• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Nanostructured material with interferometric properties for visual detection of biorecognition assays without labeling. The present invention relates to a nanomaterial that allows chemical and biochemical analysis to be carried out and its result determined by direct visual detection, without using markers (label-free) and without requiring any type of instrumentation. The material is formed by a layer of bioreceptors that selectively interact with the analytes, and which is arranged on an interferometric layer deposited on a substrate. Incubation of samples on the material generates changes in its interferometric response, which result in color changes directly observable visually under lighting with natural or artificial light, and whose magnitude depends on the concentration of the analyte present in the sample. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2745337A1
    申请号:ES202030023
    申请日:2020-01-16
    公开日:2020-02-28
    发明作者:Oliver José Miguel Avellà；Fornes Gabriel Sancho；Rubio Javier Carrascosa；Catala Angel Maquieira
    申请人:Universidad Politecnica de Valencia；
    IPC主号:
    专利说明:

    [0001]
    [0002] Nanostructured material with interferometric properties for visual detection of biorecognition tests without dizziness
    [0003]
    [0004] The present invention relates to a biosensor that allows chemical and biochemical analysis to be carried out and its result determined by direct visual detection, without using markers ( label-free) and without requiring any type of instrumentation. The material is made up of a layer of bioreceptors that selectively interact with the analytes, and which is arranged on an interferometric layer deposited on a substrate. The incubation of samples on the material, generates changes in its interferometric response, which result in color changes directly observable visually under lighting with natural or artificial light, and whose magnitude depends on the concentration of the analyte present in the sample.
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007]
    [0008] Biosensors are chemical and biological analysis devices based on generating a response (signal) due to a biointeraction. They are characterized by their sensitivity and selectivity, and are generally designed to be simple, compact, portable, fast and economical. One of the key objectives of biosensors is to allow obtaining analytical results at the same time and place where they are required, by non-specialized users, what is known as point of care. This gives them great potential in many fields such as clinical diagnosis, food safety and agri-food control, among others. Some paradigmatic examples of biosensors with great commercial impact are the glucometer and the human fertility test.
    [0009]
    [0010] The capacity of the point of care biosensors is strongly compromised by the complexity and price of the instrumentation involved in these developments (detectors, radiation sources, batteries, electronic circuits, etc.). Another important issue in this regard is to minimize the number of operations (dilutions, sample preparation, incubations, etc.) necessary to perform the analysis, in order to simplify the analytical process as much as possible, as well as to minimize the required instrumentation. to automate the trials. In this line, the systems for detection without labeling ( label-free ) they allow the direct analysis of samples, thus avoiding additional stages related to incubations of marked secondary bioreactives and signal development. This brings important advantages because it greatly simplifies the analytical process. However, label-free systems are usually based on materials with non-scalable nanostructures at the industrial level, as well as on complex instrumentation for measuring bio-interactions.
    [0011]
    [0012] Articles have been found that report the use of this type of materials together with specific instrumentation to quantify the interferometric changes generated by biorecognition events ( ACS Nano, 2008, 2, 1885-1895; Anal. Chem. 2009, 81, 4963- 4970), highlighting that in both the developments described do not contemplate direct visual detection, but through specific instrumentation.
    [0013]
    [0014] On the other hand, EP0727038B1 describes methods for analyzing an optical surface for an analyte of interest in a sample and related instruments. The method involves the use of a thin film optical immunoassay device whereby an analyte of interest is detected in a sample through spectral changes in the light that affect the surface before and after the binding of the analyte to the layers of reactive substrate. The device includes a substrate that has a first color in response to the light that falls on it.
    [0015]
    [0016] Also, WO9403774A1 belonging to the same family claims a tool to detect the presence of a certain analyte, as well as to measure its concentration. The result can be determined by a color change detectable to the naked eye or by devices intended for that purpose. This utensil is made up of a multilayer structure. The outermost layer contains molecules capable of binding to the analyte. The inner layers have such optical characteristics that they will show a color change in the presence of bound analyte.
