WO2003045558A1

WO2003045558A1 - Support for the use in the assay of particles in a sample and method for the fabrication of such a support and method for the assay of particles in a sample

Info

Publication number: WO2003045558A1
Application number: PCT/NL2002/000768
Authority: WO
Inventors: Richard Bernardus Maria Schasfoort; Gerardus Henricus Maria Engbers
Original assignee: Ibis Technologies B.V.
Priority date: 2001-11-28
Filing date: 2002-11-24
Publication date: 2003-06-05
Also published as: NL1019875C2; AU2002354361A1

Abstract

The present invention provides a support (10,20,30) for use in the assay of at least one type of particle in a sample, characterised in that: - a first part of the surface (11) of the support is suitable for receiving at least one microbead (3-3'') as used in known diagnostic techniques, the microbead (3-3'') being suitable for binding at least one particle of a specific type to its surface, and - the first part of the surface (11) is suitable for being inspected by means of an optical technique. The present invention also provides a method for the fabrication of such a support. Furthermore, the present invention provides a method for the assay of at least one type of particle in a sample, comprising the steps of: K. receiving at least one microbead (3-3'') as used in known diagnostic methods, on a first part of the surface (11) of a support (10,20,30), the microbead (3-3'') being suitable for binding at least one particle of a specific type to its surface, and L. inspecting the first part of the surface (11) by means of an optical technique.

Description

SUPPORT FOR THE USE IN THE ASSAY OF PARTICLES IN A SAMPLE AND METHOD FOR THE F ABRICATION OF SUCH A SUPPORT AND METHOD FOR THE ASSAY OF PARTICLES IN A SAMPLE

Field of the Invention The present invention concerns a support for use in the assay of at least one type of particle in a sample. The invention also concerns a method for the fabrication of such a support. Furthermore, the invention concerns a method for the assay of at least one type of particle in a sample.

Description of the Prior Art

The use of microbeads or microspheres in (bio)chemical analysis is known. These microbeads exists in countless types and dimensions, marked or unmarked. Many microbeads can bind ligands such as proteins and antibodies to their surface. These proteins in their turn can be marked, e.g. by means of a colour, a fluorescent or radioactive particle or by conjugation with gold, see e.g. US 5,981,180 or US 6,268,222. A specific ligand can form a selective bond with a specific (bio)chemical particle to be determined in a sample. A so-called 'reporter molecule' that also has been marked can in its turn selectively bind to the particle to be assayed. The microbeads and reporter molecules are generally mixed with the sample after which the markings are determined qualitatively and quantitatively, from which the presence and concentration of one of more particles to be assayed can be deduced. An important advantage of this 'microbead technology' is the enormous diversity of markings on the microbeads, ligands and reporter molecules, as a result of which the (bio)chemical assay of a large number of particles in a sample can be carried out simultaneously. Due to the relatively large surface of the microbeads and the concomitant small difiusion lengths and high interaction speeds, the required analysis time is relatively short.

Furthermore, flow cytometry is known for the assay of marked particles whereby these particles flow through a channel, see e.g. US 6,046,807, US 6,139,800 or US, 6,268,222. With this technique, the simultaneous analysis of several samples is not possible, and it is a 'wet' technique whereby the particles are distributed in a fluid which hampers storage and a renewed analysis on a later moment. An alternative method comprises the application of various types of optically active microbeads on the separate ends of a bundle of optic fibres. The end of the bundle is then immersed in the sample to be analysed. By measuring the change in the optical response of each individual fibre, it can be determined which types of particles are present in the sample. This method is relatively expensive and its implementation is relatively complicated, as it is not based on a generally known and accepted technology. It is marked by relatively long analysis times due to the relatively long diffusion lengths.

