JPH0224459B2 - - Google Patents

Description

Claim 1: A specific reactive sampling and testing device having one or more cavities, each having dimensions sufficiently small to allow sample liquid to be drawn into the cavity by capillary action, comprising:
the surface of the cavity carries immobilized reagents suitable for the tests to be carried out in the device, the surface being the surface of a transparent fixation plate that acts as a light transmission waveguide and forms the walls of the cavity; The device wherein the plate is substantially optically smooth and has an edge transverse to the plane of the plate.

2. The device of claim 1, wherein the remaining edges of the waveguide are coated with a light-absorbing material.

3. The waveguide surface carrying the immobilized reagent has a thin layer of material for enhancing the movement of light across the waveguide boundary by extremely small waves located close to the interface. 2. A device according to claim 1 or 2.

4. The device of claim 3, wherein the thin layer material comprises magnesium fluoride or silica.

5. The device according to claim 1 or 2, wherein the waveguide surface carrying the immobilized reagent further comprises a thin film of conductive material capable of exhibiting a surface plasmon resonance effect.

6. The device of claim 5, wherein the thin film of conductive material is a coating of silver with a thickness of about 50 nm or less.

7. The device according to any one of claims 1 to 6, wherein the immobilized reagent supported on the waveguide surface includes a biochemical specific binding agent.

8. The biochemical specific binding agent is an antigen, an antibody,
8. A device according to claim 7 selected from derivatives thereof or non-immunological binding agents.

9. The device of claim 8, wherein said antigen, antibody or derivative thereof is covalently bonded to the surface of a transparent sheet material which acts as a light transmission waveguide.

10 Claims 1 to 10 in which the auxiliary reagents are added separately or carried in releasable dry form in the part of the device that contacts the sample liquid during use.
10. The device according to any one of clauses 9.

11. The device of claim 10, wherein at least one of the auxiliary reagents is labeled with a fluorophore.

12. A device according to any one of claims 1 to 11, wherein the waveguide comprises a glass or plastic material, such as soda glass or acrylic plastic.

13. Claims 1 to 12 consist of a bonded structure of transparent plates joined at such a spacing as to leave a thin flat cavity of capillary dimensions between the plates.
A device according to any one of paragraphs.

14. The device according to any one of claims 1 to 13, further comprising an operating piece or a holder.

15. A device according to any one of claims 1 to 14, further comprising a sample concentration means or selective barrier, such as a filter or dialysis membrane, located within the capillary cell or in the inlet passageway to said cell. .

16. A device according to any one of claims 1 to 15, wherein a light absorbing wall or an opaque wall or a reflective wall is attached to the surface of the cavity facing the waveguide surface holding the reagent.

17. Claim 1 further includes an electrode structure for electrically measuring the liquid content of the capillary cell.
17. The device according to any one of clauses 16 to 16.

18 A method for manufacturing a specific reactive sample collection and testing device, comprising: (a) forming a specific reactive immobilization coating on the surface of a transparent sheet material forming part of a number of devices; ) of a plurality of devices each of which is coated with said sheet material and of capillary dimensions for collecting and retaining by capillary action a volume of sample liquid in contact with said specific reactive coating; (c) separating said sheet material into portions each providing one or more sample collection and testing devices, and (c) separating said sheet material into portions each providing one or more sampling and testing devices; Dimensions such that the step results in at least one edge on each device that is essentially optically smooth and transverse to the plane of the plate.

19. A method according to claim 18, wherein the specific reactive coating is divided into a pattern of discrete parts, such as a two-dimensional array of patches.

20. Claim 19, wherein the patterned patch is manufactured by first forming a continuous coating and then removing portions thereof to leave a desired pattern, such as an array of discrete portions. Method described.

21. Any of claims 18 to 20, wherein the specific reactive coating is a specific binding agent that is immobilized on the surface of a transparent sheet material that acts as a light transmission waveguide to form an immunosorbent. The method described in paragraph (1).

22. The method of claim 21, wherein the specific binding agent is selected from an antigen, an antibody, a derivative thereof or a non-immunological binding agent.

23. The method of claim 22, wherein said antigen, antibody or derivative thereof is covalently attached to the surface of a transparent sheet material which acts as a light transmission waveguide.

24. Claims 18 to 24, in which the auxiliary reagents are added separately or carried in releasable dry form in the part of the device that contacts the sample liquid during use.
The method according to any one of paragraph 23.

25. The method of claim 24, wherein at least one of the auxiliary reagents carried in releasable dry form is labeled with a fluorophore.

26. The method according to any one of claims 18 to 25, wherein the immobilized reagent is overlaid on a thin film of a conductive material supported on the waveguide surface and capable of exhibiting a surface plasmon resonance effect.

27. The method of claim 26, wherein the thin film of conductive material is a coating of silver about 50 nm thick or less.

28. A method according to any one of claims 18 to 27, wherein an immobilized reagent is overlaid on a thin dielectric layer deposited on the surface of the transparent sheet material.

