Method and kit having layered device for detecting biological component by interference color

What is claimed is:

1. A method for detecting a component of a biological system, which comprises providing a biological component detecting device composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (II), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III); contacting said device with a solution containing the biological component to be detected; then forming a light-transmitting reflecting layer (IV) on its surface, wherein said layer (IV) is a layer of a metal; and thereafter detecting the color of light interference or the brightness of the reflected light on the surface of the device.

2. A method for detecting a component of a biological system, which comprises providing a biological component detecting device composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (II), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III); contacting said device with a solution containing the biological component to be detected; further reacting another substance capable of reacting with another reaction site in the biological component to be detected after or while reacting the detecting substance with the biological component to subject thc surface layer of the device to contrast enhancement treatment; then forming a light-transmitting reflecting layer (IV) on its surface, wherein said layer (IV) is a layer of a metal; and thereafter detecting the color of light interference or the brightness of the reflected light on the surface of the device.

3. A method for detecting a component of a biological system, which comprises providing a biological component detecting device composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a hydrophobizing agent layer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III); contacting said device with a solution containing the biological component to be detected; then forming a light-transmitting reflecting layer (IV) on its surface, wherein said layer (IV) is a layer of a metal; and thereafter detecting the color of light interference or the brightness of the reflected light on the surface of the device.

4. A method for detecting a component of a biological system, which comprises providing a biological component detecting device composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a hydrophobizing agent layer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III); contacting said device with a solution containing the biological component to be detected; further reacting another substance capable of reacting with another reaction site in the biological component to be detected after or while reacting the detecting substance with the biological component to subject the surface layer of the device to contrast enhancement treatment; then forming a light-transmitting reflecting layer (IV) on its surface, wherein said layer (IV) is a layer of a metal; and thereafter detecting the color of light interference or the brightness of the reflected light on the surface of the device.

5. A method for detecting a component of a biological system, which comprises providing a biological component detecting device composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a reactive interlayer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III); contacting said device with a solution containing the biological component to be detected; then forming a light-transmitting reflecting layer (IV) on its surface, wherein said layer (IV) is a layer of a metal; and thereafter detecting the color of light interference or the brightness of the reflected light on the surface of the device.

6. A method for detecting a component of a biological system, which comprises providing a biological component detecting device composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a reactive interlayer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III); contacting said device with a solution containing the biological component to be detected; further reacting another substance capable of reacting with another reaction site in the biological component to be detected after or while reacting the detecting substance with the biological component to subject the surface layer of the device to contrast enhancement treatment; then forming a light-transmitting reflecting layer (IV) on its surface, wherein said layer (IV) is a layer of a metal; and thereafter detecting the color of light interference or the brightness of the reflected light on the surface of the device.

7. The method of claim 2, 4 or 6 wherein the other substance used for the contrast enhancement treatment is a secondary antibody, an enzyme-labelled secondary antibody, a secondary antibody fixed to an emulsion, or a secondary antibody fixed to a latex.

8. The method of any one of claims 1 to 6 wherein the substance in the biological component detecting substance layer (III) is an antibody, and the biological component to be detected is an antigen.

9. The method of any one of claims 1 to 6 wherein the substance in the biological component detecting substance layer (III) is an antigen, and the biological component to be detected is an antibody.

10. The method of any one of claims 1 to 6 wherein the substance in the biological component detecting substance layer (III) and the biological component to be detected are the same antigen, and an antibody added to the solution containing an antigen to be detected reacts competitively with both antigens.

11. The method of any one of claims 1 to 6 wherein the light-transmitting reflecting layer (IV) is a layer of a metal formed by a colloidal particle coating method.

12. The method of claim 11 wherein the metal is gold.

13. The method of claim 11 wherein the light-transmitting reflecting layer (IV) is a layer of metal colloid particles coated at a pH which is up to 1.0 higher than the isoelectric point of the biological component to be detected and lower than, or more than 1.0 higher than, the isoelectric point of the biological component detecting substance.

14. The method of claim 13 wherein the metal is gold.

15. The method of any one of claims 1 to 6 wherein the light reflectivity of the substrate is high such that the reflection on the substrate is balanced on the reflection of the light-transmitting reflecting layer.

