WO1998004743A1

WO1998004743A1 - Polymeric assemblies for sensitive colorimetric assays

Info

Publication number: WO1998004743A1
Application number: PCT/US1997/013253
Authority: WO
Inventors: Deborah Charych
Original assignee: The Regents Of The University Of California
Priority date: 1996-07-29
Filing date: 1997-07-28
Publication date: 1998-02-05
Also published as: AU3897397A

Abstract

The present invention relates to a method for direct detection of analytes using color changes in liposomes which occur in response to selective binding to analytes to their surface. The placement and selection of the polymerizable group in the monomer utilized as a precursor in colorimetric film and liposome production improves sensitivity and also provides a final color change reaction which is specific to an exact analyte concentration.

Description

POLYMERIC ASSEMBLIES FOR SENSITIVE COLORIMETRIC ASSAYS
The present application is a continuation in part of priorfiled U.S. Patent Application No. 08/389,475 filed February 13th, 1995, which is a continuation in part of U.S. Patent Application
Nos. 08/289,384 filed August 11th, 1994, and 08/328,237 filed
October 24th, 1994.

This application also claims priority to the provisional application number 60/022,942 filed July 29, 1996
This invention was made with Government support under
Contract No DE-AC03-76SF00098 between the U.S. Department of
Energy and the University of California for the operation of
Lawrence Berkeley Laboratory. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION
The present invention relates to a method for direct detection of analytes using color changes in liposomes which occur in response to selective binding of analytes to their surface.

Analytical Chemistry Analytical chemistry techniques have been used for many years to determine such medical parameters as hematocrit levels. While useful in their own right, analytical chemistry methods are of limited or no practical applicability to many biological parameters in which rapid and direct assessment would be valuable. Unless expensive and cumbersome gas or liquid
MISSING UPON TIME OF PUBLICATION chromatographic methods are used, measurement of biological analysis is difficult. Often, quantitative results are limited or not available. However, such techniques have been, used for such basic chemical tests as creatinine assays.

Microbioloqical and Pathology Methods are another approach to medical-biological systems analysis by direct microscopic observation using various cell-staining and classic pathology techniques. Augmenting these capabilities have been well developed microbiological techniques, such as culturing, colony characterization, and observation of metabolic and nutrient limitations. Most of medical science has been developed using this basic arsenal of analytic techniques. While culturing and direct tissue observation techniques have served as the bulwark of medical detection processes for many years, they have considerable limitations. Pathological analysis of patient tissues to determine the development of a disease state and the identification of the causative pathogen generally requires an invasive procedure.

On the other hand, culturing the pathogen from various body fluid or other samples is time consuming and expensive.

Immunoassavs A breakthrough in medicine occurred with the development of immunoassay techniques. In these methods, an antibody is developed which will specifically bind to a target of interest. While costly in both their development and production, antibodies from animals allowed a very accurate analysis of a number of analytes which had previously been virtually inaccessible in both research and particularly clinical situations.

An important technical advancement in immunoassay was the development of monoclonal antibodies. Instead of subjecting an animal to an analyte and harvesting its whole range tf antibodies, in this technique a single spleen cell of a sensitized animal is rendered immortal and multiplied many times. The resulting cell line is then cultured to produce a very specific and pure antibody product.

Because the antibody itself is a small molecule, it must be labeled in some way so that the binding event can be detected.

This can be done with a dye, fluorescent, radioactive or other label. Conversely, if binding inhibition occurs between a known amount of introduced, labeled analyte and the material to be analyzed, the diminution of the signal will indicate the presence of test analyte. If the agglutination of the antibody particles is of sufficient volume and density, the formation of a precipitant can also serve to signal the presence of an analyte.

In recent years, the research and medical communities have come to rely heavily on immunoassay techniques to detect and quantify biological materials. While successful in many respects, the indirect nature of immunoassay methods as well as their dependence on antibody materials, results in a variety of complications, problems, and assay limitations. Briefly, the development and production of antibodies remains expensive, and these molecules are sensitive to environmental changes. Also, only those materials to which antibodies can be produce can be detected by these systems.

Lanomuir-Blodaett Film Assavs
The techniques of molecular self-assembly, such as that described by Swalen et al., (Lanomuir, Vol. 3, page 932, 1987) as well as Langmuir-Blodgett (LB) deposition (Roberts, Ed. Lanamuir
Blodaett Films, Wiley, New York, 1966) have been used for coating surfaces with a well-defined, quasi two-dimensional array of molecules. The initial use for this new advancement was for materials science applications such as wetting (hitesides, et al., Lanomuir, Vol. 6, p. 87, 1990) and friction (Novotny et al., Lansmuir Vol. 5, p. 485, 1989). These bilayer films are also used as immobilizing supports for analytic reactions. Bio-sensors based on LB films can detect molecules of diagnostic significance such as glucose (Okahata, et al., Thin Solid Films, Vol. 180, p.

65, 1989) and urea (Arisawa, et al., Thin Solid Films, Vol. 210, p. 443, 1992). In these cases, classic analytical chemistry systems are immobilized on the films in order to improve the readout of the test results and otherwise simplify and improve the detection capabilities of the test procedure.

The detection of receptor-ligand interaction is generally accomplished by indirect assays such as the enzyme-linked immunosorbent and radio-labeled ligand assay. Although biotechnological functionalized films have led to elegant examples of molecular recognition at an interface, the problem of directly transducing the molecule recognition event into a measurable signal has remained a difficulty until the advent ,of the subject invention -
In the case of biosensor devices, detection- is generally carried out by coupling the LB films to a secondary device such as an optical fiber (Beswick, Journal Colloid Interface Science, Vol.

124, p. 146, 1988), quartz oscillator (Furuki et al., Thin Solid
Films, Vol. 210, p. 471, 1992), or electrode surfaces (Miyasaka, et al., Chemical Letters, p.627, 1990)
Some of the analyte bound films provide for fluorescent label, where the fluorescence or its quenched state indicate the occurrence of a binding event (Beswick, Journal Colloid Interface
Science, Vol. 124, p. 146, 1988). In some cases, these detection materials have been embedded in the surface of the supporting bilipid layer (Tieke, Advanced Materials, Vol 3, p. 532, 1991).

Polydiacetylene films are known to change color from blue to red with an increase in temperature or changes in pH due to conformational changes in the conjugated backbone (Mino, et al.,
Lanomuir, Vol. 8, p. 594, 1992; Chance, et al., Journal of
Chemistry and Physics, Vol. 71, p. 206, 1979; Shibutag, Thin Solid
Films, Vol. 179, p. 433, 1989; Kaneko, et al., Thin Solid Films,
Vol. 210, p. 548, 1992).

Functionalized Liposomes
Unpolymerized liposomes expressing sialic acid residues have been extensively used as model systems to study the interaction between influenza virus and cell surfaces (Ott, et, al., European
Journal of Pharmacological Science, Vol. 6, p 333, 1994). These liposomes are typically made of such lipid materials as cholesterol and egg phosphatidylcholine (Kingery-Wood, et al,
Journal of the American Chemical Society, Vol. 114, p 7303, 1992).

The standard in the field is to progress with the polymerization procedure until the materials are fully red, indicating that the polymerization is complete
While it has been a goal of the research community to exploit this characteristic in the detection of binding events, researchers have yet to develop a method using this phenomenon in practical applications. Ideally, such a development would include the potential for these materials to be in a liposome form.

SUMMARY OF THE INVENTION
The present invention provides several innovative chemical design approaches which provides highly sensitivity of colorimetric films and liposomes, with excellent control of analyte concentration specificity. The Inventor has discovered unexpectedly that controlling the placement of the polymerizable group in the monomer used as a precursor in colorimetric film and liposome production provides dramatically imprqved sensitivity.