29. The method of claim 28, wherein said thin dielectric layer comprises magnesium fluoride or silica.

30 The additional structure is another sheet material bonded to the first sheet material by a suitable bonding adhesive, e.g. 30. A method according to any one of claims 18 to 29, characterized in that the sheet material is spaced by the amount of capillary voids.

31. A method according to any one of claims 18 to 30, wherein the device has an external surface into which a sample liquid can be loaded or applied and from which it can be drawn into the cavity. .

32. Claims 18 to 31, in which a light-absorbing wall or an opaque wall or a reflective wall is attached to the surface of the cavity facing the waveguide having a specific reactive coating.
The method described in any one of paragraphs.

Description The present invention relates to a device used in chemical (particularly biochemical or clinical) testing methods and a method for manufacturing the same.

In one embodiment, the device is intended for use in specific binding assay methods, an important group of which comprises immunoassay methods. Examples of such immunoassays, in particular enzyme-linked immunoassays, are cited in EP 0042755, GB 2074727, GB 2086041 and GB 1548741.

In the past, immunoassay methods have often been carried out using so-called microtiter wells, customarily having a working volume of about 0.5 ml, among a variety of other liquid containers for the assay reaction liquid. Other devices and apparatus for handling immunoassay materials are described, for example, European Patent No. 031993, British Patent No.
1571872, UK Patent No. 1584129 and UK Patent No.
It is described in the specification of No. 1414479.

In particular, many analytical devices for manipulating and measuring small test samples have been disclosed in the prior art.

British Patent No. 2090659 [Instrumentation Laboratory Ltd.
Laboratory, Inc.] is constructed with a self-filling metering channel and a lip or inlet on which a sample of, e.g. ) describes a test strip in which 10μ is sucked up and reacted with reagents contained in a fibrous pad on a layer of filter beneath a transparent window. The results can be seen with the naked eye, for example as a color reaction.

British Patent No. 2036075 [H.E.
HEMennier], British Patent No. 1104774 [JP
Gallagher], European Patent No. 0057110, no.
No. 0034049, No. 0010456 [Kodak] all describe several other aspects of the use of capillary channel or chamber dimensions for manipulating biological or test body fluids. There is.

British Patent No. 1530997 [Monsanto] describes coated optical fibers that can be used in tests where the light transmission capacity of a waveguide is changed by a reaction of the coating, such as an antigen-antibody reaction. It describes the use of. WO/81/00912 [Buckles] also describes fiber engineering devices in which the surface or periphery of the fiber alters light transmission through the core.

U.S. Pat. No. 3,939,350 describes the optical measurement of fluorescent substances bound to the surface of a transparent solid prism by a method involving interaction of the bound substance with extremely small waves at the prism surface and a single total internal reflection. It is listed.

European Patent No. 0075353 [Battelle] describes the relationship between the coating and the external radiation, which is exponentially attenuated due to the logarithmically amplified light in the fiber (very small). Specific reference is made to interactions, and this principle is also addressed in the Inomassay test device of EP 0103426 [Block]. In this device, a substance coated on a fiber or plate, containing a fluorescent.tagged binding partner, is brought into contact with a capillary-sized sample liquid volume separated by a tube or another plate. The fluorescence excitation wave is enhanced along with the emission wavelength inside an optical fiber or plate coated with the antigen or antibody.

In accordance with the invention described herein, a capillary-filled cell device is provided that can be conveniently manufactured and particularly facilitates specific binding assays using extremely small volumes of liquid sample.

According to the invention, there is provided a specific reactive sample collection and testing device having one or more cavities, each having dimensions sufficiently small to be able to aspirate a sample liquid into the cavity by capillary action. , the surface of the cavity carries immobilized reagents suitable for the test to be carried out in the device, said surface being the surface of a transparent solid plate which acts as a light transmission waveguide and forms the walls of the cavity. and the plate has edges that are substantially optically smooth and transverse, for example at some transverse angle or most preferably perpendicular to the plane of the plate. Provide the device.

The device allows convenient sample collection and enables in situ optical analysis of reactive components of the sample with one or more reagents contained in the device. The waveguide plate is infrared,
One method of using a device that is capable of transmitting visible and/or ultraviolet light is to prepare a fluorescent substance that binds to an immobilized reagent, with varying ranges depending on the desired assay and sample material, and then The method is to optically measure the resulting bound fluorescent substance.

The immobilized reagent in the device can be, for example, an immobilized antigen or antibody capable of binding fluorescent or luminescent or colored components of the assay mixture. However, it is understood that any immobilized material capable of specifically interacting with another component of the test reactant in a manner that provides an optically measurable result can be used, depending solely on the type of test in question. be done.

The surface of the cavity opposite the waveguide surface holding the reagents is provided with a second surface, as shown in the examples below.
may be formed from a similar transparent plate. However, this is not necessary and for some types of testing it may be preferable to use a light absorbing wall or an opaque wall or a reflective wall on the side opposite the capillary cavity.

