Modified micro-lens array, micro-lens chip, kit, manufacturing method, and use

Abstract

The present invention provides a modified micro-lens array, a micro-lens chip, a kit, a manufacturing method, and a use. The modified micro-lens array of the present invention comprises a plurality of micro-lenses, and a capture interface which is formed on the surface of a first side of the micro-lenses and comprises a first specific binding agent, wherein the first specific binding agent can specifically bind to an object to be tested. The modified micro-lens array and the micro-lens chip of the present invention are configured such that: testing and analysis do not require a complex optical path, a high-numerical-aperture objective lens, and complex image processing, thereby simplifying an optical path system and an image processing process to a great extent. An optical apparatus is simple, has low cost, and is easily integrated, for example, a white light source and a low-numerical-aperture objective lens can be used, thereby having good application prospects in clinical diagnosis. By means of the technical solution of the present invention, testing and analysis, such as an immunoassay method, do not require a washing step, and real-time counting of signal groups is achieved.

Description

Modified microlens array, microlens chip, kit, preparation method and use Technical Field

[0001] The present invention relates to the field of medical analysis, and more specifically to a modified microlens array, a microlens chip comprising the modified microlens array, a kit comprising the microlens chip, and a preparation method and use thereof. Background Art

[0002] Protein biomarkers are biological molecules whose expression levels change during the occurrence and development of diseases. The detection and quantification of protein markers are crucial in disease prevention, diagnosis, and progression monitoring. Currently, immunoassay technology is an important analytical method for detecting proteins in modern medicine and has been widely used in tumor marker detection, drug analysis, prognosis monitoring, and other fields. However, traditional immunoassay technology quantifies the relationship between the intensity change of the overall signal (or simulated signal) of the sample to be tested and the biomarker to be tested. Due to problems such as high detection limit, low sensitivity, and poor reliability in complex systems, its further application in precision medicine is limited.

[0003] In recent years, a new type of ultra-sensitive digital immunoassay technology has been rapidly developed. This technology uses high-resolution microscopic imaging technology to detect immune complexes, which can achieve accurate counting of immune complexes "one by one", thereby achieving accurate and ultra-sensitive quantitative detection of the target (AS Basu, et al. "Digital assays part II: digital protein and cell assays." SLAS Technology, 2017, 22 (4): 387-405). In digital immunoassays, commonly used microscopic imaging technologies include dark field microscopic imaging technology, surface plasmon resonance microscopic imaging technology, and interference microscopic imaging technology. However, these microscopic imaging technologies often have high requirements for the optical system, requiring a relatively complex and expensive microscope system, a strong illumination power (usually 1-10kW / cm 2 Digital immunoassays require a highly stable laser source (e.g., a laser with a laser source) and a high numerical aperture oil immersion objective lens, and in some cases, complex image processing techniques to detect optical signals. These drawbacks significantly increase the cost of digital immunoassays and result in poor temporal resolution, which in turn hinders their application in clinical diagnosis.

[0004] Therefore, it is of great research significance to develop a digital immunoassay method that is simple, low-cost and rapid.

[0005] Summary of the Invention

[0006] Based on the defects of the current digital immunoassay, the present invention provides a modified microlens array, a microlens chip, a kit, a preparation method and uses.

[0007] In a first aspect, the present invention provides a modified microlens array comprising a plurality of microlenses and a capture interface comprising a first specific binding agent formed on a surface of a first side of the microlenses, wherein the first specific binding agent can specifically bind to an analyte.

[0008] In a second aspect, the present invention provides a method for preparing a modified microlens array, comprising providing a plurality of microlenses, and forming a capture interface comprising a first specific binding agent on a surface of a first side of the microlenses, wherein the first specific binding agent is capable of specifically binding to an analyte.

[0009] In a third aspect, the present invention provides a microlens chip comprising a substrate and the modified microlens array of the present invention, wherein the microlenses are arranged on the substrate.

[0010] In a fourth aspect, the present invention provides a method for preparing a microlens chip, comprising providing a substrate and preparing the modified microlens array of the present invention, wherein the microlenses are provided on the substrate.

[0011] In a fifth aspect, the present invention provides a detection and analysis kit comprising the modified microlens array or lens chip of the present invention, and a labeling reagent comprising a second specific binding agent and a signal group connected thereto.

[0012] In a sixth aspect, the present invention provides a method for preparing a detection and analysis kit, comprising preparing the modified microlens array or microlens chip of the present invention, and preparing a labeling reagent comprising a second specific binding agent and a signal group connected thereto.

[0013] In a seventh aspect, the present invention provides a detection and analysis method, comprising contacting an analyte with the capture interface of the modified microlens array of the present invention, and contacting a labeling reagent comprising a second specific binding agent and a signal group linked thereto with the analyte.

[0014] In an eighth aspect, the present invention provides a detection and analysis device comprising the modified microlens array of the present invention or the detection and analysis kit of the present invention.

[0015] In a ninth aspect, the present invention provides use of the modified microlens array of the present invention in detection and analysis, preparation of a microlens chip, preparation of a detection and analysis kit, or preparation of a detection and analysis device; use of the microlens chip of the present invention in detection and analysis, preparation of a detection and analysis kit, or preparation of a detection and analysis device; use of the detection and analysis kit of the present invention in detection and analysis, or preparation of a detection and analysis device; and / or use of the detection and analysis device of the present invention in detection and analysis.

[0016] Compared with the prior art, the technical solution of the present invention has the following technical effects:

[0017] 1. The modified microlens array of the present invention comprises a plurality of microlenses, and a capture interface comprising a first specific binding agent formed on a first surface of each of the microlenses, wherein the first specific binding agent is capable of specifically binding to an analyte. In particular, the modified microlens array, in combination with a labeled reagent capable of forming a sandwich binding therewith, eliminates the need for complex optical pathways, high-numerical-aperture objective lenses, and tedious image processing for detection and analysis. This significantly simplifies the optical pathway and image processing process, resulting in a simple, low-cost, and easily integrated optical device. For example, a white light source and a low-numerical-aperture objective lens can be used, resulting in a promising application in clinical diagnosis.

[0018] 2. The modified microlens array or microlens chip of the present invention can make detection and analysis, such as immunoassay methods, unnecessary for a rinsing step, and can achieve real-time counting of signal groups. Through the technical solution of the present invention, it is possible to effectively achieve real-time detection of the concentration of the analyte, achieve net counting of signal groups, and improve the sensitivity and specificity of detection. Through the technical solution of the present invention, information such as the intensity and size of a single signal group can be analyzed, which helps to eliminate the interference of agglomerated signal groups or larger impurities, and ensure the net count of a single signal group bound to the chip capture interface. Through the technical solution of the present invention, image preprocessing such as filtering and noise reduction can be avoided, and the shortcomings of existing digital detection and analysis, such as digital immunoassay methods, such as cumbersome operation, multiple incubation and washing steps, can be avoided.

[0019] 3. The modified microlens array or microlens chip of the present invention can realize the quantitative detection of different biomarkers, which is of great significance in the early diagnosis and prevention of various diseases such as cardiovascular disease, neurodegenerative disease, and cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 1 to 4 are schematic diagrams of the structure of a microlens chip in some embodiments of the present invention; FIG1 is a front view, FIG2 is a top view, FIG3 is a cross-sectional view of the microlens chip taken along line A-A in FIG2 , and FIG4 is a top view of the structure of the microlens chip above line B-B in FIG1 omitting line B-B;

[0021] FIG5 is a schematic diagram of forming a "second specific binding agent-analyte-first specific binding agent" sandwich conjugate in some embodiments of the present invention;

[0022] FIG6 is a schematic diagram of a detection and analysis device in some embodiments of the present invention;

[0023] FIG7 is a method for processing and analyzing image information obtained by a microscope in some embodiments of the present invention;

[0024] FIG8 is a schematic diagram of observing the imaging of gold nanoparticles on the capture interface in Example 1 of the present invention;

[0025] FIG9 is a schematic diagram of observing the imaging of gold nanoparticles on the capture interface in Comparative Example 1;

[0026] FIG10 is a schematic diagram of observing the imaging of gold nanoparticles on the capture interface in Comparative Example 2.

[0027] The reference numerals in the figures are as follows: 10: microlens chip; 10′: modified microlens array; 100: substrate; 110: microlens; 120: coating layer; 130: capture interface; 131: first specific binding agent; 140: liquid reservoir; 150: liquid reservoir wall; 151: inlet; 152: outlet; 200: labeling reagent; 201: second specific binding agent; 202: signal group; 210: analyte; 30: optical device; 310: objective lens; 320: moving device; 330: optical path; 340: light source; 350: optical sensor; 40: data processing device; 500: method; 510, 520, 530, 540, 550, 560: steps; A-A, B-B: line segments. DETAILED DESCRIPTION

[0028] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0029] As used herein, singular terms refer to one or more than one. For example, "element" or "an element" refers to one element or more than one element. As used herein, the term "plurality" refers to at least two.

[0030] As used herein, the terms "comprise," "include," "have," "contain," or any similar terms are open-ended conjunctions intended to encompass non-exclusive inclusion, indicating that a combination (e.g., a device, composition, method, etc.) includes the listed elements (e.g., units of a device, components of a composition, substantial steps of a method, etc.), but does not exclude other elements. For example, a composition or article containing a plurality of elements is not limited to the elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent to the composition or article. Unless expressly stated to the contrary, the term "or" refers to an inclusive "or" rather than an exclusive "or." As used herein, the term "consisting essentially of" when used to define compositions and methods means excluding other elements that have any substantial effect on the combination for the purpose being described, but does not exclude other elements that do not substantially affect the basic and novel characteristics of the invention. As used herein, closed conjunctions such as "consisting of" refer to combinations (units, components, substantial steps, etc.) that exclude other elements, but do not mean to exclude trace amounts of unavoidable impurities unless otherwise specified. Embodiments defined by each of these transitional terms are within the scope of the present invention. Disclosure of a technical solution including the terms "comprising," "including," "having," "containing," or any other similar terms as specific embodiments thereof shall also be deemed to simultaneously disclose corresponding technical solutions including the terms "substantially consisting of" and "consisting of."

