount of biological material associated with type A and type B. Following incubation of at least one additional specimen of the GMDs at desired conditions, growth of both types of cells is allowed to occur. All or a portion of this speci¬ men can then be exposed to the same preparation of fluorescence-labeled antibo¬ dies, which provides a distinguishable measurent of the amount of biological material associated with type A and type B. The measured amount of each type of biological material can then be quantitatively compared with an amount of bio¬ logical material of each type in non-incubated GMDs, thereby providing a meas¬ urement of the growth of each type of cell in the sample. As demonstrated by this example, GMDs are used to contain biological entities so that growth can be measured, but the volume of the GMDs need not necessarily be measured.

Although measurement of at least one type of biological entity of a mixed population is often desired, it is possible to use the results of such measurement process to further provide the basis of physical isolation of MDs containing one type of biological entity. In this further process, the value of at least one meas- urement is used to provide the basis for applying at least one force to the corresponding MDs. This force results in physical manipulation of such MDs, and then physical isolation of such MDs. In this way MDs containing a first type of biological material are isolated from MDs not containing the first type of bio¬ logical material. Following such isolation of MDs the biological entities con- tained within the MDs can be isolated or removed from the MDs by methods described elsewhere in this disclosure, thereby providing isolation of the biologi¬ cal entities

Although measurements of a mixed population sample do not in all cases require measurement of the volume of the associated MDs, in most cases, the practice of this invention involves the measurement of MD volumes and the use of statistical analysis to determine the number of biological entities in MDs. In such cases, it is preferred to use statistical formulae such as Poisson statistics and modified Poisson statistics. These formulae provide a relation between the volume, V_MD , the particular occupation or number, n , of initial biological entities in a MD, and the average number, n, of biological entities in a MD of this volume. Thus, by applying these formulae in an iterative fashion, which is gen¬ erally well known, and which can readily by accomplished using a computer, in the process of this invention, the probability that a measured MD has individual occupation can be determined. By then further using measurements which can distinguish at least two types of biological entities, it is possible to catagorize MD measurements into at least two catagories, and which, through the use of the statistical formulae, have a high probability of relating to measurements on only one type of biological entity.

Thus, to summarize this process, as a result of statistical anlysis on measure¬ ments which distinguish at least two types of biological entities and on MD 5 volume measurements, the measurements which have a high probability of relat¬ ing to at .least one type of biological entitity from a mixed population sample can be^obtained: Such measurement results are possible because the process of this invention-provides a statistical, usually essentially random, separation of biologi¬ cal entities into MDs, such that the subsequent measurements and statistical 10 ⁾ analysis allow separated measurements of biological entities. Thus, using this and similar versions of the method described herein, it is also possible to make useful measurements on a mixed sample under conditions where measured GMDs have a high probability of containing less than two biological entities.

Although it is preferred to make measurements using MDs which are indivi- 15 dually occupied, it is possible to make useful measurements even if MDs are multiply occupied. For example, as previously described, if two cell types, type A and type B, are measured by using antibodies for surface antigens, and one antibody has a Green Fluorescence label and the second a Red Fluorescence label, the growth of each type of cell can be separately determined.

20 In another example, if the average occupation due to all types of biological entities of interest, n , is greater than about K = 0.15, the probability of initial occu¬ pation by an individual biological entity decreases, such that if n » 0.15 most MDs are multiply occupied. Even in the case of such multiple occupation, how¬ ever, it is often possible to make useful measurements on MDs formed from a

25 mixed population. For example, if W = 3, for a given size range of MDs, then as described elsewhere in this disclosure, the probability of having n > 7 is small, so that there is a low probability of having more than 6 of one type in the presence of the other type. In many cases the biological entities differ significantly in pro¬ perties such as biological material composition, or in growth, so that useful meas-

30. urements- ca be made in such cases. However, as previously described, the pre¬ ferred-, use erf this invention is to utilize GMDs which have a high probability of containing at least one biological entity.

The use of this invention with measurements which determine growth of at least one type of biological entity is particularly useful, and can be further 35 extended by exposing at least one specimen of GMDs to conditions which affect the growth of at least one type of biological entity. For example, the use of selective growth media is well established in microbiology. The use of such media allows the outgrowth, typically accomplished with long incubations which correspond to a large number of doubling times of the selected microorganisms in » the presence of large numbers of other types of microorganisms. Specifically the

5 microorganisms include those which do not grow, or grow much more slowly, than the selected type. Identical or similar selective media can be used with this invention, but it is not necessary to incubate for long periods. In this case it is preferred, but not necessary, to use Poisson statistics or other suitable statistics to identify GMDs which have a high probability of individual occupation.

10 It is often useful to practice this invention by exposing microdroplets to con¬ ditions which affect the growth of at least one type of biological entity. For example, consider the illustration wherein the MDs are GMDs, and the provision of a selective medium results in zero growth of one type of bacteria and growth of a second bacteria type such that the doubling time for the growing type is

15 t_ = 30 minutes, and that the lag time for establishing growth is only a few minutes.

In this case, following an incubation of approximately one hour the non-growing bacteria will still be present as single bacteria, such that GMDs with n = 1 or indi¬ vidually occupied GMDs will have either single bacteria if occupied by the non- growing type, or will have microcolonies of 4 bacteria if occupied by the growing

20 type. Continuing this illustration, if each type of bacteria has essentially the same magnitude of Red Fluorescence signal following the use of a propidium iodide staining protocol, the non-growing individual cells and microcolonies of 4 cells can be readily distinguised. Individually occupied GMDs with growing bacteria will have Red Fluorescence signals about four times those of individually occu-

25 pied GMDs with non-growing bacteria. _f It is also useful to practice this invention by comparing the growth of at least one type of biological material contained in microdroplets to the growth of at least one other type of biological material contained within the microdroplets. For example, consider a related illustration, wherein again two types of bacteria

30 comprise a mixed population, but now with the less optimal case wherein the selective conditions result in non-zero but different growth rates. Specifically, consider the case wherein type A has a doubling time, t_A2 = 30 minutes, and type B has a doubling time, t_B2 = 45 minutes. In such a case it is preferred, but not neces¬ sary, to use Poisson statistics or other suitable statistics to identify GMDs which

35 have a high probability of individual occupation. In this case, assuming a lag time of only a few minutes, following an incubation of approximately 60 minutes, the type A bacteria will be present in individually occupied MDs as micro¬ colonies of about 4 cells, cells, while the the type B bacteria will be present as microcolonies of about 2.5 cells. Thus, individually occupied GMDs, i.e. GMDs initially containing one cell, will have either microcolonies comprised of about 4 cells if type A, or will have microcolonies of about 2.5 cells if type B. Continu¬ ing this example, if each type of bacteria has essentially the same magnitude of Red'Huorescence signal following the use of a propidium iodide staining proto¬ col; the.microcoibnfes of about 4 cells and microcolonies of about 2.5 cells can be readily distinguised, as the Red Fluorescence signals will be proportional, in this example, to microcolony size. Using this type of measurement and analysis, the average growth rate of both types of cells can be simultaneously determined from the mixed sample. This illustrates a basic method for making rapid measure¬ ments on a mixed population. This example also serves to illustrate the use of this invention to use biological growth to distinguish at least two types of bϊologi- cal entities using biological growth Many variations of this example, including straightforward extension to more than two cell types, are possible.

A general version of this invention involves the use of measurement of bio¬ logical material with sufficient specificity that at least one type of biological material can be distinguished from at least one other type of biological material. For example, fluorescence-labeled antibodies to surface antigens provide a general basis for such specificity of biological material measurements. This has been described previously by means of an example based on a Green Fluorescence labeled antibody to a first surface antigen, and a Red Fluorescence labeled anti¬ body to a second surface antigen, with growth of both cell types then possible. The process of this invention further allows viable enumeration of one or more biological entities of a mixed population sample. As illustrated previously, the growth of different types of biological entities can be determined separately and simultaneously. By further utilizing MD volume measurements with a statist¬ ical analysis based on Poisson statistics or other suitable statistics, the number of viable biological entities per volume of sample can be obtained for one or more types o biological entities, which comprises obtaining a viable enumeration of at least, one. type? of biological entity

Differences in growth under selective medium conditions can be enhanced by exposing one or more specimens of GMDs to compounds which significantly alter growth, such that one or more incubations of GMDs are provided with such compounds present. This allows two or more types of biological entities to be measured by significantly altering the growth of at least one type of biological

* entity. Examples of suitable growth altering compounds include antibiotics, antimicrobial compounds, antifungal compounds, chemotherapeutic compounds,

* toxic compounds, cytotoxins, irreversible inhibitors, reversible inhibitors,

5 mutagenic compounds, hazardous compounds, hormones, growth factors, growth enhancers, nutrients, vitamins, food preservatives, pesticides and insecticides

In addition to making measurements of differential growth and/or measure¬ ments which measure different types of biological material, the process of this invention can be further extended by making measurements which are indicative

10 of different types of biological entity function. More specifically, it is useful to make measurements wherein at least two types of biological entities are deter¬ mined by a measurement selected from the group consisting of biological material, biochemical activity, production of molecules, degradation of molecules, secretion of molecules, metabolism, membrane integrity, enzyme activity and

15 growth. Thus, for example, if biological entities such as cells differ in their abil¬ ity to produce molecules, the resulting differences in production can be measured by using a stimulus such as electroporation to release molecules for capture at binding sites and subsequent measurement. Finally, if biological entities such as cells differ in their enzyme activity when exposed to certain conditions, and the

20 resultant differing enzyme activity is measured, this type of measurement also distinguishes types of biological entities.

As described previously, optical means are preferred for measurement of biological material and microdroplet volumes, using optical phenomena such as light scattering, light absorbance or colorimetric, fluorescence, time-delayed 25 fluorescence, phosphorescence and chemiluminescence. It is also often useful to incorporate marker entities into GMDs in order to enhance microdroplet volume measurement, using marker entities such as beads, non-biological particles, cry¬ stals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts, vesicles, stains and dyes. Alternatively, it is possible to pre- 30 treat GMDs so as to attach marker entities to at least one gel matrix constituent.