In some useful embodiments of the device of the invention, a filter or dialysis membrane may be used to ensure selectivity of the uptake of the sample liquid into the capillary-sized cavity to exclude undesired substances. A selective barrier can be installed. Such undesirable materials may include cells such as red blood cells, cell debris, or dispersed high molecular weight materials, depending on the nature of the test. A suitable filter may be, for example, a paper filter at the sample inlet of the capillary cavity or a strip or surface of a filter fixed to or within the capillary cavity or a dialysis membrane material.

The area coated with the immobilized reagent, e.g. immobilized antigen or antibody, within the capillary cavity is much smaller than the total area of the capillary cavity, and the entire sample liquid must pass through the immobilized reagent when liquid is drawn into the cavity. The scope of the present invention also includes methods that can prevent this from occurring. in this way,
A higher surface concentration of the relevant analyte (eg complementary antibody or antigen) is obtained than would result from uniform adsorption in the entire area of the capillary cell, and a sample concentration effect is achieved. This immobilized coating on a limited threshold area of the capillary cell surface actually creates a selective barrier that selectively retains the desired analyte through which the sample liquid passes. sell.

In using the device of the invention, a sample droplet may be placed on the collection surface of the test device, or the device may be immersed in a volume of liquid to be sampled. When auxiliary reagents are required, they may be added separately or they may be contained in dry releasable form on the part of the device that comes into contact with the sample liquid during use, e.g. on the surface of the capillary cavity or on the surface of the filter, if present. Good too.

Other variations and special features of the test device are illustrated below.

In accordance with the present invention, (a) a specific reactive immobilization coating is formed on the surface of a sheet material that provides a plurality of device portions; forming an additional structure on each device that provides a capillary-sized cavity for collecting and retaining a volume of sample liquid by capillary action in contact with the specific reactive coating; (c)
A method of manufacturing a specific reactive sampling and testing device is also provided, comprising dividing a sheet of material into sections each providing one or more sampling and testing devices. In this method, the specific reactive coating may be confluent, or may be continuous, or may be divided into, for example, a pattern of discrete parts, such as two-dimensional array patches. good. When forming such patches, for example, first a continuous coating is formed,
By then removing portions of the coating to leave the desired pattern, e.g., an array of sectioned portions,
Or it can be made as a patch array (eg screen printed or flexographic printed).

The auxiliary reagent may be a releasable agent, such as a releasable antigen or a coating of an antibody or derivative thereof, with a specificity suitable for the desired assay, held in a solid wetting agent, such as a sucrose glaze. It may be in the form of a reagent coating. The additional structure is
bonded to the first sheet material, e.g. by a suitable bonding adhesive, spaced apart by a capillary space of, e.g., less than about 1 mm, and preferably depositing a limited amount of sample liquid, which can be repeatedly taken, onto the sheet by capillary action. It may be another sheet of material that can be introduced in between. One or both of the sheets may be light transmissive with an optically uniform and generally smooth surface. sheet material,
For example, a glass, quartz or plastic material can be marked or broken into units and, in the example below, a sample liquid can be placed or applied to the outside from which the sample liquid can enter the cavity of the device. loading surface
The units are separated so as to leave the surface. The outer loading surface preferably has a capacity to contain or hold at least enough liquid (eg, in the form of a drop of material spread over the surface) to fully fill the cavity.

The invention also extends to the products obtained by the methods described herein and to the uses of those products.

In some embodiments of the device of the invention, the cavity of the device has two cavities facing each other forming a cell.
a thin, flat cavity between two walls;
Preferably, they are joined or made up of units. In some cases, for example, the device may contain a structure with attached transparent plates similar to the structure of an unfilled liquid crystal display device as obtained as an intermediate step in liquid crystal display manufacturing.

The device of the invention thus forms a liquid-bearing cell with an opening capable of drawing up and holding a (preferably defined) volume of liquid (usually water) by capillary action. The cells are coated with an immobilized specific binding agent having a specificity appropriate for the assay to be performed, consisting of a pair of oriented transparent plates sheeted together with a spacing of less than about 1 mm. is supported on at least one of the plurality of inner surfaces of the . Devices having translucent or transparent (eg, disposable) capillary cells that can be manufactured by the methods described herein for carrying out specific binding assays are included. By "defined volume" is meant a volume that is substantially determined by the shape and arrangement of the cell itself, and is not clearly determined from the sample volume when applied in excess.

In the device of the invention, it is important to have a cavity that is precisely defined (parallel) to the capillary gap size, and less important that the total volume wicked by the capillary gap should be defined.
Rather, the important parameter is the sample volume given per unit area of the waveguide surface holding the optical reagent, which precisely defines the space of parallel oriented walls forming the cavity within the device. It is given by This space is
Preferably within the range of about 0.01 to 1 mm, e.g. 0.1 mm
degree, i.e. 0.03-0.3 mm, which means that the waveguide surface holding the reagent has too large a space and only a small amount of sample It corresponds to the middle ground between a space that is too narrow and can only be harvested.

