Bioactive coatings for surface acoustic wave sensors

Abstract

Acoustic wave sensors often lack sensitivity and do not operate efficiently when presented with a target, which necessitates stable, high-strength immobilization of receptor molecules that can efficiently bind the biomolecules / analytes of interest to the surface, enhancing detection sensitivity. [Solution] Acoustic wave biosensor components are provided. The acoustic wave biosensor includes a piezoelectric substrate with or without a 3D matrix microstructure to increase the surface area of ​​the effective sensing area, and an anchoring substance covalently attached to the surface of the piezoelectric substrate, where the anchoring substance is capable of binding to a capture reagent. Also provided is a process for coating the surface of the piezoelectric material with a bioactive film containing the anchoring substance to create the 3D biosensor surface and components.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application is a continuation of U.S. Provisional Patent Application No. 62 / 529,986, filed July 7, 2017. and the benefit of U.S. Provisional Patent Application No. 62 / 530,735, filed July 10, 2017. and claims priority thereto, each of which is incorporated herein by reference in its entirety. .

[0002] The present disclosure generally relates to bioactive coating methods and to the use of piezoelectric surfaces in surface acoustic wave technology. 3D microfluidic single or multiplexed biosensor devices More particularly, the present disclosure relates to the modification of small molecules, nucleic acid sequences, proteins, antigens, etc. in buffer solutions. A service for the rapid detection of body and cell, biological samples from potentially infected patients or animals. To perform a sandwich assay, a three-dimensional (3D) surface is fabricated to measure the density of capture agent binding. Biocoating the surface of piezoelectric crystals or metals to enhance accuracy and improve sensitivity Regarding the method, we have created a platform technology suitable for the development of elastic surfaces based on biosensors. To manufacture. [Background technology]

[0003] Without diagnosis, medicine is blind, and so the diagnostic field must be able to quickly identify diseases and threats. It is important to accurately identify the detection techniques used to diagnose biological phenomena. Traditionally, optical and chemical sensors have been used, and recent developments in acoustic technology have enabled The possibility of using acoustic methods for biosensing is emerging. A response to an electrical signal that is accompanied by the generation of elastic waves (i.e., very high frequency sound) The sensor utilizes the ability of the piezoelectric material to generate a responsive sound. When propagating on the surface, binding of the analyte causes mass loading and / or viscosity changes in the waveguide. This leads to a change in the velocity and / or amplitude of the surface or bulk acoustic waves. These changes may correlate with the corresponding amounts bound to their surfaces. may be present and measured to provide sensing / detection of said analyte. The bond between the molecule and the sensor surface can be weak, and therefore acoustic wave sensors are often In the case of ion channels, they lack sensitivity and do not operate efficiently when the target is presented. of receptor molecules that can efficiently bind molecules / analytes to surfaces to enhance detection sensitivity; A stable, high strength immobilization is required. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the present disclosure provides a method for detecting a substrate coated with a metal; a binding protein and at least one and an anchor substance comprising a functional group having at least one sulfur atom, wherein the anchor substance comprises: Directly bond to metals via functional groups to form monolayers on metal-coated substrates; The anchor material provides a biosensor component that is configured to couple to the capture reagent. do.

[0005] In one embodiment, the metal is aluminum, gold, aluminum alloys, and any of the foregoing. and combinations thereof.

[0006] In one embodiment, the metal is aluminum.

[0007] In one embodiment, the functional group is a thiol group.

[0008] In one embodiment, the binding protein is an avidin, an oligonucleotide, an antibody, an affima The antibody may be an antibody, an aptamer, or a polynucleotide.

[0009] In one embodiment, the binding protein is neutravidin, native avidin, streptavidin, avidin (strepavidin), and any combination thereof. It's gin.

[0010] In one embodiment, the capture reagent comprises a biotinylated compound for binding to a binding protein of the anchor substance. Includes the .

[0011] In one embodiment, the capture reagent is a reagent for detecting a cell type selected from the group consisting of whole cells, bacteria, eukaryotic cells, tumor cells, viruses, fungi, It comprises a moiety that binds to a parasite, a spore, a nucleic acid, a small molecule, or a protein.

[0012] In one embodiment, the moiety is selected from the group consisting of an antibody, an affimer, or an aptamer. can be.

[0013] In one embodiment, the biosensor further comprises an acoustic wave transducer.

[0014] In one embodiment, the acoustic wave transducer generates bulk acoustic waves.

[0015] In one embodiment, the bulk acoustic waves include thickness shear modes, acoustic plate modes, and the like. plate mode, and horizontal plate mode. It is selected.

[0016] In one embodiment, the biosensor component is a film bulk acoustic wave resonator-based (FBA) It is an R-based device.

[0017] In one embodiment, the acoustic wave transducer generates a surface acoustic wave.

[0018] In one embodiment, the surface acoustic wave is a shear type surface acoustic wave, a surface transverse wave e), Rayleigh waves, and Love waves.

[0019] In one embodiment, the substrate comprises a piezoelectric material.

[0020] In one embodiment, the metal is coated onto the substrate.

[0021] In one embodiment, the substrate further comprises a dielectric layer, and the metal is coated on the dielectric layer. are.

[0022] In one aspect, the present disclosure provides a bulk wave resonator comprising any one of the preceding biosensor components. Provide the equipment.

[0023] In one aspect, the present disclosure provides a process for coating the surface of a metallic material with a bioactive film. A method for manufacturing a metal material, comprising: applying a first composition containing an anchor substance to a surface of the metal material; forming a layer, wherein the anchoring substance comprises a binding protein and at least one a second composition comprising a biotinylated capture reagent; a monolayer of anchor material, wherein the biotinylated capture reagent binds to the binding protein and binding the biotinylated capture reagent to the anchor material via the biotinylated capture reagent. A process including the steps of:

[0024] In one embodiment, the surface of the anchor material is plasma cleaned.

[0025] In one aspect, the present disclosure provides a piezoelectric substrate; and a spacer and bonded to a surface of the piezoelectric substrate. The method includes the steps of: attaching an anchor substance containing a synthetic moiety to a capture reagent via a binding moiety; and A biosensor component is provided that is linked to a capture reagent.

[0026] In one embodiment, the binding moiety is a binding protein.

[0027] In one embodiment, the binding protein is an avidin, an oligonucleotide, an antibody, an affima The antibody may be an antibody, an aptamer, or a polynucleotide.

[0028] In one embodiment, the binding protein is neutravidin, native avidin, streptavidin, avidin (strepavidin), and any combination thereof. It's gin.

[0029] In one embodiment, the binding component is a binding compound having one or more functional groups.

[0030] In one embodiment, the conjugation compound is N-hydroxysuccinimide (NHS), sulfo- Selected from the group consisting of NHS, epoxy, carboxylic acid, carbonyl, maleimide and amine The compound has one or more functional groups selected from the group consisting of methyl, ...

[0031] In one embodiment, the spacer is a polymer linker.

[0032] In one embodiment, the polymer linker is polyethylene glycol, polyvinyl alcohol, or polyacrylate.

[0033] In one embodiment, the polymer linker is polyethylene glycol.

[0034] In one embodiment, the anchoring material forms a layer on the surface of the piezoelectric substrate.

[0035] In one embodiment, the monolayer of anchoring material forms a self-assembled monolayer on the surface of the piezoelectric substrate. It is completed.

[0036] In one embodiment, the binding protein of the anchor substance is attached to the surface of the piezoelectric substance via a spacer. extending away from the surface.

[0037] In one embodiment, the piezoelectric substrate is made of quartz, lithium niobate, and lithium tantalate. 6°Y quartz, 36°YX lithium tantalate, langasite, langatate, langanite lead zirconate titanate, cadmium sulfide, berlinite, lithium iodate, tetraboron Lithium oxide, bismuth germanium oxide, zinc oxide, aluminum nitride, and gallium nitride The compound is selected from the group consisting of:

[0038] In one embodiment, the biosensor component further comprises a housing and a fluid chamber. The surface of the piezoelectric material with the anchor layer forms the wall of the chamber.

[0039] In one embodiment, the anchoring material is attached to the surface of the piezoelectric substrate via a silane group.

[0040] In one embodiment, the binding protein is an avidin, an oligonucleotide, an antibody, an affima The antibody may be an antibody, an aptamer, or a polynucleotide.

[0041] In one embodiment, the binding protein is neutravidin, native avidin, streptavidin, avidin (strepavidin), and any combination thereof. It's gin.

[0042] In one embodiment, the biosensor component further comprises a capture reagent, the capture reagent comprising an anchor. -Contains a biotin moiety for binding to the binding protein of the substance.

[0043] In one embodiment, the capture reagent is a reagent for detecting a cell type selected from the group consisting of whole cells, bacteria, eukaryotic cells, tumor cells, viruses, fungi, and a third portion that binds to the parasite, spore, nucleic acid, protein, or small molecule.

[0044] In one embodiment, the biosensor component further comprises an acoustic wave transducer.

[0045] In one embodiment, the acoustic wave transducer generates bulk acoustic waves.

[0046] In one embodiment, bulk acoustic waves include thickness shear modes, acoustic plate modes, and water modes. flat plate mode.

[0047] In one embodiment, the biosensor component is a film bulk acoustic wave resonator-based (FBA) It is an R-based device.

[0048] In one embodiment, the acoustic wave transducer generates a surface acoustic wave.

[0049] In one embodiment, the surface acoustic wave may be a shear surface acoustic wave, a surface transverse wave, a Rayleigh wave, or a Rayleigh wave. The wave is selected from the group consisting of:

[0050] In one aspect, the present disclosure provides a bulk wave resonator comprising any one of the preceding biosensor components. Provide the equipment.

[0051] In one aspect, the present disclosure provides a process for coating the surface of a piezoelectric material with a biofilm. A first composition containing an anchoring substance is applied to a surface of a metal-coated substrate. applying the anchoring agent to the binding moiety to form a monolayer on the surface; a second composition comprising a biotinylated capture reagent; applying a monolayer of material, wherein the biotinylated capture reagent binds to the binding moiety of the anchor material; binding to the anchor material via a molecule to form a layer of biotinylated capture reagent; The present invention provides a process including:

[0052] In one aspect, the present disclosure provides a method for determining the presence or amount of an analyte in a sample, comprising: contacting any one of the biosensor components with a sample; generating an acoustic wave across the substrate; and and measuring a change in amplitude, phase or frequency of the elastic wave as a function of the acoustic wave amplitude, phase or frequency. to provide.