[0031] As used herein, the terms "first", "second", "third" ... and other ordinal numbers, unless otherwise specified or further limited, are only used to distinguish objects with the same attributes in name, and are not used to describe a specific order or sequence, nor are they used to further limit these objects through these ordinal numbers themselves. It should be understood that these ordinal numbers can be interchanged under appropriate circumstances without affecting the understanding of the present invention. In particular, the terms "first antibody" and "second antibody" used herein should be understood accordingly; the terms "first antibody" and "second antibody" used herein are not intended to limit these antibodies to "primary antibody" and "secondary antibody", respectively.

[0032] As used herein, the term "specific binding" or "specific for" refers to the binding of one molecule to another molecule with an exceptionally low dissociation constant, or a dissociation constant that is significantly lower than that of other molecules. Some examples of molecules that are specific for each other include antibodies and antigens, aptamers and their targets, ligands and receptors, enzymes and substrates, and binding proteins and the molecules to which they bind.

[0033] Embodiments of the present invention provide a microlens chip. Specifically, Figures 1-4 illustrate the structure of a microlens chip 10 in some embodiments of the present invention. Specifically, Figure 1 may be a front view, Figure 2 may be a top view, and Figure 3 may be a cross-sectional view of the microlens chip 10 taken along line A-A in Figure 2 . Figure 4 is a top view of the microlens chip 10 omitting the structure above line B-B in Figure 1 .

[0034] The microlens chip 10 may include a modified microlens array 10 ′. The modified microlens array 10 ′ may include a plurality of microlenses 110 and a capture interface 130 located on the microlenses 110 , preferably on a surface of a first side of the microlenses 110 .

[0035] In some embodiments, the microlens chip 10 includes a substrate 100 , wherein the microlenses 110 are disposed on the substrate 100 .

[0036] In some embodiments, the substrate 100 may include a micro-channel array, wherein the micro-lenses 110 are disposed in the micro-channel array.

[0037] In some embodiments, the microlens chip 10 may further include a coating layer 120 disposed on the substrate 100 , wherein the coating layer 120 surrounds the microlens 110 ; preferably, the capture interface 130 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 .

[0038] In some embodiments, the first side is a side of the microlens 110 and / or the coating layer 120 away from the substrate 100 .

[0039] In some embodiments, the substrate 100 may be a transparent substrate, preferably including commonly used micro-nano processing materials such as polydimethylsiloxane (PDMS), glass, quartz, or silicon wafer, and preferably including transparent glass.

[0040] In some embodiments, the dimensions of substrate 100 may be 10 mm×10 mm×2 mm (ie, 10 mm in length, 10 mm in width, and 2 mm in height).

[0041] In some embodiments, the material used for the microlens 110 may include or consist of a dielectric material.

[0042] In some embodiments, the material used for the microlens 110 may include barium titanate, polystyrene, arsenic trisulfide, arsenic triselenide, lanthanide optical glass materials, barium oxide, silicon oxide, titanium oxide, zinc oxide, or a mixture of two or more of barium oxide, silicon oxide, titanium oxide, and zinc oxide, or consist of them.

[0043] In some embodiments, the material used for the microlens 110 may include barium titanate or polystyrene, preferably barium titanate, or consist of the same.

[0044] In some embodiments, the material used for the microlens 110 may include or consist of a high molecular polymer material.

[0045] In some embodiments, the material used for the microlens 110 may include a sulfur-containing polymer or a selenium-containing polymer, preferably a sulfur-containing polymer, more preferably polythiourea; or preferably a selenium-containing polymer.

[0046] In some embodiments, microlenses 110 are transparent.

[0047] In some embodiments, the microlens 110 may be a microball lens.

[0048] In some embodiments, the microlenses 110 are spherical microlenses.

[0049] In some embodiments, the microlenses 110 are spherical microlenses.

[0050] In some embodiments, the microlens 110 is a hemispherical microlens.

[0051] In some embodiments, the microlenses 110 are cylindrical lenses.

[0052] In some embodiments, the microlens 110 is an aspherical microlens, preferably an ellipsoidal microlens, a parabolic microlens, or a hyperbolic microlens.

[0053] In some embodiments, the diameter of the microlens 110 is 3 to 400 microns, preferably 20 to 100 microns, more preferably 40 to 70 microns, more preferably 45 to 60 microns, more preferably 50 to 55 microns, and more preferably 52 microns.

[0054] In some embodiments, the refractive index of the microlens 110 is 1.65 to 2.20, preferably 1.80 to 2.10, more preferably 1.90 to 2.00, more preferably 1.91 to 1.93, and more preferably 1.92.

[0055] In some embodiments, in the microlens chip 10 or the modified microlens array 10', the number of microlenses 110 is greater than or equal to 1, preferably greater than or equal to 10, more preferably greater than or equal to 100, more preferably greater than or equal to 10. 2 to 10 8 between 10 and 20, more preferably 10 3 to 10 6 between.

[0056] In some embodiments, the microlenses 110 are formed on a plane above the substrate 100 .

[0057] In some embodiments, the microlenses 110 are arranged in a hexagonal packing pattern on a plane, preferably in a hexagonal packing pattern on a plane above the substrate 100 .

[0058] In some embodiments, each microlens 110 contacts the substrate 100 .

[0059] In some embodiments, the diameter of the microlens 110 and the thickness of the coating layer 120 differ within ±10%, preferably within ±5%, preferably within ±1%, and preferably the diameter of the microlens 110 and the thickness of the coating layer 120 are the same.

[0060] In some embodiments, the coating layer 120 has a thickness of 3 to 400 microns, more preferably 20 to 100 microns, more preferably 40 to 70 microns, more preferably 45 to 60 microns, more preferably 50 to 55 microns, and more preferably 52 microns.

[0061] In some embodiments, the coating layer 120 may include a polymer material, preferably a polymer resin material, more preferably PDMS or epoxy resin, more preferably PDMS, or consist of the same.

[0062] In some embodiments, coating layer 120 is transparent.

[0063] In some embodiments, the microlens 110 is optically connected to the substrate 100 . Preferably, two opposite surfaces of the microlens chip 10 are optically connected to the microlens 110 through the substrate 100 .

[0064] In some embodiments, capture interface 130 may include a first specific binding agent 131 .

[0065] In some embodiments, first specific binding agent 131 is formed on the surface of a first side of microlens 110 and optional coating layer 120 .

[0066] In some embodiments, the first specific binding agent 131 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 by 3-aminopropyltriethoxysilane and glutaraldehyde, or poly-L-lysine, or diethoxy(3-glycidyloxypropyl)methylsilane (GPMS), or bovine serum albumin, glutaraldehyde, and protein A.

[0067] In some embodiments, the first specific binding agent 131 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 using 3-aminopropyltriethoxysilane and glutaraldehyde.

[0068] In some embodiments, the first specific binding agent 131 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 by poly-L-lysine.

[0069] In some embodiments, the first specific binding agent 131 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 by diethoxy(3-glycidyloxypropyl)methylsilane (GPMS).

[0070] In some embodiments, the first specific binding agent 131 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 by bovine serum albumin, glutaraldehyde, and protein A.

[0071] In some embodiments, the microlens chip 10 may further include a liquid reservoir 140 located on the capture interface 130. Preferably, the capture interface 130 is located between the liquid reservoir 140 and the microlens 110. More preferably, the capture interface 130 is adjacent to the liquid reservoir 140 on one side and adjacent to the microlens 110 on the other side. The liquid reservoir 140 may be formed in any manner that allows liquid to remain therein. For example, the liquid reservoir 140 may be defined by a liquid reservoir wall 150.

[0072] In some embodiments, the reservoir 140 is the space surrounded by the reservoir wall 150 and the capture interface 130 .

[0073] In some embodiments, the microlens chip 10 may further include a reservoir wall 150 formed on the microlens 110 and / or the optional coating layer 120; preferably, the capture interface 130 is located on one side of the space (i.e., the reservoir 140) defined by the reservoir wall 150 (i.e., the surface of the first side of the microlens 110 and the optional coating layer 120).

[0074] In some embodiments, the capture interface 130 is formed on the surface of the first side of the microlens 110 and the optional coating layer 120 within the reservoir wall 150, and preferably the capture interface 130 is completely formed on the surface of the first side of the microlens 110 and the optional coating layer 120 within the reservoir wall 150.

[0075] In some embodiments, the reservoir wall 150 can include an annular sidewall. In some embodiments, the reservoir wall 150 can include a disc-shaped top wall.

[0076] In some embodiments, the liquid reservoir wall 150 may include an inlet 151 and / or an outlet 152. Through the inlet 151 and / or the outlet 152, the liquid in the liquid reservoir 140 can flow in and out, preferably in a specified direction.

[0077] In some embodiments, the reservoir wall 150 can be transparent, preferably comprising or consisting of glass.

[0078] Based on the same inventive concept, the present invention also provides a method for preparing a microlens chip 10 or a modified microlens array 10'. Specifically, the method for preparing the microlens chip 10 or the modified microlens array 10' includes providing a plurality of microlenses 110 and forming a capture interface 130 on a first side of the microlenses 110.

[0079] In some embodiments, the method for preparing the microlens chip 10 includes providing a substrate 100 , for example, cleaning and drying the substrate 100 .