Suitable materials for forming the cavity are glass, such as soda glass in sheet form approximately 1 mm thick, silica, and plastic sheet materials such as acrylic plastic materials.

When making capillary cells using plastic materials, for example, precision molds with spacers such as ridges may be used to control the wall space that constitutes the capillary cell cavity. can.

In some cases, the cell has an outer surface portion or lip to which a sufficient amount of sample can be applied to fill the cell and to which the sample can be easily drawn into the capillary cell by capillary action. There is. Such a rim can be easily formed by stretching one of the plates, with the cell opening facing outward, to a length sufficient to provide a large enough surface area to easily place the sample. . If desired, the cell can also be equipped with a liquid conduit, for example a globe or channel, leading to the inlet of the capillary cell. An alternative form of inlet is one formed by an opening inside one wall of the capillary cell, for example a hole exposed in a region of the opposite wall of the cell in which the sample can be deposited. A selective barrier, such as a filter or dialysis membrane, as described herein may be disposed within or adjacent such an inlet opening, and the selective barrier may be provided with a dry releasable material. The above reagents may be administered in advance. It is particularly advantageous to have plastic capillary cells with these features, and in such cases, precisely shaped openings provided in the shaped plastic sheet will allow the resulting plurality of capillary cells to Vertical and optically flat capillary cell ends can be provided when dividing the collection of cells into each cell unit.

An epoxy resin line is used to create a filling opening and another opening through which air is released from the capillary cell as the cell fills, e.g. between two opposing rectangular capillary cells. Preferably, the cell can be sheeted by stretching the resin along the ivy side, leaving an opening. Preferably, the resin consists of solid particles and provides the desired spacing in the plate as the solid particles are laid down on the resin. To adjust a space as small as a ballotini or fiber diameter,
Substantially monodisperse with a diameter corresponding to the selected capillary void or a diameter of about 100μ
Ballotini (fine glass grains) or e.g. diameter
Particles such as 8μ, short glass fibers with a length of 50-100μ (eg, made by motor grinding long glass fibers and removing the long fibers left by sieving) are suitable. In general, non-limiting examples include 5-
A space with a range of 500μ is selected. Since it is easier to obtain diameters of less than about 50 microns with fibers than with monodisperse Ballotini, fibers are preferred for very narrow voids, and Ballotini are preferred for wide voids.

The specific binding agent can be selected from any commonly used for specific binding assay purposes, especially antigens or antibodies. Preferred embodiments are anti-globulin antibodies, or antibodies specific for human apolipoprotein A ₁ or B, or anti-steroid antibodies specific for e.g. estrone-3-glucuronide or pregnanediol glucuronide, or non-immunological antibodies such as concanavalin A. binder or Cibachrome Blue. The specific binding agent can be immobilized on glass or silica or plastic surfaces by any immobilization method. For example, it may be useful to simply form a film of the binder and sucrose on the surface of the carrier simultaneously or sequentially and dry it. If desired, in particular in the case of plastic materials, such as glass or silica, etc. For a wide range of support materials, including quartz materials, see ``Immobilized Enzymes for Industrial Reactors'' (Ed. Messing, Academic Press, 1975; especially Filbert, Chapter 3) or For example, U.S. Patent No.
Covalent or other immobilization may be achieved by any of the methods described in GB 3,652,761 or GB 1,530,997.

The types of immunoassays that can be carried out in these capillary cell devices, in particular the types of immunoassays such as fluorescent immunoassays, correspond to other known types of immunoassays of the prior art. For example, such an assay may be a so-called competitive assay, in which the antigen analyte competes for binding sites on a specific immunosorbent with a fluorescent competitor, such as a fluorescently labeled antigen analog. Alternatively, the analyte may be contacted and reacted with an inomusorbent in the absence of a fluorescent ligand, and then e.g. It is also possible to contact a treated immunosorbent with a fluorescent ligand.
Furthermore, for example, after forming a mixture of labeled and unlabeled immune complexes, the analyte can be prepared in the presence of immobilized antiglobulin carried on the capillary cell surface to immobilize the antibodies involved in the assay. and solution phase binding partner and fluorescently labeled competitor,
Solution phase competition can be performed between (eg, a soluble antibody and a competitive fluorescently labeled antigen).

In yet another embodiment, an immunosorbent and a fluorescent ligand (e.g. both antibodies or an antigen and an antiglobulin respectively in an antiglobulin test)
both the analyte (antigen or antibody) being tested
A Sand Deutsch test or an antiglobulin test, which has a specific affinity for the virus, can be performed.

In yet another embodiment, the more general ligand binding reaction involves an immobilized layer of concanavalin A that competitively binds, for example, glucose and fluorescently labeled dextran, or a fluorescently labeled and unlabeled albumin or other protein. A layer of dyes such as cibacrom blue, which binds to the dye, can be used.

Similarly, luminescence assays can also be performed using labeling agents that participate in chemical or biological photoreactions, such as luciferase or horseradish peroxidase.