Careful selection of the placement of the polymerizable group can also provide a final color change reaction which is specific to an exact analyte concentration. Additionally, the overall carbon chain length of the monomer and the liposome size can also be designed in a manner which provides secondary means of controlling the sensitivity and analyte concentration specificity of the inventive improved colorimetric liposomes. The multiple means of controlling the sensitivity of responses provided by the present invention allows unprecedented control of the color change reaction.

The increase of sensitivity provided by the present invention for colorimetric films and liposomes opens up whole new areas of applications for colorimetric film and liposome technology.

Dramatically extending the limits of this innovative technology, the present invention makes small materials such as biological toxins susceptible to detection using colorimetric liposomes. In the inventor's s laboratory, cholera toxin has already been established as detectable using the innovations of the present invention. Small glycoproteins, such as Anti-DNP antibodies have also been detected, as well as large analytes such as toxigenic Ecoli bacteria and viruses.

The innovations provided by the present invention also allow fine, incremental control of colorimetric film and liposome response to specific concentrations of analytes. By assembling films and liposomes from a mixture of monomers with differing polymerizable group placements, incremental sensitivity between the two homogeneous monomer constructs is achieved. Thus, the inventive method of engineering films and liposomes sensitive to a specific concentration of analyte allows customizing of these films and liposomes which will identify virtually any specific level of analyte.

Other methods of customizing sensitivity to specific analyte concentration have also been discovered by the inventor which can be employed in concert with control of carbon chain length to further augment customization of sensitivity ranges. For instance, larger sized liposomes, as well as those which form aggregates, have been found to be more sensitive in their color response to an analyte. Incorporating destabilizing materials into the films and liposomes can also serve to modify the level of concentration to which there will be a reaction to an analyte.

Some such methods are described in U.S. Patent Application 08/609,312 filed March 1st, 1996, to which the present inventor is a co-inventor.

It is an object of the invention to provide a colorimetric film or liposome assay for small analytes, large analytes with limited valance, or analytes at low concentrations.

It is a further object of the invention to provide a colorimetric film or liposome assay which will provide specific quantitation of an analyte.

The present inventive assay means and method provide for an improved direct colorimetric detection of a receptor-ligand interaction using novel polymeric films and liposomes. Using the inventive method of producing these original films and liposomes, a ligand or its derivative is incorporated into a polymeric assembly. Some of these aspects of the present invention are described in the inventor's published communication of which the inventor is one author, incorporated by reference herein, (Reichert et al, J. Am Chem. Soc., Vol. 117, p 829, 1995).

The presence of an analyte which binds to the incorporated ligands can be detected by observing changes in the spectral characteristics of the inventive colorimetric films or liposomes.

The colorimetric liposome of the present invention thus encompasses a molecular recognition site and a detection site, all within a single molecular assembly.

In one embodiment of the invention, chromatic polydiacetylene liposomes are produced, and placed in a liquid. The test sample is added. The color change which occurs indicates the presence of the analyte, and the degree of the color change allows a quantification of the analyte's concentration. Multiple wells of varying analyte concentration further refine the quantitation capacity of the present invention.

The inventor has prepared synthetic, polymerized liposomes that resemble the organization and functionalization of cell membranes and have employed them as simple colorimetric sensors.

The liposomes can specifically bind to cholera toxins and other pathogenic toxins and, bacteria and virus in addition, report the binding event by undergoing a visible color change. In effect, these colorimetric liposomes mimic cell surface molecular recognition as well as signal transduction.

In order to impart both molecular recognition and detection functions to the liposomes, the inventor combined a known ligand receptor interaction with the unique optical properties of certain polymers. The conjugated backbone of alternating double and triple bonds gives rise to intense absorption into the visible spectrum. In the case of polydiacetylenes in sing-le crystals or
Langmuir-Blodgett films, the inventive liposomes are known to undergo blue to red color transitions due to a variety of environmental perturbations including heat, mechanical stress, pH, and exposure to solvent.

In one embodiment of the invention, influenza virus particles are enveloped by a lipid bilayer to which the hemaglutinin (HA) lectin is anchored. HA binds to terminal alpha glycosides of sialic acid on cell-surface glyco-proteins and glycolipids, initiating cell infection by the virus. As described in the prior art section of the subject application, liposomes expressing sialic acid residues have been extensively used as model systems to study the interaction between influenza virus and cell surfaces. The polymerized liposomes of the subject invention, however, are composed of molecules that allow direct visualization of this specific interaction.

Advantaaes of the Invention
The present invention represents an advancement over the limitation of analytical chemistry techniques. Analytical chemistry techniques are the only assay system prior to the advent of the subject invention that allows direct detection.

Unfortunately, analytical chemistry methods have limited applicability to many biological system's assay needs. Often, quantitative results from such methods are limited or not available. However, such techniques have been used for tests such as hematocrit analysis, and creatinine assays.

Analytical chemistry methods are virtually unavailable for most biological molecules due to the destruction of the analytes characteristics during preparation and analysis steps, and the typically small amount of the analyte present in the test sample.

For these reasons, the advent of immunoassay techniques were revolutionary in the biological sciences.

The present invention also represents an advancement beyond the limitations of immunoassay techniques. Many small biological molecules are notoriously difficult to assay in a direct manner due to the severe limitation of environmental ranges which they can tolerate without losing their specific characteristics. For these among other reasons, immunoassays have been heavily relied upon to assay these classes of materials. While successful in many respects, the indirect nature of immunoassay methods results in a variety of interference, complications, problems, and assay limitations and expense.

The requirement that an antibody be developed and produced for each possible target limits the efficacy of immunoassay methods in such applications as designer drug development and screening. Ironically, while allowing testing within a portion of biological environmental ranges, large glycoproteinaceous antibodies are often highly sensitive to degradation outside of a small testing parameter environmental range. Thus, the susceptibilities of antibodies to environmental challenges rigorously limit the environmental testing range, available in these assay systems. In addition, immunoassays require multiple binding and washing steps and secondary reagents to visualize a binding event (i.e. "sandwich" assay). The inventive assay is one step.

A subtle disadvantage to immunoassay systems occurs in rapidly evolving pathogens such as the influenza virus. In such organisms, especially in the case of viruses, the external coat which is available for immune reactions constantly shifting in its antibody recognition elements. Thus, despite a full blown immunity response to an influenza strain, within months an individual can again develop flu, but from a pathogen with an external coat so modified that it is immunologically unrecognizable by the victims memory cells. This is the reason individuals can develop flu year after year.

The present invention enjoys the unique advantage over both immunoassay and analytical chemistry techniques of directly detecting biological analytes. In contrast to assays requiring binding to immunoglobulins, in one embodiment of the present invention, the host attachment site on the pathogen is exploited for recognition function. This site, generally in an immunologically inaccessible valley on the pathogen surface, is highly genetically conserved over time. The minimal variability of this site is necessary for the pathogen to maintain its infectivity. As a result, a single assay system of the present invention will provide effective assays for a panoply of influenza strains, many of which may be very newly evolved.

The inventive films and liposomes exploit the genetically conservative host binding site to identify the pathogen. Even in comparatively large parasites, the host binding site tends to be held constant over time throughout the generations of pathogens.

Additionally, parasites are usually present in the body in a large number of diverse life stages. In well established parasites, the immune accessible sites often vary considerably from stage to stage, the advantage being that the host organism is unable to mount a immunological response with sufficient rapidity to avoid the entrenchment of the parasite. There are times when antibody is desired. In this case, the inventive assay is still superior to ELISA because it is one-step and direct.