16. The method of any one of claims 1 to 6 wherein the light-transmitting reflecting layer (IV) is a layer of a metal formed by a physical vapor deposition method.

17. The method of claim 16 wherein the metal is gold.

18. The method of any one of claims 1 to 6 wherein the interference color is detected by a color difference value calculated in accordance with the equation ##EQU3## X.sub.o, Y.sub.o, and Z.sub.o are tristimulus values of an illuminating light source, and X, Y and Z are tristimulus values of the spectrum of the calorimetric standard observer stipulated by International Committee of Illumination (1931).

19. A kit for detection of a component of a biological system, comprising (I) a pack of a device for detecting the biological component composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (II), wherein the light interference layer (a) does not have substantially reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III), and (2) a pack of a metal colloid, and optionally (3) a pack of a contrast enhancement agent.

20. A kit for detection of a component of a biological system, comprising (1) a pack of a device for detecting the biological component of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a hydrophobizing agent layer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III), and (2) a pack of a metal colloid, and optionally (3) a pack of a contrast enhancement agent.

21. A kit for detection of a component of a biological system, comprising (1) a pack of a device for detecting the biological component composed of a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a reactive interlayer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III), and (2) a pack of a metal colloid, and optionally (3) a pack of a contrast enhancement agent.

22. A device for detecting a component of a biological system, comprising a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (II), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III).

23. A device for detecting a component of a biological system, comprising a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a hydrophobizing agent layer (P) formed on the layer (II), and a layer (III) of a substance for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800 nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III).

24. A device for detecting a component of a biological system, comprising a light reflecting substrate (I), made of a metal or metal alloy itself or having a surface of a metal or metal alloy, and being substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), a reactive interlayer (P) formed on the layer (II), and a layer (III) of a substrate for detecting said biological component provided at least in a region on the layer (P), wherein the light interference layer (a) does not have substantial reflecting characteristics to visible light, namely a wavelength of 300 to 800nm, (b) has a thickness and refractive index controlled such that an increase in the thickness of the layer (III) appears as a change in interference color, and (c) has a surface with sufficient affinity for the layer (III).

25. The device of claim 22, 23 or 24 wherein the substance in the biological detecting substance layer (III) is an antibody.

26. The device of claim 22, 23 or 24 wherein the substance in the biological component detecting substance layer (III) is an antigen.

27. The device of claim 22, 23 or 24 wherein the light interference layer (II) is a layer of an inorganic substance, and the biological component detecting substance layer (III) is a layer of an antibody protein which is formed by forming a complex of the antibody protein with a substantially water-insoluble mono- or bi-molecular film, and laminating it to the light interference layer whose surface is optionally subjected to a hydrophobizing treatment.

28. The device of claim 22, 23 or 24 wherein the light interference layer (II) is a layer of an inorganic substance, and the biological component detecting substance layer (III) is a layer of an antibody protein which is formed by spreading a monomolecular film of a longchain fatty acid having 24 to 32 carbon atoms, a polyvalent metal salt thereof and/or an ester thereof, or a monomolecular film of a polyvalent metal salt of a longchain fatty acid having 14 to 23 carbon atoms and/or an ester of said fatty acid on a water phase, contacting the film with a water-soluble antibody protein dissolved in the water phase to form an antibody protein-monomolecular film complex on the interface of the aqueous phase, and laminating it onto the light interference layer (II).

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

This invention relates to a very simple and convenient immunological detecting method for detecting a component of a biological system, above all for immunological diagnosis, and to a device for immunological detection. More specifically, this invention relates to a method and a device for detecting an antigen or an antibody protein on the basis of an antigen-antibody reaction on a reflecting substrate.

Immunological diagnosis has been performed by utilizing an antigen-antibody reaction which is a very specific biochemical reaction. Radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA) and latex agglutination settling analysis (LSA), for example, are known and used in practice as specific methods of immunological diagnosis. These methods still have technical problems to be solved. RIA has very high detection sensitivity, but requires special facilities for handling radioactive elements. EIA requires a long period of time (usually several hours to one day) for the completion of detection. FIA does not have sufficient detection sensitivity. LSA cannot avoid a non-specific agglutination reaction, and has low reliability in detecting trace components.