Furthermore, marker entities can be incorporated into microdroplets after creation of the microdroplets.

Alternative measurement means include optical, weighing, sedimentation, field flow sedimentation fractionation, acoustic, magnetic, electrical and thermal 35 means. Further, as described elsewhere in this disclosure, it is useful to use opti¬ cal measurements are selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphores¬ cence and chemiluminescence This process of this invention can also involve the additional step of exposing microdroplets to at least one compound which affects biological material prior to measurement, thereby enhancing the distinction ? between at least two types of biological entities.

This invention can be used with mixed popultions in order to make measure¬ ments of biological entities such as small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of 0 molecules, and it is preferred to make measurements on cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells , and also cells such as normal human cells, human cancer cells, pathogenic bac¬ teria, pathogenic yeast, mycoplasms, parasites, and pathogenic viruses.

Furthermore, although this invention can be carried out by measuring the - amount of biological material in at least one gel microdroplet by measuring a naturally occuring optical signal associated with the biological material, such as optical signals selected from the group consisting of light scattering, light absor¬ bance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence, it is preferred to utilize at least one staining process -' involving at least one stain for biological material. Representative types of stains which can be used are stains such as stain indicative of biological composition, stain indicative of enzyme activity, and stain indicative of cell membrane integrity.

It is also useful to practice this invention in an embodiment wherein at least ^'- one stain is used for biological entity identification and at least one stain or a naturally occuring optical signal is used to determine biological entity growth. A particularly useful general version of this embodiment involves the use of at least one fluorescence-labeled antibody is used for biological entity identification.

Thus, this invention can be used to make a variety of measurements on '- mixed biological populations which cannot be readily made by prior methods.

Provision of External Influence on Biological Entities

In many tests and assays the interaction of biological entities with external sources of chemicals, biological factors or physical fields are extremely important, and it is highly desirable to provide means for extending MD measurement and isolation processes to include the effects of such influence. In order to provide such external influence on biological entities, the biological entities are incor¬ porated into MDs , so that MDs which contain biological entities can be intro- duced into position of proximity to a source, maintained in position in proximity to a source or removed from proximity to a source. In the general practice of this invention, sources of influence include biological sources of influence, chemi¬ cal sources of influence, physical sources of influence, and combinations of bio¬ logical, chemical and physical sources of influence are provided by using MDs, In general, however, it is often preferred to provide influence by utilizing

MDs which are GMDs, particularly GMDs which are surrounded by an aqueous medium such that the aqueous medium contacts the aqueous interior medium of GMDs, thereby allowing chemical and small biological entities to be exchanged between the GMDs and the aqueous medium. It is also possible to provide influence by using LMDs and/or GMDs surrounded by a non-aqueous fluid, as most types of physical influence is readily provided, but the chemicals which can be exchanged is more limited, and relatively few biological entities can be exchanged. For aqueous surrounded GMDs, exemplary surrounding aqueous media include aqueous growth medium, physiological fluids, human body fluids, animal body fluids, organ perfusates, suspensions of cells, suspension of small multicellular organisms, animal tissue homogenates, plant tissue homogenates, cell culture medium, culture medium for microorganisms containing biological material, defined culture medium for microorganisms, defined culture medium for mammalian cells, blood, blood plasma, urine, cerebral spinal fluid and interstitial fluid. Upon exposure of GMDs to one or more such aqueous media or environ¬ ments, and depending on the molecular filteration characteristics of the gel matrix, most chemical compounds and some small biological entities can enter the GMD by partitioning into the aqueous environment of the gel matrix. These can be transported, often by diffusion, but also by drift and/or convection, within the GMD, thereby exposing biological entities contained within GMDs to chemical compounds and/or small biological entities contained within the aqueous environ¬ ment.

In the case of such GMDs, the present invention provides means for influencing biological entities to external influence though the general means of providing physical forces which allow manipulation of GMDs, such that GMDs can be moved into and within an aqueous environment. Such manipulation of GMDs allows GMDs to be positioned in proximity to sources of influence which are extemal to GMDs, such that chemical compounds of the aqueous environment in proximity to one or more extemal sources can enter GMDs and thereby interact with biological entities within GMDs, and thus provide influence on the ^" biological entiles. More specifically, this process provides a general means for providing external influence on biological entities by utilizing physical manipula¬ tion .of GMDs.,_..containing at least one biological entity, so as to affect the prox¬ imity of atieast-cme_GMD to an influence source which is external to the GMDs. Thus, it is particulalry useful to provide influence on biological entities by utiliz- 0 ing GMDs in an aqueous fluid.

However, because of the greater generality of this invention, it is appropriate to describe most of this invention in terms of the use of the more general case of MDs surrounded by either aqueous fluids or non-aqueous fluids. Following the provision of influence, it is generally useful to determine the effect of the influence on biological properies of biological entities contained within the MD.

This is particularly relevant for biological properties such as biological material, biochemical activity, production of molecules, degradation of molecules, secretion of molecules, metabolism, membrane integrity, enzyme activity and growth, as these are directly or indirectly involved in many assays and tests In order to flexi- bly provide influence due to one or more biological sources it is useful to provide such biological influence by selecting a source from the group consisting of cells, feeder cells, animal tissues, plant tissues, animal tissue homogenates, plant tissue homogenates, perfused organs, whole animals, plants, cell suspensions, biological entities within gel microdroplets, cell cultures, body fluids. ^" As used herein, the term biological influence describes the influence associ¬ ated with chemical compounds and small biological agents such as phage, which have biological origin and can be present in the aqueous environment in proxim¬ ity to a biological source. Representative biological sources include cells, feeder cells, animal tissues, plant tissues, animal tissue homogenates, plant tissue homo- genates, perfused: organs, whole animals, plants, cell suspensions, biological enti¬ ties within geliπ^ήcrodroplets, cell cultures, body fluids. Chemical sources include any entity which provides to an aqueous environment one or more chemical com¬ pounds, and can therefore include biological sources. Physical sources include any entity which is the source of physical conditions in an aqueous environment. These include physical processes such heat, electrical energy, magnetic energy, optical energy, radioactivity or ionizing radiation, and acoustic energy which alter chemical composition of chemical compounds in an aqueous environment. Physi¬ cal sources also include any entity which directly provides heat, electrical energy, magnetic energy, optical energy, radioactivity or acoustic energy to MDs, thereby providing influence on biological entities contained within MDs. By using the process of this invention in which MDs can be manipulated, including specifically manipulation which results in MDs being near, maintained near and removed from one or more sources of external influence, external influence can be caused on biological entities such as small multicellular organ¬ isms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules It is particularly useful to provide influence on cells such as animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells , and such as normal human cells, human cancer cells, patho¬ genic bacteria, pathogenic yeast, mycoplasms, parasites, and pathogenic viruses In the important case of mammalian cells it is preferred to provide influence on cells such as of normal human cells, cancerous human cells and hybridoma cells

In order to provide complex biological influence, wherein many different and complex biochemical pathways may be involved, and wherein different tissues may participate, it is possible to reversibly position MDs, particularly GMDs, within whole animals such as mouse, immunodeficient mouse, rat, rabbit, pri¬ mate, goat, dog, horse, pig, guinea pig and and human being. These are impor¬ tant biological sources of external influence, and additional related sources comprise one or more perfused organs from such animals Thus, for example, GMDs containing magnetic force-coupling entities in addition to biological enti- ties can be inserted into an animal, so that complex biological influence can be provided. If desired, the biological entities within the GMDs can be protected from an immune response by the animal by providing an appropriate molecular weight cutoff property for the gel matrix of the GMDs. An exemplary means for accomplishing this protection is to use composite GMDs wherein an outermost gel region surrounding one or more biological entity-containing regions is used with a gel with a moderately low molecular weight cutoff.

Additional flexibility can be provided by supplying influence due to one or more sources which are themselves contained within MDs, and in this general case it is the relative position of the influencing MDs and the influenced MDs which is important. Thus either or both types of MDs can be manipulated The flexibility of this invention can also be increased by providing and utilizing gel material for MDs which are GMDs wherein the gel matrix which allows selective passage of molecules

In order to physically manipulate MDs with respect to one or more sources of influence, the process of this invention provides physical forces such as electri- 5 cal force, magnetic force, field flow sedimentation fractionation force, acoustic force, optical, pressure force, gravitational force, sedimentation force, non- rotational! acceleration force, centrifugal force and centripetal force, wherein the means fσrgeneration of such forces is well-known or is described elsewhere in this disclosure In order to more effectively provide such forces it is useful to sup-

100 ply one or more types of coupling entities, or force-coupling entities, wherein such entities are selected from the group consisting of beads, non-biological parti¬ cles, bubbles, and non-aqueous fluid inclusions with force coupling properties ' selected from the group consisting of ferromagnetic properties, diamagnetic pro¬ perties, paramagnetic properties, dielectric properties, electrical charge properties,

155 electrophoresis properties, mass density properties, and optical pressure properties

It is generally preferred to provide magnetic force by incorporating magnetic cou¬ pling entities into MDs , and an exemplary type of magnetic force coupling enti¬ ties are those comprised of magnetite

Forces for MD manipulation near sources of influence can also be provided 20 by incorporating coupling entites with a mass density different than the gel den¬ sity, so that physical forces such as field flow sedimentation fractionation force, acoustic force, gravitational force, centripetal force, centrifugal force and non- rotational acceleration force can be utilized

An illustration of a source of physical influence relates to manipulations and 25.^" measurements involving the use of heat and ionizing radiation for treatment of cancer, wherein cancer cells can be killed by application of heat and or ionizing radiation. The use of ionizing radiation is well established as a therapeutic modality for the treatment of cancer, and the use of elevated temperature, or hyperthermia, either alone or in combination with ionizing radiation, has been 30 more recently established as a therapeutic modality. In such treaments, a general tendency of increased cancer cell killing relative to normal cell killing is exploited. In order to carry out in vitro tests of the sensititivy, and the variability of sensitivity, of cancer cells to heat and/or ionizing radiation, cancer cells can be incorporated into GMDs. The GMDs are then exposed to the physical influence 35 comprising elevated temperature and/or exposure to ionizing radiation, and then, if desired, incubated. The MDs are then measured for changes in the amount of biological material or biochemical activity. It is preferred to measure the amount " of biological material relative to one or more controls, thereby providing a meas¬ ure of growth of the cells exposed to physical influence relative to the growth of ? control cells in which are not exposed to the physical influence. Further, staining

5 protocols can be used which test for membrane integrity. Thus, the fluorescent stain propidium iodide, and additional or separate staining with vital stains such as FITC diacetate can be used as the basis of a short term indicator of viability.