The subject invention represents a dramatic advancement over both prior art direct chemical and immunoassay systems, achieving advantages which, prior to the present invention, where available exclusively in only one or the other of these analytic art methods. Much as the advent of immunoassay techniques revolutionized medical and research analytical capacities, the subject invention represents a critical advance in the analytical arts.

The present invention allows the advantages of both immunoassay and chemical analysis in a single system. The present invention enjoys the direct assay advantages of analytical chemistry methods, with many of the advantages inherent in such systems. The inventive assay technique also has a substantial environmental range of testing beyond that of immunoassays. This allows the accommodation of various analytes in their most advantageous environmental parameters. Additionally, the present invention allows rigorous, direct analysis to occur even in very narrow environmental ranges, previously unavailable with analytical chemistry techniques. The speed and simplicity of the color change indicator of the subject invention are its hallmark advantages. Large, expensive bulky equipment is not required.

The assay can be carried out by a lay-person.

Ana lvtes
One of the unique advantages of the subject invention is the wide range of target materials, binding events, and biochemical reactions amenable to analysis using the inventive techniques.

Many of these materials previously could not be detected using a straightforward, practical assay. The present invention allows many of the advantages of immunoassay systems, without the complications of immunoglobulin generation or indirect analysis.

In general, the present invention requires no pre-analysis purification step. This feature of the subject invention is due to the high specificity of the ligands incorporated into the detecting polymeric assembly. Additionally, the inventive direct assay system avoids the expense, complications, and increased inaccuracies inherent in the indirect systems currently available.

The inventive liposomes can employ ligands and analytes which are stable or enjoy appropriate binding characteristics within a limited in vitro or environmental range of conditions. Within in vitro range conditions, the present invention is useful in that stringent limitations even within this narrow range of conditions can be met. This allows, for instance, three dimensional conformations of sensitive biochemicals and biomolecules to be maintained throughout the testing procedure. Can be used to detect infectious diseases sucy as respiratory diseases and sexually transmitted diseases. The inventive assays also can be applied to environmental monitoring, food pathogens, food processing packaging, manufacturing, and home health monitoring.

The present invention functions well even in carefully limited conditions. Thus, conditions such as pH, salinity, and temperature can be carefully controlled by feedback controls, titration and other techniques without interfering with the accuracy or sensitivity of the analysis.

Because of this wide experimental range advantage of the present invention, intact cells or sensitive sub-cellular inclusions can be assayed without disturbing their structural integrity. The color change when the inventive assemblies bind to a surface will pinpoint the location of an analyte, such as in a tissue sample.

Subtle cellular development stages can be monitored by the present invention, such as the various stages of malaria infection. Additionally, the association between various factors can be tested or monitored even during the interaction process using the method of the subject invention.

A structural linker of sufficient length and conformability aids in allowing binding of multiple sites on the analyte even when they are conformationally separated on a curved surface. As a result of these special features, the present invention can detect many ligands previously unsuitable for assay evaluation.

The main criteria for effective indication of the presence of analyte is that the surface of the liposomes be sufficiently perturbed to produce the requisite spectral change. Binding the analyte to an immobilizing particle will serve this purpose, as it concentrates the analyte in a small area, and further provides a three-dimensional aspect over a relatively large area to even a small analyte.

A large variety of ligands can be employed in the subject invention, allowing great flexibility in detecting a test target.

Ligand selection can be based on the most advantageous binding and steric characteristics, rather than compromising these factors to accommodate the test system. Thus, the most advantageous ligand can be selected based on such factors as hydrophobicity and hydrophilicity, size, position of binding site, and conflicting affinities. Ligands which can be employed in the subject invention can include carbohydrates, peptides, nucleotides, heterocyclic compounds, and other organic molecules.

In cases where specific binding ligands are not known, specific antibodies can be attached to the liposome surface or the (Fab)2 fragments can be attached. Any antibodies raised against the analyte can be used for bio-recognition.

The rigor and outstanding advantages of the inventive assay system allows the direct and rapid detection and quantitative evaluation of materials which have been previously unachievable because of the limitations of the prior art methods.

The inventive liposomes and assay method can also assay very small biological or other molecules for which antibodies can not be developed These target materials can include organic solvents or pollutants present at extremely low levels. There are special opportunities made available by the advances achieved by the subject inventors for drug screening in both forensic and clinical applications. Inhibition techniques applied to the subject invention can allow the testing of materials which are of a tiny size or have a small number or single valiancy.

While applicant is not bound there by, it is hypothesized by the inventors that the unexpected spectral signal achieved by the present invention is due to a physical perturbation of the liposomes which occurs as a result of the binding event. It is the case that multivalent materials, such as viruses and cell membrane fragments, can be very easily detected using the subject inventive method. Thus, multivalent materials generally elicit a particularly strong response in the subject system. This may be the case because of conformational changes introduced into the lipid bi-layer as a result of binding causing physical reconfiguration of structure. In addition, materials which can intercalale into the lipid bilayer also illicit a strong response.

Signal Observation
Various spectral changes to the inventive bi-layer films and liposome can be used to detect the presence or absence of the target material. Means of amplifying the spectral signal well known in the art, such as scintillators, can also be employed when low levels of analyte are present. Because of the colorimetric nature of the signal, there are many opportunities for automating the read out of the present inventive assay system.

In one particular embodiment of the present invention, a blue to pink color shift can be observed simply by visual observation by the testing technician. Because of the simplicity of the observation, this function can easily be accomplished by an untrained observer such as an at-home user. Alternatively, spectral test equipment well known in the art can be employed to determine a change in spectral qualities beyond the limits of simple visual observation, including optical density to a particular illuminating light wavelength.

The subject liposomes can also be optimized in assays by binding them to any one of a number of immobilizing materials and objects. Bonding to sephedex beads, for instance, would allow flow-through and washes to be possible during the assay procedures. The inventive assemblies could even be embedded in a gel, with the analyte defusing through it, possibly with an electrical gradient.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a chemical structural representation of cholera toxin Ge and PDA monomer 5,7-Docosadiynoic acid.

Fig. 2 is a chemical formula representation of the polymerization of 5,7-Docosadiynoic acid monomer into a liposomic polymer.

Fig. 3 is a chemical structural representation of some of the variants of the inventive diacetylene monomer structures.

Fig. 4 is a chemical structural representation of Gx1 assembled with 5,7-Docosadiynoic acid on a liposome.

Fig. 5 is a graph of the absorption spectra of a 5% GM1 95% matrix lipid liposomes as a function of W irradiation time.

Fig. 6 A & B are graphs of the absorption spectrum of the inventive liposomes alone and with cholera toxin.

Fig. 7 is a yraph of the colorimetric response of polymerized liposomes after successive additions of cholera toxin.

Fig. 8 is a visible absorption spectrum of the polymeric liposomes containing 5% GM1 ligand and 95% 5,7 docadiynoic acid (DCDA).

Fig. 9 is the visible absorption of the same polymeric liposomes as in Fig. 8, after the addition of 40 uL of lmg/ml E. coli enterotoxin.

DETAILED DESCRIPTION OF THE INVENTION
The present invention provides several innovative chemical design methods to increase and control the sensitivity of colorimetric liposomes assays previously developed by present inventor along with other researchers, as set forth in U.S. parent application No. 08/389,475, filed February 13, 1995. It has been found unexpectedly that by positioning the polymerizable group, such as the diacetylene group on the precurse monomer closer or farther from the head group end of the monomer, the final liposomes product will have an increased or decreased color change sensitivity to analyte concentrations. This allows the engineering of liposomes which react to lower valency analytes or only at a specific level of analyte concentration.

Overall carbon chain length, and liposome size also influence sensitivity present invention provides for the detection of large, low valence analytes, weak binding analytes, and analytes present at very low concentrations.