[0080] In some embodiments, in the method for preparing the microlens chip 10, the microlenses 110 are formed on the substrate 100, preferably including providing, preferably dropping, a suspension including microlens 110 microspheres and a solvent onto the substrate 100, and removing the solvent, preferably by drying.

[0081] In some embodiments, a method for preparing a microlens chip 10 includes forming a coating layer 120 on a substrate 100, including spin-coating a material for the coating layer 120 on the substrate 100. The thickness of the coating layer 120 can be controlled by adjusting the coating speed and coating time and the ratio of the material for the coating layer 120.

[0082] In some embodiments, forming the coating layer 120 on the substrate 100 further includes curing the material of the coating layer 120 .

[0083] In some embodiments, the material of the coating layer 120 is, for example, a mixture of a PDMS prepolymer and a PDMS curing agent.

[0084] In some embodiments, the microlens 110 is first formed on the substrate 100 , and then the coating layer 120 is formed on the substrate 100 , preferably on the substrate 100 and the microlens 110 , preferably by spin coating.

[0085] In some embodiments, the coating layer 120 is first formed on the substrate 100 , preferably by spin coating, and then the microlenses 110 are formed on the substrate 100 , preferably on the substrate 100 and the coating layer 120 .

[0086] In some embodiments, the method for preparing the microlens chip 10 further includes forming a capture interface 130 on the surface of the first side of the coating layer 120; specifically, the method for preparing the microlens chip 10 includes forming a capture interface 130 on the surface of the microlens 110 and optionally on the surface of the first side of the coating layer 120.

[0087] In some embodiments, forming the capture interface 130 includes: contacting the surface of the first side of the microlens 110 and the optional coating layer 120 with a 3-aminopropyltriethoxysilane solution to obtain a first modified surface; contacting the first modified surface with a glutaraldehyde solution to obtain a second modified surface; contacting the second modified surface with a first specific binding agent 131; and preferably, blocking the capture interface 130 obtained after the second modified surface is contacted with the first specific binding agent 131, preferably using serum protein, more preferably using bovine serum albumin.

[0088] In some embodiments, the method for preparing the microlens chip 10 sets a reaction pool 140 on the capture interface 130, preferably forming a sidewall 150 on the surface of the first side of the microlens 110 and / or the coating layer 120, and the space surrounded by the sidewall 150 and the capture interface 130 is the reaction pool 140.

[0089] In some embodiments, in the microlens chip 10 , the sidewall 150 is formed on the surface of the first side of the microlens 110 and / or the coating layer 120 by an adhesive, such as PDMS glue.

[0090] In some embodiments, the microlens chip 10 of the present invention can be used together with a labeling reagent 200 comprising a second specific binding agent 201 and a signaling group 202 linked thereto (eg, as a detection and analysis kit).

[0091] Figure 5 is a schematic diagram illustrating the formation of a "labeling reagent 200 - analyte 210 - first specific binding agent 131" sandwich conjugate in some embodiments of the present invention. As shown in Figure 5 , the digital detection and analysis method of the present invention may include contacting the analyte 210 with the capture interface 130 of the microlens chip 10, and contacting the analyte 210 with the labeling reagent 200 comprising a second specific binding agent 201 and a signaling group 202 linked to the second specific binding agent 201.

[0092] In some embodiments, the first specific binding agent 131 can specifically bind to the analyte 210 .

[0093] In some embodiments, the second specific binding agent 201 is capable of specifically binding to the analyte 210 .

[0094] In some embodiments, both the first specific binding agent 131 and the second specific binding agent 201 can specifically bind to the analyte 210 .

[0095] In some embodiments, the first specific binding agent 131 and the second specific binding agent 201 can be simultaneously

[0096] Detector 210 specifically binds to analyte 210. In some embodiments, analyte 210 is multivalent.

[0097] In some embodiments, the first specific binding agent 131 may include an antibody or an aptamer, preferably an antibody or an aptamer that can specifically bind to the analyte 210 .

[0098] In some embodiments, the second specific binding agent 201 may include an antibody or an aptamer, preferably an antibody or an aptamer that can specifically bind to the analyte 210 .

[0099] In some embodiments, the first specific binding agent 131 may include an antibody, preferably an antibody that can specifically bind to the analyte 210 .

[0100] In some embodiments, the second specific binding agent 201 may include an antibody, preferably an antibody that can specifically bind to the analyte 210 .

[0101] In some embodiments, the first specific binding agent 131 may include an aptamer, preferably capable of binding to the analyte 210

[0102] In some embodiments, the second specific binding agent 201 may include an aptamer, preferably

[0103] An aptamer is selected that can specifically bind to the analyte 210 .

[0104] In some embodiments, a molecule, and a molecule that specifically binds thereto, has a low dissociation constant (K D ), for example, 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less, more preferably 10 -9 M or less, more preferably 10 -10 M or less, more preferably 10 -11 M or less, more preferably 10 -12 M or less; or having a dissociation constant much lower than that for other molecules, for example, the quotient of the dissociation constant of the molecule for the molecule specifically binding thereto and the dissociation constant of the molecule for other molecules is less than 10 -1 Below, preferably 10 -2 Less than, more preferably 10 -3 Less than, more preferably 10 -4 Less than, more preferably 10 -5 Less than, more preferably 10 -6 the following.

[0105] In some embodiments, the analyte 210 may include an antigen, a cell, a bacterium, or an exosome, preferably an antigen, more preferably a multivalent antigen, and the first specific binding agent 131 may include an antibody or an aptamer that specifically binds to the antigen, cell, bacterium, or exosome.

[0106] In some embodiments, the analyte 210 may include an antigen, a cell, a bacterium, or an exosome, preferably an antigen, more preferably a multivalent antigen, and the second specific binding agent 201 may include an antibody or an aptamer that specifically binds to the antigen, cell, bacterium, or exosome.

[0107] In some embodiments, the first specific binding agent 131 and the second specific binding agent 201 may include antibodies or aptamers that can specifically bind to the same analyte (eg, analyte 210 ).

[0108] In some embodiments, the analyte 210 may include an antigen, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or an aptamer that specifically binds to the antigen.

[0109] In some embodiments, the analyte 210 may include a multivalent antigen, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or an aptamer that specifically binds to the multivalent antigen.

[0110] In some embodiments, the analyte 210 may include a biomarker, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or aptamer for the biomarker.

[0111] In some embodiments, the analyte 210 may include a disease marker, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or aptamer for the disease marker.

[0112] In some embodiments, the analyte 210 may include a protein biomarker, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or aptamer to the protein biomarker.

[0113] In some embodiments, the analyte 210 may include an acute myocardial infarction marker, a cancer marker, or a neurodegenerative disease marker, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or aptamer for an acute myocardial infarction marker, an antibody or aptamer for a cancer marker, or an antibody or aptamer for a neurodegenerative disease marker.

[0114] In some embodiments, the analyte 210 may include cardiac troponin I, myoglobin, creatine kinase isoenzyme, C-reactive protein, N-terminal pro-brain natriuretic peptide, alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, carbohydrate antigen 19-9, beta-amyloid protein, serum amyloid A, monocyte chemoattractant protein, human monocyte chemoattractant protein, matrix metalloproteinases (e.g., MMP-2, MMP-3, or MMP-9), tumor necrosis factor alpha, vascular endothelial growth factor, transforming growth factor, interferon, procalcitonin, viral nucleocapsid protein p24, growth-associated protein, glial fibrillary acidic protein, neurofilament light chain protein, Angiogenin, interleukin, human epididymis protein 4, α-L-fucosidase, S100 calcium-binding protein, alveolar protein, neuron-specific enolase, squamous cell carcinoma antigen, lipoprotein-associated phospholipase, heart-type fatty acid-binding protein, D-dimer, human brain semaphorin 4D, human growth differentiation factor 15, exosomal synaptophysin, exosome surface protein, α2-macroglobulin, angiogenic factor family (e.g., ANG-2, FGFB, HB-EGF, HGF, PLGF, or VEGF), fibroblast growth factor 7 (fibroblast growth factor 7), growth factor 7), brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 9 (BMP-9), C-chain insulin (C-peptide), CD-14 protein, chemokine protein family (e.g., IP-10, ITAC, MCP-1, MIP-3B, IL-8, MIP-3β / CCL19, MIP-3α / CCL20, MIP-4 / CCL18, RANTES / CCL5, SDF-1 / CXCL12, CXCL9, MDC, MIP-2, MIP-1α, or MIP-1β), chemokine ligands (e.g., CXCL 1, CXCL 11, or CXCL 13), human cytokines (e.g., IFN-γ, IL-1β, IL-2, IL-6, IL-10, KC, or TNF-α), epidermal growth factor family (e.g., epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), or epiregulin), endoglin, eosinophil chemotactic protein (Eotaxin 1,CCL-11), apoptosis-related factor ligand (FasL), fibrinogen, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), glycoprotein 130 (GP130), granzyme B protein (Granzyme B), I309 / CCL-1 protein, IgE protein, insulin, interferon-inducible protein 10 (IP-10), neuropsychiatric disease-related markers (e.g., tau protein, β-amyloid 40 (Aβ40), β-amyloid 42 (Aβ42), neurofilament protein (Nf-L), glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), or TDP-43 protein), osteoprotegerin (OPG), osteopontin (OPN), PD-1 protein, PD-L1 protein, platelet-derived growth factor (PDGF), placental growth factor The first specific binding agent 131 and / or the second specific binding agent 201 may include an antibody or aptamer thereof.

[0115] In some embodiments, analyte 210 may include cardiac troponin I, and first specific binding agent 131 and / or second specific binding agent 201 may include a cardiac troponin I antibody or aptamer.