    [0017]
    [0018] Finally, Maier Irene et al. ( Anal. Chem., 2008, 80 (8), 2694-2703) describe biosensing chips, which are used for the detection of a certain analyte. The chips comprise a support containing gold nanoparticles. On this layer there is another one that contains biomolecules capable of recognizing and binding to the analyte. This union translates into a color change in the chip, which can be seen with the naked eye. However, for the detection they use an interferometric configuration to amplify the signal based on surface plasmon resonance, and also use a marking (colloidal gold nanoparticles conjugated to antibodies), which differentiates this biosensor from the present invention where the biorecognition test generates color changes by itself, without using markers , thanks to the design of interferometric materials.
    [0019]
    [0020] DESCRIPTION OF THE INVENTION
    [0021]
    [0022] Taking into account these antecedents, the present invention consists of a material that allows chemical and biological tests to be carried out and to detect the result through observable color changes directly with the eye, without the need for marking, without requiring any type of instrumentation for the process. of analysis, and the material being manufactured on an industrial scale. These capabilities result in a simple and portable biosensor, very suitable for point of care applications in which end users perform chemical and biochemical analyzes in a single step (incubate the sample on the material) and determine the result visually by color change . Thus, the present invention comprises a substrate on which an interferometric layer is arranged, on which a recognition layer composed of bioreceptors is immobilized for the recognition of analytes.
    [0023]
    [0024] Therefore, in a first aspect the present invention relates to a biosensor that allows direct visual detection of analytes in a sample, comprising:
    [0025] i) A substrate (a) that acts as a support for the interferometric layer (b);
    [0026] ii) An interferometric layer (b) consisting of one or several superimposed films, with an arrangement designed to generate constructive and / or destructive interferometric phenomena in the visible range and to modify this interferometric response depending on the properties (thickness, index of refraction, density, etc.) of the recognition layer;
    [0027] iii) A recognition layer (c) composed of biological receptors that are attached to the last film of the interferometric layer (b), and that specifically recognize the analytes present in the sample.
    [0028]
    [0029] In a preferred embodiment the substrate (a) is a solid, typically flat material, which acts as a support for the interferometric layer (b).
    [0030] In another more preferred embodiment, the substrate (a) is selected from glass sheets, metals, synthetic polymers, silicon, membranes and gels. Also, in some embodiments the substrate may be omitted.
    [0031]
    [0032] In another preferred embodiment the metal of the substrate (a) is selected from aluminum, steel, gold, silver, platinum, chromium and iron. In a more preferred embodiment the metal is steel.
    [0033]
    [0034] In another preferred embodiment the synthetic polymer of the substrate (a) is selected from polycarbonate, polyethylene, polystyrene, polymethylmethacrylate, polydimethylsiloxane and cyclic olefin polymers (COP). In a more preferred embodiment the polymer is polycarbonate.
    [0035]
    [0036] In another preferred embodiment the thickness of the substrate layer (a) is between 50 micrometers and 5 millimeters, and more preferably between 500 micrometers and 1.2 millimeters.
    [0037]
    [0038] In another more preferred embodiment, the substrate (a) consists of a polycarbonate sheet, and more preferably a polycarbonate sheet with a thickness of between 100 micrometers and 3 millimeters, and even more preferably a 600 pm thick polycarbonate sheet.
    [0039]
    [0040] In another preferred embodiment the interferometric layer (b) is constituted by one or several superimposed films that independently consist of a material that is selected from metals, alloys, metal compounds, silicon, and silicon compounds;
    [0041] with the proviso that when the substrate (a) of the biosensor and the first film of the interferometric layer (b) consist of a metal, these metals are different.
    [0042]
    [0043] In another preferred embodiment the metals of one or more of the films of the interferometric layer (b) are selected from gold, silver, platinum and chromium. In a more preferred embodiment the metal is gold.
    [0044]
    [0045] In another preferred embodiment, when a polished steel sheet is used as a substrate, the lower gold film is omitted.