By assembling liposomes from a mixture of monomers with differing polymerizable group placement, incremental sensitivity between the two homogeneous monomeric constructs is achieved.

Thus, the inventive method of engineering liposomes with a color change reaction limited to a specific concentration of analyte allows customizing of liposomes which will react to virtually any incremental level of analyte concentration.

The other methods of customizing sensitivity discovered by the inventor can be employed in concert with control of polymerizable group placement to further augment customization of analyte concentration sensitivity. Another factor in providing control of sensitivity is regulating the overall carbon number in the monomer.

By providing a series of analytical units of progressive sensitivities, quantitation of an analyte can be directly and very quickly achieved. By example, a series of analytical wells or films of incremental sensitivity can be provided in the same unit.

When a single sample to be analyzed is washed over the group, the presence of the analyte can be quantitated. The series of wells which displayed a positive reaction provide a continuum indicating the concentration of analyte contained in the sample, while providing the quality assurance of multiple reactions.

Differences in color hue can then be used to fine-tune the quantitation or provide a secondary method of assuring selectivity based on the pattern produced.

This multiple well method provides the quality control advantage of providing both positive and negative controls. This approach would avoid problems should any single well maltunction, potentially providing inaccurate information.

The present invention is particularly suitable for detecting analytes which were undetectable by the previous colorimetric liposome detection methods because of their small size. Various specific pathogen toxins are good candidates for detection using the present invention.

A case in point is the cholera toxin from Vibriocholerae, which is about 38,000 MW. Specifics as to the protocol used to obtain the detection of this small molecule are set forth below as
Example 1. Other toxins, such as pertussis toxin or entero toxins from enteropathic bacteria such as E. coli are also detectable using the present invention.

Using the approach of the present invention, a great variety of small molecules will be susceptible to detection using colorimetric liposomes. By example, the inventor has detected anti-DNP antibody using the inventive technology. This glycoprotein has a molecular weight of 150,000. Other small proteins and other small analities are equally detectable using the present invention.

Molecular recognition sites for specific analytes are excellent sources of ligands for the inventive liposomes.

Molecular recognition sites on cell membranes serve as the main communication channels between the inside of a cell and its surroundings. Upon receptor binding, cellular messages such as ion channel opening or activation of enzymes are triggered. The inventive liposomes serve as artificial cell membranes made from conjugated lipid polymers such as polydiacetylene which can on a simple level, mimic membrane processes of molecular recognition and signal transduction. The surface of a cell membrane is a mosaic of highly specific molecular recognition receptor sites.

When specific ligand binding occurs at these sites, the binding event is often transduced into a cellular message. Cell membrane recognition sites may trigger, for example, the opening of ion channels or the activation of intracellular enzymes. From the materials science point of view, the cell membrane may be considered a completely self-contained biosensing system wherein molecular recognition is directly linked to signal transduction.

The inventor has been interested in the design of synthetic membranes that attempt to mimic, on a very simple level, the complex molecular choreography of cell membranes. The simplified constructs allow the study of fundamental receptor-ligand interactions and, in a more practical sense, the application of receptor-ligand binding to biosensor design.

The synthetic membranes of the present inventive liposomes are organized supramolecular structures that resemble natural cell surfaces at the interfacial region but possess a chromophoric conjugated polymer at its interior. The latter part serves as an optical "transducer" of molecular recognition events occurring at the interface. Signaling occurs by a simple color change of the chromophoric unit from blue to red.

The inventors and other fellow researchers demonstrated that polydiacetylene (PDA) thin films and liposomes functionalized with sialic acid molecular recognition groups can bind and colorimetrically detect influenza virus (Reichert, et al J. Am.

Chem. Soc., Vol 117, p 829, 1995; and Berman, Science V. 259, p 515, 1995). The multivalent nature of viral binding at the interface triggered large conformational changes in the polymer side chains followed by disruption of conjugation in the chromophoric polymer backbone . The result is a visible color change from blue to red, similar to color changes previously observed in PDA induced by heat (thermochromism) and mechanical stress (mechanochromism).

For the viral binding study, the ligand molecule for the biotarget was a synthetic diacetylenic lipid compound derivatized with the binding ligand. The ligand-lipid could be cross-linked with the remaining diacetylene groups forming the conjugated polymer backbone. More recently, the inventor and other researchers showed that naturally derived lipophilic molecules can be incorporated into polydiacetylene Langmuir-Blodgett films.

(Charych, Chem. Biol. V 3, p 113, 1996). The present invention demonstrates that these molecules can also be formed into liposomes when mixed with a polymerizable monomer lipid.

Gangliosides are a complex subclass of sphingolipids that are derivatives of ceramide. The large polar head is made up of several carbohydrate units. The membranes of the human nervous system contain at least 15 different gangliosides of which little is known about their function. However, in addition to its natural role in animal cells, the ganglioside GMl, as shown in Fig.

1, is the point of attachment of cholera toxin as it attacks the cell. This interaction provides a useful model to demonstrate molecular recognition between the protein toxin and the lipidpolymer matrix.

Polvmerizable GrouP Placement in Monomer Carbon Chain The carbon chain length positioning the head group a specific distance from the polymer backbone in the final polymerized liposome is dependent on the position of the polymerizable group in the unassembled monomer. It has been discovered by the inventor that, in the case of diacetylene liposomes a diacetylene group positioned from between the 18-20 positions to the 3-5 position in the monomers will produce progressively more sensitive liposomes.

Liposomes produced from monomers with the diacetylene groups from the 10-12 position to the 4-6 position provides particularly efficient control of sensitivity. Diacetylene groups positioned in about the 5-7 position are preferred, such as in cholera toxin detection. The production protocol for the monomer determines at which position the diacetylene group will be placed in the final monomer product.

Total Carbon Chain Lenath The total carbon chain length in the unassembled monomer will also influence the level of sensitivity of the liposome product, although to a lesser extent than the position of the polymerizable group in the monomer carbon chain. The shorter chain length typically provides for greater sensitivity. The monomers which are useful in construction of the inventive colorimetric liposomes can range from between C12 to C25 in length. A preferred range of monomer carbon chain length in the present invention is C20 to C23. The most preferred carbon length for monomers in the present invention is C22.

The synergistic influence of monomer chain lengths and positioning of the polymerizable group on the chain has been concretely demonstrated in experimental work completed by the inventor. It was shown that in the case of 10,12 derivatives, that the C23 chain provides final colorimetric liposomes product which changes color at a lower analyte level than those produced from monomers with a C2 chain. In the case of 5,7 derivatives, the C22 length chain provides a greater sensitivity than the C24 length chain.

Analytical Devices The present invention provides a special opportunity to provide an instant and continuous reading of the level of analyte in a sample. This capacity of the present invention has important applications in monitoring materials present in a feed stream or an environmental area of concern. As an example, personal safety of waste management and cleanup workers is an important factor at various facilities, and this technologic advancement would have special applications in such situations.

A good method of displaying incremental levels of analyte using the present invention is by a series of wells which will react at different titrations of analyte. The series of wells which displayed a positive reaction provide a continuum to indicate the concentration of analyte contained in the sample, while providing the quality assurance of multiple reactions.

Differences in color hue can then be used to fine-tune the quantitation. The multiple sequential sensitivity well method provides the quality control advantage of providing both positive and negative controls. This approach would avoid problems with any single well providing inaccurate information.

An alternative to the multiple method is to provide liposomes of incrementally advancing sensitivities in an immobilized state.

This approach provides a continuous display of analyte concentration. Column or dipstick devices are natural applications for this embodiment of the inventive technology. A wide range of production methods are also applicable to the present invention. For instance, providing layers of immobilizing gels containing liposomes of ever increasing sensitivity would allow a layered cake production. Careful slicing would provide inexpensive production of single strip units capable of detecting multiple levels of analyte.