[0116] In some embodiments, the analyte 210 may include myoglobin, and the first specific binding agent 131 and / or the second specific binding agent 201 may include a myoglobin antibody or aptamer.

[0117] In some embodiments, the analyte 210 may include creatine kinase isoenzymes, and the first specific binding agent 131 and / or the second specific binding agent 201 may include creatine kinase isoenzyme antibodies or aptamers.

[0118] In some embodiments, analyte 210 may include C-reactive protein, and first specific binding agent 131 and / or second specific binding agent 201 may include a C-reactive protein antibody or aptamer.

[0119] In some embodiments, the analyte 210 may include N-terminal pro-B-type natriuretic peptide (NTP), and the first specific binding agent 131 and / or the second specific binding agent 201 may include an N-terminal pro-B-type natriuretic peptide (NTP) antibody or aptamer.

[0120] In some embodiments, the analyte 210 may include alpha-fetoprotein, and the first specific binding agent 131 and / or the second specific binding agent 201 may include an alpha-fetoprotein antibody or aptamer.

[0121] In some embodiments, analyte 210 may include carcinoembryonic antigen, and first specific binding agent 131 and / or second specific binding agent 201 may include a carcinoembryonic antigen antibody or aptamer.

[0122] In some embodiments, analyte 210 may include prostate-specific antigen, and first specific binding agent 131 and / or second specific binding agent 201 may include a prostate-specific antigen antibody or an aptamer.

[0123] In some embodiments, the analyte 210 may include carbohydrate antigen 19-9, and the first specific binding agent 131 and / or the second specific binding agent 201 may include a carbohydrate antigen 19-9 antibody or aptamer.

[0124] In some embodiments, the analyte 210 may include β-amyloid protein, and the first specific binding agent 131 and / or the second specific binding agent 201 may include a β-amyloid protein antibody or aptamer.

[0125] In some embodiments, the analyte 210 may include tau protein, and the first specific binding agent 131 and / or the second specific binding agent 201 may include a tau protein antibody or aptamer.

[0126] In some embodiments, the aptamer is DNA, RNA, XNA, or a peptide, preferably a DNA, RNA, XNA, or peptide that can specifically bind to the analyte 210 .

[0127] In some embodiments, the aptamer is DNA.

[0128] In some embodiments, the aptamer is RNA.

[0129] In some embodiments, the aptamer is an XNA.

[0130] In some embodiments, the aptamer is a peptide.

[0131] In some embodiments, the analyte 210 may include an antibody or an aptamer, preferably an antibody or an aptamer that can specifically bind to the first specific binding agent 131 and / or the second specific binding agent 201 .

[0132] In some embodiments, the analyte 210 may include an antibody, preferably an antibody that can specifically bind to the first specific binding agent 131 and / or the second specific binding agent 201 .

[0133] In some embodiments, the analyte 210 may include an aptamer, preferably an aptamer that can specifically bind to the first specific binding agent 131 and / or the second specific binding agent 201 .

[0134] In some embodiments, the first specific binding agent 131 and / or the second specific binding agent 201 may include antigens, cells, bacteria, or exosomes, preferably antigens, more preferably multivalent antigens, and the analyte 210 may include antibodies or aptamers that specifically bind to the antigens, cells, bacteria, or exosomes.

[0135] In some embodiments, the first specific binding agent 131 and / or the second specific binding agent 201 may include a multivalent antigen, and the analyte 210 may include an antibody or an aptamer that specifically binds to the multivalent antigen.

[0136] In some embodiments, first specific binding agent 131 and / or second specific binding agent 201 may include a biomarker, and analyte 210 may include an antibody or aptamer to the biomarker.

[0137] In some embodiments, the first specific binding agent 131 and / or the second specific binding agent 201 may include a disease marker, and the analyte 210 may include an antibody or aptamer to the disease marker.

[0138] In some embodiments, the first specific binding agent 131 and / or the second specific binding agent 201 may include a protein biomarker, and the analyte 210 may include an antibody or aptamer to the protein biomarker.

[0139] In some embodiments, the first specific binding agent 131 and / or the second specific binding agent 201 may include an acute myocardial infarction marker, a cancer marker, or a neurodegenerative disease marker, and the analyte 210 may include an antibody or aptamer for an acute myocardial infarction marker, an antibody or aptamer for a cancer marker, or an antibody or aptamer for a neurodegenerative disease marker.

[0140] In some embodiments, the first specific binding agent 131 and / or the second specific binding agent 201 may include cardiac troponin I, myoglobin, creatine kinase isoenzymes, C-reactive protein, N-terminal pro-brain natriuretic peptide, alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, carbohydrate antigen 19-9, beta-amyloid protein, serum amyloid A, monocyte chemoattractant protein, human monocyte chemoattractant protein, matrix metalloproteinases (e.g., MMP-2, MMP-3, or MMP-9), tumor necrosis factor alpha, vascular endothelial growth factor, transforming growth factor, interferon, procalcitonin, viral nucleocapsid protein p24, growth-associated protein, glial fibrillary acidic protein , neurofilament light chain protein, angiogenin, interleukin, human epididymis protein 4, α-L-fucosidase, S100 calcium binding protein, alveolar protein, neuron-specific enolase, squamous cell carcinoma antigen, lipoprotein-associated phospholipase, heart-type fatty acid binding protein, D-dimer, human brain semaphorin 4D, human growth differentiation factor 15, exosomal synaptophysin, exosome surface protein, α2-macroglobulin, angiogenic factor family (e.g., ANG-2, FGFB, HB-EGF, HGF, PLGF, or VEGF), fibroblast growth factor 7 (fibroblast growth factor 7), growth factor 7), brain-derived neurotrophic factor (BDNF), bone morphogenetic protein 9 (BMP-9), C-chain insulin (C-peptide), CD-14 protein, chemokine protein family (e.g., IP-10, ITAC, MCP-1, MIP-3B, IL-8, MIP-3β / CCL 19, MIP-3α / CCL20, MIP-4 / CCL18, RANTES / CCL5, SDF-1 / CXCL12, CXCL9, MDC, MIP-2, MIP-1α, or MIP-1β), chemokine ligands (e.g., CXCL 1, CXCL 11, or CXCL 13), human cytokines (e.g., IFN-γ, IL-1β, IL-2, IL-6, IL-10, KC, or TNF-α), epidermal growth factor family (e.g., epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), or epiregulin), endoglin, eosinophil chemotactic protein (Eotaxin 1,CCL-11), apoptosis-related factor ligand (FasL), fibrinogen, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), glycoprotein 130 (GP130), granzyme B protein (Granzyme B), I309 / CCL-1 protein, IgE protein, insulin, interferon-induced protein 10 (IP-10), neuropsychiatric disease-related markers (e.g., tau protein, β-amyloid protein 40 (Aβ40), β-amyloid protein 42 (Aβ42), Neurofilament protein (Nf-L), glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), or TDP-43 protein), osteoprotegerin (OPG), osteopontin (OPN), PD-1 protein, PD-L1 protein, platelet-derived growth factor (PDGF), placental growth factor (PlGF), receptor for advanced glycation end products (RAGE), receptor activator of nuclear factor-κB ligand (RANKL), SARS-CoV-2 protein, SARS-CoV-2 spike protein, stem cell factor (SCF), 25kDa synaptosomal associated protein (SNAP-25), tumor necrosis factor receptor type II (sTNFRII), amino acid kinase receptor-2 (TIE-2), tissue inhibitor of metalloproteinases (TIMP, such as TIMP-1, TIMP-2, or TIMP-3), or thrombospondin 2 (TSP-2), and the analyte 210 may include antibodies or aptamers thereof.

[0141] Since the first specific binding agent 130 includes a first specific binding agent specific for the analyte 210 and the second specific binding agent 201 also includes a first specific binding agent specific for the analyte 210 , a “second specific binding agent-analyte-first specific binding agent” sandwich conjugate can be formed.

[0142] In some embodiments, the signal group 202 can be microparticles or nanoparticles, preferably nanoparticles.

[0143] In some embodiments, the signal group 202 may be a metal nanoparticle (e.g., a single metal nanoparticle or an alloy nanoparticle), quantum dots, carbon dots, upconversion nanoparticles, semiconductor nanoparticles, silica nanoparticles, organic polymer luminescent particles, or metal-organic framework nanoparticles, preferably a metal nanoparticle or a semiconductor nanoparticle, preferably a gold, silver, copper or palladium nanoparticle, more preferably a gold, silver, or copper nanoparticle, or preferably an alloy nanoparticle.

[0144] In some embodiments, the signaling group 202 can be a fluorescent nanoparticle.

[0145] In some embodiments, the signaling group 202 can be gold nanoparticles (AuNPs).

[0146] In some embodiments, the particle size of the signal group 202 may be 1 to 1000 nm, preferably 10 to 100 nm, more preferably 50 to 100 nm, and even more preferably 80 to 90 nm.

[0147] In some embodiments, in the labeling reagent 200, the connection between the second specific binding agent 201 and the signal group 202 can be through a protein A / G intermediate modified with polyethylene glycol or 3-(2-pyridyldithio)-propionic acid-N-succinimidyl ester (SPDP).

[0148] In some embodiments, in the labeling reagent 200 , the molar ratio of the second specific binding agent 201 to the signal group 202 is 10-1000:1, preferably 50-200:1, and preferably 100-150:1.

[0149] In some embodiments, preparing the labeling reagent 200 can include attaching a signaling group 202 to the second specific binding agent 201 .