[0053] In one aspect, the present disclosure provides a method for detecting a capture reagent comprising: a piezoelectric substrate; and a capture reagent immobilized on the piezoelectric substrate; The piezoelectric substrate may include a tertiary structure configured to increase the number of capture reagents immobilized on the piezoelectric substrate. a 3D matrix microstructure, and a capture reagent bound to the 3D matrix microstructure. The present invention provides a biosensor component immobilized on a piezoelectric substrate by

[0054] In one embodiment, the 3D matrix microstructure comprises a plurality of pores.

[0055] In one embodiment, the 3D matrix microstructure comprises a microarray of capture agents.

[0056] In one embodiment, the 3D matrix microstructure comprises a hydrogel matrix.

[0057] In one embodiment, the hydrogel matrix comprises a plurality of pores.

[0058] In one embodiment, the hydrogel matrix comprises a cross-linked polymer.

[0059] In one embodiment, the cross-linked polymer is hydrophilic.

[0060] In one embodiment, the 3D matrix microstructure comprises a dendrimer.

[0061] In one embodiment, the 3D matrix microstructure comprises microarrays of a hydrogel matrix. Including Ray.

[0062] In one embodiment, the 3D matrix microstructure comprises a layer of a hydrogel matrix.

[0063] In one embodiment, the hydrogel matrix can be used to culture whole cells, bacteria, eukaryotic cells, tumor cells, against viruses, fungi, parasites, spores, nucleic acids, small organic molecules, polypeptides, or proteins and is impermeable.

[0064] In one embodiment, the biosensor component comprises a capture reagent in a 3D matrix microstructure or It further includes an anchor material that attaches to the piezoelectric material.

[0065] In one embodiment, the capture reagent comprises a biotinylated compound for binding to a binding protein of the anchor substance. Includes the .

[0066] In one embodiment, the capture reagent is a reagent for detecting a cell type selected from the group consisting of whole cells, bacteria, eukaryotic cells, tumor cells, viruses, fungi, Moieties for binding to parasites, spores, nucleic acids, small organic molecules, polypeptides, or proteins Includes minutes.

[0067] In one embodiment, the moiety is selected from the group consisting of an antibody, an affimer, or an aptamer. can be.

[0068] In one embodiment, the biosensor component further comprises an anchoring material.

[0069] In one embodiment, the acoustic wave transducer generates bulk acoustic waves.

[0070] In one embodiment, bulk acoustic waves include thickness shear modes, acoustic plate modes, and water modes. flat plate mode.

[0071] In one embodiment, the biosensor component is a film bulk acoustic wave resonator-based (FBA) It is an R-based device.

[0072] In one embodiment, the acoustic wave transducer generates a surface acoustic wave.

[0073] In one embodiment, the surface acoustic wave may be a shear surface acoustic wave, a surface transverse wave, a Rayleigh wave, or a Rayleigh wave. The wave is selected from the group consisting of:

[0074] In one aspect, the present disclosure provides a bulk wave resonator comprising any one of the preceding biosensor components. Provide the equipment.

[0075] In one aspect, the present disclosure provides a method for forming a 3D matrix microstructure on a piezoelectric substrate to form a piezoelectric substrate. and immobilizing one or more capture reagents on the piezoelectric substrate. A method for fabricating a biosensor component is provided, comprising the steps of:

[0076] In one embodiment, the present disclosure includes forming pores on a piezoelectric substrate.

[0077] In one embodiment, the method includes the steps of forming a hydrogel matrix on a piezoelectric substrate. Includes flops.

[0078] In one embodiment, the method comprises forming a microarray of a hydrogel matrix on a piezoelectric substrate. The method includes forming a

[0079] In one embodiment, the method includes forming a layer of a hydrogel matrix on a piezoelectric substrate. Including step.

[0080] In one embodiment, the hydrogel matrix comprises a plurality of pores.

[0081] In one embodiment, the method involves using lithographic printing to print a matrix of a capture reagent onto a piezoelectric substrate. forming a microarray.

[0082] In one embodiment, the method includes forming a layer of a dendrimer on a piezoelectric substrate. nothing.

[0083] In one aspect, the present disclosure provides a method for determining the presence or amount of an analyte in a sample, comprising: contacting any one of the biosensor components with a sample; generating an elastic wave as a result of the analyte binding to the capture reagent; measuring changes in amplitude, phase or frequency of the wave.

[0084] In one embodiment, the sample is an environmental sample or a biological sample.

[0085] In one embodiment, the biological sample is blood, serum, plasma, urine, sputum, or feces.

[0086] In one embodiment, the acoustic waves have an input frequency of about 100 to 3000 MHz.

[0087] Additionally, some embodiments involve the use of a metal coated substrate and a binding protein or or a nucleotide and an anchor substance containing a functional group having at least one sulfur atom. the anchor substance is configured to link to the capture reagent via a functional group It relates to biosensor components that bind directly to metals and form monolayers on metal substrates.

[0088] Some embodiments involve applying a first composition comprising an anchoring substance to the surface of the metal / crystalline material. and spreading the anchoring agent over the surface to form a monolayer on the surface, the anchoring agent comprising a binding protein and and at least one sulfur-containing functional group, The second composition is a process for coating the crystal planes and / or the crystal planes with a bioactive film. The monolayer of anchor material contains a biotinylated capture reagent, which is capable of binding proteins. The biotinylated capture reagent binds to the anchor substance via the substrate to form a layer of the biotinylated capture reagent.

[0089] Some embodiments include a biosensor component, a piezoelectric substrate, and a piezoelectric material bonded to the surface of the piezoelectric substrate. In addition, the anchor substance includes a spacer and a binding component, and the capture reagent includes a is linked to the capture reagent via a binding moiety.

[0090] Some embodiments involve applying a first composition comprising an anchoring substance to the surface of the metal / crystalline material. and applying the anchoring agent to the surface to form a monolayer on the surface, the anchoring agent being linked to the binding component. The surface of the piezoelectric material is coated with a biofilm by the step of The second composition comprises a biotinylated capture reagent on a monolayer of anchor material. The biotinylated capture reagent binds to the anchor substance via the binding component of the anchor substance, A layer of biotinylated capture reagent is formed.

[0091] Some embodiments are directed to a method of determining the presence or amount of an analyte in a sample, comprising: contacting a biosensor component as described herein with a sample; generating an acoustic or bulk wave throughout the material; and detecting the analyte binding to the capture reagent. Measure the change in amplitude, phase, or frequency of the elastic or bulk wave as a result of coupling The method includes the steps of:

[0092] Some embodiments involve bulk wave resonators that include the biosensor components described herein. The piezoelectric substrate comprises an antenna including a spacer and a bonding component bonded to the surface of the piezoelectric substrate. The anchor substance is linked to the capture reagent via a binding moiety. There are.

[0093] Some embodiments involve applying a first composition comprising an anchoring substance to the surface of the metal / crystalline material. and applying the anchoring agent to the surface to form a monolayer on the surface, the anchoring agent being linked to the binding component. The surface of the piezoelectric material is coated with a bioactive coating by the steps including a spacer. The second composition is a process of attaching a biotinylated capture reagent to a monolayer of an anchor material. The biotinylated capture reagent, which includes the drug, binds to the anchor substance via the binding moiety of the anchor substance. This forms a layer of biotinylated capture reagent.

[0094] Some embodiments relate to methods for determining the presence or amount of an analyte in a sample. The method includes generating an acoustic or bulk wave across the coated substrate. and contacting the biosensor component with the capture reagent. measuring the change in amplitude, phase or frequency of the elastic or bulk wave as a result of the measurement; Includes.

[0095] Some embodiments include a piezoelectric substrate and a capture reagent immobilized on the piezoelectric substrate; The piezoelectric substrate may include a tertiary structure configured to increase the number of capture reagents immobilized on the piezoelectric substrate. a 3D matrix microstructure, and a capture reagent bound to the 3D matrix microstructure. The present invention relates to a biosensor component immobilized on a piezoelectric substrate by

[0096] Some embodiments may include forming a 3D matrix microstructure on a piezoelectric substrate to form a piezoelectric substrate. and immobilizing one or more capture reagents on the piezoelectric substrate. The present invention relates to a method for fabricating a biosensor component by the steps of:

[0097] Some embodiments are directed to a method of determining the presence or amount of an analyte in a sample, comprising: and contacting the biosensor component of any one of the embodiments of the present invention with the entire metal substrate. generating bulk acoustic waves across the surface of the sample as a result of the analyte binding to the capture reagent; measuring changes in amplitude, phase or frequency of the elastic or bulk wave; According to the method.

[0098] Some embodiments involve bulk wave resonators that include the biosensor components described herein. Regarding.

[0099] Some embodiments utilize poly(methyl methacrylate) (PMMA) polymer as the Love wave. and plasma etching to create 3D structures on the surface of the sensor, Increases surface area.

[0100] The following terms shall have the meanings given to them below.

[0101] "Anchor material" refers to the piezoelectric substrate (for "direct" bonding) or metal part on the sensor surface. A coating that binds both the intermediate coating on the surface and the "capture reagent" (defined below). The term refers to a binding material that is avidin and acts as a specific binding partner. Members of a family of proteins functionally defined by their ability to bind biotin (e.g., avidin, streptavidin, neutravidin), and In addition, oligonucleotides and polynucleotides having specific binding partners and Proteins that can be used to modify and thus anchor the capture reagent. -coatings that can be bonded to piezoelectric sensor materials. Also included are natural carbohydrate-binding lectins that bind to hydrate groups (e.g., antibodies and antibody fragments). fragments (i.e., Fe fragments) and nucleotide fragments such as aptamers). There is a risk of conformational changes or partial denaturation of the capture reagent, which could affect accuracy. In general, it is not preferred to use capture reagents as anchors. and polynucleotides, either directly or through an applied intermediate silver coating (e.g. By bonding to the piezoelectric material via ionic or dipole sites (e.g., by ion exchange). Their specific binding partners are complementary nucleotide molecules, which It can be used to modify the capture reagent.