    [0046] In another preferred embodiment the alloys of one or more of the films of the interferometric layer (b) are selected from Ni-Va, Ni-Fe, Ni-Cr, Fe-Co, Fe-Co-Ta-Zr, Co- Ta-Zr, Cu-Ga, Cu-In, Cu-In-Ga, Ti-Al, Al-Cu, Al-Sn-Cu and W-Ti. In a more preferred embodiment the alloy consists of Ag-In-Sb-Te.
    [0047]
    [0048] In another preferred embodiment the metal compounds of one or more of the films of the interferometric layer (b) are selected from zinc sulphide, titanium oxide, germanium oxide, phosphorus oxide, boron oxide and tantalum pentoxide. In a more preferred embodiment the metal compound is zinc sulfide.
    [0049]
    [0050] In another preferred embodiment the silicon compounds of one or more of the films of the interferometric layer (b) are selected from silicon oxide, silicon nitride, and quartz.
    [0051]
    [0052] The arrangement of the different films comprising the interferometric layer (b) is designed to generate constructive and / or destructive interferometric phenomena in the visible range, and to modify this interferometric response depending on the properties (thickness, refractive index, density, etc.) of the recognition layer (c).
    [0053]
    [0054] In a more preferred embodiment, the films that make up the interferometric layer (b) generate destructive interferometric phenomena, and consist of a 50 nm film of gold, followed by a 60 nm film of zinc sulphide, followed by a film of 15 nm of the Ag-In-Sb-Te alloy, followed by a 65 nm zinc sulphide film, and finally a 5 nm gold film.
    [0055]
    [0056] In a more preferred embodiment, the films that make up the interferometric layer (b) generate constructive interferometric phenomena, and consist of a 50 nm gold film, followed by an 85 nm zinc sulfide film, followed by a film of 20 nm of the Ag-In-Sb-Te alloy, followed by an 85 nm zinc sulphide film, and finally a 5 nm gold film.
    [0057]
    [0058] In a more preferred embodiment, configurations of constructive interferometric materials together with destructive interferometric materials are used in combination, and the signals of both systems are considered for the purpose. to increase the reliability of the results.
    [0059]
    [0060] In another preferred embodiment the biological receptors of the recognition layer (c) are selected from antibodies, enzymes, other proteins, nucleic acids, molecular imprint polymers, polysaccharides, protein-hapten complexes, bacteria, viruses and tissues.
    [0061]
    [0062] In another preferred embodiment the arrangement of the biological receptors on the interferometric layer can be performed through covalent anchoring processes or by fisisorption. Also, the recognition layer (c) can be disposed on the entire surface of the interferometric layer (b), or in discrete portions thereof.
    [0063]
    [0064] In a more preferred embodiment, a recognition layer (c) consisting of a monolayer of specific antibodies covalently immobilized on the interferometric layer is used to detect protein analytes in biological samples.
    [0065]
    [0066] In another preferred embodiment, the recognition layer (c) consists of a monolayer of proteins immobilized by fisisorption, to detect specific antibodies present in the samples.
    [0067]
    [0068] In another preferred embodiment, the recognition layer (c) is arranged according to a microarray structure, in which each point contains different biological receptors to determine multiple analytes in a sample.
    [0069]
    [0070] In another preferred embodiment, these microarray-type assays are grouped into sets of three points (test, positive control, and negative control) and the resulting combination is considered to evaluate the result of the analysis.
    [0071]
    [0072] In the present invention the term "analyte" refers to a substance or group of substances whose presence or concentration is to be determined in the sample. Examples of analytes include, but are not limited to organic molecules, biomachromolecules (proteins, enzymes, antibodies, nucleic acids, carbohydrates, etc.) and microorganisms (bacteria, viruses, cells), among others. As for their function and interest in their determination, these analytes can be drugs, metabolites, biomarkers, allergens, drugs, phytosanitary, contaminants, pathogens, weapons Chemical and biological, allergenic agents, etc.
    [0073]
    [0074] In the present invention, the term "alloy" refers to a combination or mixture of two or more chemical elements, where at least one of which is a metal.