On the other hand, an ellipsometric method was proposed in which an increase in the thickness of a protein layer which occurs with the progress of an antigen-antibody reaction on a solid substrate is detected by using ellptic polarized light (British Pat. No. 1,479,661). This method also requires an expensive device and much expertise is required for measuring the protein film thickness. There has also been proposed a method of detecting an antigen-antibody reaction simply with the unaided eye without using such an expensive device. For example, there is a method which comprises adsorbing and fixing an antibody (or an antigen) on and to the surface of gold particles deposited on a solid substrate, and visually observing changes in the color of the reflected light which occur as a result of an increase in the thickness of a layer of an immobilized antibody (or antigen) by an antigen-antibody reaction (U.S. Pat. No. 3,979,184). According to this method, the color of the complex of gold and the protein film on the solid substrate certainly changes with the antigen/antibody reaction. Since, however, the change is only slight from brown to dark brown and very obscure, the evaluation of the antigen-antibody reaction may possibly depend greatly upon the expertise of the testing personnel.

When an antigen or antibody is fixed to a dielectric layer formed on a highly light reflecting substrate such as a metallic chromium or tantalum substrate and an antigen-antibody reaction is carried out on its surface as shown, for example, by Langmuir and Blodgett, Physical Review, vol. 51, pages 964-978 (1937) or Vroman, Thromb. Diath. Haemorrhag., vol. 10, 455-493 (1964), the difference in refractive index between antigen or antibody and the air is very small and the reflectance on the surface of the antigen or antibody is as low as 5% at an incidence angle of 0.degree. to 60.degree.. On the other hand, the proportion of light reflected from the metallic substrate and coming back to the surface of the protein is higher than 50%. Accordingly, it is difficult to detect the interference color on the surface of the device. To discriminate this interference color with good efficiency, it is necessary to adjust the angle of reflection of light on the surface of the device to at least 60.degree. to 70.degree., and it is difficult to detect with the unaided eye. Another proposal for solving this problem (U.S. Pat. No. 4,558,012) states that light interference occurs efficiently by providing two types of dielectric layers on a non-metallic substrate which does not so much reflect light, and making the amount of light reflected from the surface of the substrate nearly equal to that of light reflected from the surface of the dielectric layers. However, substrates meeting such conditions are limited to those which are colored or have high light transmitting property, and those having a high reflectance cannot be used. The colored substrates affect the interference color on the surface of the device and make the detection difficult. With the substrates having a high light transmittance, the color of the device becomes dark and light interference which gives a brilliant visible light color does not easily occur. If a substrate having a relatively high surface reflectance of 60 to 90% is used, it is essential to provide a plurality of dielectric layers having different refractive indices, and the process of building the device becomes complex.

SUMMARY OF THE INVENTION

The present inventors have made extensive investigations in order to develop a device for detecting a component of a biological system, which is free from the aforesaid problems of the prior art and permits easy and highly sensitive detection of the biological component with the unaided eyed. These investigations have now led to the discovery that if a thin film of fine metallic particles is provided on the surface of the device after reacting a detecting substance with a substance to be detected, optionally after the surface is subjected to a contrast enhancement treatment, the amount of light reflected from the surface of the substrate is balanced with the amount of the light reflected from the surface of the device within ranges where these amounts are large.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view explaining light interference of a biological assay sample.

FIG. 2 is a side elevation showing an example of building a device with multiantibodies for detecting plural biological components, in the stages shown in FIGS. 2A, 2B and 2C.

FIG. 3 is a sectional view showing how a substance for detecting a biological component can be fixed to a light interference layer.

FIG. 4 is a sectional view showing an embodiment for increasing the detection sensitivity of a device.