[0102] A "capture reagent" is a reagent that specifically binds to an analyte in a biological sample and captures the analyte from the biological sample. can be used to identify and / or quantify an analyte by capturing This term includes antibodies, aptamers, and antibody fragments thereof. The capture reagent is, but is not limited to, a phosphorus-containing compound that is a specific binding partner of the anchor substance. Amino acids with or without modification with a binding group (e.g., biotinylation or complementary nucleic acid) In other words, the capture reagent is a specific binding partner of the anchor substance. is or comprises a molecule that simultaneously recognizes the analyte.

[0103] A "small organic molecule" refers to an organic molecule having an average molecular weight of more than about 10 daltons and less than about 2500 daltons, preferably about 200 less than 0 daltons, preferably between about 10 and about 1000 daltons, more preferably between about 10 and about 1000 daltons An organic compound, either natural or synthetic or recombinant, having a molecular weight between about 500 daltons Refers to the molecule.

[0104] "Avidin" is a protein derived from the egg whites of birds, reptiles, and amphibians. It is used in many biochemical reactions. The avidin family includes neutravidin These proteins include ribosomal protein, streptavidin, and avidin, all of which are biotinylated. Avidin is functionally defined by its ability to bind antibodies with high affinity and specificity. , bacterial avidins such as streptavidin, and modified avidins such as neutravidin. (Deglycosylated avidin from Thermo Scientific: www www.thermoscientific.com). They are small oligomeric proteins. Each of these molecules contains four (or two) identical subunits, each of which is It has a single binding site for biotin. In the present invention, it is attached to the surface of a biosensor. In this case, some parts may be exposed to the metal coating on the piezoelectric material surface. Therefore, it is not available for biotin binding. Some other sites are available from the piezoelectric material. The avidin binding site faces away from the surface of the antibody and is available for biotin binding. The binding affinity, although non-covalent, is so high that it can be considered irreversible. The dissociation constant (KD) of avidin is approximately 10-15 M, so this binding is the strongest. It is one of the known non-covalent bonds. In its tetrameric form, avidin has a molecular weight of 66-69 kDa. It is estimated that the size of the mannose chain is 10%. This is due to the carbohydrate content of avidin, which consists of two N-acetylglucosamine residues. The carbohydrate moiety contains at least three unique oligosaccharide structural types that are similar in structure and composition. It contains

[0105] "Biotin" refers to d-biotin or vitamin H, vitamin B7, and coenzymes. Also known as RI, it is a specific binding partner of avidin. are commercially available from several sources, including

[0106] Ranges provided herein are understood to be shorthand for all values ​​within the range, e.g. , the range of 1 to 50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4 Any of the groups including 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 Any number, combination of numbers, or subranges, as well as all decimals between the aforementioned integers, e.g. For example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1. 9 is understood to include 9. For subranges, the "in" and "out" ranges extending from either end of the range are understood to include 9. "Nested subranges" are specifically contemplated. For example, the exemplary range 1 to 50 The resulting subranges are 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or Other directions may include 50-40, 50-30, 50-20, and 50-10. [Brief explanation of the drawings]

[0107] [Figure 1] 1 shows one embodiment of biocoating an unmodified aluminum surface using a thiolated biological capture reagent. [Figure 2] Figure 2A shows the preferential binding of neutravidin (NAv) to aluminum (Al) surfaces. Figure 2A shows the results of an enzymatic assay using a biotinylated HRP / o-phenylenediamine dihydrochloride (OPD) pair. The intensity of absorbance at 417 nm was proportional to the amount of NAv bound to the sensor surface. The amount of bound NAv on the Al-coated crystal surface was significantly increased when thiolated NAv was used. Figure 2B shows a microscopy-based image (500x magnification) of biotinylated fluorescein molecules bound to surface NAv. Figure 2C shows an image (500x magnification) demonstrating the binding of 0.2 µm polystyrene biotinylated fluorescent beads. [Figure 3] FIG. 1 shows a schematic diagram of the development of a neutravidin-based biocoating for selective capture of target analytes. [Figure 4] Figure 4 shows contact angle measurements of water on the sensor. Figure 4A shows that plasma cleaning resulted in a significant decrease in the contact angle. Figure 4B shows that coating with PEG-silane significantly increased the hydrophobicity of the sensor. [Figure 5]Fluorescence images of biotinylated fluorescein (Figure 5A, 50x magnification) and fluorescent polystyrene beads (Figure 5B, 500x magnification) are shown, demonstrating uniform binding to the surface biocoating. [Figure 6] 1 shows biocoating development (without neutravidin) for selective capture of target analytes. [Figure 7] Figure 7A shows a fluorescent analyte bound to a surface biocoating immobilized via an epoxy spacer. Figure 7A is a control (500x magnification) and Figure 7B is an epoxy-coated sensor (500x magnification). [Figure 8] Figure 8 shows SEM images and contact angles of sinusoidal structures in a hydrogel matrix perforated by a picosecond laser system. Figure 8A shows a sinusoidal structure with a periodicity of 25 μm and a height of 12 μm. Figure 8B shows a sinusoidal structure with a periodicity of 35 μm and a height of 45 μm. [Figure 9] A soft lithography process for the fabrication of micro / nanopatterns is presented. DETAILED DESCRIPTION OF THE INVENTION

[0108] The present disclosure is based, at least in part, on the modification of the biosensor substrate prior to coating. or may be coated with a metal, and functionalized with at least one sulfur atom Binding groups (e.g., polypeptides, proteins, protein complexes, etc.) The anchoring material having the formula (I) is attached to the metal coated substrate to form a bioactive coating. This bioactive coating can be used in biosensor devices (e.g., For example, surface acoustic wave (SAW) sensors, bulk acoustic wave (BAW) sensors, ) sensor) to generate a signal detected by the biosensor device. It is based on the discovery that it is possible to increase the intensity and sensitivity.

[0109] In some embodiments, a metal coating of an anchoring substance, such as avidin, is applied to the piezoelectric material. Direct conjugation of can be achieved under the conditions discussed herein. The anchoring material is attached to the metal coating via strong and stable covalent or chemisorption bonds. Direct adhesion to the piezoelectric substrate surface was successfully achieved, forming a monolayer on the metal-coated piezoelectric surface. A single layer is not suitable for biosensors, as multiple layers of anchoring material can interfere with the acoustic signal. This can contribute to optimal and consistent functioning of the sensor.

[0110] The acoustic techniques described herein enable high accuracy and sensitivity biological sensing. The techniques described herein can be used to detect biologically sensitive drugs in a variety of ways, including: It can be fitted to and bonded to the surface of an acoustically transparent material, which allows for acoustic sensing applications. Some embodiments may further extend the use of the method by combining biological materials with metal coatings. Silanes, reactive amines, and carboxylates to provide strong adhesion between the crystal surface Use of chemical agents such as compounds with alkyl and epoxy residues, as well as carbohydrate-based materials In some embodiments, the crystalline surface is made of quartz and similar materials, such as niobium. Lithium tantalate and lithium tantalate, 36°Y crystal, 36°YX lithium tantalate , langasite, langatate, langanite, lead zirconate titanate, cadmium sulfide , berlinite, lithium iodate, lithium tetraborate, bismuth germanium oxide, acid The nitride may be zinc nitride, aluminum nitride, or gallium nitride.

[0111] Some embodiments provide a crystalline surface of a metal suitable for attachment of biological materials or chemical compounds. In some embodiments, the metal is aluminum, It can be aluminum alloy, gold, silver, titanium, chromium, platinum, tungsten, etc. In some embodiments, the metal may be aluminum or an aluminum alloy. The methods described herein allow the formation of a complex bond between some metal surfaces that may traditionally bond poorly to biomaterials. For example, aluminum itself may form weak bonds with biomaterials. However, the methods and materials described herein can be used to This allows for a wide range of applications for aluminum surfaces. The Minium allows for coupling without signal loss when used in conjunction with acoustic sensors. It has the advantages of not destroying materials and not forming black or purple plague. Additionally, aluminum surfaces may propagate acoustic waves more effectively. The described method involves the coupling of biological or chemical molecules with metals (aluminum or aluminum alloys). This provides a strong bond between the coating and the surface, which allows for the formation of a gold-plated SAW sensor. This allows the use of metal coated surfaces.

[0112] The methods and materials described herein are suitable for use with metal (aluminum or aluminum alloy) cores. Stable covalent biocoatings on coated and uncoated crystals These surface-bound bioactive coatings retain functional bioactivity. Furthermore, the methods and materials described herein can be used to detect and measure the electrical activity of highly sensitive electrical systems. When combined with various modifications, these compounds can be useful in providing highly sensitive biosensors. Obtainable.

[0113] The methods described herein can achieve covalently attached affinity capture reagents. Some examples of reagents include small molecules, antibodies, protein antigens, aptamers, or targeted assays. Other such molecules suitable for selective capture of entities include, but are not limited to, In some embodiments, surface attachment provides a method for selectively and specifically capturing target analytes. This results in a favorable orientation of the affinity agent on the aluminum surface. In some embodiments, the materials described herein comprise an affinity agent and an affinity agent covalently bound to an activating moiety. The combination of silanes activated with thiol functional groups can be used to attach the , activating moieties can include epoxy and other suitable adhesive chemical functional groups. In some embodiments, the activating moiety is a PEGylated carbohydrate, such as a PEGylated carbohydrate, to minimize steric hindrance. It may also be used in conjunction with a spacer to increase the signal response. In this case, the activating moiety may not be used in conjunction with a spacer.

[0114] The biological anchor materials described herein are often known to be biologically active. These include, but are not limited to, agents such as avidin. A wide range of engineered proteins, polymers, and carbohydrate moieties are available, with enhanced stability. The method described herein can be used to detect SAW sensors that are biotinylated. can be used to activate the surface of These different processes include, but are not limited to, gases such as nitrogen. Provides various treatments under the conditions of aluminum coating crystal surface of SAW sensor is activated under these conditions, which allows for strong covalent attachment of the biologically active capture reagent. These surface modifications and material combinations are used to specifically capture desired target analyte molecules. A universal platform for modifying the sensor surface with any antibody or other affinity capture agent for It functions as a platform.