Embodiments of the invention will be described, for example, with reference to the accompanying FIGS. 1 to 4 and the related description.

FIG. 1 is a schematic cross-sectional view of a disposable capillary cell device according to one embodiment of the invention.

FIG. 2 is a schematic plan view of the cell device of FIG. 1, including the line - indicating the cross-sectional line of FIG.

FIG. 3 is a schematic partial plan view showing an intermediate stage in the manufacture of a plurality of devices such as those of FIGS. 1 and 2;

3a is a schematic partial plan view corresponding to a modified example of the arrangement shown in FIG. 3; FIG.

FIG. 4 is a schematic perspective view of a specifically responsive capillary cell device according to an embodiment of the invention.

5 and 6 are schematic plan and cross-sectional views of the device of FIG. 4; FIG.

7 and 8 are corresponding schematic plan and partial cross-sectional views of another device that is a modification of the device of FIGS. 4-6.

Figures 1 and 2 show capillary cell devices of a size that is easy to handle, for example approximately 3 cm x 1.5 cm.
cell device). The device has a transparent top (e.g. plastic, glass or silica)
It consists of a plate 1 and a lower transparent (e.g. similar) plate 2 (approximately 1 mm thick), which are separated by less than 1 mm by bonding tracks 3 of suitable (e.g. epoxy) adhesive to form a capillary cell cavity 4. Moreover, they are fixed so as to face each other in parallel. Both ends of the cavity 4 are open and communicate with the outside via a first discontinuous portion of the bonding material 3 arranged to form the opening of the cell at the side 5 of the plate 1 . Another discontinuity is present at the other end of the coupling 3, providing another opening for the exclusion of air when introducing the sample liquid into the cell. Plate 2 is larger than plate 1 and has a portion 6 extending away from the opening. The portion 6 of the plate 2 serves as a platform or threshold or lip onto which a sample droplet can be applied, so that this liquid fills the capillary cell cavity 4 by capillary flow. When placed in this manner, the cavity 4 attracts and contains a constant, suitable and reproducible volume of liquid.

A layer 7 of the substance involved in the test method using the capillary cell is fixed to the inner surface of the capillary cell. In the embodiment shown in the figures, the layer 7 is a patch of material carried on the plate 2. For immunoassay purposes, it may be an immunosorbent sensitizing region, such as an immobilized antibody suitable for immunoassays.
For example, one layer may be provided on plate 1 as well as plate 2, or multiple layers may be stacked and/or side-by-side on either plate.
There may be one or more such layers, such as a side). For similar or other purposes, layer 7 or other layers lining the inner surface of the capillary cell may include one or more electrically conductive layers, as described in June 1984. As set out in the Applicant's pending application of the same date derived from UK8415018 of 13. In such a case, it may optionally pass between the bonding layer 3 and the plate surface. Conductive connections to the outside can be made by conductive tracks or connectors from the inside of the cell to the outside of the cell. These can be produced, for example, by methods which are known per se and are used, for example, for the conventional surface processing of conductive tracks, which are often used in the production of semiconductors and liquid crystal displays, as referred to below.

When the cell is to be used for optical measurements, either plate 1 or plate 2 or both must be transparent or translucent.

The cross-sectional view shown in FIG.
section) does not extend through the coupling track 3, thus representing the plates 1 and 2 being spaced apart.

The manufacture of a plurality of cells such as those of FIGS. 1 and 2 is illustrated in FIG. 3, which is a partial plan view showing an intermediate stage in the manufacture of these cells. A large plate 8 of glass or other material for making the plate 2 is cleaned and prepared in any suitable manner (for example as described below) using a track of bondable adhesive 3 and a patch 7 of any type of material described above. method) to coat plate 8. The second plate is then attached to plate 8, after forming bonding tracks corresponding to track 3, if appropriate, and any other desired patches or tracks of material on the second plate, not shown. and allow the adhesive to harden. The assembly is then sectioned or cut along the line indicated by dotted line 9 in FIG. 3 and the corresponding line on the top plate (although line 9 need not be labeled). As a result, cells such as those shown in FIGS. 1-2 are obtained.

FIG. 3a shows a schematic plan view illustrating another preferred intermediate step in the manufacture of a capillary cell device such as that of FIGS. 1 and 2. FIG. Figures 3 and 3a
The main differences between the two figures are not only the shape and details of the pattern of the device to be manufactured, but also the fact that there is no waste edge 31 in the sheet material 8.
is provided, and an adhesive track 3a that can be connected to this waste liquid edge 31 is provided. The purpose of this is to better or more conveniently adjust the spacing between the upper and lower plates. Bondable adhesive tracks 3 and 3a contain the fibers or microparticles described above to adjust the spacing between the upper and lower plates, so that when track 3a is installed, the spacing becomes irregular at the edges of the plates. can be prevented.

The arrangements in FIGS. 3 and 3a have been described using sheet glass as the substrate material. Particularly, but not exclusively, when referring to plastic sheets, it may be convenient to use other than flat sheet materials, such as spacer rods;
The inlet openings and filter arrangements described herein can be incorporated as part of such sheets prior to assembly of the capillary cells.