Furthermore, the use of (digital) imaging technology in combination with analytical (bio)chemistry is known. Especially the combination with gel electrophoresis is widely used. E.g. by means of 2-dimensional gel electrophoresis the separation of proteins can be achieved, in a first dimension by their charge and in a second dimension by their relative mobility. With this technique, thousands of proteins can be separated in a pattern of spots. For a quantification of the proteins, these are made visible after the separation, e.g. by (fluorescent) dyeing and subsequent recording by means of an imaging technique. For this a CCD (charge-coupled device) camera or a document scanner is generally used. Software is available for the processing of the recording. See e.g. W.F. Patton, Biologists perspective of analytical imaging-systems as applied to protein gel-electrophoresis, Journal of Chromatography A, 698 (1-2), 55-87, 1995. Another example of the use of imaging technology in bioanalysis are the 'DNA microarrays', amongst others extensively used in the elucidation of the human genome. With this technology, spots of different DNA fragments are arrayed on a support and then contacted with fluorescent-marked DNA/RNA, after which an image is made which is processed with either standard or custom-made software. At present, this technology is also extended towards 'proteomics' whereby instead of DNA, proteins are immobilised in an array. The resolution is determined by the size of the spots (at present typically in the order of several tens to 100 micrometers) and the pixel size of the used camera system (presently about 10 by 10 micrometers). Instead of fluorescence detection, the interaction with the DNA or protein arrays is also observed by means of imaging SP (surface plasmon resonance).

US, 5,892,577 describes an analytical device and method based on a transparent disk that can be optically scanned by means of a light source and a detector, using current CD technology, in transmission and/or reflection, whereby a change in colour or another light interfering change is measured. The possibility of profiling the surface in order to provide growth and binding sites for cells is mentioned, as well as the application of growth sites by chemical patterning (site-dependent chemical modification) of the surface. Besides that, the possibility of coating the surface with a gel is suggested in order to perform gel electrophoresis. As an application example, ELISA (enzyme-linked immunosorbent assay) is mentioned. Also mentioned are microtiter plates provided with wells and the like which can be scanned (step-wise). Thereby a CCD-array can be used as a light source, e.g. a linear CCD-array in combination with a laser line generator.

In WO 99/35499 a method is described for the qualitative and quantitative analysis of a target molecule by binding this to a non-cleavable capture molecule that is bound to the surface of a disk, which also carries registration data. The binding generates a signal that can be detected using a light beam. The signal can be a change of colour, reflection, absorption, light emission, magnetic or electrical field or interference by a precipitate (silver). Images of the surface can be made. The use of CDs and CD technology is extensively described, with many details of possible embodiments and options. Also grooves in the disk with data as well as capture molecules are mentioned. The binding of target molecules with capture molecules creates a number of pits and lands that can be read just like a regular CD. The disk can be divided in several surface sections and different capture molecules can be used simultaneously. In this document (Page 24, lines 10-20) microbeads are mentioned but then to mark the binding between a capture molecule and a target molecule (cause disturbances in the reflection of laser light). In WO 00/58735, the combinatorial synthesis on microbeads is presented as state of the art. Here compounds are synthesised on the surface of microbeads that are connected to the surface of the microbeads, optionally by means of a cleaveable linker. A method is claimed for the separation of microparticles, such as microbeads, from a suspension and the parallel arraying thereof on a plate (hydroplate) with which each microparticle can be positioned individually in a small volume reaction vessel. To this end, the surface of the plate is partitioned in hydrophobic and hydrophilic areas, thus creating non-communicating fluid retention areas. In each fluid retention area one or more wells have been applied for receiving one or more microbeads that are retained there through non-covalent interactions (e.g. electrostatic, magnetic, friction, glue). After the application of the microbeads in the wells, assay fluid, e.g. an analyte with target molecules, is applied in a wetting/dewetting procedure whereby droplets remain on the fluid retention areas. The specific binding between capture molecules ('compounds') and the target molecules takes place in the droplets. The hydroplate can subsequently be inspected optically.

Finally, in WO 99/45366 the application of two-dimensional imaging technology in multiple sample diagnostic tests is claimed. Thus multiple results, e.g. multiple dot-bots, can be analysed from a single image. The use thereby of a multiple sample reaction container plate is mentioned. The invention comprises the combination of: specific binding arrays in a disposable platform + liquid handling robotics + high-resolution imaging.