    [0075]
    [0076] Another aspect of the invention is a process for obtaining the biosensor of the invention comprising the following steps:
    [0077] i) Design of the films that make up the substrate (a) and the interferometric layer (b) from theoretical calculations based on the Fresnel equations;
    [0078] ii) Preparation of the multilayer structure of the interferometric layer (b) by sputtenng deposition of the films that comprise it;
    [0079] iii) Immobilization of the bioreceptors by covalent anchoring or by fisisorption on the surface of the interferometric layer (b), forming the recognition layer (c).
    [0080]
    [0081] On the other hand, a sample is incubated on the material recognition layer for the analysis process. The bioreceptors interact with the analytes present in the sample, which generates a change in the amount of matter that constitutes the recognition layer. This change modifies the interferometric response of the material and results in a visually detectable color change, the magnitude of which depends on the concentration of the analyte in the sample.
    [0082]
    [0083] Therefore, a third aspect of the invention is an analyte detection method comprising the following steps:
    [0084] i) Contact the biosensor of the invention with the sample to be analyzed; ii) Incubate said sample on the biosensor of the invention; Y
    [0085] iii) Visually assess the color change of the recognition layer (c) of the biosensor after removing the solution.
    [0086]
    [0087] Optionally, in order to improve the reliability of the process, these three stages can be performed in parallel on points consisting of different bioreceptors, so that a microarray consisting of groups of three points is obtained, with a point dedicated to the test of interest of according to the process described above, another point as a negative control, and a third as a positive control.
    [0088] Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0089]
    [0090] BRIEF DESCRIPTION OF THE FIGURES
    [0091]
    [0092] Fig. 1. Scheme of the configuration of superimposed films constituting the interferometric layer with destructive (I) and (II) constructive configuration, including the substrate and the recognition layer.
    [0093]
    [0094] Fig. 2. Scheme of a biosensor of interferometric material with destructive (I) and (II) constructive configuration, both with a receiver microarray, after incubating samples with different analyte concentrations. In these schemes the highest concentration of analyte has been incubated at the point located in the upper left corner, this concentration decreases sequentially point to point to the right (first) and down (second), the least concentrated point being that of the lower left corner, with a concentration of 0 pg / mL.
    [0095]
    [0096] Fig. 3. Scheme of the test configuration to discriminate false positives and false negatives quickly and intuitively by means of microarray format tests, in interferometric materials with destructive (I) and (II) constructive configuration.
    [0097]
    [0098] EXAMPLES
    [0099]
    [0100] Next, the invention is illustrated by tests carried out by the inventors, which show the effectiveness of the product of the invention.
    [0101]
    [0102] Materials and methods
    [0103]
    [0104] First, the thickness of each of the films of the material is simulated and optimized to obtain the desired response in recognition tests performed on the surface of the outermost layer of the material. These theoretical calculations use the Fresnel equations, calculate the variations of intensity of light reflected at a given wavelength, and consider the thickness and refractive index of each film, together with the changes generated by varying the thickness of the recognition layer from 0 to 20 nanometers (taking a refractive index of 1.47). From these results, the multilayer configurations to be manufactured are selected.
    [0105]
    [0106] To make the nanomaterials, 1.5 x 1.2 cm chips are first cut from 600 qm thick polycarbonate sheets, using a numerical control milling machine. These chips are cleaned with ethanol, with distilled water and dried with pressurized air. Next, the chips are coated with the different layers that make up the material, one by one and sequentially, using a sputtering equipment (ultra high vacuum magnetron) and a particular target for each deposited material. Layer thicknesses during manufacturing are characterized by X-ray reflectometry / diffractometry, ellipsometry refill indices, and the multilayer structures resulting from FIB-FESEM.
    [0107]
    [0108] To prepare the recognition layer on the surface of the interferometric layer, matrices of 4 x 3 drops of 40 nL of a bioreceptor solution are printed, in carbonate buffer, by a contactless microprinter, and incubated 16 hours at 4 ° C so that the receiver is immobilized by fisisorción (BSA at 100 qg / mL for the BSA / anti-BSA system). After incubation, the biosensors are washed with PBS-T buffer, rinsed with water and air dried.