DETAILED DESCRIPTION OF THE INVENTION

According to this invention, there is provided a method for detecting a component of a biological system, which comprises (i) contacting a device for the detection of a biological component composed of a light reflecting substrate (I) substantially free from diffused reflection, a light interference layer (II) formed on the substrate (I), and a layer (lIl) of a substance for detecting said biological component provided at least in a region on the layer (II), with a solution presumed to contain the biological component to be detected, (ii) then forming a light-transmitting reflecting layer (IV) on it, optionally after subjecting it to a contrast enhancement treatment, (iii) irradiating light onto it, preferably at an incidence angle of 0 to 50 degrees, and (iv) detecting the color of light interference or the brightness of the reflected light on the surface of the device.

In the accompanying drawings:

FIG. 1 is a view for explaining the concept of light interference of a biological assay sample, and FIG. 2 is a side elevation showing an example of building a device wih multiantibodies for detecting plural biological components. In the drawings, I represents a light reflecting substrate; II, a light interference layer; III, a layer of a substance for detecting a biological component; III', a complex layer formed by reaction of the layer III with the biological component to be detected; IV, a light transmitting reflecting layer; n.sub.1, the refractive index of the light-transmitting reflecting layer; n.sub.2, the refractive index of the biological component to be detected; n.sub.3, the refractive index of the substance for detecting the biological component; n.sub.4, the refractive index of the light interference layer; .theta..sub.1 and .theta..sub.1 ', angles of incidence; and .theta..sub.2 and .theta.'.sub.2, reflection angles.

The light reflecting substrate (I) used in this invention may be made of an ordinary metal such as iron, nickel, cobalt, zinc, titanium and bismuth, an alloy thereof, or a metal having a high reflectance such as gold, silver, copper and aluminum. The substrate (I) may be made of such a material itself in a plate form, or may be made by forming a thin layer of such metals or alloys, either singly or to improve adhesion to the substrate, in combination, by vapor deposition or sputtering on a solid substrate such as a glass plate or a plastic plate. The reflectance of this layer is at least 50%, preferably at least 70%, when white light is allowed to fall upon it at an incidence angle of 0 to 50 degrees, preferably 0 to 30 degrees.

The light interference layer (II) used in this invention should meet the following requirements (1) to (3). (1) It should not have substantial reflecting characteristics to visible light (wavelength 300 to 800 nm). (2) The thickness and refractive index of the light interference layer (II) should be controlled such that an increase in the thickness of the layer (III) of a substance for detecting a biological component with the biological component detecting reaction appears as a change in interference color. (3) Its surface preferably has sufficient affinity for the layer (III).

The light interference layer (II) may be made of an organic or inorganic material. The organic material may be any which does not substantially have reflecting or absorbing characteristics in a visible light region (300 to 800 nm) and are film-forming. Preferably, it may be an organic material which permits control of its film thickness to the order of 50 to 100 .ANG. so as to induce efficient changes in the color of light interference with an increase in the thickness of a protein film by a biological component detecting reaction such as an antigen-antibody reaction to be described in detail hereinafter. Such organic materials may, for example, be compounds capable of forming a stable condensed monomolecular film on a water surface, such as long-chain carboxylic acids and metal salts and esters thereof, and materials capable of forming films having a thickness of not more than 2,000 .ANG. by coating or vapor deposition. Specific examples of the former include long-chain saturated and unsaturated carboxylic acids such as palmitic acid, stearic acid, lignoceric acid, oleic acid and omega-tricosanoic acid, esters thereof, and salts thereof with mono- to tri-valent metals. Examples of the latter include vinyl polymers such as poly(methyl (meth)acrylate), polystyrene, poly(meth)acrylonitrile and polyvinyl chloride; polyolefins such as polyethylene, polypropylene and poly-4-methyl-pentene-1; and condensation polymers such as polyamides and polyesters. Since these substances induce effective light interference according to their refractive indices, their film thickness is controlled. The inorganic material which may constitute the light interference layer should likewise have no reflection and no absorption in the visible light region, and the thickness of a film thereof should be controlled to the order of 50 to 100 .ANG.. Examples of the inorganic material having such properties include metal oxides such as silicon oxide, aluminum oxide, tin oxide, lead oxide, tungsten oxide, magnesium oxide, cobalt oxide, molybdenum oxide, titanium oxide, zirconium oxide, zinc oxide and tantalum oxide; metal fluorides such as calcium fluoride, magnesium fluoride and lithium fluoride; intermetallic compounds such as galliumarsenic; and silicon nitride. Such a material may be formed into a film of the desired thickness according to its refractive index by vapor deposition or sputtering and provided as the light interference layer (II) on the reflecting substrate (I).