Among other devices manufactured according to the invention:
The capillary cell device of FIGS. 1-2 may have any convenient form of handling portion as desired.
piece) or a holder, and if such holder is not formed in one operating part with the cell device, a fixed piece for mounting such a holder for the above purpose. Alternatively, a simple removable connection device may be provided.

To facilitate optical measurements, it is desirable to make one or more edges of plates 1 and/or 2 substantially optically smooth and perpendicular to the plane of the plates. If desired, the remaining edges may be coated with black paint or other light absorbing material. Alternatively or additionally, a light-absorbing pigment such as carbon black may be incorporated into the cement, such as the epoxy, used to bond the two plates together.

In use, the device of FIGS. 1 and 2 can be connected to a plate 1 such as a waveguide or optical fiber.
Fluorescence from a fluorescent substance adsorbed along one or both of 2 and 2 can be transmitted. Effective light across plate boundaries can be transmitted by evanescent waves located very close to the interface. If desired, a thin layer of e.g. silica or magnesium fluoride, on the order of 1/4 wavelength thick at the wavelength, can be formed on the plate prior to depositing the biochemical reagents to facilitate transmission of light across the interface. It can also be improved. For example, in the plate in the path corresponding to total internal reflection near (and slightly above) the critical angle of the interface between the plate material and the sample liquid, which is usually in the range of about 1° to 5° above the critical angle. Preferably, the thickness and refractive index of the dielectric layer are selected together to maximize the intensity of said extremely small waves that couple with the transmitted light. The optimum refractive index of the dielectric layer is as close as possible to that of the sample liquid.
In practice, the most common choice when using glass is a dielectric layer of magnesium fluoride (n=1.38) or silica (n=1.46). The optimum thickness of the layer is set by the angle of incidence P of the light in the plate at the plate-dielectric layer interface and the refractive index of the medium.
If the sample liquid is _n1 , the plate is _n2 , and the dielectric layer is _n3 , and L is ^the wavelength of the light used, then t=L/4・1-2/πarccos〓( _n32 ⁻ _n22si
n ² p/n ₃ ² −n ₁ ² )/〓(n ₃ ² −n ₂ ² sin ² p).

In a further derived embodiment, a thin metallic layer of silver or indium, continuous or discontinuous, is deposited on one or both plates, preferably with a very thin corrosion-resistant layer, such as silica, vacuum-deposited thereon. for example B. Liedberg et al., Sensors and Actuators, 4 (1983) 299.
It is also possible to use this device for other corresponding optical analysis methods known per se that make use of such layers, such as the surface plasmon resonance phenomenon as described in US Pat. In any of these examples, the immobilized layer of biochemical reagents can be supplemented with a releasable reagent coating formed, for example, by air-drying a protein-sucrose mixture into a thin film on a plate. This is selected and combined with the chemistry of the particular test being performed in the device. The chemical or binding reactions that may form part of the test to be carried out range from known binding reaction tests, including any type of enzyme binding based on solid phase immunosorbents or other specific binding adsorbents. , fluorescence, luminescence, binding and quenching reactions.

Details of procedures and materials for manufacturing cell devices, such as the capillary cell device of FIGS. 1-2, are provided in the following examples which further illustrate embodiments of the invention.

EXAMPLE A sheet of glass, e.g. about 1 mm thick (e.g. soda), large enough to contain a two-dimensional array of cell areas, with several cell units in each direction, is prepared by a suitable method, e.g. detergent and ultrasonic treatment. and if necessary degreasing with solvent vapor by known methods or successive treatments with ammonia/hydrogen peroxide and hydrochloric acid/hydrogen peroxide (80°C), washing with water, e.g. air at 115°C for 30 minutes. Clean by drying. A pattern of patches of the desired protein or other coating is then formed by the following or equivalent techniques. The antigen or antibody or other protein is covalently bound by first reacting the glass with a silane-based coupling compound in a known manner. As used herein, a suitable such reagent is a terminal amino-alkyltrimethoxysilane such as a 3-aminopropyl compound used at a suitable concentration of about 2% v/v in acetone. . In other methods, other reagents substantially as described in USP 3,652,761 may be used instead. After reaction with the aminosilane reagent, the amino terminus immobilized on the flass is sequentially reacted with glutaraldehyde (e.g., 2%, pH 7), excess reagent is removed, and the activated glass with immobilized aldehyde groups is prepared. , in a manner known per se, with a protein in solution (for example, 1 mg/ml antibody immunoglobulin). other proteins
A concentration of about 0.1 to 1 mg/ml can be freely selected and applied. A treatment of 2 hours at 37° C. and approximately PH 9.5 has been found to be suitable here. A suitable loading rate of final active protein onto a glass surface can be, for example, about 0.5 μg/cm ² . The proteins are thought to constitute continuous or near-continuous layers. The amount or density or inactivity of the immobilization layer is determined by the sensitivity requirements of the particular assay chemistry and does not itself form part of the invention. For example, a strong buffer (0.1M acetate, 0.5M NaCl, PH
4-5), and then washed in a neutral buffer (PH7).
~7.4), then washed at pH 9-10, and then neutralized with, for example, a neutral Tris buffer to remove excess reagent.