In the methods described in the four mentioned documents (US 5,892,577; WO 99/35499; WO 00/58735; WO 99/45366) the specific binding reactions take place on the surface of a carrier which causes relatively long analysis times due to the relatively long diffusion lengths. Thus, the advantage of the relatively large surface of microbeads in a homogeneous phase and the corresponding short diffusion lengths, high interaction speeds and a relatively short analysis time, is not exploited to the full.

Thus there is a need for a system for the (bio)chemical analysis of samples that combines the advantages of known (bio)chemical analysis technologies based on the use of microbeads with the advanced possibilities of known modern optical techniques such as powerful (digital) laser technology e.g. as known and current in compact disk technology, or (digital) imaging technology e.g. using a CCD-camera or a scanner. The purpose of the present invention is to provide such a system.

Summary of the Invention

The present invention provides a support for use in the assay of at least one type of particle in a sample, characterised in that:

- a first part of the surface of the support is suitable for receiving at least one microbead as used in known diagnostic techniques, the microbead being suitable for binding at least one particle of a specific type to its surface, and

- the first part of the surface is suitable for being inspected by means of an optical technique. The optical technique can comprise e.g. laser technology e.g. as known and current in compact disk technology, or imaging technology e.g. with the use of a digital camera or scanner. Thus, the major advantages of analytical (bio)chemical techniques based on the use of microbeads can be combined with the advanced possibilities of modern optical techniques.

The microbead can be marked. By applying several types of binding sites on the microbeads, several types of particles in a sample can be assayed simultaneously. The bond between support and microbead can be electrostatic, magnetic or hydrophobic. The microbead can be provided with a suitable affinity ligand such as an antibody, DNA or a receptor. The affinity ligand can bind the particle to be assayed. Next, a marker molecule with a fluorescent label can be bound to the particle to be assayed thus creating a double identification possibility. On the one hand by means of the colour of the microbead with the affinity ligand that is characteristic for a particular test, on the other hand by the fluorescent marker that indicates whether there actually has occurred a bond between the affinity ligand and the particles to be assayed.

In the first part of the surface of the support at least one recess, such as a groove or well, can be made for receiving at least one microbead,. This allows aligned binding of the microbeads on the surface of the support. E.g. a recess can have the shape of a cup-like well for receiving one or more microbeads, or that of a groove for fixing the microbeads in an array.

In a first embodiment, the support has a shape that is substantially equal to a compact disk as known and current in compact disk technology. This makes the unit compatible with current compact disk technology. In a second preferred embodiment, at least a part of the support is substantially equal to a current standard microtiter plate. Thus, a link can be made to current microtiter plate technology.

Preferably: - a second part of the surface of the support is suitable for storing data, and

- the second part of the surface is suitable for being scanned and read e.g. by means of laser technology e.g. as known and current in compact disk technology. In this way data concerning the sample and the analysis results can be recorded on the same support. Thus, an erroneous link between sample data and analysis results is virtually impossible.

The present invention also provides a method for the fabrication of a support for use in the assay of at least one type of particle in a sample, comprising the steps of: A. preparation of a first part of the surface of the support for receiving at least one microbead as used in known diagnostic techniques, the microbead being suitable for binding of at least one particle of a specific type to its surface, and B. preparation of the first part of the surface for being inspected by means of an optical technique. The microbead can be marked. The particle to be bound can be e.g. the particle to be assayed, a marked protein molecule that selectively binds the particle to be assayed, or a ligand that selectively binds the particle to be assayed. The optical technique can comprise laser technology as known and current in compact disk technology, or imaging technology e.g. with the use of a digital camera or a scanner.

The method can also comprise the step of:

C. making in the first part of the support of at least one recess, such as a groove or well, for receiving at least one microbead.

This can be done with great precision and relatively cheaply by means of current etching, die, punch or laser methods.