    [0109]
    [0110] Example 1. Design of the materials
    [0111]
    [0112] Flat polycarbonate supports (600 qm thick) were selected as a substrate, as it is a material manufactured on an industrial scale in a very cheap and homogeneous way. On the other hand, gold films are chosen for the interferometric layer, due to the versatility of this metal to immobilize bioreceptors, as well as its high reflexivity and easy deposition in nanometric layers of controlled thickness. Zinc sulphide (ZnS) and the Ag-In-Sb-Te alloy are also selected, as they are two materials known in the state of the art that have very different refractive indices and allow manufacturing structures that generate the multilayer reflections necessary for the response desired interferometric.
    [0113] Theoretical calculations resulted in a structured interferometric layer according to the configuration shown in Figure 1.
    [0114]
    [0115] Example 2. Biosensor with destructive interferometric response
    [0116]
    [0117] 1 x 1 cm square chips with the multilayer structure shown in Figure 1 (I) were manufactured by sputtering and materials were experimentally obtained with the interferometric response shown in Figure 2 (I).
    [0118]
    [0119] In a preferred embodiment, a 600 pm thick polycarbonate sheet is used as a substrate, onto which an interferometric layer consisting of a 50 nm gold film is deposited by sputtering , followed by a 60 nm zinc sulfide film, followed by a 15 nm film of the Ag-In-Sb-Te alloy, followed by a 65 nm zinc sulphide film, and finally a 5 nm gold film. This configuration is designed to generate a destructive interferometric response to recognition tests, so that the material interacts with white light and reflects a light purple color that changes to a deep blue color in positive tests.
    [0120]
    [0121] The biosensing capacity of these materials was experimentally demonstrated with an immunochemical model system based on the BSA protein (bovine serum albumin) as a bioreceptor, and specific anti-BSA G immunoglobulins as analytes. After manufacturing the interferometric layer on the substrate, the receptor was immobilized by passive adsorption on the upper gold surface, according to a microarray arrangement (12 points per chip, ~ 1 mm in diameter per point). Then, a solution containing the analyte was incubated on the material, and the color change was visually evaluated after removing the solution.
    [0122]
    [0123] The interaction between the bioreceptor and the analyte increases the amount of matter that makes up the receptor layer. Thus, part of the interface that was previously occupied by air (refractive index = 1), is now occupied by biomacromolecules (refractive index between 1.35 and 1.47). This increase in the refractive index modifies the interferometric response of the material, which changes the polychromatic response of the radiation reflected by it, and as a consequence a color change is observed in the points with positive tests. In this case, the material is designed to generate color changes from light purple for negative tests, to dark blue in positive tests.
    [0124]
    [0125] Figure 2 (I) shows the scheme of a chip after incubating the analyte (anti-BSA IgG) at different concentrations on the points of a microarray in which the receptor (BSA) has been immobilized. The expected color change from purple to blue is observed, directly detectable with the eye for positive tests. All the individuals surveyed were able to identify as positive the four most intense analyte concentrations analyzed in this experiment (upper row of the microarray), which represents a detection limit of around 30 pg / mL of analyte for this immunochemical system and This material configuration.
    [0126]
    [0127] This invention allows the visual detection of analytical tests, in a single stage, without labeling and without the need for any type of instrumentation for the analysis process. On the other hand, this invention is compatible with a strategy that allows to increase in a very simple way the reliability of the results obtained by direct visual detection. This strategy consists in creating microarrays made up of groups of three points, with a point dedicated to the test of interest, another point as a negative control, and a third as a positive control. In this way, as shown in Figure 3 (I), false negatives and false positives can be discriminated quickly and intuitively.
    [0128]
    [0129] In this exemplary embodiment, for the point dedicated to the test of interest BSA is printed, for the negative control the ovalbumin protein is printed, and for the positive control a rabbit anti-immunoglobulin antibody is printed (40 pL at 100 pg / mL in carbonate buffer in all cases). In this way, as shown in Figure 3, false negatives and false positives can be discriminated quickly and intuitively.