The process of this invention relating to provision of physical influence can also be carried out under conditions that approximate in vivo conditions. For

10 example, GMDs containing cells to be tested can be placed within the body of an animal, such as a immunocompromised mouse, and the animal then exposed to conditions which approximate hyperthermia and/or ionizing radiation treatment in animal species, including humans. This provides extemal influence on the cells which is a combination of physical influence relating to heat and/or ionizing radi-

15 ation, and also external biological influence relating to the biochemical environ¬ ment within the so treated animal. Following such provision of external influence on cells, measurements such as those relating to cell death, cell survival, cell growth and biochemical activity can be carried out using aspects of this invention described elsewhere in the present disclosure. Thus, exposure to heat influence

20 can be provided and utilized to determine cell death and cell growth under condi¬ tions relating to cancer hyperthermia treatment.

Similarly, the effect of the physical influence of ionizing radiation on biolog¬ ical entities, particularly normal mammalian cells and cancerous mammalian cells, can be tested by the process of this invention, either under completely in vitro 25 conditions, or, by inserting MDs, particuarly GMDs, into a test animal, or even a human being, under essentially in vivo conditions. Thus, this invention can be used to determine cell death and cell growth under conditions relating to cancer radiation treatment

The process of this invention can also be applied to combinations of cancer 30 treatment which include sequential or simultaneous combinations of physical treatment such as ionizing radiation and hyperthermia, and chemical treatment, including established chemotherapy and newer therapy based on monoclonal anti¬ bodies and the like.

In the preferred embodiment of this invention, one or more sources of exter- 35 nal influence are selected from the group consisting of mammalian cells, yeast cells and bacterial cells. In the case of mammalian cell culture, biological entities such as cultured mammalian cells can be exposed to "feeder cells". These "feeder cells" comprise the external source of influence, which in this case, are compounds which enhance the growth of cells. In addition, it is often preferred to influence biological entities, particularly cells, to the complex biochemical influence which is be provided by whole animals, especially animals selected from the group consisting of mouse, immunodeficient mouse, rat, rabbit, primate, gpat;.dogr„ horse and and human being. For example, the complex metabolic processes" of whole animals can provide activation and degradation of chemical compounds which is generally difficult to duplicate in cell culture. Thus, the use 0 of the present invention provides a general means for reversibly exposing biologi¬ cal entities such as cells to complex extemal influence resulting from whole animal activation and metabolism of chemical compounds.

In the preferred embodiment of this invention, biological entities are selected from the group of bacterial cells, yeast cells and mammalian cells, particularly ^" normal human cells, cancerous human cells and hybridoma cells.

This invention can also be used in a version wherein one or more sources of external influence are incorporated into microdroplets, thereby providing a means for influencing biological entities wherein the source of influence can be manipu¬ lated by altering the position of the source with respect to the influenced biologi- cal entities which are in other MDs In this general case, different types of force can be used with the influencing MDs, for example magnetic force, while another force, for example, a force depenedent on the mass density of MDs, can be used with the influenced MDs Finally, in a related embodiment MDs sources of influence can be incorporated into MDs, and the resulting MDs manipulated so as - to provide influence on biological entities which are not contained in MDs For example, feeder cells can be incoporated into GMDs with are also provided with magnetic force coupling entities such as magnetite, so that the feeder cells can be positioned in proximity to cultured cells which are not in GMDs, and, following any desired incubation, the GMDs containing the feeder cells can be readily removed.

Ebrces suitable for manipulation of MDs surrounded by a non-aqueous fluid are described elsewhere in this disclosure. Any of a variety of physical forces can be used to introduce GMDs surrounded by an aqueous fluid into close prox¬ imity to a source of influence, maintain GMDs in close proximity to a source of influence, and to remove GMDs from close proximity to a source of influence.

Generally, the physical force is selected from the group consisting of . Electrical force can be provided by interaction of an applied electric field, including an electric field with field gradients, with dielectric particles provided within the gel matrix of GMDs, or more generally by interaction with charge ? groups associated with the gel matrix or coupling entities provided within GMDs.

5 Gravitational force, non-rotational force, centripetal force or centrifugal force and sedimentation force can all be applied by utilizing gel matrix composition having different mass density than the surrounding aqueous medium and providing the corresponding physical force field or acceleration. Magnetic force can by applied by providing coupling entities within GMDs, said coupling entities having

10 diamagnetic, paramagnetic or ferromagnetic properties different from the aqueous medium which surrounds the GMDs. Representative coupling entities for apply¬ ing a magnetic force are magnetic particles, magnetic granuals, ferrofluid inclu¬ sions and the like. A preferred embodiment comprises magnetite (Fe₂0₄) particles within GMDs. Alternatively, magnetic force can be applied directly in those

15 cases wherein the diamagnetic, paramagnetic, ferromagnetic or electrical conduc¬ tivity properties of GMDs differs from the diamagnetic, paramagnetic, ferromag¬ netic or electrical conductivity properties of the aqueous medium which surrounds the GMDs. Acoustic forces can be applied by applying sound or acoustic fields which preferentially interacts with the gel matrix of GMDs, or with coupling enti-

20 ties contained within GMDs, such that the inclusion of said coupling entities results in GMDs having a different mass density, or different mechanical compli¬ ance, than the aqueous medium which surrounds the GMDs. Optical pressure force can be applied by utilizing optical radiation which interacts with the gel matrix or coupling entities contained within the GMDs, or with the larger biologi-

25 cal entities contained within the GMDs (see, for example, Ashkin et al, Nature

330: 769 - 771, 1987). Sedimentation field flow fractionation force can be pro- * vided by simultaneously utilizing a hydrodynamic force and a sedimentation force

(see, for example, Levy and Fox, Biotech. Lab. 6:14 - 21, 1988), and can be used to separate GMDs.

30 After one or more exposures to one or more compounds, and following one or more incubation periods with or without controls, the MDs, that is LMDs and GMDs, are exposed to one or more staining processes, which are described else¬ where in this application, but, as also described elsewhere, if naturally occuring signals are adquate, the staining processes are omitted.

35 Following exposure of the GMDs to suitable stains, individual GMDs are individually measured, or measured in small groups, provided that the probability of finding more than one cell-containing GMD in the group is small. In the exemplary case of cells stained by fluorescent compounds, optical analysis such as digital fluorescence microscopy or flow cytometry is used to analyze individual GMDs, using a wavelength band sufficiently different from that used for any : detection of measurement of GMD properties so that simultaneous, or serial, measurement of. GMD properties and of cells with said GMD are possible. The associated .fluorescence signals are acquired and analyzed, with correction for spectral overlap- i necessary, by conventional means.

The magnitude of the optical signal due to the cell stain in each GMD, or ' small group of GMDs, is compared to the fluorescence of individual cells, whether or not such individual cells are entrapped in GMDs, thereby providing a calibration. Comparison of the GMD signal magnitude to that of individual cells provides the basis for determination of growth of individual cells, for which the growth determination can often be made within about one generation time, but ^' without a need for significant prior culture to obtain large numbers of cells, and growth can also be determined over several generations if desired.

By making a large number of such individual cell growth determinations, the distribution of growth rate, distribution of lag time, and the plating efficiency caused by the exposure to one or more compounds or agents can be automatically determined by computer calculation. Other measurements relating to cell survival and cell death, particularly vital stains such as transmembrane potential stains, membrane exclusion stains and intracellular enzyme activity responsive stains, can also be used. Manual or visual inspection and scoring of GMDs can also be used, but is relatively labor intensive and therefore more prone to error. Thus, the preferred process is that conducted using the automated measurement means.

Similarly, measurements, assays, tests and isolation procedures directed towards the use of biological entities for the production of desirable compounds, or the use of biological entities to provide processes directed towards the degrada¬ tion or modification of undesirable compounds, such as toxic wastes, can benefit from biological or biochemical activation of the biological entities, which activa¬ tion comprises a form of influence on the biological entities.

Homogeneous Specific Binding Assay

This invention can be used to provide measurement of certain types of bio¬ logical entities, herein refered to as analyte entities, capable of reacting with and binding two or more labeled specific binding molecules, wherein the labeled specific binding molecules are measured directly by measuring one or more labels which have been attached to the individual labeled specific binding molecules, or are measured indirectly through the subsequent binding of additional, labeling molecules which can bind to, and thereby label, the labeled specific binding molecule. Examples of suitable specific binding molecules are antibodies, antigens, nucleic acids, avidin-biotin, enzyme inhibitors and Iectins A key pro¬ perty of analyte entities is that the analyte entities have two or more specific binding sites which can bind labeled specific binding molecules during the time required to from MDs from a sample containing the labeled specific binding molecules.

Although the process of this invention is general, applicable to many types of specific binding molecules, this invention is most readily described or illus¬ trated in terms of its application to immunoassays. For example, the process of this invention is illustrated by considering a analyte entity with two distinct epi- topes. In the case of labeled labeled specific binding molecules, a sample con¬ taining the analyte entity is exposed to two different labeled antibodies, with one antibody specific for each of the two distinct epitopes. Following mixing, if desired, and after waiting for diffusion, encounter and binding of the labled anti¬ body molecules, the analyte entities have a high probability of being specifically labeled with two labels because of the binding of the two labeled antibodies.