[0150] In some embodiments, preparing the labeling reagent 200 may include contacting the signal group 202 with polyethylene glycol to obtain a modified signal group; contacting the modified signal group with a second specific binding agent 201; and optionally, blocking the labeling reagent obtained after the modified signal group contacts the second specific binding agent 201, preferably using serum protein, more preferably using bovine serum protein.

[0151] Based on the same inventive concept, the present invention further provides a detection and analysis device. Specifically, FIG6 is a schematic diagram of a detection and analysis device 1 in some embodiments of the present invention, wherein the detection and analysis device 1 includes a microlens chip 10 .

[0152] In some embodiments, the detection and analysis device 1 may further include an optical device 30 .

[0153] In some embodiments, the optical device 30 may include an objective lens 310 .

[0154] In some embodiments, the optical device 30 may further include an optical path 330 , a light source 340 , and an optical detector 350 , wherein the optical path 330 is optically connected to the objective lens 310 , the light source 340 , and the optical detector 350 .

[0155] In some embodiments, the optical detector 350 may be electrically connected to a data processing device 40 for processing data received by the optical detector 350 .

[0156] In some embodiments, light may originate from the light source 340 , pass through the optical path 330 and the objective lens 310 , reach the microlens chip 10 , and pass through the microlens chip 10 , preferably the substrate 100 and the microlens 110 , to reach the solution in the liquid reservoir 140 .

[0157] In some embodiments, light can originate from the solution in the reservoir 140 , preferably the signal group 202 , through the microlens chip 10 , preferably the microlens 110 and the substrate 100 , to the objective lens 310 , and through the objective lens 310 and through the optical path 330 to the optical detector 350 .

[0158] In some embodiments, optical detector 350 is configured to receive light emitted from signal group 202. The light emitted from signal group 202 can be of the same wavelength or a different wavelength than the light emitted from light source 340, preferably of the same wavelength or a higher wavelength. The light emitted from signal group 202 can be backscattered light (e.g., Rayleigh scattered light or Mie scattered light) or fluorescent light.

[0159] In some embodiments, the objective lens 310 may be an air objective lens, for example, an air objective lens with a magnification of 40 times and a NA of 0.55.

[0160] In some embodiments, the light source 340 may be an LED light source, a halogen light source, a laser light source, or a xenon light source.

[0161] For example, it is an LED white light source. In some embodiments, the optical device 30 of the present invention is imaged by a bright field imaging method.

[0162] In some embodiments, the optical path 330 is a reflective optical path.

[0163] In some embodiments, optical path 330 can include a filter.

[0164] In some embodiments, the optical sensor of the present invention comprises a complementary metal oxide semiconductor (CMOS) or a charge coupled device (CCD), preferably a CMOS.

[0165] In some embodiments, the optical device may further include a moving device 320 connected to the objective lens 310 or the microlens chip 10, and configured to move the objective lens 310 or the microlens chip 10 to adjust the distance between the objective lens 310 and the microlens chip 10. Light emitted from the signal group 202 in the liquid in the liquid reservoir 140 may be collected by the microlens 110, captured by the objective lens 310, and imaged onto the optical sensor 350 through the optical path 330.

[0166] In some embodiments, after the optical signal of the signal group 202 is received by the optical sensor 350, the data processing device 40 can generate image information based on the signal and calculate the number of signal groups 202 per unit area (e.g., the field of view of a microlens 110) in the capture interface 130 based on the image information, thereby quantifying the amount of the analyte 210 and further calculating the concentration of the analyte 210. Since a "labeling reagent 200 - analyte 210 - first specific binding agent 131" sandwich is formed, the amount of the analyte 210 can be further calculated based on the number of signal groups 202.

[0167] The optical signal of the signal group 202 located on the sandwich conjugate can generate a stronger scattered light signal through the microlens 110, which is clearly and intuitively displayed as individual bright spots. This allows the signal groups 202 to be easily counted, for example, using existing image processing software such as ImageJ, thereby improving the sensitivity and specificity of the detection. In contrast, the signal group 202 in the labeling reagent 200 that is not located on the sandwich conjugate and is freely moving or free is difficult to continuously observe through the microlens 110 and can only be observed briefly when passing through the focusing range of the microlens.

[0168] For example, the position of each individual signal group 202 in a sequence of images per unit area of ​​the capture interface 130 can be tracked, thereby excluding freely moving or continuously dissociating signal groups 202 from the count range; this can eliminate the need for a rinse step in the detection and analysis method and achieve real-time counting of signal groups 202. Alternatively, the positional information of the signal groups 202 can be analyzed to further confirm whether each signal group 202 is specifically or nonspecifically interacting. Alternatively, the number of bright spots corresponding to the signal groups 202 in the initial image can be subtracted from the number of bright spots corresponding to the signal groups 202 in the subsequent image, thereby removing the signals from freely moving or continuously dissociating signal groups 202 (if any), and obtaining the count of signal groups 202 in the image. This can effectively achieve real-time detection of the concentration of the analyte 210, achieve a net count of signal groups 202, and improve the sensitivity and specificity of the detection. Alternatively, analysis of information such as the intensity and size of individual signal groups 202 can be performed to help eliminate interference from aggregated signal groups 202 or larger impurities, ensuring a net count of individual signal groups 202 bound at the chip capture interface. Moreover, these methods do not require image preprocessing such as filtering and noise reduction, and also avoid the shortcomings of existing digital detection and analysis methods, such as cumbersome operation and multiple incubation and washing steps.

[0169] The microlens chip 10 of the present invention, including the first specific binding agent 131, preferably in combination with a labeling reagent 210 capable of forming a sandwich conjugate therewith, eliminates the need for complex optical paths, high-numerical-aperture objective lenses, and tedious image processing for detection and analysis. This greatly simplifies the optical path system and image processing process, resulting in a simple, low-cost, and easily integrated optical device. For example, a white light source and a low-numerical-aperture objective lens can be used, which has great application prospects in clinical diagnosis.

[0170] In some embodiments, the microlens chip 10 of the present invention may include multiple first specific binding agents 131, for example, first specific binding agents 131 specific for different analytes 210. Combined with multiple labeling agents 210, for example, labeling agents 210 also specific for these different analytes 210, quantitative detection of different biomarkers can be achieved. This is of great significance in the early diagnosis and prevention of various diseases, such as cardiovascular disease, neurodegenerative disease, and cancer.

[0171] In some embodiments, the microlens chip 10 of the present invention may have multiple liquid reservoirs 140 , each providing a different first specific binding agent 131 . In some embodiments, the microlens chip 10 of the present invention may provide multiple different first specific binding agents 131 for one liquid reservoir 140 .

[0172] The technical solutions of the present invention are further described below with reference to embodiments.

[0173] Example

[0174] Preparation of microlens chip

[0175] Ultra-white glass with a size of 10 mm × 10 mm × 2 mm (length × width × height) was selected as the substrate, which was washed with anhydrous ethanol and deionized water in sequence, and then dried with nitrogen.

[0176] A suspension of barium titanate glass (BTG) microspheres is added dropwise to a clean glass substrate and dried at a predetermined temperature. As the solution evaporates, the BTG microspheres self-assemble on the glass substrate, forming a monolayer array of microspheres. The drying temperature is not limited, as long as it allows the solution to evaporate, and can be, for example, 50-90°C, preferably 70°C.

[0177] To further secure the microsphere array, a PDMS glue prepared by mixing and stirring a PDMS prepolymer and a PDMS curing agent in a specific mass ratio is spin-coated onto the glass substrate. The mass ratio of the PDMS prepolymer to the PDMS curing agent in the PDMS glue is not limited, as long as it allows for curing after baking; for example, this ratio ranges from 20:1 to 5:1, with a preferred ratio of 10:1.

[0178] After removing bubbles from the spin-coated PDMS glue, the PDMS is cured by baking at a certain temperature. The baking temperature is not limited as long as it can cure the PDMS, for example, 50-90 degrees, preferably 70 degrees.

[0179] Reservoir formation

[0180] The cleaned glass ring is tightly attached to the glass substrate prepared above by adhesive to form a liquid storage tank.

[0181] Preparation of capture interface

[0182] The surface of the microsphere lens-assisted chip manufactured above was cleaned using a plasma cleaner for several minutes.

[0183] Next, a 3-aminopropyltriethoxysilane (APTES) solution is added dropwise to the liquid reservoir formed on the microsphere lens-assisted chip, reacted at room temperature for several minutes (e.g., 15-60 minutes, preferably 30 minutes), rinsed with anhydrous ethanol, and dried with nitrogen gas; then, incubated at a certain temperature for several hours (e.g., at 50-90°C, preferably 70°C, for 0.5-2 hours, preferably 1 hour).

[0184] Glutaraldehyde (GA) solution is added dropwise into the reservoir, reacted at room temperature for several hours (eg, 0.5-2 hours, preferably 1 hour), then rinsed with phosphate buffered saline (PBS), and dried with nitrogen gas.

[0185] The capture antibody specific to the analyte is added dropwise into the reservoir, reacted at room temperature for several hours (eg, 6-24 hours), and then rinsed with deionized water and dried with nitrogen.

[0186] Bovine serum albumin blocking buffer (BSA-BB) was added dropwise to the reservoir of the modified capture antibody, incubated at room temperature, and then rinsed with deionized water to obtain a capture interface.

[0187] Preparation of labeling reagents

[0188] An aqueous solution of gold nanoparticles (AuNPs) of a certain size (e.g., 40-150 nm, preferably 50-100 nm, preferably 80 nm) is mixed with an excess amount (e.g., 10-1000 times, preferably 100 times on a molar basis) of polyethylene glycol (PEG) linker and stirred continuously at room temperature for several minutes.

[0189] Add the detection antibody specific to the analyte to the above solution and incubate at a certain temperature for several hours (eg, 6-24 hours).