[0115] Biosensor Components The surface of the sensor is a metal layer (aluminum or aluminum) deposited on a piezoelectric crystal material. The sensor may be a non-coated piezoelectric material without a metal layer. In some embodiments, the surface of the SAW sensor may be deposited on a piezoelectric crystal material. In some embodiments, the insulating layer may be a metal layer (aluminum or aluminum alloy). The SAW sensor sections may contain alternating metal coatings with crystals or In some embodiments, the dielectric layer is a polymer. In some embodiments, the dielectric layer may be a silicon dioxide (SiO2), a polyimide (PCB), or a ceramic layer. May contain (methyl methacrylate) (PMMA), zinc oxide, or aluminum nitride In some embodiments, suitable crystals can be used with various crystal cuts. In some embodiments, sections of the sensor may be made of dielectric material deposited on a piezoelectric substrate. In some embodiments, the sensor section may be deposited on a metal layer. In some embodiments, the piezoelectric substrate may include a dielectric layer, and a metal layer may be deposited on the piezoelectric substrate. In an embodiment, the section of the sensor may include a metal layer deposited on a dielectric layer, and Additionally, a dielectric layer is deposited on the metal layer. In some embodiments, the sensor section The semiconductor device may include a first metal layer deposited on a dielectric layer, the dielectric layer further comprising a second metal layer. A second metal layer is deposited on the piezoelectric substrate. All preferred approaches to using sensors for detection are described in the preferred embodiments herein. The detection of biomolecules can be based on the ability to modify the sensor surface with suitable coatings. To this end, the sensor surface is made of a suitable material capable of selectively capturing the desired target analyte. In some embodiments, the sensors described herein can be immobilized or modified with The sensor may be a SAW sensor. In some embodiments, the sensors described herein may be a BAW sensor.

[0116] Some embodiments include a substrate coated with a metal layer, a binding protein, and at least The anchor substance includes a functional group having at least one thiol group, and the anchor substance is The anchor substance is configured to be linked to the capture reagent. In some embodiments, the anchoring material is a metal. After bonding the layers, a monolayer is formed on the metal layer.

[0117] In some embodiments, the metal is aluminum, gold, an aluminum alloy, silver, titanium , chromium, platinum, tungsten, and or any combination thereof In some embodiments, the metal is deposited on a piezoelectric or dielectric substrate. In some embodiments, the metal is deposited on a dielectric substrate, and further is deposited on another metal layer.

[0118] In some embodiments, the substrate comprises a piezoelectric material. It further includes a dielectric layer disposed directly over the piezoelectric material.

[0119] In some embodiments, the functional group on the binding protein is a thiol group.

[0120] In some embodiments, the binding protein is avidin, an oligonucleotide, or In some embodiments, the binding protein is an affinity antibody, such as an antibody. In some embodiments, the binding protein can be neutravidin, Natural avidin, streptavidin, and any combination thereof In some embodiments, the binding protein is an avidin selected from the group consisting of: These may include antibodies, affimers, and aptamers.

[0121] The capture reagent may be an antibody, an aptamer, or a biotinylated oligonucleotide, a nucleotide, or , nucleic acids, proteins, peptides, and antibodies, including IgA, IgG, IgM, and IgE, enzymes enzymes, enzyme cofactors, enzyme inhibitors, membrane receptors, kinases, protein A, poly U, poly A, poly Ligand receptors, polysaccharides, chelators, carbohydrates or other specific ligands formed from sugars The enzyme may be a receptor or a nucleotide.

[0122] In some embodiments, the capture reagent is a reagent for detecting a target cell, such as a whole cell, a bacterium, a eukaryotic cell, a tumor cell, a virus, or a protein. may contain moieties for binding to fungi, parasites, spores, nucleic acids, proteins or small molecules. In some embodiments, the moiety may be an antibody, a protein fragment, a peptide, a polypeptide, or the like. The antibody is selected from the group consisting of an antibody, an affimer, an antibody fragment, an aptamer or a nucleotide. In some embodiments, the moiety is selected from the group consisting of an antibody, an affimer, or an aptamer. be selected.

[0123] The capture reagent can be modified with a specific binding partner to the binding protein. In some embodiments, the capture reagent comprises a biotinylated molecule for binding to a binding protein of the anchor substance. It further comprises a chin portion.

[0124] Some of the exemplified biosensors and detection methods involve the use of attached antibodies as capture reagents. However, biosensors are shown as surfaces with The present invention is not limited to the use of proteins, but may also include the use of proteins, such as protein fragments, affimers, antibody fragments, and antigens, on the sensor surface. to immobilize other capture agents, including but not limited to, peptides or nucleotides. can be adapted to

[0125] The biosensor components described herein may include acoustic or bulk wave transducers. In some embodiments, the acoustic wave transducer generates bulk acoustic waves. In some embodiments, the bulk acoustic waves may be thickness shear modes, acoustic plate modes, or other modes. In some embodiments, the beam splitter is selected from the group consisting of a beam splitter, a horizontal plate mode, and a horizontal plate mode. The iosensor component is a film bulk acoustic wave resonator-based (FBAR-based) device. is.

[0126] In some embodiments, the acoustic wave transducer generates a surface acoustic wave. In some embodiments, the surface acoustic waves include shear surface acoustic waves, surface transverse waves, Rayleigh waves, and Rayleigh waves. The wave is selected from the group consisting of:

[0127] Some embodiments comprise a crystal layer, a binding protein, and at least one thiol group. The anchor substance includes an anchor material having a functional group having a specific molecular structure, and the anchor material is directly attached to the crystal layer via the functional group. The biosensor portion is configured to bind to the anchor material and to link to the capture reagent. Regarding the product.

[0128] Embodiments in which bonds between metal surfaces / materials and functional groups (e.g., thiol groups) are described In some embodiments, the metal surface / material can be replaced with a crystalline or other piezoelectric material.

[0129] Spacer-containing anchor substance Some embodiments include a metal coated substrate and a spacer bonded to the metal. and an anchor substance comprising a binding component, and a capture reagent, Some embodiments relate to a biosensor component that is coupled to a capture reagent via a molecule. In some embodiments, the substrate may comprise a piezoelectric material. The silane groups are capable of forming covalent bonds on the metal coating. In some embodiments, the anchoring material is attached to the surface of the metal-coated piezoelectric substrate via a silane group. Attach to the face.

[0130] Some embodiments include a crystalline material and a spacer and a linking moiety attached to the crystalline material. and a capture reagent. is linked to the capture reagent via a binding moiety. In some embodiments, the spacer is , which contain silane groups that can form covalent bonds on crystalline materials. Thus, in some embodiments, the anchoring substance is attached to the surface of the crystalline material via a silane group. Combine.

[0131] In some embodiments, the binding moiety is, for example, avidin, an oligonucleotide, or In some embodiments, the binding protein is a binding protein such as a polynucleotide. The substances are neutravidin, natural avidin, streptavidin, and and any combination thereof.

[0132] In some embodiments, the linking moiety is N-hydroxysuccinimide (NHS), sulfonyl succinimide (SF6), or sulfonyl succinimide (SF6). Fluoro-NHS, epoxy, carboxylic acid, carbonyl, maleimide and / or amine The linking compound has one or more functional groups selected from the group consisting of:

[0133] In some embodiments, the spacer is a polymer linker, wherein the polymer The linker may be polyethylene glycol, polyvinyl alcohol, or polyacrylate. In some embodiments, the polymer linker is from about 50 to about 10,000, about 1 00 to approx. 10,000, approx. 200 to approx. 8000, approx. 300 to approx. 8000, approx. 400 to approx. 8 000, about 500 to about 6000, about 600 to about 6000, about 700 to about 6000, about 80 0 to approx. 5000, approx. 900 to approx. 5000, approx. 1000 to approx. 5000, approx. 500 to approx. 400 0, approx. 600 to approx. 4000, approx. 700 to approx. 4000, approx. 800 to approx. 4000, approx. 900 and above Approximately 4000, approximately 1000 to approximately 4000, approximately 500 to approximately 3000, approximately 600 to approximately 3000, Approximately 700 to approximately 3000, approximately 800 to approximately 3000, approximately 900 to approximately 3000, approximately 1000 to approximately 3000, approx. 500 to approx. 2000, approx. 600 to approx. 2000, approx. 700 to approx. 2000, approx. 8 00 to about 2000, about 900 to about 5000, or about 1000 to about 2000 molecules It is a linear polyethylene having a high molecular weight.

[0134] In some embodiments, the polymer linker has more than about 10, more than about 50, more than about 100, Over 200, over 300, over 400, over 500, over 600, over 700, over 800 , over 900, over 1000, over 1200, over 1400, over 1600, around 1800 In some embodiments, the polyethylene is a linear polyethylene having a molecular weight of greater than about 2000. In the above, the polymer linker is less than about 500, less than about 600, less than about 700, or less than about 800. , less than about 900, less than about 1000, less than about 1200, less than about 1400, less than about 1600, Less than about 1800, less than about 2000, less than about 2200, less than about 2400, less than about 2600, Less than about 2800, less than about 3000, less than about 3500, less than about 4000, less than about 4500, Less than about 5000, less than about 5500, less than about 6000, less than about 6500, less than about 7000, Less than about 7500, less than about 8000, less than about 8500, less than about 9000, less than about 9500, Or it is a linear polyethylene having a molecular weight of less than about 10,000.