    [0130]
    [0131] Example 3. Biosensor with constructive interferometric response
    [0132]
    [0133] In another preferred embodiment, a 600 pm thick polycarbonate sheet is used as a substrate, on which an interferometric layer consisting of a 50 nm gold film is deposited by sputtenng , followed by an 85 nm zinc sulfide film, followed by a 20 nm film of the Ag-In-Sb-Te alloy, followed of an 85 nm zinc sulfide film, and finally a 5 nm gold film, as shown in Figure 1 (II). This configuration is designed to generate a constructive interferometric response against recognition tests, so that the material interacts with white light and reflects a silver color that changes to red in positive tests, as outlined in Figure 2 (II) .
    [0134]
    [0135] The biosensing capacity of these materials was experimentally demonstrated with an immunochemical model system based on the BSA protein (bovine serum albumin) as a bioreceptor, and specific anti-BSA G immunoglobulins as analytes. After manufacturing the interferometric layer on the substrate, the receptor was immobilized by passive adsorption on the upper gold surface, according to a microarray arrangement (12 points per chip, ~ 1 mm in diameter per point). Then, a solution containing the analyte was incubated on the material, and the color change was visually evaluated after removing the solution.
    [0136]
    [0137] The interaction between the bioreceptor and the analyte increases the amount of matter that makes up the receptor layer. Thus, part of the interface that was previously occupied by air (refractive index = 1), is now occupied by biomacromolecules (refractive index between 1.35 and 1.47). This increase in the index of refraction modifies the interferometric response of the material, which changes the polychromatic response of the radiation reflected by it, and as a consequence a color change is observed in the points with positive tests. In this case, the material is designed to generate color changes from silver for negative tests, to red in positive tests.
    [0138]
    [0139] Figure 2 (II) shows the scheme of a chip after incubating the analyte (anti-BSA IgG) at different concentrations on the points of a microarray in which the receptor (BSA) has been immobilized. The expected color change from silver to red is observed, directly detectable with the eye for positive tests.
    [0140]
    [0141] This invention allows the visual detection of analytical tests, in a single stage, without labeling and without the need for any type of instrumentation for the analysis process. On the other hand, this invention is compatible with a strategy that allows to increase in a very simple way the reliability of the results obtained by direct visual detection. This strategy consists of creating shaped microarrays by groups of three points, with a point dedicated to the essay of interest, another point as a negative control, and a third as a positive control. In this way, as shown in Figure 3 (II), false negatives and false positives can be discriminated quickly and intuitively.
    [0142]
    [0143] Example 4. Analyte Detection
    [0144]
    [0145] To perform the tests, samples dissolved in PBS-T (phosphate buffered saline with polysorbate 20) are incubated on the surface of the chip for 20 minutes at room temperature. To incubate a single sample, between 60 and 120 microliters of sample are dispensed on the biosensor. Individual drops can also be incubated on the different points (1 microliter / point) to analyze different samples in a single biosensor. After incubating the sample, the biosensor is washed with PBS-T, followed by water, and dried in a stream of air. The result of the analysis is known by visual inspection of the resulting color at the matrix points.
    权利要求:
    Claims (21)
    [1]
    1. A biosensor that allows direct visual detection of analytes in a sample, comprising:
    i) A substrate (a) that acts as a support for the interferometric layer (b);
    ii) An interferometric layer (b) consisting of one or more superimposed films, with an arrangement designed to generate constructive and / or destructive interferometric phenomena in the visible range and to modify this interferometric response based on the properties of the recognition layer ;
    iii) A recognition layer (c) composed of biological receptors that are attached to the last film of the interferometric layer (b), and that specifically recognize the analytes present in the sample.
    [2]
    2. Biosensor according to the preceding claim, wherein the substrate (a) is selected from glass sheets, metals, synthetic polymers, membranes and gels.
    [3]
    3. Biosensor according to the preceding claim, wherein the substrate metal (a) is selected from aluminum, steel, gold, silver, platinum, chromium and iron.