The surface of the light interference layer (II) should also be required to have affinity for substances (such as antigens or antibodies) for detecting biological components in the layer (III). For this purpose, the surface of the light interference layer (II) may be treated by a hydrophobizing agent such as an alkyl- or aryl-silane, or chemically modified by a reactive compound which can chemically fix the substance for the detection of biological component to interference layer (II). The hydrophobizing agent layer and the reactive compound layer are shown as layer (P) in FIG. 3 (a sectional view similar to FIG. 2). The hydrophobizing agent layer is essential when the light interference layer is composed of an inorganic material.

Examples of the alkyl- or aryl-silane as the hydrophobizing agent are C.sub.12 -C.sub.20 alkyltrichlorosilanes such as octadecyltrichlorosilane, mono-, di- or tri-alkoxysilanes, dimethyldichlorosilane, dimethylphenylchlorosilane and methyldiphenylchlorosilane.

The treatment of forming the reactive interlayer is carried out, as required, in order to increase the affinity of the surface of the light interference layer (II) for the biological component detecting substance in the layer (III).

In most known devices for detecting an immunological reaction utilizing light interference, antigen molecules are fixed, and there is no example in which an antibody is fixed in such devices. Since generally there are many recognition sites in antigen molecules, the alignment of the antigen molecules is not of much significance in fixing them. On the other hand, since the recognition sites of antibody molecules are strictly limited, they have to be arranged so that the recognition sites are effectively exposed to the surface of the detecting device. By an ordinary physical adsorption method or a chemical fixing method, it is extremely difficult to fix antibody molecules without impairing their activity.

In view of the above background, the present inventors have extensively worked on a method and a device for detecting an antigen-antibody reaction with good sensitivity by a simple procedure within a short period of time, and consequently found that an immunological detecting device of high sensitivity and free from delamination can be obtained by using a device consisting of a light reflecting substrate and a light interference layer having an optimized thickness and an optimized refractive index and chemically bonding an antibody layer to the surface of the light-interference layer selectively at sites other than the recognition sites of the antibody.

As one embodiment, the present invention provides a simple immunological detecting device comprising a light reflecting substrate (I) substantially free from diffused reflection, a light interference layer (II) laminated to the surface of the substrate (I), a reactive interlayer (P) formed on the layer (II) and composed of a compound capable of selectively reacting the carboxyl group or thiol group contained in antigen or antibody molecules or fragmented antibody molecules mainly by pH adjustment, and a layer (III) of an antigen substance and/or an antibody protein composed substantially of a monomolecular layer formed on the interlayer (P); and a method of immunological detection utilizing the device.

The compound capable of reacting with the carboxyl group of the protein is preferably one which contains functional groups such as ##STR1## or --NH.sub.2 in the molecule and can be fixed at a high density to the light interference layer. It may be a low-molecular-weight or high-molecular-weight compound. Specific examples of the low-molecular-weight compounds are ##STR2## Specific examples of the high-molecular-weight compound are ##STR3## Compounds capable of reacting with the thiol group (SH) of the protein are, for example, ##STR4## (when this compound is used, the substrate is preferably pre-treated with, for example, ##STR5## N-substituted maleimide is most preferably used. Many of the above compounds can react not only with the carboxyl or thiol groups in the protein but also with the amino groups in it. Hence, the protein fixing reaction should be carried out by adjusting the pH of the reaction system. The preferred pH range is 3 to 5 for the reaction with the carboxyl groups, and 4 to 6 for the reaction with the thiol groups.

Such a compound is formed as a thin film layer (P) on the light interference layer (II). The thickness of this film should be controlled such that the interference color of the entire layer above the light reflecting substrate should be within the visible light region. Such a film thickness is selected from 25 to 5000 .ANG., preferably 30 to 3,000 .ANG..