General Linosome The inventive colorimetric liposomes allow for the direct detection of the presence of a wide range of analytes by changes in color. The- results can be read by an untrained observer, and the test can be conducted in ambient conditions. Very mild testing conditions are possible, which allows the detection of small biomolecules in a near natural state, providing information as to their interactions and avoiding the risk of modification or degradation of the analyte.

LiDid Ordering GrouDs The lipids appear to be important in structurally ordering the three-dimensional liposomes so that binding of the analyte produces a detectable color change. The inventor hypothesizes that a structuring effect of the ordering groups serves to appropriately stabilize the physical structure of the three-dimensional liposomes to facilitate color stability and polymerization. In turn, the binding of the analyte to the molecular recognition ligand groups then causes sufficient steric perturbation or stress of the structure to result in a color change. It may be that the stability and relative rigidity engendered by the ordering lipids so unites the bilayer surface, that a steric change in one area triggers a larger effect in the surface of the assemblies as a whole.

It is further hypothesized that the shortened chain lengths of the present invention decrease the stability of the structure thus providing a reaction to low levels of analyte.

It is not certain which of the many results of binding result in the observed spectral changes. Most likely the changes are due to stresses induced by binding which changes the effective conjugation length of the polymer backbone. The inventive three dimensional structures are highly color sensitive to a number of environmental parameters, such as heat, and these factors may be a component of the observed phenomena as well. However, the applicant is not bound to any of the above hypothesis which are simply attempts to explain the demonstrated effective assay method of the subject invention.

Previous studies have suggested that color transitions in polydiacetylenes arise from changes in the effective conjugation length of the polydiacetylene backbone and that the electronic structure of the polymer backbone is strongly coupled to side chain conformation. The inventor can only speculate at this point that specific analyte-liposome interactions may serve to alter side chain conformation, reducing the effective conjugation length of the enzyme backbone. Indeed, theoretical calculations suggest that very slight around the C-C bond of the polymer backbone decrease the Z electron delocalization.

Head Group Materials for use as head groups in the present invention include -CH2 OH, -CH2OCONHPh, -CH2OCONHEt, -CH2CH(Et)OCONHPh, -(CH2)90H, -CH2OCOPh, -CH20CONHMe, -CH2OTs, -CH(OH)Me,
EMI30.1

EMI31.1

EMI32.1

-CH2OCOR2, wherein R2 n-C17H35, Ph, PhO, or
-OS02R2, wherein R2 is Ph, p-MeC6H4, p-FC6H4, p-CIC6H4, pBrC6H4, p-MeOC6H4, m-CF3C6H4, 2-C10H7, or Me
CO2M, wherein M is K,HNa, or Ba/2.

The preferred materials which can be employed as head groups in the present invention are:
-CH2OCONHR2 or -CH2CONHR2 where R2 is Et, n-Bu, n-C6Hl3, n-C8Hl7, n C,2H2s, cyclo CEH, Ph, p-MeC6H4, m-MeC6H4, o-CIC6H4, m-CIC6H4, p-CIC6H4, o-MeOC6H4, 3-Thienyl, Me, Et, Ph, 1-C10H7, Et, Ph,
EtOCOCH2, BuOCOCH2, Me, Et, i-Pr, n-C6Hl3, EtOCOCH2, BuOCOCH2, Ph,
2,4(NO2)2C6H3OCH2, or CH2CH2OH.

The most preferred head groups are taken from -CH2COX, where X is OH, MeO or any salt thereof.

Lipand Grout The ligand group of the present invention can be of a wide variety of materials. The main criteria is that the ligand have an affinity for the analyte of choice. The ligand may be of a broad range, such as when a class of materials is to be assayed. Appropriate ligands include peptides, carbohydrates, nucleic acids or any organic molecules which bind to receptors.

For instance, all influenza strains share binding sites to a host receptor molecule. Thus, this molecule can successfully be employed to screen for all influenza strains, including those which have not yet been characterized.

Ligands can also be used in the present invention when they function as competitive binders to the analyte. For instance, a pathogen could be introduced with a test material which is to be the presence of receptor molecule. In absence of this molecule, the pathogen will bind to the three-dimensional polymeric structure and produce a color. To the degree that the pathogen surface is bound to the receptor molecule introduced in the test material, the binding will be diminished. In this way, the presence of receptor molecule can be detected and quantified.

Recettor-Bindina Molecules The use of sialic acid derivatives in one preferred embodiment described in the examples below is an example of the use of receptor-bindinz molecules in this capacity.

Receptor-binding molecules are materials on the surface of a host cell to which a pathogen attaches itself as a prelude to the infective event. Selecting these molecules at the ligand group in the present invention has many advantages over other receptor molecules. The recognition sites for these molecules tend to be highly genetically conserved in the pathogen because of its obvious criticality to survival. Therefore, different strains of the same pathogen will generally not produce a false negative when such molecules are selected as the ligand group in the subject invention. Also, receptor molecules tend to be smaller and less complex, and often less hydrophobic, then antibodies to the same analyte.

An increasing number of receptor molecules are being recognized, identified, isolated, and synthesized for a large number of pathogens. Many have been improved for use in various analytic and treatment systems. An example of this trend in research is the sialic acid derivative used in the example below of the subject invention. Examples of the receptors for a number of pathogens are provided in the application as Table 3. All of these, as well as many more, could be exploited by. the method of the subject invention.

Livid Polvmerization Groups Many different polymerizing groups have been incorporated into lipids and are shown to be effective in monolayer polymerizations. Such moieties include: acetylenes, diacetylenes, alkenes, thiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydride, urethanes, allylamines, siloxanes or vinylpyridinium etc. Lipids containing these groups can be made into homopolymers or mixed polymers. The preferred group for use in this invention is the diacetylene due to its unique optical properties in the polymerized form: Polydiacetylene. However, other polymerizing groups could be used when they provide an observable change in properties upon a binding event.

Detection of Cholera Toxin
Cholera toxin is an enterotoxin of the Gram-negative bacterium Vibrio cholerae that causes potentially lethal diarrheal disease in man. The cholera-GMl interaction is we]l-characterized and the GN1 lipid can be easily incorporated into liposomes.

Cholera toxin is composed of two subunits: A (27 kDa) and B (11.6 kDa) with the stoichiometry AB5. The B components bind specifically to Gg gangliosides on cell surfaces, ultimately leading to translocation of the Al fragment through the membrane.

Previous studies have shown that cholera toxin could be recognized by GM1-containing supported lipid membranes and polymerized
Langmuir-Blodgett films containing GM1 and a carbohydrate "promotor" lipid. The ganglioside GM1 was mixed at 5 mol % with the diacetylene "matrix lipid" monomers, 2. Liposomes were prepared using the probe sonication method and polymerized by W irradiation (254 nm). The solid-state polymerization proceeds as a 1,4 addition controlled by the packing of the monomers.

The time course of the polymerization is shown in Figure 5.

The visible absorption arises from the conjugated ene-yne system that comprises the polymer backbone. (The monomer absorption occurs at wavelengths less than 300 nm.) The absorption intensity increases with the W irradiation time and nearly saturates after a total energy dose of 7.2 J/cm2.

The absorption peak at 620 nm is designated as the PDA blue form. The appearance of the colored polymer provides a sensitive and simple test of molecular order in the self-assembled nanostructure. "Looser" structures such as micelles would not form the conjugated polymer due to the topochemical nature of the polymerization reaction. The formation of liposomes in sonicated samples of amphiphilic diacetylenes has been previously demonstrated by electron microscopy. Transmission electron microscopy of the liposomes composed of 5% G,, and 95% 2 indicate an oblong shape with a mean length of 600 nm.