The method can also comprise the step of:

D. shaping the support substantially equal to a compact disk as known and current in compact disk technology.

To this end, existing cheap (mass) production methods in compact disk technology can be used.

The method can also comprise the step of:

E. shaping at least part of the support substantially equal to a current standard microtiter plate.

The production of microtiter plates is cheap because of the large scale on which this technology is used. Preferably, the method also comprises the step of:

F. preparing a second part of the surface of the support for storage of data and for scanning and reading of data e.g. by means of laser technology e.g. as known and current in compact disk technology. Thus, tried and tested laser technology can be used for the storage and reading of data like sample and analysis data.

The present invention also provides a method for the assay of at least one type of particle in a sample, comprising the steps of: K. receiving at least one microbead as used in known diagnostic methods, on a first part of the surface of a support, the microbead being suitable for binding at least one particle of a specific type to its surface, and

L. inspecting the first part of the surface by means of an optical technique.

Thus, the major advantages of (bio)chemical analytical methods based on the use of microbeads can be combined with one or more proven powerful optical methods, such as compact disk technology or imaging technology e.g. using a digital camera or a scanner.

For imaging and studying larger surfaces, a camera can be moved relative to the support, e.g. by means of an X-Y plotter, and thus the surface involved can be scanned.

Furthermore, in the case of a microtiter plate, the camera can individually image each well in the microtiter plate. Also, the support or the microtiter plate can be rotated. For a more accurate image with a higher resolution, microscopic methods can be used. Thereby the measurement of a plurality of fluorescent markers is possible. For reading the image, a document scanner can also be used.

Preferably, the method also comprises the step of:

M. receiving at least one microbead in a recess, such as a groove or well, the recess been made in the first part of the surface of the support. In a first preferred embodiment, a support is used for this that has a shape substantially equal to a compact disk as known and current in compact disk technology. In a second preferred embodiment, use is made of a support that is at least partly substantially equal in shape to a current standard microtiter plate. Thus, the method is at least partially compatible with current (digital) laser technology or current (digital) imaging technology, respectively. Preferably, the method also comprises the steps of: X. storing data on a second part of the surface of the support, and

Y. scanning and reading of the second part of the surface of the support, preferably by means of laser technology e.g. as known and current in compact disk technology. Thus, known (bio)chemical analytical microbead technology with its enormous diversity of possible markings of the beads, ligands and reporter molecules, can be used, allowing the

(bio)chemical assay of a large number of types of particle in a sample, e.g. in combination with current powerful laser technology. With this, the reading of the markings on the first part of the support as well as the storage and reading of data and analysis results on the second part of the surface of the support can be performed fast and efficiently.

The method can also comprise the steps of:

I. application onto the support of at least a part of the sample, and

J. spreading of the applied part of the sample over at least a part of the first part of the surface of the support by means of a flat plate by moving the flat plate on a relatively small distance to the support in such a way that the applied part of the sample is spread over the involved part of the first part of the surface of the support.

Moving can imply translation or rotation. Thus, one or more samples can be spread over the surface, e.g. in concentric circles or each in its own compartment.

Description of Preferred Embodiments

The present invention will be further clarified in the following by means of three non- limitative examples of embodiments of a support according to the present invention and two non-limitative examples of applications of a method according to the present invention. To this end:

Fig. 1 shows a schematic top view of a first embodiment of a support according to the invention;

Fig. 2 shows a schematic first measurement set-up to be used in a first embodiment of a method according to the invention ; - Fig. 3 shows a schematic top view with partial magnification of a second embodiment of a support according to the invention;

Fig. 4 shows a schematic top view of a third embodiment of a support according to the invention, and Fig. 5 shows a schematic second measurement set-up to be used in a second embodiment of a method according to the invention.