[0190] Add bovine serum albumin blocking buffer and react at room temperature for several minutes.

[0191] The obtained solution was centrifuged at room temperature for several minutes, the supernatant was discarded, and the solution was washed several times with deionized water. The obtained solid was resuspended in deionized water to obtain a labeling reagent.

[0192] Biomarker testing

[0193] A certain volume of sample solution containing the analyte is mixed with a certain volume of labeling reagent (the volume ratio of sample solution to labeling reagent is, for example, 1:10, preferably 1:5), and reacted at room temperature for several minutes (for example, 5-15 minutes, preferably 10 minutes) to form an "analyte-labeling reagent" complex.

[0194] Subsequently, the microlens chip was placed on the microscope observation table, and the above-mentioned complex solution was dripped into the liquid reservoir of the chip. The process of the sandwich complex being captured at the chip capture interface was observed using an ordinary optical microscope (LED white light source, bright field imaging method, and objective lens with a magnification of 40 times and NA 0.55).

[0195] Data collection and analysis

[0196] FIG7 is a flow chart showing a method 500 for collecting image information of the microscope imaging and performing processing and analysis.

[0197] In step 510, images of the detection area are captured at certain time intervals to obtain an image sequence consisting of multiple frames of images continuously captured within the capture time.

[0198] A CMOS camera may be used to collect image information in real time, and for example, the detection area may be collected for 20 minutes at a time interval of 2 seconds and an exposure time of 2 ms, thereby obtaining an image sequence including 600 frames of images.

[0199] In step 520 , signal groups 202 in the image are identified.

[0200] The recognized image can be a frame of an image acquired by image capture or a partial region thereof, for example, the field of view of a single microlens 110. The partial region can be identified by cutting out the partial region, for example, using software such as Image J.

[0201] In step 530, the specifically bound signal groups 202 in the image are determined and counted. The "specifically bound state" herein refers, for example, to the signal group 202 specifically binding to the analyte 210 via the second specific binding agent 201, while the same analyte 210 is also bound by the first specific binding agent 131.

[0202] By tracking the position information of the specific signal group 202 in the continuous image sequence, the movement trajectory, or binding and dissociation events of the specific signal group 202 can be identified, and it can be determined whether the signal group 202 in the image is in a specific binding state, a nonspecific binding state, or an unbound state.

[0203] For example, the signal group 202 in the unbound state (including freely moving and non-specifically bound signal groups) may move freely in the form of Brownian motion, while the signal group 202 in the specific or non-specific binding state may not move freely. The signal group in the unbound state can be excluded from the counting of the signal group 202.

[0204] The positions of the signal groups 202 in the preceding and following frames can be compared. For example, when a signal group 202 is detected, preferably a signal group 202 that has not moved in the previous plurality of consecutive images, and disappears in the next frame, it indicates that the signal group 202 may have undergone a dissociation event, and the signal group 202 is included in the count of the signal groups 202. Conversely, when a signal group 202 is detected that does not appear in one frame but appears in the next frame, preferably without moving in the subsequent plurality of consecutive images, it indicates that the signal group 202 may have undergone a binding event, and the signal group 202 is included in the count of the signal groups 202.

[0205] Through the modified microlens array of the present invention, freely moving or free signal groups 202 can only be observed briefly when passing through the focusing range of the microlens. Therefore, these freely moving or free signal groups 202 can be identified particularly easily by comparing continuous image sequences, thereby screening out signal groups 202 in an unbound state or a non-specifically bound state.

[0206] Alternatively, the number of signal bright spots corresponding to the signal group 202 in the initial image may be simply subtracted from the number of signal bright spots corresponding to the signal group 202 in the subsequent image to obtain the signal group 202 count in the image.

[0207] The signal can be analyzed by focusing on its intensity and size. Specifically, the signal can be compared with the average signal intensity and size of signals that are likely to belong to the majority of signal groups 202 in the image to identify whether the signal belongs to a signal group 202. This helps eliminate interference from aggregated signal groups 202 or larger impurities, ensuring a net count of individual signal groups 202 bound at the chip capture interface.

[0208] The Trackmate plug-in of Image J can be used to track the position information of the signal group 202, or to analyze the binding and non-dissociation events, or to analyze the intensity and size of individual signals.

[0209] In step 540, the relationship between the signal group 202 count and time is obtained based on the signal group 202 counts in each image. At different analyte concentrations, the relationship between the signal group count and time at each concentration can be obtained based on each acquired image sequence.

[0210] In step 550, a representative signal group 202 count is obtained based on the relationship between the signal group 202 count and time. The representative signal group 202 count can be, for example, the signal group 202 count at a specific image acquisition time point, or the signal group 202 count when the signal group 202 count reaches equilibrium or is below a specific growth ratio.

[0211] In step 560 , a standard curve is established based on the relationship between the counts of the representative signal groups 202 and the concentration of the analyte 210 for quantitative analysis of the concentration of the analyte 210 .

[0212] The standard curve can be established, for example, by obtaining representative signal group 202 counts of multiple samples with known concentrations of the analyte 210 according to steps 510 to 550, and performing regression analysis based on the representative signal group 202 counts of these concentrations to obtain a standard curve.

[0213] The present invention will be further described in detail below in conjunction with Examples and Comparative Examples. This section further illustrates content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this area.

[0214] The BTG microspheres in the following examples were purchased from: Microspheres-Nanospheres (Cold Spring, New York, USA)

[0215] Cardiac troponin I (cTnI) was purchased from Wuxi OriGene Biotechnology Co., Ltd. (OriGene China)

[0216] cTnI capture antibody (D021) was purchased from Wuxi OriGene Biotechnology Co., Ltd. (OriGene China)

[0217] PDMS prepolymer and PDMS curing agent were purchased from Sigma-Aldrich

[0218] Example 1

[0219] The microlens chip of the present invention was used in conjunction with an optical device to observe the imaging of 80 nm AuNPs on the capture interface and detect 1 ng / mL cardiac troponin I (cTnI). The specific experimental steps are as follows:

[0220] (1) Chip production

[0221] First, a glass substrate was cleaned with anhydrous ethanol and deionized water, followed by nitrogen drying. Then, 30 μL of a 52 μm BTG microsphere suspension (dissolved in water) was dropped onto the cleaned glass substrate and dried at 70°C for 10 minutes. As the solution evaporated, the BTG microspheres self-assembled on the glass substrate, forming a monolayer microsphere array. To further secure the microsphere array, PDMS glue, prepared by mixing a 10:1 mass ratio of PDMS prepolymer and a PDMS curing agent, was spin-coated at 1000 rpm for 18 seconds and then at 5000 rpm for 5 minutes. After removing air bubbles from the spin-coated PDMS glue, the resulting glass substrate was cured at 70°C for 1 hour. A cleaned glass ring was then tightly bonded to the BTG microsphere-immobilized glass substrate using PDMS glue to form a reservoir, thereby fabricating a BTG microsphere-assisted chip. The solution was then stored at room temperature (25°C) until further use.

[0222] (2) Capture interface preparation

[0223] First, the BTG microsphere-assisted chip prepared above was treated with a plasma cleaner for 2 min at 30W to make the surface of the chip carry -Si(OH) x group; then, 100 μL, 5% APTES (v / v, dissolved in anhydrous ethanol) was added dropwise to the above-mentioned reservoir, reacted at room temperature (25°C) for 30 minutes, rinsed the surface with anhydrous ethanol three times to remove excess APTES, and dried with nitrogen; then, the above-mentioned glass slide was placed in an oven and incubated for 1 hour at 70°C; then, 100 μL, 2.5% glutaraldehyde (v / v, dissolved in 1×PBS, pH 7.2) was added dropwise to the above-mentioned reservoir, reacted at room temperature for 1 hour, rinsed with PBS, and dried under N2; then, 40 μL, 50 μg / mL cTnI capture antibody (D021, dissolved in 1×PBS, pH 7.2) was added dropwise to the above-mentioned reservoir and incubated overnight; then, the above-mentioned reservoir was rinsed with deionized water and dried under N2; finally, 50 μL of 2% BSA (v / v, dissolved in deionized water) was added dropwise to the above-mentioned reservoir of modified capture antibody to prevent nonspecific adsorption of proteins; finally, the modified capture interface was stored at 4°C for subsequent cTnI detection.

[0224] (3) cTnI detection

[0225] A 10 μL sample solution of 1 ng / mL cTnI (dissolved in 1× PBS, pH 7.2) was mixed with 50 μL of the prepared labeling reagent and gently stirred at room temperature for 10 minutes. The glass slide with the prepared capture interface was then placed on a microscope observation stage, and the mixed solution was dripped onto the capture interface of the chip. The binding of AuNPs to the capture interface surface was observed using a 40×, NA 0.55 air objective lens, and image data was captured using a CMOS sensor, as shown in Figure 8.

[0226] As can be seen from Figure 8 , a large number of single AuNPs were observed to bind to the capture interface surface within the focusing range of the microspheres.

[0227] Comparative Example 1

[0228] The capture interface and microlens were placed on opposite sides of the reservoir, and an optical device was used to observe the imaging of 80nm AuNPs on the capture interface to detect 1ng / mL cTnI. The specific experimental steps are as follows:

[0229] (1) Fabrication of thin films containing microsphere lenses

[0230] First, a glass substrate was cleaned with anhydrous ethanol and deionized water, followed by nitrogen drying. Then, 30 μL of a 52 μm BTG microsphere suspension (dissolved in water) was dropped onto the cleaned glass substrate and dried at 70°C for 10 minutes. As the solution evaporated, the BTG microspheres self-assembled on the glass substrate, forming a monolayer microsphere array. To further secure the microsphere array, a PDMS glue (a mixture of a PDMS prepolymer and a curing agent in a 10:1 mass ratio) was spin-coated at 1000 rpm for 18 seconds and then at 5000 rpm for 5 minutes. After removing air bubbles from the spin-coated PDMS glue, the resulting glass substrate was cured at 70°C for 1 hour. Finally, the PDMS film containing the BTG microspheres was peeled from the glass substrate, completing the microsphere lens film, which was stored at room temperature until further use.