Another and sometimes preferred method for attaching proteins to glass is DPHerman et al., J. Chromatogr. Sci, (1981), 19 (9). 470
-6, a method using an epoxy-silane reagent, in particular glycidyloxypropyltrimethoxysilane (e.g. 2% v/v in toluene, 2 and a half hours at 70°C). Since epoxysilylated glasses can react directly with proteins, the use of aldehyde reagents can be omitted for this reagent.

It is usually desirable to apply a stabilizing coating, such as a coating of a solid wetting agent such as sucrose, to such layer-coated surfaces. Suitable examples of such coatings include:
For example, spin-coat the plate with a sucrose solution.
8 micron thick solid sucrose coating applied by coating) and air drying.

Releasable coatings can be applied using compositions known per se, eg mixed with solid wetting agents such as sucrose glazes. Particularly if the active substance to be released is itself a protein, it is advisable to incorporate detergents or inert proteins (or soluble salts or buffer substances) into such coatings so that they are compatible with the test reactions involved. In this context, the inventors have found it particularly desirable to avoid large excesses of reagent in such releasable coatings.

The uniformity of the coating process is important and can be checked, if appropriate, in a test procedure in which the radiolabeled and/or fluorescent protein is coated and then reacted with an optionally further and preferably separately labeled binding agent. The uniformity of the coating and its binding capacity can then be checked using surface fluorescence measurements and/or surface radioactivity measurements, for example using a research gamma scanner.

If it is desired to subsequently etch the coating on the glass, place the coated glass in a confined substantially moisture-free atmosphere or in an air draft, e.g. to reduce the air gap on the coated side to less than about 1 mm. Another flat inert surface can be approached. The glass is then illuminated with an image patterned with ultraviolet light in a pattern corresponding to the areas to be etched away, such as a grid pattern (with light in a narrow wavelength band that can be implemented around C280 nm). ), leaving a pattern of residual protein patches. Irradiation is carried out for about 5 to 50 minutes using, for example, a GE7-Watt mercury lamp a few centimeters from the plate. The illumination pattern can be produced by masking close to the plate or by a solid imaging system. The ultraviolet etching used here is based on the same principle as the UV etching process described by JAPanitz and I. Giaver in Surface Science, 97 (1980) pp. 25-42. and refers to this document.

The desired pattern is then printed onto the coated, eg patch coated, glass plate with a UV curable epoxy adhesive for bonding with the plates spaced on top. The epoxy adhesive is applied by silk-screening techniques which are themselves conventional but which do not form part of this invention.

Epoxy resin (made, for example, by grinding long glass fibers in a motor and sieving to remove remaining long fibers) is approximately 20 microns in diameter and approximately 20 microns long.
Contains a small amount of short glass fibers of 100-200 microns. Instead of galan fiber pieces, preference is given to Balloteini in epoxy resin, which is used as described below. For example, to produce a 100 micron gap, a correspondingly sized balloteini is incorporated into the epoxy. slightly thicker than the desired space between the plates e.g. 10% thicker, e.g.
A layer of approximately 110 microns of epoxy can be screen printed for the desired spacing of 100 microns, and another plate is gently pressed into place to lightly spread the epoxy.

If desired, apply a mirror image of the first pattern of epoxy adhesive as a pattern to a second similar glass sheet patchwise coated or otherwise uncoated with the same or a different protein or other coating material. The two sheets can then be brought together, subjected to vacuum or deoxidized conditions if necessary for curing, and cured by ultraviolet radiation. As an image, ultraviolet light is applied in a pattern that prevents patches of coated protein or other material that should remain in active form.

After curing the adhesive, any convenient known method used in the manufacturing stage of liquid crystal devices, especially CH- for manufacturing liquid crystal display devices.
Two glass plates can be scribed with lines and divided into cell units in the manner described in US Pat. Nos. 627559 and 629002. These specifications and the corresponding steps of the method of the invention may be carried out in a similar manner mutatis mutandis.

A convenient form of cell obtained by this process consists of two substantially parallel opposing glass layers having a gap of approximately 5 to 500 microns and an incomplete bonding material interposed between the layers. together with a frame (having at least one opening for the inward passage of liquid and possibly the outward passage of air);
Forms a capillary cell that can draw up a defined amount of aqueous solution. Preferably, one glass layer extends beyond the opening of the cell so that all or part of a droplet placed on its surface can enter the cell.