MDs are then formed, using methods described elsewhere in this disclosure, such that at least some of the MDs have a high probability of being individually occupied by the so labeled analyte entities. One or more measurements of the amount of label in each MD is then made, such that the measurement is capable ^" of resolving the difference of one label from two labels, and other measurements are made which allow the measurement of each MD volume, or the volume of eac group of MDs It is preferred to measure individual MDs for volume and enzyme activity , but in some cases two or more MDs can be measured together Such measurement is used to characterize each MD according to the number of labels contained, for example 0, 1, 2 or more than 2 labels. Statistical analysis, such as that based on Poisson statistics, is then employed with the measured frequency-of-occurrence distribution for n_F = 0 and n_F = 1 to predict the number of MDs in each volume range which should have n_F = 2 because of a random distri¬ bution.

This random prediction is then compared to the measured frequency-of- occurrence, and the excess over random is attributed to the binding of two labeled antibodies to the analyte entity. This excess frequency-of-occurrence is then used with statistical analysis to compute the concentration of analyte entities in the solution or suspension from which MDs were formed, and is further corrected by i computation, if necessary, for any dilution that was made while preparing the solution or suspension, thereby measuring the concentration of the analyte entities imthe± sample.

Although a variety of suitable ^ measurement processes are described elsewhere in this disclosure, in some cases it is useful to measure one or more '^' positive optical signals associated with molecules contained within microdroplets and/or one or more negative optical signals associated with molecules contained in a fluid surrounding microdroplets, such as has been partially described previ¬ ously as "negative fluorescence" (Gray et al (Cytometry 3: 428 - 434, 1983)

In order to describe this invention we use the following notation.

An Subscript denoting analyte (molecule, virus, cell, etc.)

BS Subscript denoting binding sites on the analyte.

F Subscript denoting free or unbound LSBMs (Labeled

Specific Binding Molecules)

L Subscript denoting label of LSBMs H_A. Particular number of analyte entities in a MD.

Average or mean number of analyte entities in a MD.

LSBM Labeled specific binding molecule (e.g. labeled antibody). n_F Particular number of unbound LSBMs in a MD. n_F Average or mean number of unbound LSBMs in a MD. J N_BS Number of binding sites on analyte capable of binding

LSBMs. jotat Total number of labels found within a MD (= n_F + N^n^) ^~L,to .ι Average or mean number of labels found within a MD.

S Signal obtained from a particular MD.

'^ A property of many solutions and suspensions is that LSBMs and analyte entities are distributed randomly within the solutions and suspensions if the LSBMs and analyte entities are free. As used herein, the term free means that the LSBMs and analyte enti¬ ties are not bound, and includes the absence of binding of LSBMs to analyte entities. For the general case in which the formation of MDs divides the solution or suspension from which MDs are formed randomly into the small volumes of MDs, the probability of unbound or free LSMBs occupying MDs is well described by the Poisson formula.

More specifically, if unbound LSBMs are randomly distributed, the probability of finding exactly n_F unbound LSBMs in any MD is,

(n_F)"^Fe-

P_F{n_{F r}n_F) = n_F\ (9)

where the mean occupation is n_F, n_F = V_MDp_F with V_MD a microdroplet volume, and ρ_F is the concentration of all the free or unbound labeled specific binding molecules in the general case wherein N_BS different binding sites (e.g. epitopes) are exploited, N_BS different LSBMs are used at the same concentration, and therefore the concentration of unbound label in a MD with no analyte entitity is N_{BS F}.

Likewise, for randomly distributed analyte entities

In ^{' nΛn} _e~"^A' fo Λ.) - ^{i i} "A-j- ⁽10⁾

so that H_A. = V_m p_M where V_MD is a microdroplet volume, and p_An is the concentration of the analyte. The variable n_Λn is random for a well mixed system, and is therefore independent of n_F.

The total number of measurable labels remains constant, unless one or more degra- dative reactions occur, thereby modifying labels so as to effect the measureable proper¬ ties of the labels. Further, it is well known that non-specific binding can sometimes occur if macroscopic solid surfaces or microscopic entities such as certain cell surfaces, virus surfaces and/or molecules are present. Such undesirable effects can also decrease the amount of free label. Generally, however, the combination of label degradation and non-specific binding of LSBMs can be made small by avoiding the use of macroscopic solid surfaces, since the occurrence of label degradation is relatively rare. Thus, although a general condition is that total amount of LSBM is conserved. In many cases essentially all of the LSBMs exist free in solution or are specifically bound to analyte entities.

Although it is not generally necessary, it is often preferred to carry out an addi¬ tional step following the reaction of LSBMs with analyte entities. This additional step comprises reducing the concentration of the free LSBMs, so that the concentration of free LSBMs relative to the concentration of LSBM-analyte is reduced prior to formation of MDs. In this way, a relatively high concentration of LSBMs can be first employed in order to more rapidly drive the specific binding reactions which lead to the LSBM- analyte entity complexes Following addition and mixing of LSBMs to a sample solution or suspension, and a subsequent incubation during which LSBMs are allowed to bind to analyte entities, it is, often desirable to reduce the concentration of the remaining unbound or free LSBMs. This can be accomplished by the additional step of adding purging entities, such that free LSBM encounters and binds to the purging entities. Suitable purging entities _^: include beads, non-biological particles, crystals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts and vesicles, which have sur¬ faces with tightly bound molecules capable of tightly binding labeled specific binding molecules . By using one or more additional signals (e.g. light scattering, fluorescence) to distinguish the purging entities, the label signals associated with the purging entities : can be ignored during analysis of the measurements. Alternatively, if the purging enti¬ ties bind significantly larger numbers of label than analyte, the analysis of measurements can distinguish such larger numbers of labels, and will not confuse such labels with labels associated with LSBM-analyte complexes.

A general attribute of the process of this invention is that it is not necessary to accurately know the concentration of the LSBMs, since the concentration of LSBM- analyte complexes is the information sought. More specifically, although the provided concentration of the LSBMs is readily known, the concentration following reaction with and binding to analyte is generally not known. This is further less known following the use of any purging entities. However, it is often desirable to know that concentration of label in order to provide corrections to the measurements. An advantage of this inven¬ tion is that the frequencies-of-occurrence of label is determined using many MDs, often of significantly different volumes, V_UD, such that subsequent analysis of such frequencies-of-occurrence allows computation of p-sw

The process of this invention is illustrated by considering MDs with a range of - volumes from V_MD to V_UD + AV_MD , such that following exposure of the sample to LSBMs and the subsequent step, if desired, of adding purging entities, this range is characterized by 7T_F = 0.15, n_An = 0.15 and N_BS = 2. In this case the Poisson formula predicts the follow¬ ing probabilities.

n_F P(.n_FJT_F=0.15) n_AΛ P(n_{An r}n_An=QΛ5) 0 0.8607 0 0.8607 1 0.1291 1 0.1291

2 0.00968 2 0.00968

3 0.000484 3 0.000484

In this illustration a MD with zero analyte thus has a probability of about 0.0097 of having two labels through random occupation by LSBMs, and the same volume MD has a-φrobability of 0.1291 of having two labels through occupation by one analyte entity. The excess probability of two labels resulting from LSBM-analyte occupation compared to random occupation by two free LSBMs is

AP = P(n_An=2,n_An~~0Λ5) - P(n_F=2ji_F=0Λ5) = 0.119 (11)

Thus, if a sufficiently large number of occupied MDs are measured, the difference in probabilities can be measured by measuring the corresponding difference in frequency- of-occurrence of the measured and that predicted based on the measured frequency-of- occurrence of n_F = 0 and n_F = 1.

Continuing this illustration, the Poisson probabilities for n_F = 0, n_F = l and n_F = 2 are

P{n_F~-0,n_F) = --"^F (12)

P(n_F=lJT_F) = ne^~"^F (13)

The probability of measuring two labels resulting from random occupation is thus related to the probabilities for measuring zero randomly occuring label, and for measur¬ ing one randomly occuring label through the equation

The mean or average occupation, n_F, of randomly occuring label can be obtained from the mathematical relation

The experimentally measured frequency-of-occurrence of n_F = 0, n_F = 1 and n_F = 2 can now be. quantitatively compared with the probabilities obtained by computation using the Poisson probabilities. More specifically, the experimentally measured frequency-of- occurrence, f (jip) and the Poisson formula are utilized to compute the difference between measured frequency-of-occurrence and the theoretical random frequency-of- occurrence. This can be used to interpret the difference, if statistically significant, to the occurrence of MDs with complexes of analyte and N_BS LSBMs. This is accomplished by measuring the frequencies-of-occurrence and then computing first the mean value n_F associated with random occurrence, i.e.

^{nF =} T^≡όT ⁽¹⁷⁾

and then using this in computing the difference

Δf (n_F =2) (18)

This measured difference is used with the total volume, V_{UD total}, of measured MDs in the volume range AV_MD to compute the concentration of analyte. More specifically,

^YMD, total = ∑ V M, DJ (19)

wherein N MDs are measured within the volume range V_MD to V_MD + AV_MD . Continuing - this illustration further, the analyte concentration, p_An, is computed from

and thereby achieves the desired result of measuring the analyte concentration.

Continuing this illustration still further, the error in the measurement of p_An can be estimated by using well-known methods of error analysis. As shown above, the deter¬ mination of p^ depends on measured frequencies-of-occurrence and measured MD volumes. Thus, the error in determining p^ can be computed using well-known propagation-of-error methods, so that the overall result of the process of this invention is a measurement of p_An, and also a determination of the error in the measurement.

More generally the process of this invention utilizes measurement of frequencies- of-occurence in MDs, and also measurement of MD volumes, such that comparison between random occupation of MDs by label and non-random occupation of MDs by label can be made. One general process, useful in cases wherein the average occupation by free label, n_F, is less than one, utilizes the mathematical recursion relation for the Poisson formula, here used with the abbreviated notation P(n) ≡ P(n -n). so that the recur¬ sion relation is

P(_n-<-\) = = ^■P in)

(N + l) (21)

so that on average the measured frequency-of-occurrences for random events will be related by

^{<D,+,W =} T cS W'* ⁽²²⁾

and can be straightforwadly used to compute the excess over random for occupation of MDs by label over a wider range of occupation.