The conjugated ene-yne backbone of poly(diacetylene) liposomes results in the appearance of a deep blue/purple solution. The visible absorption spectrum of the freshly prepared purple liposomes is shown in Figure 6A. The spectrum can be analyzed by determining the initial percentage of blue phase (%B) in the preparation by comparing the intensity of the peak at 620 nm to the red absorption maxima at 490 nm. Typically, tB-50 for the initial liposome preparation. When cholera toxin. is added to the liposomes composed of 5% GM1 and 958 2, the solution immediately changes to an orange color, and the "red phase" absorption of poly(diacetylene dominates, Figure 6B with By=18.

The colorimetric response (%CR) is measured as the percent change in the absorption at 620 nm (blue phase polydiacetylene) relative to the total absorption maxima at 620 and 490 nm. A positive response is obtained if the %CR is greater than 7%. These color changes are easily seen with the naked eye, particularly if the liposome solution is placed in a white 96-well microtiter plate.

If the ganglioside GM1 was mixed with a matrix lipid composed of 10,12 pentacosadiynoic acid instead of 5,7 docosadiynoic acid, (2), the colorimetric response was significantly reduced. The enhanced sensitivity of the system composed of matrix lipid 2 most likely arises from the positioning of the optical reporter group nearer to the interface (three methylene units compared to eight).

It has been previously shown by Fourier transform IR spectroscopy that small rotations about the C-C bond b to the polymer backbone are sufficient to change the effective conjugated length. These conformational changes are more easily transduced through shorter alkyl chain length.

A negative response was observed if the ganglioside, GMl ligand was removed from the liposomes (for example, for 233 Crg/ml cholera toxin the %CR was -6 compared to =43 with the ganglioside present). Similarly, negative responses were obtained when comparable quantities of other proteins besides cholera toxin were added to the GM1-containing liposomes. These include human serum albumin, avidin and wheat germ agglutinin.

Kinetic experiments indicate that greater than 95% of the color change occurs within the first two minutes of adding the toxin. The color transition is not an all or nothing effect but depends on the quantity of toxin titrated into the solution,
Figure 7. The sigmoid behavior suggests cooperativity of the colorimetric transition. This may indicate that the binding itself is cooperative in the sense that binding of toxin to the GM1 ligand makes the binding of subsequent toxins more favorable.

Alternatively this result might more appropriately be understood in terms of the lipid-polymer side chain conformation and its result on the effective conjugated length of the polydiacetylene backbone. Once the effective conjugated length is reduced as a result of toxin binding, subsequent perturbation of the remainder of the lipid-polymer backbone becomes more favcrable. This might be explained as a reduced activation barrier of the blue to red conversion. Temperature-dependent studies of the blue to red transition induced by molecular recognition as well as by heat (thermochromism) may shed light on the relative energetics of the blue-red transition. In addition, the effects of liposome size and GMl mole density on the absolute sensitivity of this approach will be examined.

The inventor has demonstrated that protein-ligand molecular recognition occurs at the interfacial region of polymerized liposomes and that molecular recognition can be directly linked to signal transduction. Such artificial membranes resemble the organization and functionalization of cell membranes but have the added benefit of a built-in synthetic 'trigger' that signals molecular recognition events by an easy to measure color change.

Non-specific adsorption if it occurs, does not appear to effect the color of the liposome solutions. These results establish that polymerized supramolecular assemblies offer an alternative approach to investigating molecular recognition at tailored interfaces.

Example 1 Cholera Toxin Detection
Ganglioside, G,,, cholera toxin from Vibrio Cholerae, human serum albumin, and wheat germ agglutinin were purchased from Sigma. 5,7
Docosadiynoic acid was synthesized. Deionized water was obtained by passing distilled water through a Millipore F ultrapurification train. Solvents used were reagent grade.

In the protocol for the formation of the liposomes, compounds 1 and 2 as seen in Fig. 1 were dissolved in methanol and chloroform, respectively. The solutions were mixed in appropriate volumes to achieve a lipid mixture of 5% by mole of GMI and total lipid content of 2 fool. The solvent was evaporated by rotary evaporation and 2 mL of deionized water added to the dried lipid.

The suspension was probe sonicated, cooled, and polymerized for 60 min. using a hand-held W lamp (254 nm) as shown in Fig. 2. The resulting blue/purple liposome (as shown in Fig. 4) suspension was stored in the dark at 40 C.

For the colorimetric assay, cholera toxin was diluted to 1 mg/mL in 50 mM Tris buffer, pH 7.C. In a 500 AL glass cuvette, blue phase liposomes produced as above were diluted 1:5 in 50 mM
Tris buffer, pH 7.0. The liposomes were preincubated in the buffer for 15 - 30 min to ensure stability of the blue phase prior to the addition of cholera toxin. No color changes were observed during this period.

Cholera toxin was added to the cuvette by the method of successive additions. After each addition, the contents were mixed and the visible absorption spectrum was recorded as a function of time. Typically, 95% of the absorption changes were observed to occur within the first 2 min after addition of toxin.

After each experiment, the contents of the cuvette were transferred to a single well of a white microtiter plate. The pink-orange color of the cholera-treated liposomes was verified visually with a blue negative control.

Results
Figure 5 shows the visible absorption spectra of poly(diacetylene) liposomes composed of 5% GM1 ligand, 1, and 95% matrix lipid 2 as a function of W irradiation time. The liposomes were exposed to a total energy dose of 7.2 J/cm2. Each spectrum (in order of increasing absorption) corresponds to a dose of 0.8, 1.6, 2.4, 3.2, 4.0, 5.6, and 7.2 J/cm2.

Figure 6 shows the results of the colorimetric detection of cholera toxin by polymerized diacetylene liposomes (5% GM1 and 95% 2). (A) Visible absorption spectrum of blue/purple liposome solution prior to addition of cholera toxin. Liposomes were diluted in Tris buffer, pH 7.0, to a final concentration of AM total lipid. (B) Visible absorption spectrum of liposomes after the addition of cholera toxin to a final concentration of 310 WG/mL. The incubation time with the liposomes was 2 min.

Figure 7 shows the colorimetric response (%CR) of polymerized liposomes liposomes (58 GM1 and 95% 2) after successive additions of cholera toxin. The liposomes were incubated with toxin for 2 min after each addition and the spectrum recorded as in Figure 6.

In order to quantify the response of a liposome solution to a given amount of toxin, the visible absorption spectrum of the liposome solution without the toxin was analyzed as Bo=I620/(I620+I490)
The same value was calculated for liposome solutions exposed to cholera toxin (but) The colorimetric response (%CR) is defined as the percentage change in B upon exposure to toxin:
CR=[Bo-Bct)/Bo]x100%
Example 2 E.coli toxin detection Ganglioside, GMI, cholera toxin from Vibrio Cholera, human serum albumin, and wheat germ agglutinin were purchased from Sigma. 5, 7 Docosadiynoic acid was synthesized.

The formation of the liposomes was accomplished as in Example 1, above, with 5% by mole of G,,
For the colorimetric assay, E.coli toxin (Sigma) was spun through a 30 K molecular weight cutoff filter at 2000 x g, 15 degree C to remove salts. The protein was re-diluted in 50 mM
Tris buffer pH 7.0 to a final concentration of lmg/ml.

Controls
The liposomes (lmM) in water was diluted with 50 mM Tris buffer, pH 8.0 to a final concnetration of 0.2 mM. (40uL of liposomes plus 160 uL of buffer). The absorption spectra of the diluted liposomes was recorded in a plastic cuvette.