[0135] The binding compound has one or more functional groups (e.g., N-hydroxysuccinimide (NH S), sulfo-NHS, epoxy, carboxylic acid, carbonyl, maleimide, and / or In embodiments having a spacer having a hydroxyl group, the spacer may have a length of about 0.1 to 50, 0.5 to 50, , 1~50, 1.5~50, 2~50, 2.5~50, 3~50, 4~50, 5~50, 0.1~40, 0.5~40, 1~40, 1.5~40, 2~40, 2.5~40, 3~ 40, 4~40, 5~40, 0.1~30, 0.5~30, 1~30, 1.5~30, 2 ~30, 2.5~30, 3~30, 4~30, 5~30, 0.1~20, 0.5~20, 1~20, 1.5~20, 2~20, 2.5~20, 3~20, 4~20, 5~20, 0 .1~10, 0.5~10, 1~10, 1.5~10, 2~10, 2.5~10, 3~1 0, 4~10, 5~10, 0.1~8, 0.5~8, 1~8, 1.5~8, 2~8, 2. 5~8, 3~8, 4~8, 5~8, 0.1~5, 0.5~5, 1~5, 1.5~5, 2~ 5, 2.5~5, 3~5, 4~5, 0.1~3, 0.5~3, 1~3, 1.5~3, 2~ 3, 2.5~3, 0.1~2.5, 0.5~2.5, 1~2.5, 1.5~2.5, and In some embodiments, the spacer is in the range of 0.05 More than nm, More than 0.1 nm, More than 0.2 nm, More than 0.3 nm, More than 0.4 nm, More than 0.5 nm, 0 More than .6nm, more than 0.7nm, more than 0.8nm, more than 0.9nm, more than 1nm, more than 1.2nm, 1 More than .4nm, more than 1.6nm, more than 1.8nm, more than 2nm, more than 2.5nm, more than 3nm, 4nm Ultra, More than 5nm, More than 6nm, More than 7nm, More than 8nm, More than 9nm, More than 10nm, More than 20nm, 3 More than 0nm, more than 40nm, more than 50nm, more than 60nm, more than 70nm, more than 80nm, more than 90nm or greater than 100 nm. In some embodiments, the spacer has a length in the range of Less than 1 nm, less than 1.5 nm, less than 2 nm, less than 2.5 nm, less than 3 nm, less than 4 nm, Less than 5nm, Less than 10nm, Less than 20nm, Less than 30nm, Less than 40nm, Less than 50nm , less than 100nm, less than 150nm, less than 200nm, less than 250nm, or less than 500nm It has a length in the range of less than m.

[0136] In some embodiments, the anchoring material is a monolayer on the surface of the metal coated piezoelectric material. In some embodiments, the anchoring material forms a surface of the metal coated piezoelectric material. A self-assembled monolayer is formed on the surface. In one embodiment, the binding protein of the anchor substance extends away from the surface of the metal via a spacer.

[0137] In some embodiments, the piezoelectric substrate is made of lithium niobate (LiNbO), tantalum It consists of lithium tungsten oxide (LiTaO3), silicon dioxide (SiO2), and borosilicate In some embodiments, the metal coating is selected from the group consisting of aluminum or It may also be an aluminum alloy.

[0138] In some embodiments, the biosensor components described herein include a housing and The fluid chamber further includes a chamber wall coated with an anchor substance and a capture reagent. The piezoelectric substrate is formed on the surface thereof.

[0139] Bulk Acoustic Wave Resonators A bulk acoustic wave (BAW) resonator consists of at least one piezoelectric material sandwiched between two electrodes. The electrodes apply an alternating electric field to the piezoelectric material, which creates a stress. Depending on the design, layers with high and low acoustic impedances are used. Layers are added to build Bragg reflectors and / or suspend these layers. The resonator contains multiple layers, piezoelectric substrates (AlN, PZT, quartz, LiNbO3, Langasite ), electrodes (gold, aluminum, copper, etc.), Bragg reflectors (high acoustic impedance or or low acoustic impedance material), a layer for capturing analytes (bioactive layer, antibody, antigen , gas sensitive layer, palladium, etc.) layer, or any material capable of propagating acoustic waves The BAW sensor may be a mixture of the various layers described herein. The sensing layer (the layer that captures the analyte) may be in direct contact with the electrode (A) or may be in contact with a black The acoustic wave may be on a reflector or on any material that can propagate acoustic waves. Good too.

[0140] Some embodiments relate to BAW resonators that include the biosensor components described herein. The construction of BAW sensors for liquid or gas sensing involves the surface of the BAW sensor being It works on the principle that anything that passes through it changes its resonant frequency. By tracking and decoding the frequency (measurement or phase frequency) of the sensor, The mass loading and viscosity of particles attached to a surface can be measured.

[0141] Bio-coating method Some embodiments involve a process for coating the surface of a metallic material with a bioactive film. A method for manufacturing a metal material, comprising: applying a first composition containing an anchor substance to a surface of the metal material; forming a layer, wherein the anchoring substance comprises a binding protein and at least one a second composition comprising a biotinylated capture reagent; applying the material to a monolayer of anchor material, wherein the biotinylated capture reagent is attached to the binding protein. and binding the biotinylated capture reagent to the anchor substance via the protein to form a layer of the biotinylated capture reagent. and

[0142] Some embodiments are processes for coating crystalline materials with bioactive films. A first composition containing an anchoring substance is applied to the surface of the crystalline material to form a monolayer on the surface. the anchor substance comprises a binding protein and at least one thiol a second composition comprising a biotinylated capture reagent; a monolayer of anchor material, wherein the biotinylated capture reagent binds to the binding protein and binding the biotinylated capture reagent to an anchor material via the biotinylated capture reagent to form a layer of the biotinylated capture reagent. Hmm, process-related.

[0143] Some embodiments involve coating aluminum surfaces with bioactive films. a first composition including an anchoring material applied to an aluminum surface to form an aluminum coating; forming a monolayer on the silicon surface, the anchoring substance comprising a binding protein and and a thiol functional group; and a second composition comprising a biotinylated capture reagent. - applying a monolayer of the material, wherein the biotinylated capture reagent binds to the material via a binding protein. and binding the biotinylated capture reagent to an anchor material to form a layer of biotinylated capture reagent. This process involves the coating of crystalline surfaces or surfaces of dielectric materials. can also be used.

[0144] Some embodiments involve coating the surface of a metal-coated piezoelectric material with a bioactive film. A process for treating the surface of a metal coated piezoelectric material to activate the metal surface. and depositing a layer of anchoring material directly onto the activated surface of the metal-coated piezoelectric substrate. and applying the anchoring substance to the surface of the substrate. Some of the capture reagents have the property of binding to a specific binding partner. In embodiments, the anchoring material comprises a silane functional group. The silane functional group is attached to the metal coating. In some embodiments, the method can react with the piezoelectric surface. In some embodiments, the metal is aluminum. This process can also be used to coat crystalline or dielectric surfaces. It is possible.

[0145] In some embodiments, the method comprises covalently bonding chemisorbed ammonium ions onto a metal surface. The method includes forming a Kerr layer.

[0146] Some embodiments provide a method for determining the presence or amount of an analyte in a biological fluid sample, contacting the biosensor component with a composition comprising a capture reagent, The agent comprises or comprises a specific binding partner for the anchor substance and the analyte. and binding the capture reagent to an anchor substance to form a capture reagent layer. forming a piezoelectric surface while contacting the bound capture reagent layer with a sample of biological fluid; generating an acoustic wave across / throughout the capture reagent layer as a result of the analyte binding to the capture reagent layer. measuring the change in amplitude, phase, time delay, or frequency of the wave as Regarding the method.

[0147] Some embodiments involve a process for coating the surface of a metallic material with a bioactive film. A method for manufacturing a metal material, comprising: applying a first composition containing an anchor substance to a surface of the metal material; forming a layer, wherein the anchor material comprises a spacer linked to a binding moiety; and applying a second composition comprising a biotinylated capture reagent to the monolayer of anchor material. a step of attaching the biotinylated capture reagent to the anchor substance via a binding moiety of the anchor substance; and binding the biotinylated capture reagent to the substrate to form a layer of the biotinylated capture reagent. do.

[0148] In some embodiments, the methods described herein activate the surface of the anchoring material. In some embodiments, the step of activating the surface of the anchoring material further comprises: In some embodiments, the plasma cleaning is performed using oxygen or acid. In some embodiments, the method includes treating the surface with a hydrogen / argon mixture. The plasma cleaning lasts for 1-10 minutes, 1-20 minutes, 1-30 minutes, or 1-60 minutes. In some embodiments, the plasma cleaning is performed for 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, Lasts longer than 40 minutes, 50 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, or 4 hours. In some embodiments, the plasma cleaning is performed for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or more. Lasts less than 10 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, or 4 hours. In plasma cleaning, treatment involves 50 to 150 KHz and 50 to 200 watts.

[0149] In some embodiments, the method described herein is direct coating. In some embodiments, direct coating is a simple process that can be performed in seconds or minutes instead of hours. It contains a pure and fast coating chemistry with the precision and power required to deposit a single layer of material. Scalable, continuous, minimal operator intervention, such as inkjet printing, The coating process is produced using an in-line method that is easily automated. This coating method results in fewer rejects and less hazardous waste. , the anchoring substance is deposited directly on the piezoelectric surface without an intermediate layer of material.

[0150] In some embodiments, the preparation methods described herein include a step of cleaning the piezoelectric substrate surface. The cleaning step involves acid treatment, UV exposure, and the generation of highly reactive species. and a plasma treatment that can substantially remove all organic contaminants on the surface of the piezoelectric substrate. This can be achieved in multiple ways, including but not limited to various methods of In some embodiments, the preparation method includes a step of plasma cleaning.

[0151] Binding of analyte to coated biosensor In some embodiments, the bound avidin on the piezoelectric substrate surface binds the analyte of interest. Activation requires the addition of a specific antibody, such as an antibody, specific for the analyte antigen of interest. Antibodies or other agents can be attached to avidin-coated chips. The antibody is biotinylated before being attached to the avidin substrate. The antibody can be analyzed either before or after being attached to the avidin substrate. The analyte-biotinylated antibody complex is formed on the outside of the sensor. The complex can be contacted with a sensor, which allows the biotinylated antibody on the antibody to bind to the complex. The antibody binds to an avidin-coated chip. The preferred method of the two is determined by the analysis. Both methods are within the scope of this disclosure. Analysis of the surface coating with specific antibodies bound to DNA, again using AFM Depths of 6–9 nm were measured, indicating that the antibody was indeed bound to the avidin layer.