    [4]
    4. Biosensor according to the preceding claim, wherein the metal of the substrate (a) is steel.
    [5]
    5. Biosensor according to claim 2, wherein the synthetic polymer of the substrate (a) is selected from polycarbonate, polyethylene, polystyrene, polymethylmethacrylate, polydimethylsiloxane, and cyclic olefin polymers.
    [6]
    6. Biosensor according to the preceding claim, wherein the polymer is polycarbonate.
    [7]
    7. Biosensor according to any of the preceding claims, wherein the thickness of the substrate layer (a) is between 50 micrometers and 5 millimeters.
    [8]
    8. Biosensor according to claim 7, wherein the thickness of the substrate (a) is 600 pm.
    [9]
    9. Biosensor according to any of the preceding claims, wherein the interferometric layer (b) is constituted by one or several superimposed films that independently consist of a material that is selected from metals, alloys, metal compounds, silicon, and silicon compounds;
    with the proviso that when the substrate (a) of the biosensor and the first film of the interferometric layer (b) consist of a metal, these metals are different.
    [10]
    10. Biosensor according to the preceding claim, wherein the metal of one or more of the films of the interferometric layer (b) is selected from gold, silver, platinum and chromium
    [11]
    11. Biosensor according to the preceding claim, wherein the metal is gold.
    [12]
    12. Biosensor according to claim 9, wherein the alloy of one or more of the films of the interferometric layer (b) is selected from Ni-Va, Ni-Fe, Ni-Cr, Fe-Co, Fe-Co-Ta -Zr, Co-Ta-Zr, Cu-Ga, Cu-In, Cu-In-Ga, Ti-Al, Al-Cu, Al-Sn-Cu and W-Ti.
    [13]
    13. Biosensor according to the preceding claim, wherein alloy consists of Ag-In-Sb-Te.
    [14]
    14. Biosensor according to claim 9, wherein the metal compound of one or more of the films of the interferometric layer (b) is selected from zinc sulphide, titanium oxide, germanium oxide, phosphorus oxide, boron oxide and Tantalum pentoxide.
    [15]
    15. Biosensor according to the preceding claim, wherein the metal compound is zinc sulphide.
    [16]
    16. Biosensor according to claim 9, wherein the silicon compound of one or more of the films of the interferometric layer (b) is selected from silicon oxide, silicon nitride and quartz.
    [17]
    17. Biosensor according to any of the preceding claims, wherein the films of the interferometric layer are arranged to cause constructive interferometric phenomena together with destructive interferometric phenomena.
    [18]
    18. Biosensor according to any of the preceding claims, wherein the biological receptors of the recognition layer (c) are selected from antibodies, enzymes, proteins, nucleic acids, molecular imprint polymers, polysaccharides, protein-hapten complexes, bacteria, viruses and tissues.
    [19]
    19. Procedure for obtaining a biosensor according to any of the claims 1 to 18, comprising the following stages:
    i) Design of the films that make up the substrate (a) and the interferometric layer (b) from theoretical calculations based on the Fresnel equations;
    ii) Preparation of the multilayer structure of the interferometric layer (b) by sputtenng deposition of the films that comprise it;
    iii) On the surface of the interferometric layer (b) the bioreceptors are immobilized by covalent anchoring or by fisisorption, forming the recognition layer (c).
    [20]
    20. Analyte detection method comprising the following steps: i) Contacting the biosensor described in any of claims 1 to 18 with the sample to be analyzed;
    ii) Incubate said sample on the biosensor; Y
    iii) Visually assess the color change of the recognition layer (c) of the biosensor after removing the solution.
    [21]
    21. Method according to the preceding claim, where steps i), ii) and iii) are carried out in parallel on points constituted by different bioreceptors, so that a microarray consisting of groups of three points is obtained, with one point dedicated to the test of interest according to the process described above, another point as a negative control, and a third as a positive control.
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    公开号 | 公开日
    WO2021144487A1|2021-07-22|
    ES2745337B2|2021-10-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1994003774A1|1992-07-31|1994-02-17|Biostar, Inc.|Devices and methods for detection of an analyte based upon light interference|
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