Fig. 1 and 2 schematically show a support (10) in the shape of a compact disk, with a first part of the surface (11) subdivided in a number of compartments (12). A liquid sample with particles to be assayed is mixed with suitable colour marked microbeads (3-3'") and colour-marked reporter molecules (4-4'"). The specific binding reactions take place in a homogeneous phase. Next, a part of this mixture is applied to the support (10) in one of the compartments (12). This application can be done e.g. by simply poring or by means of spin coating, whereby a homogeneous spreading can be facilitated by a suitable surface treatment. It can also be done by means of a flat device (not shown) such as a flat plate, whereby the flat device is moved at a small distance above the support (10) so that the applied quantity is spread over the surface, e.g. in concentric circles. After drying, the surface is scanned by means of a laser (15) and a detector (16) whereby the optical signals (17) are processed to qualitative and quantitative data concerning the particles to be assayed in the original sample. The simultaneous analysis of various samples and the assay of several types of particles simultaneously are possible, thus allowing a high through-put. It has to be noted that it is not necessary to analyse all particles in the applied volume but at least a sufficient number in order to gain statistically relevant results. Superfluous liquid can flow to a collecting space (not shown) provided on the support (10), or non-bound microbeads (3-3"') etc can be rinsed off, after which e.g. the support is spun dry and dried to the air.

A second part of the surface (13) of the support (10) is suitable for the recording of data, e g concerning samples and results. This makes it possible to link the sample data and the analysis data physically inseparable, with all advantages mentioned. The recording and reading can be done by means of known and current compact disk technology. This combination of methods provides a unique, fast and advantageous analysis system, suitable for multiple and simultaneous (bio)chemical analyses. Being based on current and accessible laser technology e.g. as known and current in compact disk technology, the implementation threshold is low.

Fig. 3 schematically represents a second embodiment (20) of a support according to the invention in the shape of a compact disk. Grooves (22) have been made in the surface (21) for the spiral alignment of colour-marked microbeads (3-3'"). To achieve an even distribution of the microbeads (3-3'") in the grooves (22), the sides (23) of each groove (22) have been provided with cup-like slots (24). A well-defined distribution of the microbeads (3-3'") over the surface (21) facilitates the analysis and increases the quality of the results.

It is remarked that the alignment of the microbeads (3-3'") can also take place by making wells in the surface of the support (20). The making of the wells or grooves can be carried out with great precision and relatively cheaply with current etching, die, punch or laser methods.

Fig. 4 schematically shows a support (30) in the shape of a microtiter plate, with a first part of the surface (11) subdivided in a number of compartments (12). In an application of a method according to the present invention, a liquid sample with the particles to be assayed is mixed with suitable colour-marked microbeads (3-3"') and colour-marked reporter molecules (4-4'"). Again, the specific binding reactions take place in a homogeneous phase. Next, a part of this mixture is applied on the support (30) in one of the compartments (12). This application can be done e.g. simply by pouring. After an optional drying step the surface is imaged by means of a light source (35) and a CCD-camera (36), as schematically represented in Fig. 5, whereby the image is processed into qualitative and quantitative data concerning the particles to be assayed in the original sample. The simultaneous analysis of several samples and the assay of various types of particles simultaneously are possible, thus allowing a high throughput.

Again, it is not necessary to analyse all particles in the applied volume but at least a sufficient number in order to gain statistically relevant results. Again, superfluous liquid can flow to a collecting space (not shown) provided on the support (30), or non-bound microbeads (3-3'") etc can be rinsed off, after which the support is spun dry and dried to the air. Here too, a second part of the surface (13) of the support (30) can be prepared for the storage of data, to be recorded and read using current laser technology. This combination of methods provides a unique, fast and advantageous analysis system, suitable for multiple and simultaneous (bio)chemical analyses. Being based on current and accessible laser technology e.g. as known and current in compact disk technology, again the implementation threshold is low. When microtiter plates are used, the analysis can be performed according to current rinsing programmes and using current pipetting stations. It will be clear to a person skilled in the art that the invention is limited in no way to the described and shown embodiments and that many variations and combinations are possible within the scope of the invention.