Figure 8 shows the visible absorption spectrurn of the polymeric liposomes containing 5% GM1 ligand and 95% 5,7 docadiynoic acid (DCDA). The liposomes were diluted in 50mM Tris buffer, pH 8.0 to a final concentration of .2 mM in a plastic disposable cuvette. The solution in the cuvette appears purple to the naked eye.

E.coli toxin
To the liposome solution in the cuvetted, 40uL of the above E.coli toxin was added and the sample allowed to incubated for 10 minutes. The visible absorption spectrum was again recorded, in
Fig. 9. The solution in the cuvette appears pink to the naked eye after the addition of the toxin compared to a purple color before the addition. The absorption spectra of Fig. 8 and Fig. 9 confirm the color changes observed.

Figure 9 shows the visible absorption of the same polymeric liposomes as in Figure 8, however after the addition of 40 uL of lmg/ml E.coli enterotoxin in 50mM Tris buffer pH 7.0. The spectrum was recorded 10 minutes after exposure to the toxin without stirring. The solution in the cuvette appears 'pink' to the naked eye.

Pathogen Receptor Molecule
HIV CD4@@; Vasoactive Intestinal Peptide7, Peptide T8, Sialic Acid12
Vaccinia Epidermal Growth FactorÚ
Rabies Acetylcholine receptorÚ
Epstein Barr Complement Receptor@@@
Rheo Beta-adrenergic receptor@
Rhinovirus ICAM-16.10.11; N-CAM, myelin-associated glyeoprotein MAb13
Polio viruses Polio viruse receptor9
Influenza Sialic Acid15
Cytomegalovirus Glycoprotein (not Sialic Acid) 16 17 18
Coronaviruses 9-OAC Sialic Acid & Sialic Acid
Encephalomyelitis 9-OAC Sialic Acid
Rubella Virus - - - - - - - - - - - - - - - - - - - - - - - - - - 19
Measles Virus Glycoprotein (not Sialic Acid) 20.21 22.23
Herpes Oligosasaccharioes Glycoprotein 24 25 26
Chlamydia Sialic Acid27. 28. 29 @0
Rhinovirus Glycosylated Proteins31 32
Rotavirus 9-OAC Sialic Acid
Polyomavirus Sialic Acid
Reovirus Sialic Acid
Streptococcus Suis Sialic Acid &alpha;

2 # 3 Poly-N-Acetyllactosamine
Salmonella Sialic Acid
Typhimurium
Paramyxovirus
Sendi Virus Sialic Acid
Mumps Sialic Acid
Newcastle Sialic Acid
Disease Virus
Myxoviruses Sialic Acid
Escherichia Coli Oligomannose, Galactose &alpha; 1 # 4 Galactose, Sialic Acid &alpha; 2 # 3 Galactose
Encephalomyocarditis Sialic Acid
Virus
Cholera Toxin GMI (A Gangliosial of Sialic Acid, Galactose, Glucose, N-Acetyl Galacto
Meningitis Sialic Acid 1 Epsteisn, et al. Nature, Vol. 318, p. 663, 1985 2 Lentz, et al. Science, Vol. 215, p. 182, 1982 3 Fingeroth, et al., Proc Natl. Acad. Sci. USA. Vol, 81, p. 4510 1984 4 Carel, et al., J. of Biol. Chem., Vol. 265, p. 12293.1990 5 Co. et al. Proc. Natl. Acad. Sci. USA, Vol. 82, p. 1494, 1985 6 Marlin, et al.

Nature, Vol. @44, p. 70, 1990 7 Sacerdote, et al., J. of Neuroscience Research, Vol. 18, pp. @02-107, 1987 8 Ruff, et al., FEBS Letters, Vol. 211, pp. 17-22, 1987 9. Mendelsohn, et al., Cell, Vol. 56, pp. 855-865, 1989 10 Greve, et al., Cell, Vol, 56, pp. 839-842, 1989 11 Staunto, et al., Cell, Vol. 56, pp. 849-853, 1@89 12 Wies, et al. Nature, Vol. 333, pp. 426-431, 1988 13 Shephey, et al., Proc. Natl. Acad. Science, USA, Vol. 85, pp. 7743-47, 1988 14 Khstzman, et al. Nature, Vol. 312, pp. 763-770, 1985 15 W@@te et al Cell Vol 56 pp 725-728 1989 17 Inas, Vol. 86, p. 10100, 19@9 18. Virol, Vol. 176, p. 337, 1990 19. Med Microbio Imm, Vol. 179, p. 105, 1990 20. Infect Imm, Vol. 74, p. 65, 1979 21. Proc. Soc. Exp. Bio Med, Vol. 162. p. 299, 1979 22. Virol, Vol. 172, p. 386, 1989 23. J. ClinInv, Vol. 86, p. 377, 1990 24. J.

Virol, Vol. 64, p. 2569, 1990 25 Sci, Vol. 248, p. 1410, 1990 26. Febs Lect, Vol. 277, p. 253, 1990 27. Infec Imm, Vol. 57, p. 237@, 1989 28. Fams Microb Lett, Vol. 57, p. 65, 1989 29. Infect Imm, Vol. 40, p. 1060 30 Infect Imm. Vol. 25, p. 940, 1983 31 Med Viro Vol @ p 211 19@9

Claims

CLAIMS 1. Quantitative colorimetric assay liposomes that change color in the presence of a specific concentration of analyte, comprising; a) ordering hydrophilic head groups, b) a polymer backbone linked to the head groups by a carbon chain, c) hydrophobic tail groups linked to the polymer backbone by a carbon chain, and d) ligands associated with the polymeric backbone.

2. The liposomes of Claim 1, wherein the carbon chains are hydrocarbon or fluorocarbon chains.

3. The liposomes of Claim 2, wherein the hydrocarbon chains are alkyl.

4. The liposomes of Claim 1, wherein the hydrophilic head group is attached to the polymeric backbone by a chain of from about 2 to 18 carbons.

5. The liposomes of Claim 4, wherein the hydrophilic head group is attached to the polymeric backbone by a chain of from about 4 to 10 carbons.

6. The liposomes of Claim 5, wherein the hydrophobic tail group is attached to the polymeric backbone by a chain of from 9-16 carbons.

7. The liposomes of Claim 5, wherein the hydrophilic head group is attached to the polymeric backbone by a chain of about 5 carbons.

8. The liposomes of Claim 7, wherein the hydrophobic tail group is attached to the polymeric backbone by a chain of about 14 to 17 carbons.

9. The liposomes of Claim 4, wherein the carbon chain attaching the head groups to the polymer backbone are heterogeneous in length.

10. The liposomes of Claim 1, wherein the ordering head groups are selected from the group comprising carboxylic acid, amine, alcohol, phosphate, sulfate, ester, glycine, lysine, mercapto, benzoic acid, azobenzene, cysteine, alanine, maleimide, ethylene diamine, glycine sulfate, ethanolamine, disodium phosphate, and EMI46.1 EMI47.1 EMI48.1 11.The liposomes of Claim 1, wherein the ordering head groups are selected from the group comprising -CH2OH, -CH20CONHPh, -CH2OCONHEt, -CH2CH(Et)OCONHPh, -(CH2)9OH, -CH2OCOPh, -CH2OCONHMe, -CH2OTs, and CH(OH)Me, CH2OCOR2, wherein R2 is Ph, PhO, or o-(HO2C)C6H4, -OSO;R2, wherein R2 is Ph, p-MeC6H4, p-FC6H4, p-CIC6H4, pBrC6H4, p-MeOC6H4, CF3C6H4, 2-C10H7, or Me CO2M, wherein M is K, HNa, Ba/2, Ca/2, Cd/2, or NH4+.