The thickness of the light interference layer (II) should be selected such that when the incidence angles .theta..sub.1 and .theta.'.sub.1 are 0 to 50 degrees, the light path differences of the incident light at the device as shown in FIG. 1, ##EQU1## become the product of the wavelengths of incident lights multiplied by integers. Furthermore, for the discrimination of an antigen-antibody reaction site, it is convenient that the light path difference -1 differs from the light path difference -2. For example, the optical thickness of the light interference layer (II) should be controlled to about 500 to 5,000 .ANG., preferably 700 to 3,000 .ANG., when this layer has a refractive index of 1.4 to 2.0. Examples of such an accurate film thickness controlling method are the Langmuir-Blodgett method (a monomolecular film on a water surface is accumulated on a solid substrate), the spin coat method, the vapor deposition method and the sputtering method.

The biological component detecting substance in the layer (III) to be fixed to the light interference layer (II) may preferably be antibodies, antigens, etc. which are involved in immunological reactions, nucleic acids, viruses, bacteria, etc. Of these, the antigens and antibodies are preferred.

Examples of the antigens are immunoglobulins such as IgG, IgA, IgE and IgM, human chorionic gonadotropin (HCG), and carcinoembryonic antigen (CEA). As the antibodies, polyclonal or monoclonal antibodies to these antigens are used.

These antigens or antibodies may be fixed to the surface of the light interference layer (II) [the term "light interference layer (II)", to be used hereinbelow, means one optionally having the aforesaid hydrophobizing agent layer or reactive interlayer (P) on its surface]by immersing the device in an aqueous solution of an antigen or antibody for 0.5 to 20 hours, and then fully washing it with water to remove the antigen (or antibody) molecules physically adhered to it. As a result of this adsorption treatment, the antigen (or antibody) is fixed onto the light interference layer (II) as a monomolecular layer (III).

One or more kinds of antibodies and/or antigens may be adsorbed on the light interference layer (II). To fix two or more kinds of antibodies (or antigens), the depth of chips (I+II) composed of the reflecting substrate (I) and the light interference layer (II) formed thereon, to which they are immersed in solutions (X, Y and Z) of the antibodies (or antigens), is progressively increased. By so doing, it is possible to fix a plurality of antibodies (or antigens) onto the same chip as a monomolecular layer since generally, another antibody (or antigen) is not adsorbed on that part to which one antibody (or antigen) has adhered. This procedure enables expensive monoclonal antibodies, for example, to be effectively fixed.

According to another preferred embodiment of this invention, the antibody protein layer (III) can be fixed to the light interference layer (II) in a form oriented so that it does not lose activity, by spreading (1) a monomolecular film of a long-chain fatty acid having 24 to 32 carbon atoms, a salt thereof with a polyvalent metal and/or an ester thereof or (2) a monomolecular film of a polyvalent metal salt of a long-chain fatty acid having 14 to 23 carbon atoms and/or an ester of the long-chain fatty acid on an aqueous phase surface, and contacting a water-soluble antibody protein dissolved in the aqueous phase to form an antibody monomolecular mixed film on the interface of the aqueous phase, and laminating the complex on the light interference layer (II).

The antibody protein generically denotes a water-soluble protein which can induce an antigen-antibody reaction, and contains an antigen recognition site (Fab for short) and a hydrophobic terminal site (Fc for short).

Specific examples of the antibody protein are immunoglobulins G (abbreviated IgG), IgE, IgM and antibodies to them, human chorionic gonadotropin (HCG) antibody and carcinoembryonic antigen (CEA) antibody.

In fixing these antibody proteins, care should be taken not to denature the Fab portion. In conventional fixing procedures by a chemical reaction, the Fab portion is also involved in the reaction to cause a decrease in the activity of the antibody protein. According to the above method in accordance with this invention, the antibody protein is incorporated at a high density into the monomolecular film while hydrophobically interacting at the Fc site or adsorbed on and fixed to the monomolecular film while maintaining high immunological activity.

The monomolecular film preferably remains a condensed monomolecular film on a solid on a water surface and does not substantially dissolve in water. Examples of the long-chain fatty acid having 24 to 32 carbon atoms, its polyvalent metal salt and/or its ester include lignocer