Claims

1. A support (10,20,30) for use in the assay of at least one type of particle in a sample, characterised in that: - a first part of the surface (11) of the support is suitable for receiving at least one microbead (3-3'") as used in known diagnostic techniques, the microbead (3-3'") being suitable for binding at least one particle of a specific type to its surface, and

- the first part of the surface (11) is suitable for being inspected by means of an optical technique.

2. A support according to claim 1, characterised in that the optical technique comprises laser technology e.g. as know and current in compact disk technology.

3. A support according to claim 1 or 2, characterised in that the optical technique comprises imaging technology e.g. with the use of a digital camera or scanner.

4. A support according to any of the preceding claims, characterised in that in the first part of the surface (11) of the support at least one recess, such as a groove (22) or a well, for receiving at least one microbead (3-3"') has been made.

5. A support according to any of the preceding claims, characterised in that the support has a shape substantially equal to a compact disk as known and current in compact disk technology.

6. A support according to any of the preceding claims, characterised in that at least a part of the support is substantially equal to a current standard microtiter plate.

7. A support according to claim 1, characterised in that: a second part of the surface (13) of the support is suitable for storage of data, and

- the second part of the surface (13) is suitable for being scanned and read e.g. by means of laser technology e.g. as known and current in compact disk technology.

8. A method for the fabrication of a support (10,20,30) for use in the assay of at least one type of particle in a sample, comprising the steps of:

A. preparation of a first part of the surface (11) of the support for receiving at least one microbead (3-3"') as used in known diagnostic techniques, the microbead being suitable for binding of at least one particle of a specific type to its surface, and

B. preparation of the first part of the surface (11) for being inspected by means of an optical technique.

9. A method according to claim 8, characterised in that the optical technique comprises laser technology e.g. as know and current in compact disk technology.

10. A method according to claim 8 or 9, characterised in that the optical technique comprises imaging technology e.g. with the use of a digital camera or scanner.

11. A method according to any of claims 8- 10, also comprising the step of:

C. making in the first part of the support (11) at least one recess, such as a groove (22) or well, for receiving at least one microbead (3-3'").

12. A method according to any of claims 8-11, also comprising the step of:

13. A method according to any of claims 8-12, also comprising the step of:

14. A method according to any of claims 8-13, also comprising the step of: F. preparing a second part of the surface (13) of the support for storage of data and for scanning and reading of data e.g. by means of laser technology e.g. as known and current in compact disk technology.

15. A method for the assay of at least one type of particle in a sample, comprising the steps of: K. receiving at least one microbead (3-3'") as used in known diagnostic methods, on a first part of the surface (11) of a support (10,20,30), the microbead (3-3'") being suitable for binding at least one particle of a specific type to its surface, and L. inspecting the first part of the surface (11) by means of an optical technique.

16. A method according to claim 15, characterised in that the optical technique comprises laser technology e.g. as know and current in compact disk technology.

17. A method according to claim 15 or 16, characterised in that the optical technique comprises imaging technology e.g. with the use of a digital camera or scanner.

18. A method according to any of claims 15-17, comprising the step of:

M. receiving at least one microbead (3-3'") in a recess, such as a groove (22) or well, the recess been made in the first part of the surface (11) of the support.

19. A method according to any of claims 15-18, characterised in that a support is used that has a shape substantially equal to a compact disk as known and current in compact disk technology.

20. A method according to any of claims 15-19, characterised in that a support is used that is at least partly substantially equal in shape to a current standard microtiter plate.

21. A method according to any of claims 15-20, characterised in that the method also comprises the steps of: X. storing data on a second part of the surface (13) of the support, and

Y. scanning and reading of the second part of the surface (13) of the support, preferably by means of laser technology e.g. as known and current in compact disk technology.

22. A method according to any of claims 15-21, characterised in that the method also comprises the steps of:

I. application onto the support of at least a part of the sample, and

J. spreading of the applied part of the sample over at least a part of the first part of the surface (1 1) of the support by means of a flat plate by moving the flat plate on a relatively small distance to the support in such a way that the applied part of the sample is spread over the involved part of the first part of the surface (11) of the support.