[0231] (2) Capture interface preparation

[0232] First, the glass ring was fixed on a clean glass sheet to form a liquid reservoir; then, plasma treatment was performed for 2 min at 30W to make the surface of the ring carry -Si(OH) xThen, 100 μL of 5% APTES (v / v, dissolved in anhydrous ethanol) was added dropwise to the above-mentioned reservoir, reacted at room temperature for 30 minutes, washed with anhydrous ethanol to remove excess unbound APTES, and dried under N2; then, the above-mentioned glass slide was placed in an oven and incubated for 1 hour at 70°C; then, 100 μL of 2.5% glutaraldehyde (v / v, dissolved in PBS) was added dropwise to the above-mentioned reservoir, reacted at room temperature for 30 minutes, and rinsed with PBS. , dried under N2; then, 40 μL, 50 μg / mL cTnI capture antibody (D021) was added dropwise to the above reservoir and incubated overnight; then, the above reservoir was rinsed with deionized water and dried under N2; finally, 50 μL, 2% BSA (v / v, dissolved in deionized water) was added dropwise to the above modified capture antibody reservoir, incubated at room temperature for 1 hour, rinsed with deionized water, and dried under N2 to prepare the chip capture interface, which was stored at 4°C for future use.

[0233] (3) cTnI detection

[0234] A 10 μL, 1 ng / mL cTnI sample solution was mixed with 50 μL of the prepared labeling reagent and gently stirred at room temperature for 10 minutes. Subsequently, the mixed solution was added dropwise to a reservoir, and a film containing microsphere lenses was placed on the reservoir. The binding of AuNPs to the reservoir capture interface surface was observed under a 40×, NA 0.55 air objective lens, and image information was captured by CMOS, as shown in Figure 9.

[0235] As can be seen from Figure 9 , within the focusing range of the microspheres, almost no binding of AuNPs on the capture interface was observed.

[0236] Comparative Example 2

[0237] Using a liquid reservoir without a microlens, an optical setup was used to observe the imaging of 80nm AuNPs at the capture interface and detect 1ng / mL cTnI. The specific experimental steps are as follows:

[0238] (1) Capture interface preparation

[0239] First, a glass ring was fixed on a clean glass sheet as the side wall of the reservoir to form a reservoir; then, it was plasma treated for 2 minutes at 30W to make the surface of the ring carry -Si(OH) xThen, 100 μL of 5% APTES (v / v, dissolved in anhydrous ethanol) was added dropwise to the above-mentioned reservoir, reacted at room temperature for 30 minutes, washed with anhydrous ethanol to remove excess unbound APTES, and dried under N2; then, the above-mentioned glass slide was placed in an oven and incubated for 1 hour at 70°C; then, 100 μL of 2.5% glutaraldehyde (v / v, dissolved in PBS) was added dropwise to the above-mentioned reservoir, reacted at room temperature for 30 minutes, and washed with PBS. Rinse and dry under N2; then, 40 μL, 50 μg / mL cTnI capture antibody (D021) was added dropwise to the above reservoir and incubated overnight; then, the above reservoir was rinsed with deionized water and dried under N2; finally, 50 μL, 2% BSA (v / v, dissolved in deionized water) was added dropwise to the above modified capture antibody reservoir, incubated at room temperature for 1 hour, rinsed with deionized water, dried under N2, thereby preparing the capture interface and storing at 4°C for future use.

[0240] (2) cTnI detection

[0241] A 10 μL, 1 ng / mL cTnI sample solution was mixed with 50 μL of the prepared labeling reagent and gently stirred at room temperature for 10 minutes. Subsequently, the glass slide with the prepared capture interface was placed on a microscope observation stage, and the mixed solution was dripped onto the capture interface. The binding of AuNPs to the capture interface surface was observed using a 40×, NA 0.55 air objective lens, and image data was captured using a CMOS sensor, as shown in Figure 10.

[0242] As can be seen from Figure 10, almost no binding of AuNPs on the capture interface was observed.

[0243] Furthermore, it can be seen from Example 1 and Comparative Examples 1-2 that the microlens chip of the present invention can be used in conjunction with an optical device to observe the imaging of 80 nm AuNPs on the capture interface to detect cardiac troponin I (cTnI).

[0244] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A modified microlens array, characterized in that: include: multiple microlenses; and a capture interface formed on a surface of a first side of the plurality of microlenses and comprising: The first specific binding agent is capable of specifically binding to the analyte.

2. A method for preparing a modified microlens array, characterized in that: The following steps are involved: providing a plurality of micro lenses; and forming a capture interface, the capture interface being located on a surface of a first side of the microlens and comprising: The first specific binding agent is capable of specifically binding to the analyte.

3. The modified microlens array or the method for preparing the same according to claim 1 or 2, wherein: The analyte includes an antigen, and the first specific binding agent includes an antibody or an aptamer; Preferably, the analytes include cardiac troponin I, myoglobin, creatine kinase isoenzymes, C-reactive protein, N-terminal pro-brain natriuretic peptide, alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, carbohydrate antigen 19-9, β-amyloid protein, serum amyloid A, monocyte chemoattractant protein, human monocyte chemoattractant protein, matrix metalloproteinases, tumor necrosis factor α, vascular endothelial growth factor, transforming growth factor, interferon, procalcitonin, viral nucleocapsid protein p24, growth-associated protein, glial fibrillary acidic protein , neurofilament light chain protein, angiogenin, interleukin, human epididymis protein 4, α-L-fucosidase, S100 calcium binding protein, alveolar protein, neuron-specific enolase, squamous cell carcinoma antigen, lipoprotein-associated phospholipase, heart-type fatty acid binding protein, D-dimer, human brain semaphorin 4D, human growth differentiation factor 15, exosomal synaptophysin, exosome surface protein, α2-macroglobulin, angiogenic factor family, fibroblast growth factor 7, brain-derived neurotrophic factor, bone morphogenetic protein 9 , C-chain insulin, CD-14 protein, chemokine protein family, chemokine ligand, human cytokine, epidermal growth factor family, endoglin, eosinophil chemotactic protein, apoptosis-related factor ligand, fibrinogen, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, glycoprotein 130, granzyme B protein, I309 / CCL-1 protein, IgE protein, insulin, interferon-induced protein 10, neuropsychiatric disease-related markers, Osteoprotegerin, osteopontin, PD-1 protein, PD-L1 protein, platelet-derived growth factor, placental growth factor, receptor for advanced glycation end products, receptor activator of nuclear factor-κB ligand, SARS-CoV-2 protein, SARS-CoV-2 spike protein, stem cell factor, 25kDa synaptosomal-associated protein, tumor necrosis factor receptor type II, amino acid kinase receptor-2, tissue inhibitor of metalloproteinases, or thrombospondin 2, and the first specific binding agent comprises an antibody or aptamer thereof; Preferably, the matrix metalloproteinases include MMP-2, MMP-3, or MMP-9; Preferably, the angiogenic factor family includes ANG-2, FGFB, HB-EGF, HGF, PLGF, or VEGF; Preferably, the chemokine protein family includes IP-10, ITAC, MCP-1, MIP-3B, IL-8, MIP-3β / CCL19, MIP-3α / CCL20, MIP-4 / CCL18, RANTES / CCL5, SDF-1 / CXCL12, CXCL9, MDC, MIP-2, MIP-1α, or MIP-1β; Preferably, the chemokine ligand comprises CXCL 1, CXCL 11, or CXCL 13; Preferably, the human cytokines include IFN-γ, IL-1β, IL-2, IL-6, IL-10, KC, or TNF-α; Preferably, the epidermal growth factor family includes epidermal growth factor, heparin-binding epidermal growth factor-like growth factor, or epiregulin; Preferably, the neuropsychiatric disease-related markers include tau protein, β-amyloid 40, β-amyloid 42, neurofilament protein, glial fibrillary acidic protein, ubiquitin carboxyl-terminal hydrolase L1, or TDP-43 protein; Preferably, the tissue inhibitor of metalloproteinases comprises TIMP-1, TIMP-2, or TIMP-3; Preferably, the analyte comprises an antibody or an aptamer, and the first specific binding agent comprises an antigen.

4. The modified microlens array or the method for preparing the same according to any one of claims 1 to 3, wherein: The material used for the microlens includes or consists of a dielectric material. Preferably, the material used for the microlens includes barium titanate, polystyrene, arsenic trisulfide, arsenic triselenide, lanthanide optical glass material, barium oxide, silicon oxide, titanium oxide, zinc oxide, or a mixture of two or more of barium oxide, silicon oxide, titanium oxide, and zinc oxide, preferably barium titanate; and / or The material used for the microlens includes or consists of a high molecular polymer material. Preferably, the material used for the microlens includes a sulfur-containing polymer, preferably polythiourea, or preferably a selenium-containing polymer; Preferably, the microlens is a spherical microlens; Preferably, the microlens is a spherical microlens; Preferably, the microlens is a hemispherical microlens; Preferably, the microlens is a cylindrical lens; Preferably, the microlens is an aspherical microlens, preferably an ellipsoidal microlens, a parabolic microlens or a hyperbolic microlens.

5. A microlens chip, characterized in that: include: substrate; as well as The modified microlens array according to any one of claims 1 and 3 to 4, wherein the microlenses are arranged on the substrate.