An alternative method for providing statistical analysis of the MD measurements is useful in the case wherein significant measurement error, δf(n_F), in the frequency-of- occurrence is expected. This alternative method utilizes well-known averaging methods, which reduce error. The occurrence of free LSBMs and analyte entities within a MD are statistically independent, so that the probability of having a particular combination of n_F free label or free LSBMs and n^ analyte entities such that the total amount or number of labels is n_L = n_F + N_BSn_An. This emphasizes that different combinations of n and n_An can result in the same n_F , and indicates that in order to compute the probability of a given value of n_L a summation over the different possibilities giving the same n_L is needed.

A basic property of this approach is the recognition that n_L is a linear combination of n_F and n_A_, and that therefore the probability function for n_F is given by the convolu¬ tion of Pp μ_F,W_F) and P_Anin_Amji_A-). Thus, using well-known results of fundamental proba- bility theory, the average values W_L. n_F and n^ are related by

n_L = /_ +N₅₅^ (23)

and the variances of these same three parameters are related by

var(n ) = var{n_F) + N^var^) (24)

where the variance is a measure of the spread or statistical error which is expected. By ^" using the above two mathematical relations, and measurements of frequencies-of- occurrence to obtain determinations of K_L and var(n_L), the parameter W_An can be com¬ puted. This computed value is used in Poisson statistics formulae with measured values of V_MD to compute p_Λ„.

The statistical error in the average and the variance is well characterized according to fundamental probability theory, and can be used with propagation-of-error analysis to compute the error in p^.

Both monoclonal and polyclonal antibodies with labels can be used as LSBMs in this invention. If monoclonal antibodies are used, N_BS is known exactly, as is the aver¬ age number of labels per LSBM. However, in the related case wherein LSBMs are polyclonal antibodies, somewhat different numbers of antibodies,, may bind, with varying avidities, to individual analyte entities, such binding by different numbers, however, can generally be characterized by an average or mean number of binding sites, N_BS. The value of N_BS is determined from initial, calibrating experiments, as it is recognized that there may be some variation in the actual number of bound LSBMS, that is, there may 5 be a_ distribution, N_BS ~~ N_BS ± δN_BS around N_BS. In this case, measurement of frequencies-of-occurrence results in a superposition of (1) a random distribution, con¬ sistent with the Poisson formula for free label, and (2) a peaked distribution located at N_BS with width δN_BS.

A general computational process can be utilized with the measurements of the ^■ frequencies-of-occurrence and MD volumes, as both N^~ _BS and δN_BS are first determined in a calibrating process. The frequency-of-occurence measurements are then compared self-consisitently, by well known computational means, to a general probability distribu¬ tion. This comprises a linear superposition of a Poisson function for free label and for analyte wherein the average analyte has bound N_BS labels. More specifically, the meas- ured distribution is fit to

P(L)_fit = PpQir Tr) + N_BSP_An(n_{An r}n_An) (25)

Subtraction of the Poisson formula P_F thereby results in determination of P_An. The value of W_An is obtained from P sub bound, and in combination with V_UD for the range of MD volumes used, yields the desired analyte concentration p_An. This process is further illustrated by the case wherein the analyte biological entity is a macromolecule with three distinct, non-overlapping epitopes for which three non- cross reacting antibody molecules are assumed. It is preferred to supply each of the three antibody molecules at a concentration approximately equal to ten times the largest expected analyte concentration. Thus, if the largest expected analyte concentration is about p_M = 10^-9 Molar, then each of the three antibodies is provided at a concentration of about 1(T⁸ Molar . Following addition of the three antibodies to the sample, the resulting preparation is mixed, and an incubation time of about 10 seconds to 3 hours, preferably about 2 minutes to 20 minutes , is utilized. This allows diffusional encounter and an oppor¬ tunity for the antibodies to bind to the analyte. Following this specific binding incuba- tion, any reagents or gelable material, if desired, are added. The resulting preparation is mixed, and MDs are formed by any of the several methods described elsewhere in this disclosure, but preferably by dispersion into a non-aqueous fluid. Generally, it is pre¬ ferred to measure small MDs, for which the probability difference for specifically bound label and randomly distributed label is largest. That is, the difference

ΔP = P_AΛ _An,n_An) - P_F(n_Fmn_F) (26)

is the probablistic basis for measuring analyte through the highly improbable association o N_BS labels within a MD compared to the probability of random occurrence.

Although it is preferred to carry out the process of this invention by using equili¬ brium conditions, or close to equilibrium conditions following exposure of analyte to LSBMs, it is also possible to utilize conditions far from equilibrium. In such non- equilibrium cases, a sample containing analyte can be exposed to LSBMs for a significantly shorter time than needed for equilibrium, or for being close to equilibrium, and fewer completed complexes of analyte and N_BS LSBMs are formed. That is, not only are fewer complexes with exactly N_BS LSBMs formed, there are more incomplete ^' complexes formed wherein less than N_BS LSBMs are bound to each analyte entity. In spite of these less desirable attributes, a non-equilibrium measurement can be accom- pfi^'shediby the. further step of making quantitative comparison, with otherwise the same nσπ=equilibrium conditions, to one or more calibrating measurements employing known concentrations of analyte. ^: The measurement process of this invention can be applied to very low concentra¬ tions of analyte molecules, as analyte molecules can be actually counted. In addition, the measurement can be made in solution without the use of a solid phase which must be washed. Further, because the measurement process can involve a counting process, the invention provides means for measurements over a large dynamic range of analyte concentrations, that is, from high concentrations to orders of magnitude lower concentra¬ tions.

Prior to the carrying out of the process of this invention, two or more labeled specific binding molecules are obtained, using means well known in the art, such that two or more labeled specific binding molecules are prepared, which are capable of bind- ing to two or more binding sites on the analyte. In the important case wherein LSBMs are antibodies, this requirement corresponds to using antibodies which bind to at least two non-overlapping epitopes on the analyte, such that at least two antibodies can be simultaneously and specifically bound to the analyte. Examples of such labeled specific binding molecules include (a) monoclonal antibodies with about one label molecule bound to each antibody molecule, (b) antigen molecules with about one label molecule bound to each antigen entity, (c) monoclonal antibodies with about two label molecules of the same type are bound to each antibody molecule, (d) antigen molecules with about two label molecules of the same type bound to each antigen entity, and (e) polyclonal antibodies containing at least two antibodies capable of binding to at least two non- overlapping eptiopes of the analyte entity.

Many, different: types of antlyte entities can be measured by the process of this invention. More specifically, analyte entities with at least two non-overlapping and non-competing specific binding sites can be measured. In the important general class of analyte entities consisting of antigens for which the LSBMs are labeled antibodies, antigenic analyte entities capable of independently binding antibodies at two or more different sites can be measured. Examples of such analyte entitiies with two such sites include all antigens capable of assay by a sandwich assay, for example creatine kinase and hCG (human chorionic gonadotrophin). Examples of such analyte entities with three such sites include proinsulin and the β-subunit of TSH (thyrotropin). In the case of small molecules such as haptens, the assayed analyte entity with two or more specific binding sites may consist of a hapten-carrier molecule complex.

Alternatively, if an analyte entity has multiple occurrence of one or more binding sites, LSBMs with the same or different labels can be used to multiply bind to each analyte entity. For example, an antigenic polymer may have one or more multiple epti- topes, such that the same antibody can specifically bind at multiple sites on the polymer, so that such an antibody can be used in the process of this invention. Continuing this illustration, one or more labeled specific binding molecules in the form of one or more labeled antibody molecules are exposed to the analyte solution or sample, such that the antibody molecules can bind at two or more repeated specific binding sites on the poly¬ mer, thereby associating two or more labeled specific binding molecules with each analyte molecule. Other analytes with repeated binding sites include cells and viruses.

In the general practice of this invention, analyte entities such as cells, organelles, viruses, nucleic acids, antibodies, enzymes, structural proteins, hormones and drugs can be measured First, a sample containing such analyte entities is converted into a liquid solution or liquid suspension by any of the standard, well known means for preparing analyte samples. The resulting analyte preparation is then exposed to labeled specific binding molecules, such that there is a high probability, following mixing and waiting for diffusion to occur, that at least two LSBMs can bind to each analyte entity, thereby forming analyte entity-complexes which contain at least two LSBMs. Second, some or all of this-reacted preparation can then be used to form MDs, such that there is a high probability that at least one MD, and preferrably at least 10³ MDs, is individually occu¬ pied by an analyte entity-LSBM complex. Third, measurement means capable of measuring at least one LSBM is preferably utilized to measure MDs, and also the volumes of the MDs, such that by using statistical analysis, such as Poisson statistics the occupation by LSBMs, and the associated volumes of the MDs, can be determined. Computation can then be used to compare the measured frequency-of-occurrence of

LSBMS for LSBMs occuring singly and for LSBMs occuring multiply, such that the excess of multiple occurances above that due to random occurrence is attributed to the specific binding of LSBMs to analyte entities. The number of such above-random occurrences is then used with statistical analysis to compute the number of analyte enti- ties with bound LSBMs. This is combined with the volume of analyzed MDs to yield the number of analyte entities per volume in the preparation from which MDs were formed, which number per volume is the concentration of the analyte entities.