-CH2OCONHR2 or -CH2CONHR2 where R2 is Et, n-Bu, Ph, p-MeC6H4, m-MeC6H4, o-MeOC6H4, 3-Thienyl, Me, Et, Ph, 1-C10H7, Et, Ph, EtOCOCH2, BuOCOCH2, Me, Et, i-Pr, n-C6H13, EtOCOCH2, BuOCOCH2, Ph, 2,4(NO2)2C6H3OCH2, or CH2CH2OH, . CH3-, CH3O-, neo-C5H11O -, Cyclo-C6H11O-, PhCH2O-, p-AcC@H4O-, pBzC6H4O, p-BrC6H4COCH2O-, p-(PhCH=CHCO) C6H4O-, p- (PhCOCH=CH)C6H4O oBzC6H4NH-, p-BzC6H4NH-, MeOCH2CH2NH-, n-C6H13NH-, and EtO-.

12. The liposomes of Claim 1, wherein the hydrophobic tail group is attached to the polymeric backbone by a carbon chain of 0 to 22 carbons.

13. The liposomes of Claim 12, wherein the hydrophobic tail group is attached to the polymeric backbone by a carbon chain of 10 to 18 carbons.

14. The liposomes of Claim 13, wherein the hydrophobic tail group is attached to the polymeric backbone by a carbon chain of about 11-14.

15. The liposomes of Claim 12, wherein the carbon chains attaching the tail groups to the polymer backbone are heterogeneous in length.

16. The liposomes of Claim 1, wherein the tail group is selected from the group comprising hydrocarbons or fluorocarbons.

17. The liposomes of Claim 1, wherein the molecular weight of the analyte is from about 100 to about 300,000.

18. The liposomes of Claim 17, wherein the molecular weight of the analyte is from about 10,000 MW to about 150,000 MW.

19. The liposomes of Claim 18, wherein the molecular weight of the analyte is about 38,000 MW.

20. The liposomes of Claim 1, wherein the analyte is selected from the group comprising viral and bacterial pathogens, enzymes, drugs, toxins, viruses proteins, hormones, and bacterial enzymes.

21. The liposomes of Claim 20, wherein the analyte is a pathogenic toxin.

22. The liposomes of Claim 21, wherein the toxin is a cholera toxin, pertussis toxin, enterotoxin, or toxin A.

23. The liposomes of Claim 1, wherein the ligand is oligosaccharide for cholera or entero toxin mannnse sugar for pathogenic E. coli, pepstatin for enzymes of Candida Albicane, dinitophenral for anti-DNP IgG antibody, biotoxin for-streptococcus, GABA for GABA binding protein, dopamine or spiperon for dopamine D2 receptor, phospholipid substrate for phospholipase A2 enzyme, substrate for catalytic antibodies, serotonin for serotonin receptors, galactose ( ) toxin, monoclonal antibodies for Gonnorhoea, E.coli, Bianthracis, and Chlamydia.

24. The liposomes of 23, wherein oligosaccharide, epidermal growth factor, GABA, Dopamine, or serotonin are used for drug screening. [put in spec] 25. The liposome of Claim 1, where said ligand is selected from the group comprising, epidermal growth factor for vaccinia analyte, acetylcholine analyte for Rabies, complement receptor for Epstein Barr analyte, beta-adrenergic receptor for reovirus receptor for polio virus analyte, tetrasaccharide for neutrophil analyte, and derivatives and analogues thereof capable of associating with an analyte.

26. The liposomes of Claim 1, wherein said ligand is sialic acid and its derivatives and analogs which will bind to colronaviruses, influenza virus, encephalomyelitis, chlamydia, sendi virus, mumps, newcastle disease, myxovirus, -encephalo-mycarditis virus, meningitis, or malaria.

27. The liposome of Claim 1, wherein the liquid: analyte pair are oligosacchands and neutrophiles, cell adhesion peptides and target cells, oligosacchands and bacterial toxins, or transmembrane receptors and hormones.

28. The liposomes of Claim 1, wherein the ligand provided to detect HIV analytes is selected from the group comprising monoslonal antibodies, sCD4, CD26, vasoactive intestinal peptide, peptide T, and derivatives and analogues thereof capable of associating with HIV.

29. The assemblies of Claim 1 wherein said polymer backbone cojugated and comprised of monomers selected from the group comprising acetylenes, diacetylenes, alkenes, thiophenes, imides, siloxanes anilines, pyrroles and vinylpyridinium.

30. The assemblies of Claim 29, wherein said polymer backbone is comprised of diacetylene monomers.

31. A method of making the liposomes of Claim 1, comprising, a) combining monomers containing a polimerizable group with bipolar ligands in an organic solvent, b) evaporating the solvent, c) adding an aqueous solution, d) heating the solution above the main-phase transition temperature of the monomers, e) agitating the solution and cooling it to at least 4 C, f) depositing the monomer-ligand mixure in a polymerization chamber, g) polymerizing the monomer-ligand mixture short of the red phase.

32. The method of Claim 31, wherein in step a), the solvent is selected from chloroform, benzene, alcohol, cyclohexame, hexane, methylene cholride, acetonitrile, and carbontetrachloride.

33. The method of Claim 31, wherein the aqueous solution of step c) is selected from water, buffer solution, cell media, physiological saline, phosphate buffered saline, Trizma buffer, HEPES, and MOPS.

34. The method of Claim 31, wherein before the cooling in step e), the solution is filtered.

35. The method of Claim 31, wherein the diyne-ligand mixture is cooled at between 40 and -200C for between 5 minutes and 24 hours.

36. The method of Claim 35, wherein the mixture is cooled at between 0 and -15 C for between 5 and 20 minutes.

37. The method of Claim 36, wherein the mixture is cooled at between 0 and -S"C for between 5 and 12 minutes.

38. The method of Claim 31, wherein the diyne-ligand mixture is cooled during polymerization to between 1" and 22"C.

39. The method of Claim 38, wherein the diyne-ligand mixture is cooled during polymerization to between 16 -190C.

40. The method of Claim 31, wherein the polymerization is achieved by U.V. irradiation using a pen ray lamp, hand-held lamp, or a U.V. chamber with energy senser.

41. The method of Claim 31, wherein the polymerization is achieved by gamma radiation, electron beam or X-rays, or other low energy ionizing source.

42. The method of Claim 3i, wherein the polymerization accomplished with an energy dose of .1-10 joules/cm2.

43. The method of Claim 31, wherein the polymerization continues until the liposomes are in the blue or purple phase.

44. The method of Claim 36, wherein the polymerizable group in the monomer is positioned from between about the 18-20 positions to about the 3-5 position.

45. The method of Claim 44, wherein the polymerizable group in the monomer is positioned from about the 10-12 position to about the 4-6 position.

46. The method of Claim 44, wherein the overall chain length of the monomer is C23.

47. The method of Claim 44, wherein the diacetylene group in the monomer is positioned in about the 5-7 position.

48. The method of Claim 46, wherein the overall chain length of the monomer is C22.

49. The method of Claim 31, wherein the monomers used to assemble the liposomes are a mixed species with varying diacetylene group placement.

50. The method of Claim 31, wherein the total carbon chain of the monomer is about C,6 to C2 in length.

51. The method of Claim 50, wherein the total carbon chain length is about C20 to C23.

52. The method of Claim 51, wherein the total carbon chain length is about C22.

53. The method of Claim 31, wherein the molecular weight of the analyte is from about 100 to about 300,000.

54. The method of Claim 53, wherein the molecular weight of the analyte is from about 38,000 MW to about 150,000 MW.

55. The liposomes of Claim 54, wherein the molecular weight of the analyte is about 60,000 MW.

56. The liposomes of Claim 31, wherein ligands lacking a hydrophobic group in their natural state are associated with an exogenous hydrophobic group.