[0152] Apply antigen-specific biotinylated capture reagents to capture sucrose, trehalose, glycerol, The excess free bound bismuth in a non-drying medium also contains a protein stabilizer known in the art, such as A second layer consisting of a biotinylation reagent is formed. Many drugs can be biotinylated, and The most commonly used of these is a biotinylated antibody that specifically recognizes the analyte of interest. Protein capture reagents can be chemically or enzymatically biotinylated. Chemical biotinylation utilizes a variety of known conjugation chemistries to conjugate amines, carboxylates, and This results in nonspecific biotinylation of silylates, sulfhydryls, and carbohydrates. -hydroxysuccinimide (NHS) coupling to any primary It is understood that primary amines are biotinylated. Enzymatic biotinylation is achieved by using bacterial biotin. This results in the biotinylation of specific lysines within a specific sequence by ligase. Biotinylation reagents consist of a reactive group attached to the valerate side chain of biotin via a linker. Enzymatic biotinylation is most often used to bind proteins of interest to their N-terminus, C-terminus, or , or in the internal loop, AviTag or acceptor peptide This is done by binding to a 15 amino acid peptide called ATP (anticoagulant peptide). These biotinylation techniques are known in the art.

[0153] Once bound, the capture reagent is briefly exposed to heated air to remove moisture from the applied fluid. partially removed to form a protective and stabilizing gel, thereby preventing the formation of a non-dried gel layer. This ensures long-term stability of binding protein binders such as antibodies, which This allows for essentially complete, time-dependent formation of a second, antigen-specific binder layer. The glass-like layer is made of silica or molecular sieve desiccant pellets within the cartridge pouch. The cartridge is optionally dehydrated for storage in the presence of The cartridge with the upper chamber is then sealed in a plastic The container is sealed in a storage pouch made of PET, preferably in a N2 atmosphere.

[0154] The binding of the anchor substance (such as avidin) to the biotinylated capture reagent creates a second Before use, any remaining unbound biotin in the protective gel layer is removed. The ionized capture reagent and other components are added in the assay buffer or in the test fluid during the analytical procedure. These sensors detect antigens, molecules, and other substances, and can be easily removed by simply washing them away. Detection of precipitate binding and the use of biosensors for disease detection have been demonstrated. .

[0155] The biosensors described herein may be suitably configured with a capture agent that specifically binds to an analyte of interest. Appropriate biofilm coatings can be applied to detect a variety of drugs and biochemical markers. Possible uses for this integrated biosensor include human diagnostics and veterinary diagnostics. The analyte is present or found in an infectious agent. or any substance produced thereby that can be used for detection These include small molecules, oligonucleotides, nucleic acids, proteins, peptides, pathogen fragments, fragments, lysed pathogens, and antibodies, including IgA, IgG, IgM, and IgE, enzymes, and enzyme cofactors , enzyme inhibitors, toxins, membrane receptors, kinases, protein A, polyU, polyA, polylysine Antigens and antibodies include, but are not limited to, polysaccharides, aptamers, and chelators. The detection of the interaction has been previously described (U.S. Pat. No. 4,236,893, 4 ,242,096, and 4,314,821, all of which are incorporated herein by reference. and the like.) In addition, whole cells (prokaryotic cells such as pathogenic bacteria, eukaryotic cells, nuclear cells, mammalian tumor cells), viruses (retroviruses, herpes viruses, adenoviruses, viruses, lentiviruses, etc.), fungi, parasites, and spores (serotypes The detection of phenotypic variations of infectious agents, such as serotypes or vars, is , are within the scope of this disclosure.

[0156] Some embodiments provide a method for determining the presence or amount of an analyte in a sample, comprising: contacting the sensor assembly with the sample; generating an acoustic wave; and measuring a change in the amplitude, phase or frequency of the acoustic wave as a result of binding of the capture reagent to the The present invention relates to a method comprising the steps of:

[0157] In some embodiments, the sample is an environmental sample or a biological sample. In some embodiments, the biological sample is blood, serum, plasma, urine, sputum, or feces.

[0158] In some embodiments, the metal coated substrate is a piezoelectric substrate. In some embodiments, the acoustic waves have an input frequency of about 10 to 3000 MHz. In embodiments, the acoustic waves are about 1 to 10,000, 1 to 8,000, 1 to 6,000, 1 to 500 0, 1 to 4000, 1 to 3000, 1 to 2000, 1 to 1000, 10 to 8000, 10 ~6000, 10~5000, 10~3000, 10~2000, 10~1000, 50 ~8000, 50~6000, 50~5000, 50~3000, 50~2000, 50 ~1000, 100~8000, 100~6000, 100~5000, 100~300 0, 100-2000, 100-1000, 200-8000, 200-6000, 20 0~5000, 200~3000, 200~2000, 200~1000MHz range In some embodiments, the acoustic wave has an input frequency of about 1, 5, 10, 20, 30 , 40, 50, 60, 70, 80, 90, and 100 MHz or more. In some embodiments, the acoustic waves are Input frequencies below 000, 6000, 7000, 8000, 9000, and 10000 MHz It has.

[0159] The embodiments disclosed herein are of the two sensor classes disclosed above. Provide individual elements and sensors that combine the independent advantages found in each. For example, a first embodiment using an anchor substance with thiol functional groups to bond to a piezoelectric substrate. This embodiment can be combined with the embodiment of an anchor substance having a spacer.

[0160] 3D surface for improved anchoring agent coating density Many sensor designs involve the use of a single matrix, such as a functional enzyme hydrogel matrix. The term "matrix" refers to the matrix (or matrices) that in or from which something else arises, develops, is formed, and / or is discovered The term "common" is used herein in accordance with its art-accepted meaning of "commonly used" or "used" Exemplary enzyme hydrogel matrices are typically loaded with a biosensing enzyme (e.g., For example, glucose oxidase or lactate oxidase), and glutaraldehyde Human serum albumin protein cross-linked with any cross-linking agent to form a polymer network This network is then swollen with an aqueous solution to form an enzyme hydrogel matrix. The degree of swelling of this hydrogel frequently increases over a period of several weeks, This is likely due to the breakdown of network crosslinks. Regardless of its cause, the observed swelling The results showed that the protrusion of the hydrogel outside the pores or the "windows" cut into the outer sensor tube This causes the sensor dimensions to exceed the design specifications, adversely affecting analytical performance.

[0161] Some embodiments utilize multiple different micropatterns to detect target analytes (e.g., conjugation to antigens (e.g., free or expressed antigens on the surface of cells or viral particles) Immobilized capture reagents attached without disrupting the conformation of the binding moiety (e.g., antibody) available for the target. The effective surface area of ​​the drug is increased. To determine the performance of the sensor, the bound molecules are The surface density of the deposit (e.g., antigen) is crucial to allow diffusive transport of the target analyte. and an anchor substance (e.g., a biotin-conjugated antibody) to which a capture reagent (e.g., a biotin-conjugated antibody) binds. a relatively open three-dimensional matrix corresponding to the size of the binding moieties on the avidin molecule. Some embodiments involve the use of microorganisms, including viruses and bacteria. It concerns the detection of intact pathogen species, which requires the use of a capture reagent (e.g. allowing the transport of these species through matrices containing activated biotin molecules (e.g., activated biotin molecules). To achieve this, the three-dimensional (3D) matrix is ​​penetrated by a thin film of approximately 0.05 microns to approximately 10 microns in width. A passage of .

[0162] Some embodiments provide biosensor elements and the like with enhanced material properties. The present disclosure also relates to biosensors constructed from such elements. Some embodiments relate to acoustic wave sensors, but the present invention also provides methods for making and using the same. The various elements disclosed herein (e.g., piezoelectric substrate and 3D matrix microstructure) The structural design may be used with any one of a wide variety of sensors known in the art. The analyte sensor elements, structures, and Methods for fabricating and using these elements can be used to establish a variety of layered sensor structures. Such sensors exhibit remarkable sensitivity and accuracy. The sensors have a high degree of flexibility, versatility, and characteristics, which allows them to be used with a wide variety of sensors. -Configurations can be designed to investigate a wide variety of analytes.

[0163] Compared to conventional SAW sensors, the sensitivity of the biosensors described herein is for the detection of biological analytes and also for the detection of potentially small numbers of infectious particles in biological fluids. The sensors described herein are sufficient for detecting bacterial or viral infections. It has sufficient sensitivity even in situations where the volume of the liquid is limited.

[0164] The detection and quantification methods described herein detect biological analytes in the picomolar range. may be sensitive enough to detect small numbers of infectious particles in biological fluids (i.e., <10 particles / m L) It may also have sufficient sensitivity to detect possible bacterial or viral infections. The detection methods described herein, due to their enhanced sensitivity, are also limited in volume of biological fluid. It can also be used in cases where the volume is too small (e.g., 10 to 250 microliters).

[0165] 3D matrix microstructures can include 3D gels or nanostructured surfaces, 3D supramolecular architecture can be utilized to create 3D matrix microstructures with biosensors. Integrating the sensor into a cell to increase the effective surface area and the number of capture agents that can be immobilized on the surface of the biosensor Dendrimers have a branched structure that provides a high density of functional groups in a 3D space, allowing for species Individual dendrimers can be used to create 3D matrix microstructures. The functional groups facilitate antigen / antibody conjugation on the sensor surface. Their branched structure also reduces steric hindrance for antigen-antibody binding, thus increasing the target molecule Promotes capture of particles. Polyamidoamine (PAMAM) and / or polypropylene Piezoelectric substrates can be coated with PPI dendrimers. Dendrimers can be coated onto the surface of a piezoelectric substrate either covalently or non-covalently. Sialinization can be used to add aminosilanes to the sialic acid. Silanes, cyanosilanes, epoxysilanes, etc. can be used to functionalize the piezoelectric surface. Dendrimers can be covalently conjugated to functionalized elastic surfaces The height of the dendrimer layer can be between 5 and 20 nm. Domain antibodies, small molecules, DNA or antigens can be immobilized around the dendrimer surface to target It is possible to capture molecules.

[0166] Method for fabricating three-dimensional matrix microstructures Using lithography techniques to form 3D matrix microstructures on piezoelectric substrates Photomasks and molds can be used to create fine patterns during the photopolymerization process. After the lithography process, an anchor material is attached to the microstructure. After the anchoring material is attached to the 3D matrix microstructure, the capture reagent can then be attached to the 3D matrix microstructure. can be immobilized on the microstructure by linking it to an anchor substance.