In another version of this process, it is only required that measurement means be capable of measuring N_BS labels, as this measurement sufficies if the occurrence of free label within MDs obeys K_F <N_BS. Labels for LSBMs suitable for use with this invention include enzyme activity, bio¬ logical activity and fluorescence. Fluorescence can be readily measured when large numbers; of fluorescent molecules are present, but it becomes increasingly more difficult for smaller' numbers. Thus, the present detection limit is about 10³ molecules for fluores¬ cein measured in a flow cytometer, but still lower for measurement apparatus such as quantitative fluorescence microscopy. A fundamental limit appears to relate to the number of fluorescence emission photons emitted before photodamage occurs, but it may be possible to measure individual fluorescent molecules such as phycoerythrin (see Mathies et al in Fluorescence in the Biomedical Sciences, Liss, pp. 129 - 140, 1986). Thus, measurement based on singly or multiply labeled specific binding reagents wherein an analyte molecule specifically binds several labeled specific binding molecules is feasible. For example, by attaching an average of three fluorescent labels to each of three different antibodies, nine fluorescent label molecules become associated with each reacted analyte molecule. These can be readily measured by methods directed towards measurement of individual fluorescent molecules. In general, however, the magitude of fluorescence signal associated with measure¬ ment of a fluorescence label is much too small. For this reason, the preferred embodie- ment of this invention involves the use of an active label, such as analyte activity related to vesicles, phage or biochemical activity. It is preferred to utilize one or more active enzymes, such that at least one enzyme label is measured through the use of opti- cal measurements such as light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemiluminescence, but particularly fluorescence In much of the following description the invention is described in terms of an enzyme label, wherein the activity of at least one type of enzyme molecule provides the basis of measurement of the label, and is generally well known for use in macros- copic or non-microdroplet cases Such enzyme activity measurement is accomplished generally by accumulating fluorescent product of one or more enzyme catalyzed reac¬ tions within a MD containing the enzyme, and generally requires an incubation period during which the fluorescent product or fluorescent products can accumulate It is pre¬ ferred to provide conditions for a kinetic analysis, wherein substrates, co-factors, etc. are provided under conditions that will allow the enzyme catalyzed reaction to proceed at the maximum reaction velocity, which is at a rate equal to the turnover number for the enzyme. Under these conditions, product accumulation occurs linearly with time until product inhibition, substrate depletion, or other well-known enzyme reaction effects occur. Generally it is preferred to utilize measurements of MDs which have a high pro¬ bability of being unoccupied or individually occupied by analyte entities, that is, con- taining less than two analyte entities

In addition to utilizing enzyme activity measurement directly, wherein one or more enzyme labels are measured by measuring one or more reactants of the corresponding enzyme catalyzed reactions, larger signals can generally be obtained by using enzyme channeling and or enzyme cycling, both of which methods are well established for bulk solution use but have not previously been suggested or demonstrated for use in MDs to measure individual, or small numbers of, enzyme molecules. Enzyme channeling, or use of linked enzyme reactions, consists of providing additional types of enzymes, such that a product of a first enzyme catalyzed reaction serves as a substrate for a second enzyme catalyzed reaction, and a product of the second enzyme catalyzed reaction serves as a substrate for a third, and so on. Likewise, the well established method of enzyme cycling provides amplification for measuring enzyme labels by utilizing a cyclic reaction process wherein the product of a first enzyme catalyzed reaction is a substrate or cofactor for a second enzyme catalyzed reaction, and a product of the second reaction is in turn a substrate for the first (see, for example, Siddle in Alternative Immunoassays, Collins (Ed.), Wiley, 1985). In either case, larger amounts of reaction product can be obtained within a MD. In order to utilize this version of the invention, additional enzymes, substrates, cofactors and the like are provided in the sample, if not already present, so as to ensure that these additional enzyme reactions reactants and enzyme will be present in each MD A sample containing particular analyte molecules whose analysis is desired, can be converted into an aqueous solution or suspension by any of a number of well known means, such as tissue homogenization, stirring, dissolution, and the like.

During or after the above treatment which yields a liquid solution or liquid suspen¬ sion form of the sample, labeled specific binding molecules with enzyme labels are added to, and thoroughly mixed with the sample. The enzyme labeled specific binding molecules are provided in sufficient quantity, based on the expected maximum amount of the analyte molecules, so that essentially all of the analyte entitys will have reacted with and thereby bound the enzyme labeled specific binding molecules within about 10 seconds to 3 hours, and, under more optimal conditions, about 2 minutes to 20 minutes. Although substrates and/or cofactors for the enzymes catalyzed reactions can be add in ri r ste s it is referable to add these reactants us ri r to creation of li uid or gel microdroplets, in order to minimize the amounts of enzyme products are formed which would be distributed into essentially all of the liquid or gel microdroplets, and which would result in an undesireable high background fluorescence signal in all liquid or gel microdroplets. ^' A variety of methods can be used to measure the volumes of the MDs with and without LSBMs. The generation of some background, that is, fluorescence present uni¬ formly,- in. essentially all liquid or gel microdroplets, is sometimes desirable, as such background' provides a means of measuring the volume of microdroplets. In this case, all MDs have a low level of fluorescence, while MDs containing LSBMs, either ^; unreacted or reacted so as to have bound with analyte analyte entities, have increased fluorescence. This is associated with the enzyme label catalyzing a reaction which has one or more fluorescent products, or is coupled to one or more reactions which increase fluorescence.

In much of the measurement process it is possible to measure individual MDs, or ^" to measure groups of MDs, with the latter often preferable if the sample concentration of analyte entity is low, as in that case relatively few MDs are occupied by analyte enti¬ ties. In the following section the quantity V_group refers to the total volume of a group of MDs which are measured together, and it is understood that a group of MDs can contain a number of MDs which is in the range 1 to 100 MDs, preferably 1 to 10 MDs. As an alternative to utilizing background fluorescence from one or more enzyme reactions for detection of, and measurement of V_groιφ for each analyzed MD group, one orrmore. fluorescent molecules can also be provided at low concentration in the solution orrsuspension prior to creation of MDs. This background concentration is selected to correspond, in the largest MD groups, to an amount which can be distinguished from

produced by one or two enzyme molecules within the volume of the largest MD groups used in the analysis.

As another alternative, a fluorescent molecule type with fluorescence properties dis¬ tinct from those used in enzyme assays within MD groups can be used to provide detec¬ tion of, and measurement of V_group of each MD group. This is useful for subsequent mathematical processing of the measurement data from a number of individual MD groups..

The aqueous solution/suspension form of the sample is then used to create LMDs or GMDs by any of several methods described elsewhere in this disclosure. A preferred method is to add agarose, any tracer entities, reagents for enzyme assays, and the like, and to disperse the resulting solution/suspension in mineral oil or silicone fluid. The resulting MD preparation is then incubated to allow the enzyme catalyzed reaction(s) to proceed, such that fluorescent product(s) accumulate preferentially in those MDs which contain enzyme labeled specific binding molecules. Depending on the capa¬ bilities of the optical measurement apparatus, and on the turnover number of the 5 enzyme(s) used, the incubation time can range from about 30 minutes, or less, up to several hours.

There are a variety of choices of enzymes and measurement apparatus attributes. However, all are subject to the condition that either individual enzyme molecule activity is measured, or that a sufficiently large number of enzymes are bound to individual 100 analyte entities by enzyme labeled specific binding molecules that only this largeer number is measured within each analyte occupied MD group. Desirable properties of an enzyme include high turnover number, stability, and specificity.

Following an incubation period some or all of the MD groups are measured opti¬ cally, preferably using in an appratus with quantitative image analysis capability. 15.^' Representative suitable apparatus includes flow cytometry apparatus, flow-through- microfluorimetry apparatus, optical particle analyzer apparatus, fluorescence microscopy apparatus, light microscopy apparatus,image analysis apparatus and video recording apparatus.

An important aspect of detection and measurement of one or more individual 20... enzyme molecules was not explicitly mentioned or discussed in the prior art (see Rot¬ man, PNAS 47: 1981 - 1991, 1961). Specifially, it has not been disclosed that it is important to use conditions wherein the spontaneous rate of fluorescence production is sufficiently small that the catalytic effect of a single enzyme molecule in a small volume can be distinguished against the background, generally increasing with time, due to the 55 spontaneous rate of fluorescence production. Such a spontaneous rate generally occurs for fluorgenic substrates, wherein the non-fluorescent fluorogenic substrate spontaneously decays, and generates the same fluorescent molecules as the enzyme catalyzed reaction. Methods for avoiding singificant spontaneous fluorescence have not been generally appreciated, or even explicitly acknowledged. Therefore, suitable methods for minimiz- 0^'i ing spontaneous fluorescence are described as part of the present invention.

One- general method is to determine, and then utilize, enzyme catalyzed reactions for which the fluorogenic substrates under the conditions of the assay yield fluorescence sufficiently small so that the fluorescence due the product molecules catalyzed by one enzyme molecule is detectable. In this approach, it is fundamental to consider the 5 volume, V_group, which contains the enzyme labeled specific binding molecule. The spon¬ f flu resence accumulation is ro ortional to V while the enz me catalyzed rate is, to a good approximation, independent of v_groιψ.

It is preferred to use a quantitative fluorescence microscope with image analysis capability wherein the amount of fluorescence emission from individual MDs, or groups of MDs, can be automatically measured. Alternatively, a flow cytometer with capability of measuring fluorescence at low levels can be used, such that the fluorescence associ¬ ated with the accumulated product in a MD group due to one enzyme molecule can be detected ,and:distinguished from background fluorescence. Optical measurement apparatus which- allows- detection of, for example, 10⁴ to 10⁵ fluorescein molecules is well known (see, for example, Shapiro, Practical Flow Cytometry, Liss, New York, 1985).

It is preferred to use an enzyme such as β-galactosidase, with the fluorgenic sub¬ strate fluorescein-di-b-D-galactopyranoside, fluorescein is the fluorescent product, has a large quantum yield, and is readily detected and measured at the level of 10⁴ to 10^s fluorescein molecules (see, for example, Shapiro Practical Flow Cytometry, A. R. Liss, New York, 1985). Another useful substrate is FTTC-diacetate (fluorescein isothiocyanate-diacetate), which has the further desirable property of binding strongly and non-specifically to proteins. Thus, by providing a suitable amount of inexpensive protein such as BSA (bovine serum albumin) mixed into the sample before GMDs are created, and subsequently coating the GMDs with a gel or other coating sufficient to retain BSA, a significant fraction of the FTTC is intercepted and bound by the protein, so that an enzyme occupied GMD accumulates detectable GF.