FIG. 4 shows in a schematic perspective view another embodiment of a specific reactive capillary cell device according to an embodiment of the present invention. The illustrated device is a disposable, single-use test device for microchemical testing of very small liquid samples. Fifth
The figure shows a corresponding plan view, and FIG. 6 shows a schematic cross-sectional view along line 6'--6' of FIG. 5 (not to scale). The device of FIGS. 4-6 consists of an upper glass plate 1 and a lower glass plate 2, which relate to plates 1 and 2 of FIG. A reaction layer such as 7 in FIG. 1 is not shown in FIGS. 4-6, but is present on the surface of the plate 2.
If required for specific test purposes, auxiliary reagents may be placed as releasable coatings on the opposite walls of the cell, ie on the surface of the plate 1, so that they are dissolved in the sample liquid drawn into the cell. The user operates the device of FIGS. 4-6 with a support and operating frame handlepiece 42, generally designated 41, made of any conventional plastic melting material. The arm 43 is
It serves to support the cell assembly and position it in conjunction with the operating portion of the optical instrument for optically analyzing the contents of the sample cell. The end 44 of the plate 2 is optically transparent, flat and perpendicular to the plane of the plate 2, from which light emanating from the contents of the capillary cells between the plates 1 and 2 is emitted. In the embodiment, the other edges of plates 1 and 2 are painted black for practical purposes, and the epoxy bond track 3, corresponding to track 3 in FIG. ) and also contains carbon black granules to minimize stray light. A rectangular piece of paper 45 is attached to a portion 46 of plate 2 extending beyond the length of plate 1 to form a sample receiving inlet.
position it above. The adjacent open end 47 of the capillary gap between plates 1 and 2 is the entrance to the capillary cell bounded by plates 1 and 2 and the connecting track 3, and a filter 45 of a sufficiently fine grade to prevent the passage of red blood cells is provided. open entrance 47
in contact with plate 2, preferably at said end 47.
overlaps slightly. The filter 45 is optionally held by adhesive along the parallel continuous track 3, if desired by e.g.
It can also be impregnated with releasable/auxiliary reagents such as buffer salts. The portion 47 of plate 2 extending beyond plate 1 to optical end 44 of plate 2 may be coated with a hydrophobic coating if desired.

In use, a liquid sample to be examined, for example a drop of whole blood, is applied to the inlet zone formed by the filter 45 and from such a drop,
A volume of relatively cell-free fluid is drawn into the capillary cell. Here, suitable to the nature of the required test and the nature of the corresponding reagent prepared in a dry state and pre-loaded on one or both of the inner walls of the opposing cells formed by plates 1 and 2, A binding or other reaction, e.g. an enzymatic reaction, takes place and the loaded cell is then used for optical measurements, e.g. as described in co-pending UK Patent Application No. 8415019. It can be incorporated into the container.

7 and 8 are variations of the device of FIGS. 4-6. The device has two snap-on operating and support pieces, one
One is designated 41 and corresponds to 41 in Figures 4-6 except that it is removable, and the other handling and support piece is a side rib 73 at the optical end of the device.
Fit of the upper removable snap-on
The optical end of the device consists of similar optical end surfaces 44 and 74 of spaced apart plates 1 and 2. Handle 42 is a stiffer snap-on fit than handle 72.

The device of FIGS. 7 and 8 has a fixed handle 7
2 and a separate handle 42. In this condition, the cellulose nitrate or acetate fine filter or dialysis membrane 75 is disposed between the plates 1 and 2 and the coupling track 3, as described above, and further towards the ends 44 and 74 of the plates 1 and 2. Exposed at the inlet end of the capillary cell is also defined by another lateral blackened epoxy bond track 3a which limits the forward diffusion, so that in the forward part of plates 1 and 2 there is a An air-bounded waveguide section may be constructed. Two side gaps remain between the tracks 3 and 3a, and two corresponding openings 76 in the frame 73 allow air to escape when the capillary cell is filled with sample liquid. Thus, in use, the device is held by the handle 72 and dipped into a sample liquid source to fill the capillary cell with sample through the filter 75. In this embodiment, plate 1
and 2 each carry an immobilized reaction layer 7 and 77, such as a suitable antibody layer prepared as described above.
If necessary, the area of reaction layers 7 and 77, indicated by diagonal lines in FIG. passes over the layer and therefore the concentration at its surface can be concentrated in the shaded area.

After loading the sample, the separate handle 42 is snapped into place and the weaker attached handle 72 is removed, thereby freeing the optical ends 44 and 74 of plates 1 and 2 for measurements as described above. exposed, in this case the separate optical waveguides 1 and 2 of the device and each reaction layer 7.
and 77, two different optical properties can be measured at once. Therefore, the corresponding optical measuring device is indicated by the dotted line 7
The partition plate is located at the position indicated by 9, and separates each light beam emitted from plates 1 and 2.

The invention described herein is susceptible to many modifications and variations within its scope, in particular those that utilize one or more of the foregoing description, the accompanying drawings and equivalents thereof and any one or more of the numerous features. It also extends to

A number of devices manufactured in accordance with the above description are disclosed in UK Patent Application No. 8415019 (3 June 1984).
The disclosures of the aforementioned patent applications are also incorporated into this application by reference. There is.