[0167] Some embodiments relate to a method for forming a 3D matrix microstructure on a piezoelectric substrate. The method includes applying a suspension to the piezoelectric substrate to form a suspension layer; applying a mask to the layer of suspension; and exposing the photomask to an ultraviolet light source; This reacts the portions of the suspension layer that are not covered by the photomask. removing an unreacted suspension layer from the substrate and forming the 3D matrix microstructure. is formed on the substrate.

[0168] Some embodiments relate to a method for forming a 3D matrix microstructure on a piezoelectric substrate. The method further comprises providing a microfluidic device comprising at least one microchannel on the piezoelectric substrate. forming a network of microchannels; and filling the microchannels with a hydrogel precursor solution. exposing the hydrogel precursor to an ultraviolet light source; The chromofluidic network is removed from the piezoelectric substrate to form the three-dimensional leaving behind a hydrogel microstructure.

[0169] In some embodiments, a portion of the suspension layer is not covered by the photomask. .

[0170] In some embodiments, the suspension is applied to the substrate by spin coating. can be.

[0171] In some embodiments, the suspension is subjected to a flow of the suspension through a microfluidic channel. , is applied to the substrate.

[0172] In some embodiments, the unreacted suspension layer is dissolved in a solvent. The solvent is removed from the substrate by heating, and the solvent is water, saline, phosphate buffered saline, or In some embodiments, the unreacted suspension layer can be removed by washing. and then removed from the piezoelectric substrate.

[0173] In some embodiments, the methods described herein provide a 3D matrix microstructure that is In some embodiments, the method further comprises exposing the antibody to a solution containing a binding reagent. is biotin.

[0174] In some embodiments, the methods described herein provide for the preparation of 3D matrix microstructures. The method further comprises exposing the 3D matrix microstructure to a solution of a binding reagent. The drug-containing 3D matrix microstructures are attached to the anchoring material via a binding reagent.

[0175] In some embodiments, the methods described herein provide for incorporating anchoring materials into a 3D matrix. exposing the 3D matrix microstructure to a solution of a capture agent after attachment to the microstructure. Further includes:

[0176] Some embodiments may include forming a 3D matrix microstructure on a piezoelectric substrate to form a piezoelectric substrate. and immobilizing one or more capture reagents on the piezoelectric substrate. The present invention relates to a method of fabricating a biosensor component, comprising the steps of:

[0177] In some embodiments, the fabrication methods described herein involve forming pores on a piezoelectric substrate. The method further includes the steps of:

[0178] In some embodiments, the fabrication methods described herein involve depositing a hydrogel matrix on a piezoelectric substrate. The method further includes forming a binder.

[0179] In some embodiments, the fabrication methods described herein involve depositing a hydrogel matrix on a piezoelectric substrate. The method further includes forming a microarray of the nucleotides.

[0180] In some embodiments, the fabrication methods described herein involve depositing a hydrogel matrix on a piezoelectric substrate. The method further includes forming a layer of brix.

[0181] In some embodiments, the methods described herein provide a hydrogel comprising a plurality of pores. It further comprises a matrix.

[0182] In some embodiments, the fabrication methods described herein use lithographic printing. Further comprising forming a microarray of capture reagents on the piezoelectric substrate.

[0183] In some embodiments, the fabrication methods described herein involve using a laser to form pores. In some embodiments, the laser is a picosecond or femtosecond laser. It is a pulsed laser.

[0184] [Example 1] Thiolated neutravidin was used as the first layer, which was attached directly to the sensor surface. Figure 1 shows the biocapture of unmodified aluminum surfaces using thiolated biological capture reagents. Although aluminum is used as an example, it can also be used on crystals. The attachment was based on thiol chemistry. Thiols have high binding activity towards gold surfaces. Figure 2A shows that thiolated neutravidin also binds to aluminum. The crystals showed high binding activity to cellulose and unexpectedly showed preferential binding to aluminum. Preferential binding of thiolated neutravidin to aluminum metal Any desired area on the sensor can be selectively created using the capture agent. , thiolated neutravidin, and includes aptamers, nucleotides, and antibodies. The method described herein can be substituted with any thiolated scavenger, including titanium dioxide. It can be used for other materials used for the transmission of elastic waves, such as

[0185] Low microliters containing 10-0.01 mg / mL neutravidin in ddHO A small amount of liquid in the range of 1 / 4 of a milliliter is applied to the target surface in the sensor area, and the crystal or aluminum surface is The samples were air-dried for a period of time that varied from a few minutes to several hours, depending on the conditions of the samples. The remaining unabsorbed neutravidin was thoroughly washed away. Figure 2A shows the results of the optical assay. Then, surface-coated neutravidin and thiolated neutravidin were used. The data compares the binding of a biotinylated enzyme probe to a thiolated neutron. The absorption of avidin to aluminum was approximately six times greater than that to the crystal surface. The absorption of neutravidin was greater than that of thiolated neutravidin.

[0186] Figures 2A-2C show the preferential binding of neutravidin (NAv) to aluminum surfaces. Figure 2A shows the results of the biotinylated HRP / o-phenylenediamine dihydrochloride (OPD) peptide. The results of the enzyme assay using Blk LT are shown. The intensity of absorbance at 417 nm represents the amount of neutravidin bound to the sensor surface. The amount of bound neutravidin on the surface of the aluminum or crystal was proportional to the amount of neutravidin. The increase was significant when thiolated neutravidin was used. Microscope-based images of biotinylated fluorescein molecules bound to avidin are shown (magnification). Figure 2C shows the binding of 0.2 μm polystyrene biotinylated fluorescent beads ( (500x magnification). S using biotinylated fluorescein and polystyrene fluorescent beads. The presence of surface-bound fluorophores on the AW sensor allows the surface neutravidin biocoating to We confirmed that the ribonucleotides were functionally active.

[0187] [Example 2] The aluminum or crystal surface is first activated by plasma cleaning (minutes to hours). By exposing the sensor surface to plasma cleaning, functional groups were generated, which These can be easily measured by evaluating the water contact angle. A contact angle significantly smaller than 90° was optimal for subsequent deposition of activated surfaces. Afterwards, the coated sample was exposed to thiolated neutravidin, forming a layer on the surface. The sensor is then washed to remove excess thiolated nitrogen from the activated aluminum or crystal surface. The neutravidin was removed, and the coated device was then allowed to dry.

[0188] [Example 3] Figure 3 shows the use of neutravidin-based biocoating for selective capture of target analytes. The aluminum or crystal surface is first plasma cleaned (several times). The sensor was activated by exposing it to plasma cleaning (minutes to several hours). Functional groups are generated, which can be easily measured by evaluating the water contact angle. Subsequent deposition of reagents onto the activated surface is optimal at contact angles significantly less than 90°. Figures 4A and 4B show contact angle measurements of water on the sensor. Figure 4B shows plasma cleaning resulting in a significant reduction of PEG-silane coating. This indicates that the hydrophobicity of the sensor is significantly increased.

[0189] The activated surface was then coated with silanes (spacers) attached to PEGylated compounds of various lengths. The biotin was covalently attached to the top of the spacer. The concentration was important to ensure a monolayer and depended on the reaction conditions used. After rinsing, the sensor was washed to remove excess unabsorbed PEG-biotin from the activated aluminum surface. The PEG-biotin was then removed. The coated device was then allowed to dry. The integrity of the coating was confirmed by water contact angle measurements. The length of the PEG spacer was 1. The molecular weight may be between 00 and 2000 and may be adjusted to suit the particular binder of interest.

[0190] Figures 5A and 5B show the fluorescence images of biotinylated fluorescein and fluorescent polystyrene. Figure 5A shows the fluorescence images of biotinylated fluorescein beads (50x magnification). Figure 5B shows fluorescent polystyrene beads (magnification 500x) and their application to surface biocoatings. Figures 5A and 5B show that biotinylated polystyrene beads bind to the sensor. NeutrAvidin adhered to the activated surface, demonstrating that the resulting surface was fully functional. Similarly, any biotinylated antibody, including a biotinylated antibody or fragment thereof, an aptamer, etc. Substances can be fabricated onto the biocoating. In the example shown, biotin Iodofluorescein was used as a probe for surface-bound neutravidin. Under conditions where all neutravidin binding sites are taken up, e.g., by biotinylated antibodies So, it is possible to observe the subsequent addition of biotinylated fluorescein onto the biocoating. (Data not shown). The data indicate that the coating is suitable for capturing target analytes. Therefore, the binding of the analyte to the sensor surface led to the SA Microscopic viscosity and mass transfer on the sensor surface can be quantitatively detected using W technology There will be changes in the load.

[0191] [Example 4] Examples of this include surface activation and derivatization followed by aluminum and / or crystalline bilayers. The process involves coating a short NHS or epoxy spacer. Direct covalent attachment of biological capture agents (e.g., antibodies) to surfaces.

[0192] Figure 6 shows the development of biocoatings for selective capture of target analytes (Neutralization). Direct binding of antibodies or other analyte capture molecules attached to the sensor surface (without vidin) is shown. a functional group attached to a spacer (e.g., PEG or carbohydrate chain) that can This direct approach avoids the use of neutravidin and thereby In this way, the overall thickness of the bilayer and the number of process steps involved are reduced. N-hydroxysuccinimide with sarcosine (PEG or carbohydrate chains of 2-20 nm length) NHS, sulfo-NHS, maleimide, -COOH or -NH2 or 3-glycol In each case, either a hydroxypropyl (epoxy) functional group or a hydroxypropyl (epoxy) functional group can be selected. Silane was used as an anchor molecule to connect functional groups separated by a spacer. The functionalized spacer reacts with the capture molecule (e.g., antibody) to reduce density and steric hindrance. The aluminum or crystal surface of the sensor was activated by plasma cleaning. Next, silane molecules (5-10% concentration - weight / volume) were applied to the sensor surface. Incubation was allowed to continue (several minutes to several hours). Excess silane was washed away with solvent. For the purpose of incorporation, the antibody / protein (1-10µg) was applied directly to the sensor and allowed to incubate at room temperature. Following either NHS or epoxy functionalization, a fluorescent analyte was attached. This allows the functionality of the surface biocoating to be confirmed.

[0193] Figures 7A and 7B show surface biocoatings immobilized via epoxy spacers. Figure 7A shows a control (500x magnification) and Figure 7B shows a fluorescent analyte bound to an epoxy resin. Coated sensor (500x).