Another useful enzyme catalyzed reaction is alkaline phosphatase utilized with the fluorgenic substrate 4-methylumbelliferyl phosphate, which catalyzes the degradation of this substrate into the fluorescent product 4-methyl umbelliferone plus the non- fluorescent product inorganic phosphate (see, for example, Guilbault Handbook of Enzy¬ matic Methods of Analysis, Marcel Dekker, New York, 1976).

Following measurement of the amount of fluorescence(s) and volume, V_group, of each.MD group in a preparation, the resulting data is analyzed in the following way, preferably using a computer. The individual values of V.-_oup are summed, thereby pro- vidihg a determination of the sample actually analyzed. The amount of fluorescence(s) in.each MD group) is determined, with respect to previously carried out standard calibra¬ tions, so that the. number of enzyme molecules in each range of volumes of MDs is determined. Typically, the MDs have volume which range from about 5 x 10^~10 to about 5 x 10"⁷ ml , so that an analysis MDs of in the size (volume) ranges of typically 5 x 10^~10 to 7.9 x 10^-10 ml, 8 x 10^"10 to 1.19 x 10^"9 ml, 1.2 x lT⁹ to 1.59 x 10^"9 ml, 1.6 x 10^"10 to

2 x 10^→ ml, and so on, is carried out mathematically. Such an analysis first makes use of the individual V_group determinations, and divides the MD group volume range into volume intervals such that there are a significant number of MD groups in each interval. Typically, MDs created by dispersion in mineral oil have a volume distribution which rises sharply and then falls moderately with increasing V_MD , with the consequence that most MD groups have volumes just above an approximate cutoff size (e.g. 10 micron diameter, V_group - 5 x 10^~10 ml), and that it is useful to select volume intervals of approx¬ imately a half order of magnitude in V_group, as indicated above.

The Poisson distribution, is then used with iterative computations, to find the best self-consistent fit to each of the volume ranges. In the present case, if the concentration, or number density, of particular molecules is p, then the average number of such molecules in MD groups of volume V_gτoup is n = ρV_group. The Poisson distribution is used to describe the statistical distribution of small entities such as cells or molecules, such that the probability of finding n cells or molecules in a volume V_group is given by P (n ,n). For purposes of interpreting measurements on a number of MD groups, n is the number of enzyme molecules found in each MD group, and n is the subsequently com¬ puted average number of enzyme molecules for each value of V_gr0Iψ Measurements of MD groups without enzyme molecules (n = 0) and MD group with one enzyme molecule (π = 1) are used with the Poisson distribution equation to predict the statisti¬ cal frequency with which multiple enzyme occupation of MD groups will occur due to random occupation. That is, the number of randomly occuring cases of n = 2, n = 3, n = 4, etc. are computed.

The number of MD groups found by measurement of fluorescence of individual MD groups to contain n = 2, n = 3, etc. enzyme labeled specific binding molecules is then used to compute a best value of n for each range (interval) of V_group values, and then to compute the best value of p by applying the equation

P = ^- . (27)

' group

which is the intermediate of the assay, thereby being the number of analyte entities per volume in the diluted sample. It is straightforward to then compute a correction factor, o ,

for the dilution during the MD creation process, and thereby, to obtain the concentra¬ tion, p_oj_uΛyu.. of analyte entity in the (original) sample.

In this way, the process of this invention provides a determination of the amount of analyte in the original sample, and is based on the determination of individual analyte 55 entities within individual MD groups, as revealed by measurement of individual enzyme molecule activity. In order to obtain increased fluorescence it is generally preferred to provide at least one incubation, wherein the enzyme catalyzed reactions which result in altered fluorescence, usually increased fluorescence, can proceed.

Finally, in addition to providing and utilizing LSBMs, it is also possible to provide

100 and utilize one or more types of intervening molecules, such that the intervening molecules bind to one or more sites on the analyte entity, and one or more LSBMs are subsequently used. For example, a polyclonal preparation of unlabeled mouse antibo¬ dies can be used as intervening molecules, wherein these intervening molecules are mixed with the sample so as to allow binding of these unlabled intervening molecules to

15 binding sites on the analyte entities. A goat anti-mouse antibody with an enzyme label, or other suitable label, can also be added, such that the goat anti-mouse antibody is a LSBM.

It is often desirable to remove or purge the unbound intervening molecules, which can be accomplished by adding beads with surface-bound and unlabeld goat anti-mouse

20 antibodies. Such a purging step is generally useful, and consists of supplying purging entities such as beads, non-biological particles, crystals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts and vesicles, which have surfaces with tightly bound molecules capable of tightly binding labeled specific binding molecules , which will remove, but not necessarily all, of the unbound intervening

25 molecules, so as to greatly reduce the occurrence of complexes of intervening molecule-LSBM which are not associated with the analyte entities.

One or more purging steps can also be used in the case that only LSBMs are used, so as to bind much, but not necessarily all, of the free or unbound LSBMs which remains after the sample containing analyte entities has been exposed to the LSBMs, but 30. before MDs are formed.

Significantly, it is not generally necessary to remove the purging entites from the sample before forming MDs and carrying out the remainder of the process. In either the case of removing LSBM, wherein mostly free or unbound LSBMs are removed, or in the case in which unlabled intervening molecules are removed, the association of the 35 removed LSBMs and or intervening molecules with the purging entity provides a gen¬ General means for distinguishing the purging entities includes measurements based on optical properties, mass density properties, acoustic properties, magnetic properties, electrical properties and thermal properties, and it is preferred to use light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphores- cence and chemiluminescence. Thus, MDs which contain purging entities can be readily distinguished from MDs without purging entities, so that the analysis of meas¬ ured MDs can utilize measurements only from MDs without purging entities as the basis for carrying out the desired assays and tests.

The invention will now be more specifically illustrated in the following non- limiting examples.

Example I: GMD(Green FluorescenceVGMDs(Red Fluorescence)

First agarose GMDs were created by dispersion of an aqueous solution containg fluorescent microbeads with Red Fluorescence (Polysciences, 0.1 micron) into mineral oil (equivalent to parafin oil, heavy; Fisher; Saybold 162 min) using molton agarose (Sigma type IX). After dispersion to form LMDs, and transient cooling to form GMDs, the first, Red Fluorescent GMDs were transfered an aqueous medium containing Green Fluorescent microbeads (Polysciences, 2 micron) and molten agarose. This second suspension, containing both the first Red Fluorescent GMDs and the Green Fluorescent microbeads, was then used to form GMDs by the same dispersion-in-mineral oil pro- cess. The resulting composite GMD/GMDs were then transfered into an aqueous medium, and place under a fluorescence microscope for inspection. Many GMD(Green Fluorescence)/GMDs(Red Fluorescence) were found, in which the first Red Fluorescence GMDs were either partially surrounded or completely surrounded by Green Fluorescene agarose gel. This example demonstrates the ability to create GMD/GMDs with gel regions of different properties, exemplifed by entrappment of Red Fluorescence micro¬ beads in one gel region, and Green Fluorescence microbeads in the other gel region.

Example II: GMD(Red FluoresenceVGMDs (Green Fluorescence¹)

A process very similar to that of Example I was carried out, but with the Red Fluorescence and Green Fluorescence reversed, in order to demonstrate that similar types of agarose GMD/GMDs could by formed, except for the difference in position of the two types of fluorescent microbeads. Again, many GMD/GMDs were observed, with both complete and partial surrounding of the first-formed gel region by the - Example HI: GMDsfaon fluorescentVGMD(mammalian cells:Green Fluorescent)

First GMDs were created by dispersing, into mineral oil, a Chinese Hamster Ovary (CHO) cell suspension in an aqueous cell culture medium to which 37°C molten agarose (Sigma Type DC; 0.5%) was added, and transiently cooling the resulting suspension of - liquid microdroplets in order to obtain a suspension of first GMDs, many of which con¬ tained cells. The first GMDs were transfered from mineral oil into the aqueous growth medium (McCoys- 5A. modified minus calcium plus 10% fetal calf serum plus 0.05% pleuronic F86) by gentle agitation so as to obtain first GMDs suspended in an aqueous medium. In order to better visualize them, the first GMDs were exposed to bovine ? serum albumin (BSA) labeled with FTTC (Green Fluorescence), which diffused into the first GMDs, wherein the labeled BSA was cross-linked so as to entrap the BSA-FTTC within the first GMDs, thereby providing marker entities (Green Fluorescence molecules) within the first GMDs in the same gel region that contained cells.

These first GMDs were then suspended in an aqueous medium containing molton agarose (Sigma type C; 0.5%), and the dispersion process used to create GMD/GMDs wherein the second gel region contained no cells and contained no BSA-FTTC, such that the first and second gel regions could be readily distinguished. Inspection under a fluorescence microscope revealed many GMD/GMDs with a Green inner gel region con¬ taining CHO cells, and an outer clear shell of gel without cells. This demonstrates the ^• formation of composite GMD(agarose,non-fluorescent)/GMDs(agarose,Green Fluorescent +τcells).

Example* TV: GMD Manipulation Using Magnetic Force Coupling Entities

In order to demonstrate the ability to alter the position of GMDs containing force coupling.entities, GMDs containing a large number of magnetic particles of iron (2,3) oxide (Aesar Johnson Matthey, Inc.; 99.6% pure) were formed in a test tube by disper¬ sion in mineral oil using agarose (Sigma type DC). A solution with 3% molton agarose was prepared, and an equal volume (3%) of magnetic particles was also added. Follow¬ ing dispersion to form LMDs, and transient cooling to form GMDs, the GMDs were left in the mineral oil (equivalent to parafin oil, heavy; Fisher; Saybold 162 min), which has ^' a±igh viscosity and. therefore presents a large drag force which opposes motion. As a qualitative demonstration that such GMDs could be moved by an applied magnetc field, a permanent magnet was held near the outer wall of the test tube, and then slowly moved back and forth (frequency about 0.5Hz) and the large number of GMDs near the was followed this motion, which had an amplitude of about 0.3 cm, and thereby demon- strated an ability to physically manipulate the GMDs. Equivalents

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

1. A gel microdroplet having a plurality of non-biological regions, at least one non- biological region having different properties than at least one other non-biological region. 2. A gel microdroplet as in Claim 1 wherein said gel microdroplet is characterized by a plurality of non-biological regions further characterized by having at least one non-biological region having a first property surrounding substantially, or entirely, all of at least one non-biological region having a second property.