6. The microlens chip according to claim 5, wherein: The microlens chip further includes a liquid reservoir located on the capture interface; and / or The microlens chip further includes a coating layer disposed on the substrate and surrounding the microlens; Preferably, the microlens chip further comprises a liquid reservoir wall formed on the microlens and / or the optional coating layer, and the space surrounded by the liquid reservoir wall and the microlens and / or the optional coating layer serves as the liquid reservoir; Preferably, the capture interface is formed on the surface of the first side of the microlens and optionally the coating layer; Preferably, the substrate is a transparent substrate, preferably comprising polydimethylsiloxane, glass, quartz, or silicon wafer, more preferably comprising glass; Preferably, each of the microlenses is arranged in a hexagonal stacking pattern on a plane above the substrate; Preferably, the material used for the coating layer includes a polymer material, preferably a polymer resin material, more preferably polydimethylsiloxane or epoxy resin, more preferably polydimethylsiloxane, or consists of the same; Preferably, forming the capture interface comprises: contacting the surface of the first side of the microlens and the optional coating layer with a 3-aminopropyltriethoxysilane solution to obtain a first modified surface; contacting the first modified surface with a glutaraldehyde solution to obtain a second modified surface; contacting the second modified surface with the first specific binding agent; and preferably, blocking the capture interface obtained after contacting the second modified surface with the first specific binding agent, preferably, using serum protein, more preferably bovine serum albumin.

7. A method for preparing a microlens chip, characterized in that: include: Setting the base; as well as The modified microlens array is prepared according to the method according to any one of claims 2 to 4, wherein the microlenses are arranged on the substrate.

8. The method for preparing a microlens chip according to claim 7, wherein: Also includes: Disposing a liquid reservoir on the capture interface; and / or Disposing a coating layer on the substrate so that the coating layer surrounds the microlens; Preferably, the method further comprises forming a liquid reservoir wall on the microlens and / or the optional coating layer, wherein the space surrounded by the liquid reservoir wall and the capture interface is the liquid reservoir; Preferably, the capture interface is formed on the surface of the first side of the microlens and optionally the coating layer; Preferably, the substrate is a transparent substrate, preferably comprising polydimethylsiloxane, glass, quartz, or silicon wafer, more preferably comprising glass; Preferably, each of the microlenses is arranged in a hexagonal stacking pattern on a plane above the substrate; Preferably, the material used for the coating layer includes a polymer material, preferably a polymer resin material, more preferably polydimethylsiloxane or epoxy resin, more preferably polydimethylsiloxane, or consists of the same; Preferably, forming the capture interface comprises: contacting the surface of the first side of the microlens and the optional coating layer with a 3-aminopropyltriethoxysilane solution to obtain a first modified surface; contacting the first modified surface with a glutaraldehyde solution to obtain a second modified surface; contacting the second modified surface with a first specific binding agent; and preferably, blocking the capture interface obtained after contacting the second modified surface with the first specific binding agent, preferably using serum protein, more preferably bovine serum albumin.

9. A detection and analysis kit, characterized in that: include: The modified microlens array according to any one of claims 1 and 3 to 4, or the microlens chip according to claim 5 or 6; and The labeling reagent includes a second specific binding agent and a signal group connected to the second specific binding agent.

10. A method for preparing a detection and analysis kit, characterized in that: include: Prepare a modified microlens array according to the preparation method according to any one of claims 2 to 4, or prepare a microlens chip according to the method according to claim 7 or 8; and Prepare a labeling reagent, the labeling reagent comprising: a second specific binding agent capable of specifically binding to the analyte; and A signal group is connected to the second specific binding agent.

11. A detection and analysis method, characterized in that: include: contacting the object to be tested with the capture interface of the modified microlens array according to any one of claims 1 and 3 to 4, or the microlens chip according to claim 5 or 6; as well as The labeling reagent is contacted with the analyte, wherein the labeling reagent comprises: a second specific binding agent capable of specifically binding to the analyte; and A signal group is connected to the second specific binding agent.

12. The modified microlens array, preparation method, or detection and analysis method according to any one of claims 9 to 11, wherein: The analyte comprises an antigen, and the second specific binding agent comprises an antibody or an aptamer; Preferably, the analytes include cardiac troponin I, myoglobin, creatine kinase isoenzymes, C-reactive protein, N-terminal pro-brain natriuretic peptide, alpha-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, carbohydrate antigen 19-9, β-amyloid protein, serum amyloid A, monocyte chemoattractant protein, human monocyte chemoattractant protein, matrix metalloproteinases, tumor necrosis factor α, vascular endothelial growth factor, transforming growth factor, interferon, procalcitonin, viral nucleocapsid protein p24, growth-associated protein, glial fibrillary acidic protein , neurofilament light chain protein, angiogenin, interleukin, human epididymis protein 4, α-L-fucosidase, S100 calcium binding protein, alveolar protein, neuron-specific enolase, squamous cell carcinoma antigen, lipoprotein-associated phospholipase, heart-type fatty acid binding protein, D-dimer, human brain semaphorin 4D, human growth differentiation factor 15, exosomal synaptophysin, exosome surface protein, α2-macroglobulin, angiogenic factor family, fibroblast growth factor 7, brain-derived neurotrophic factor, bone morphogenetic protein 9 , C-chain insulin, CD-14 protein, chemokine protein family, chemokine ligand, human cytokine, epidermal growth factor family, endoglin, eosinophil chemotactic protein, apoptosis-related factor ligand, fibrinogen, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, glycoprotein 130, granzyme B protein, I309 / CCL-1 protein, IgE protein, insulin, interferon-induced protein 10, neuropsychiatric disease-related markers, Osteoprotegerin, osteopontin, PD-1 protein, PD-L1 protein, platelet-derived growth factor, placental growth factor, receptor for advanced glycation end products, receptor activator of nuclear factor-κB ligand, SARS-CoV-2 protein, SARS-CoV-2 spike protein, stem cell factor, 25kDa synaptosomal-associated protein, tumor necrosis factor receptor type II, amino acid kinase receptor-2, tissue inhibitor of metalloproteinases, or thrombospondin 2, and the second specific binding agent includes an antibody or aptamer thereof; Preferably, the matrix metalloproteinases include MMP-2, MMP-3, or MMP-9; Preferably, the angiogenic factor family includes ANG-2, FGFB, HB-EGF, HGF, PLGF, or VEGF; Preferably, the chemokine protein family includes IP-10, ITAC, MCP-1, MIP-3B, IL-8, MIP-3β / CCL19, MIP-3α / CCL20, MIP-4 / CCL18, RANTES / CCL5, SDF-1 / CXCL12, CXCL9, MDC, MIP-2, MIP-1α, or MIP-1β; Preferably, the chemokine ligand comprises CXCL 1, CXCL 11, or CXCL 13; Preferably, the human cytokines include IFN-γ, IL-1β, IL-2, IL-6, IL-10, KC, or TNF-α; Preferably, the epidermal growth factor family includes epidermal growth factor, heparin-binding epidermal growth factor-like growth factor, or epiregulin; Preferably, the neuropsychiatric disease-related markers include tau protein, β-amyloid 40, β-amyloid 42, neurofilament protein, glial fibrillary acidic protein, ubiquitin carboxyl-terminal hydrolase L1, or TDP-43 protein; Preferably, the tissue inhibitor of metalloproteinases comprises TIMP-1, TIMP-2, or TIMP-3; Preferably, the analyte comprises an antibody or an aptamer, and the second specific binding agent comprises an antigen.

13. The modified microlens array, preparation method, or detection and analysis method according to any one of claims 9 to 12, wherein: The signal group is a micron or nanoparticle; Preferably, the signal group is a metal nanoparticle, quantum dot, carbon dot, upconversion nanoparticle, semiconductor nanoparticle, silica nanoparticle, organic polymer luminescent particle, or metal organic framework nanoparticle, preferably gold, silver, copper or palladium nanoparticle, more preferably gold nanoparticle, or preferably alloy nanoparticle; Preferably, the signal group is a fluorescent nanoparticle.

14. A detection and analysis device, characterized in that: include: The modified microlens array according to any one of claims 1 and 3 to 4; The microlens chip according to claim 5 or 6; or The detection and analysis kit according to any one of claims 9 and 12 to 13, Preferably, the detection and analysis device further comprises an optical device, and the optical device comprises an objective lens; Preferably, the optical device further comprises an optical path, a light source, and an optical detector, wherein the optical path optically connects the objective lens, the light source, and the optical detector; Preferably, the optical detector is electrically connected to a data processing device, and the data processing device is used to process data received by the optical detector; Preferably, the optical device further comprises a moving device, wherein the moving device is connected to the objective lens or the modified microlens array and is used to move the objective lens or the modified microlens array to adjust the distance between the objective lens and the modified microlens array; Preferably, the light source is an LED light source, a halogen light source, a laser light source, or a xenon light source, preferably an LED white light source.

15. Use of the modified microlens array according to any one of claims 1 and 3 to 4, or the modified microlens array prepared by the method according to any one of claims 2 to 4, in detection and analysis, preparation of a microlens chip, preparation of a detection and analysis kit, or preparation of a detection and analysis device; Use of the microlens chip according to claim 5 or 6, or the microlens chip prepared according to the method of claim 7 or 8, in detection and analysis, preparation of a detection and analysis kit, or preparation of a detection and analysis device; Use of the detection and analysis kit according to any one of claims 9 and 12 to 13, or the detection and analysis kit prepared according to the method according to any one of claims 10 and 12 to 13, in detection and analysis, or in preparing a detection and analysis device; And / or, use of the detection and analysis device as claimed in claim 14 in detection and analysis.

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