[0194] [Example 5] The three-dimensional matrix in the piezoelectric substrate provides larger pores on the surface for the capture agent. This structure can be formed by exposing active areas of the piezoelectric substrate. Increasing the depth of each pore increases the area of ​​avidin exposed to the analyte per pore. To achieve this, it is preferable that the pores be as deep as possible. However, as the depth of each pore increases, The aspect ratio of the pores also increases, which may make it more difficult for the analyte to diffuse to the bottom of the pore. An aspect ratio greater than 10 (h / R) is required for the analyte to cover the entire area of ​​the pore. This is undesirable because it excessively increases the time required and causes the equipment response time to increase. The aspect ratio of the piezoelectric substrate coated with the scavenger is preferably 1 / R or less. 00mm 2 When adding pores with a diameter of 20 microns and a depth of 100 microns to the piezoelectric substrate, , contact area is 6280 microns per pore 2 Therefore, the total contact area is doubled. To make it 1.6 x 10 4 pores are required, and the contact area is 10 3 needed to double The number of pores is 1.6 x 10 7 1.6×10 7 The surface area occupied by each pore is 50 square meters. mm. Therefore, the area density of the pores is 50%, and the picosecond pulse laser This is quite achievable with a scan of the 3 For surface area increases above For example, a 100 micron diameter pore with a depth of 50 microns is preferred, which doubles the contact area. The number of 0 micron pores is 1.3 x 10 2 1.04mm 2 The total surface area of do.

[0195] Figures 8A and 8B show the holes drilled by a picosecond laser system operating at 1.04 μm. Figure 8A shows SEM images and contact angles of sinusoidal structures within the hydrogel matrix. , shows a sinusoidal structure with a periodicity of 25 μm and a height of 12 μm, and FIG. 8B shows a sinusoidal structure with a periodicity of 35 μm. , showing sinusoidal structures with a height of 45 μm. Pores of these dimensions in the hydrophilic cross-linked matrix are Preferably, the holes are drilled using a picosecond or femtosecond pulsed laser.

[0196] [Example 6] A microarray of biotin-streptavidin complexes is immobilized on a piezoelectric substrate. Preferably, each dot may have a diameter of 10 to 25 microns and a height of 5 to 20 microns. Preferably, the protein can be detected using conventional protein microarray technology. 0% surface packing density can be achieved. Protein microarrays are Highly inert coating material for the adsorption of avidin- and biotin-containing proteins It is easily fabricated using polyethylene glycol (PEG) as a material. Using imaging techniques, the PEG coating is patterned, biotin is attached, and then First, biotin is conjugated to streptavidin, and then the biotin conjugated to the antibody is Combine the following.

[0197] [Example 7] The hydrogel matrix is ​​a hydrophilic monolayer incorporating a water-soluble inert diluent such as PEG. The hydrogel matrix is ​​formed by polymerization and crosslinking of the polymer compound on the piezoelectric substrate. A layer of the monomer formulation is applied, and then the monomer layer is exposed to UV light to activate the photoinitiator therein. The hydrogel matrix can be formed by polymerization and cross-linking. Depending on the thickness of the matrix, its hydrophilicity (equilibrium water content), and the processing temperature, Immerse in deionized water or PBS for several minutes to several hours. This process removes the diluent. The matrix is ​​then filled with water, which replaces the water and introduces microcavities, enhancing the free volume. This allows for local mobility of segments of protein molecules attached to the hydrogel. To bind biotin to the gel matrix, a solution of biotin (i.e., phosphate The matrix is ​​eluted with NHS-LC-biotin dissolved in buffered saline. The hydrogel matrix was further eluted with a solution of streptavidin, The functionalized hydrogel matrix is ​​then , activated with biotin conjugated to antibodies that target specific pathogens or other biological agents It is in a state where it is being turned into a

[0198] [Example 8] The micropatterns and 3D matrices described in Examples 5-7 all contain piezoelectric substrates. The material can be prepared using soft lithography processes. This approach is particularly suitable for automation. In the soft lithography process for the fabrication of the sphere, polydimethylsiloxane (PDM) S) molds can be used.

[0199] [Example 9] Hydrogel matrix microarrays can also be prepared by the polymerization step described in Example 7. and crosslinking steps. The chromarrays can also be functionalized using the procedures described in the examples above.

[0200] [Example 10] The hydrogel matrix can be prepared to form a layer. Trix is ​​a small granule of water-soluble polymer such as polyvinyl alcohol or polyvinyl acetate. Once the matrix is ​​formed, the diluent is removed. The particles are washed with water or saline to dissolve any particles present. 2 ~10 6 micron 3 leaving a specific volume space in the range of Distributed within a maximum of 10 6 particles can be packed into 1 mL of monomer formulation Each microcavity thus formed allows biotin bound to an avidin molecule to be inserted. This allows access to the antibody sites present on the

[0201] [Example 11] A dendrimer (e.g., PMMA, PPI, or a combination thereof) is prepared to form a layer. Dendrimers can be formed to include functional groups. Once the trix is ​​formed, the diluent is removed and the particles, if any, are dissolved. Then, wash with water or saline. 2 ~10 6 micron 3 A specific range of Randomly and uniformly distributed within the matrix, leaving volume space. Maximum 10 6 individual particles Each microparticle formed by this process can be filled with 1 mL of the monomer mixture. The cavity allows access to the antibody site present on the biotin attached to the avidin molecule. This becomes possible.

Claims

1. A piezoelectric substrate coated with a metal layer, An anchor material bonded to the surface of the metal layer, wherein the anchor material comprises a spacer and a bonding component, and the spacer is a polymer linker configured to bond to the metal layer and self-assemble as a single layer on the metal layer, Capture reagent and A biosensor component including, The binding component is linked to the capture reagent. The aforementioned biosensor component.

2. The biosensor component according to claim 1, wherein the binding component is avidin, oligonucleotide, antibody, affimer, aptamer, or polynucleotide, or the binding component is an avidin selected from the group consisting of neutraavidin, natural avidin, streptavidin, and any combination thereof.

3. The biosensor component according to claim 1, wherein the binding component is a binding compound having one or more functional groups selected from the group consisting of N-hydroxysuccinimide (NHS), sulfo-NHS, epoxy, carboxylic acid, carbonyl, maleimide, and amine.

4. The biosensor component according to claim 1, wherein the polymer linker is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, and polyacrylate.

5. The biosensor component according to claim 1, wherein the single layer has a static water-to-water contact angle of about 50° to about 70° determined by a static droplet method.

6. The biosensor component according to claim 2, wherein the binding component extends away from the surface of the piezoelectric substrate via the spacer.

7. The biosensor component according to claim 1, wherein the piezoelectric substrate is selected from the group consisting of quartz, lithium niobate and lithium tantalate, 36°Y quartz, 36°YX lithium tantalate, langasite, langatete, langanite, lead zirconate titanate, cadmium sulfide, berinite, lithium iodate, lithium tetraborate, bismuth germanium oxide, zinc oxide, aluminum nitride, and gallium nitride.

8. The biosensor component according to claim 1, further comprising a housing and a fluid chamber, wherein the surface of the piezoelectric material supporting the anchoring material forms the wall of the fluid chamber.

9. The biosensor component according to claim 1, wherein the anchoring material is bonded to the surface of the piezoelectric substrate via a silane group.

10. The biosensor component according to claim 1, wherein the spacer contains a silane group.

11. The biosensor component according to claim 10, wherein the silane group forms a covalent bond with the metal.

12. The biosensor component according to claim 1, wherein the metal layer comprises a transition metal or post-transition metal capable of bonding anchor groups of thiol, silane, or phosphonic acid.

13. The biosensor component according to claim 12, wherein the polymer linker is linear polyethylene.

14. The linear polyethylene is 50-10,000 Da, 100-10,000 Da, 200-8,000 Da, 300-8,000 Da, 400-8,000 Da, 500-6,000 Da, 600-6,000 Da, 700-6,000 Da, 800-5,000 Da, 900-5,000 Da, 1,000-5,000 Da, 500-4,000 Da, 600-4,000 Da, 700-4,000 Da, 800-4,000 Da, 900- A biosensor component according to claim 13, having a molecular weight in the range of 4000 Da, 1000-4000 Da, 500-3000 Da, 600-3000 Da, 700-3000 Da, 800-3000 Da, 900-3000 Da, 1000-3000 Da, 500-2000 Da, 600-2000 Da, 700-2000 Da, 800-2000 Da, 900-5000 Da, or 1000-2000 Da.

15. The spacer has wavelengths of 0.1 to 50 nm, 0.5 to 50 nm, 1 to 50 nm, 1.5 to 50 nm, 2 to 50 nm, 2.5 to 50 nm, 3 to 50 nm, 4 to 50 nm, 5 to 50 nm, 0.1 to 40 nm, 0.5 to 40 nm, 1 to 40 nm, 1.5 to 40 nm, 2 to 40 nm, 2.5 to 40 nm, 3 to 40 nm, 4 to 40 nm, and 5 to 40 nm. 、0.1~30nm、0.5~30nm、1~30nm、1.5~30nm、2~30nm、2.5~30nm、3~30nm、4~30nm、5~30nm、0.1 ~20nm、0.5~20nm、1~20nm、1.5~20nm、2~20nm、2.5~20nm、3~20nm、4~20nm、5~20nm、0.1~10nm 、0.5~10nm、1~10nm、1.5~10nm、2~10nm、2.5~10nm、3~10nm、4~10nm、5~10nm、0.1~8nm、0.5~ 8nm、1~8nm、1.5~8nm、2~8nm、2.5~8nm、3~8nm、4~8nm、5~8nm、0.1~5nm、0.5~5nm、1~5nm、1.5~ A biosensor component according to claim 1, having a length in the range of 5 nm, 2-5 nm, 2.5-5 nm, 3-5 nm, 4-5 nm, 0.1-3 nm, 0.5-3 nm, 1-3 nm, 1.5-3 nm, 2-3 nm, 2.5-3 nm, 0.1-2.5 nm, 0.5-2.5 nm, 1-2.5 nm, 1.5-2.5 nm, or 2-2.5 nm.

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