3. A gel microdroplet as in Claim 1 wherein a first region comprises a fluid, and at least one other region comprises a gel.

4. A gel microdroplet as in Claim 1 wherein at least one region contains biological material.

5. A gel microdroplet as in Claim 1 wherein at least one region contains first material with optical properties selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphores¬ cence and chemiluminescence.

6. A gel microdroplet as in Claim 5 wherein a second region contains a material with a second optical property selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phos- phorescence and chemiluminescence.

7. A gel microdroplet as in Claim 6 wherein at least one gel region contains marker entities selected from the group consisting of beads, non-biological particles, cry¬ stals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts, vesicles, stains and dyes. 8. A gel microdroplet as in Claim 7 wherein the optical properties of the marker enti¬ ties are selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemi¬

Claims

9. A gel microdroplet as in Claim 8 wherein said gel microdroplet is characterized by a plurality of non-biological regions further characterized by having at least one non-biological region having marker entities surrounding substantially, or entirely, all of at least one non-biological region having a second property. 10. A method for producing gel microdroplets having a plurality of non-biological regions, at least one non-biological region having different properties than at least one other non-biological region, said method comprising: a. forming gel microdroplets of a first gel; b. suspending said gel microdroplets in a material capable of forming a second gel; and c. incorporating gel microdroplets of said first gel into gel microdroplets of said second gel, thereby forming gel microdroplets with distinct non- biological gel regions.

11. A method for producing composite gel microdroplets having a plurality of non- biological regions, at least one non-biological region having different properties than at least one other non-biological region, said method comprising: a. forming gel microdroplets; b. suspending said gel microdroplets in a material capable of forming liquid microdroplets; and c. incorporating gel microdroplets of said first gel into liquid microdroplets, thereby forming composite gel microdroplets with distinct non-biological regions.

12. A method for producing gel microdroplets having a plurality of non-biological regions, at least one non-biological region having different properties than at least one other non-biological region, said method comprising: a. forming gel microdroplets of a first gel capable of liquification; b. suspending said gel microdroplets in a material capable of forming a second gel; c. incorporating gel microdroplets of said first gel into gel microdroplets of " said second gel; and d. liquifying the first gel, thereby forming gel microdroplets with distinct non- biological liquid regions.

13. A gel microdroplet as in Claim 1 wherein at least one gel microdroplet contains at least one biological entitiy. 14. Gel microdroplets as in Claim 13 wherein the biological entity is selected from the group consisting of small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, antibody molecules, antigen molecules, and aggregates of molecules.

5 15. Gel microdroplets as in Claim 14 wherein the cells are selected from the group consisting of animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells.

16. A gel microdroplet as in Claim 1 wherein at least one region contains force- coupling entities selected from the group consisting of beads, non-biological parti-

10 cles, bubbles, and non-aqueous fluid inclusions with force coupling properties.

17. A gel microdroplet as in Claim 16 wherein the force-coupling properties of the force-coupling entities are selected from the group consisting of electrical force, magnetic force, field flow sedimentation fractionation force, acoustic force, optical pressure force, gravitational force, sedimentation force, non-rotational acceleration 5 force, centrifugal force and centripetal force.

18. A process for providing external influence on biological entities comprising the step of utilizing physical manipulation of least one microdroplet containing at least one biological entity so as to affect the proximity of said microdroplet to an influence source. 0 19. A process as in Claim 18 further comprising the step of measuring at least one bio¬ logical property of at least one biological entity, said biological property selected from the group consisting of biological material, biochemical activity, production of molecules, degradation of molecules, secretion of molecules, metabolism, mem¬ brane integrity, enzyme activity and growth. 5 20. A process as in Claim 18 wherein the external influence is selected from the group of biological, chemical and physical influence.

21. A process as in Claim 20 wherein biological influence is provided by selecting a source from the group consisting of cells, feeder cells, animal tissues, plant tissues, animal tissue homogenates, plant tissue homogenates, perfused organs, whole D* animals, plants, cell suspensions, biological entities within gel microdroplets, cell cultures, body fluids.

22. A process as in Claim 18 wherein at least one biological entity is selected from the group consisting of small multicellular organisms, groups of cells, individual cells, protoplasts, vesicles, spores, organelles, parasites, viruses, nucleic acid molecules, 5 antibody molecules, antigen molecules, and aggregates of molecules. 23. A process as in Claim 22 wherein cells are selected from the group consisting of animal cells, plant cells, insect cells, bacterial cells, yeast cells, fungi cells and mold cells.

24. A process as in Claim 22 wherein biological entities selected from the group con- ^" sisting of normal human cells, human cancer cells, pathogenic bacteria, pathogenic yeast, mycoplasms, parasites, and pathogenic viruses.

25? A process as in Claim 23 wherein mammalian cells are selected from the group consisting of of normal human cells, cancerous human cells and hybridoma cells.

26. A process as in Claim 18 wherein the source of external influence is incorporated •^"' into at least one microdroplet, thereby providing a means for influencing biological entities.

27. A process as in Claim 18 wherein microdroplets consist of gel microdroplets, such that at least one gel microdroplet contains a gel matrix which allows selective pas¬ sage of molecules. 28. A process as in Claim 18 wherein microdroplets are manipulated by means of a physical force selected from the group consisting of electrical force, magnetic force, field flow sedimentation fractionation force, acoustic force, optical pressure force, gravitational force, sedimentation force, non-rotational acceleration force, centrifugal force and centripetal force. 29. A process as iir. Claim 26 wherein coupling entities selected from the group con- sistingzof beads;, non-biological particles, bubbles, and non-aqueous fluid inclusions with- force coupling properties selected from the group consisting of ferromagnetic properties, diamagnetic properties, paramagnetic properties, dielectric properties, electrical charge properties, electrophoresis properties, mass density properties, and optical^' pressure properties are incorporated in microdroplets in order to enhance the application of force to microdroplets.

30. A process as in Claim 29 wherein magnetic force is utilized by means of incor¬ porating magnetic coupling entities.

31. A process as in Claim 29 wherein coupling entites with a mass density different than: the microdixiplet density are used with a physical force selected from the group consisting of field flow sedimentation fractionation force, acoustic force, gravitational force, centripetal force, centrifugal force and non-rotational accelera¬ tion force.

32. A process as in Claim 18 wherein influence consists of exposure of at least one microdro let to a h sical hen

m n n l fr m r u consistin of heat electric fields, magnetic fields, electromagnetic radiation, optical radiation, ionizing radiation, acoustic radiation and acceleration.

33. A process as in Claim 32 wherein exposure to heat influence is utilized to deter¬ mine cell death and cell growth under conditions relating to cancer hyperthermia treatment.

34. A process as in Claim 32 wherein exposure to ionizing radiation influence is util¬ ized to determine cell death and cell growth under conditions relating to cancer radiation treatment

35. A process as in Claim 18 comprising the additional step of incorporating biological entities into microdroplets.

36. A process as in Claim 26 wherein the influenced biological entities are not con¬ tained in microdroplets, thereby providing a means for influencing biological enti¬ ties extemal to microdroplets.

37. A process for measuring analyte entities comprising the steps of: a. adding one or more labeled specific binding reactants to a sample; b. incorporating analyte entities into microdroplets; c. measuring the label within at least one microdroplet.

38. A process as in Claim 37 wherein the label is selected from the group consisting of enzyme activity, biological activity and fluorescence. 39. A process as in Claim 38 wherein at least one enzyme activity label is measured by measuring the label enzyme activity of at least one enzyme catalyzed reaction within at least one microdroplet.

40. A process as in Claim 37 comprising the additional step of measuring the volume of at least one microdroplet which is measured for at least one label. 41. The process of Claim 37 with the additional step of using statistical analysis with measured microdroplets.

42. The process of Claim 41 wherein the statistical analysis formulae are selected from the group consisting of Poisson statistics and modified Poisson statistics.

43. The process of Claim 39 wherein at least one incubation is utilized prior to measuring enzyme activity.

44. A process as in Claim 39 wherein optical means are utilized to measure enzyme activity and microdroplet volumes. 45. A process as in Claim 44 wherein microdroplet volume measurement means is selected from the group consisting of light scattering, light absorbance or colorimetric, fluorescence, time-delayed fluorescence, phosphorescence and chemi¬ luminescence. ^" 46. A process as in Claim 37 wherein the labled specific binding reactant is selected from. the group consisting of antibodies, antigens, nucleic acids, avidin-biotin, eπzyme nhibitors and Iectins.

47. A process as in Claim 37 wherein less than two microdroplets are measured together. ^Λ 48. A process as in Claim 37 wherein at least two microdroplets are measured together.

49. A process as in Claim 39 wherein the enzyme label is selected from the group con¬ sisting of β-galactosidase, horse radish peroxidase, and alkaline phosphatase.

50. A process as in Claim 37 wherein analyte entities are selected from the group con- sisting of cells, organelles, viruses, nucleic acids, antibodies, enzymes, structural proteins, hormones and drugs.

51. A process as in Claim 37 comprising the additional step of utilizing at least one intervening, non-labled specific binding molecule.

52. A process as in Claim 51 wherein intervening molecules are selected from the group consisting of antibodies, antigens, nucleic acids, Iectins, protein A and avi- din.

53. A process as in Claim 39 comprising the additional step of providing additional enzymes and reactants suitable for more complex enzyme activity measurement selected from the group consisting of enzyme channeling and enzyme cycling. 54. A process as in Claim 37 comprising the additional step of providing purging enti¬ ties, selected from the group consisting of beads, non-biological particles, crystals, non-aqueous fluid inclusions, viable cells, dead cells, inactive cells, virus, spores, protoplasts and vesicles, which have surfaces with tightly bound molecules capable of tightly binding labeled specific binding molecules prior to forming microdro- • plet-M.