Micro-electric biochip with cell-like three-dimensional topological structure as well as preparation method and application of micro-electric biochip

Abstract

The invention discloses a micro-electric biochip with a cell-like three-dimensional topological structure as well as a preparation method and application of the micro-electric biochip. The micro-electric biochip comprises a concave-convex fluctuant cell-like three-dimensional topological structure, a metal conducting layer arranged on the surface of the cell-like three-dimensional topological structure, and at least one first binding sample solidified on the surface of the metal conducting layer with the cell-like three-dimensional topological structure, the first binding sample can be specifically bound with or dissociated from the second binding sample. According to a pure electrical detection principle, a cell-like three-dimensional topological structure is introduced to the surface of the biological chip, so that a biological microenvironment in which interaction is generated among biological molecules is more truly reduced, the anti-interference capability of a detection system is comprehensively improved, and the detection accuracy is improved. And moreover, the limitation that traditional SPR can only characterize the interaction between biomolecules on a two-dimensional plane is solved, and the method can be applied to the fields of interaction force of biomolecules, interaction force between cells or viruses and proteins, drug research and development and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biochip and detection technology, and relates to a micro-electric biochip with a cell-like three-dimensional topological structure and a preparation method and application thereof. Background Art

[0002] Surface Plasmon Resonance (SPR) is an optical, label-free detection technology that can be used to detect the binding and dissociation processes between two or more molecules in real time. The throughput, flexibility and sensitivity of the SPR instrument platform enable researchers to characterize the interactions of biomolecules in binding studies, and can be widely applied to different molecules, such as ions, molecular fragments, small molecules, proteins, viruses, etc. SPR technology can provide information such as the kinetics of binding (binding rate, dissociation rate) and affinity.

[0003] Surface plasmon is a collective oscillation phenomenon of free electrons, which usually occurs on the surface of metals. When light waves are irradiated on the metal surface, the free electrons in the metal interact with the electric field of the light waves to form an electromagnetic wave, which is called a surface plasmon wave. The resonance conditions of the surface plasmon wave depend on the thickness and refractive index of the metal layer, as well as the wavelength and angle of the incident light. The core application of SPR is to use the surface plasmon resonance phenomenon on metal surfaces (usually gold or silver) to detect molecular binding / dissociation events.

[0004] The working principle of the detection system based on SPR technology is as follows: SPR sensors usually consist of a transparent prism and a metal layer (usually gold or silver). When a beam of polarized light hits the interface between the prism and the metal layer, part of the light energy will penetrate the metal layer and excite surface plasmon waves. This resonance phenomenon will cause the reflected light intensity to change. In SPR sensors, there is usually a flow cell that allows the sample solution to flow over the metal surface. In biological detection applications, a biological molecule (such as a receptor) is first fixed on the surface of a metal film. When a solution containing another biological molecule that interacts with it (such as a ligand) flows over the surface, if the two bind / dissociate, this change will cause the refractive index near the metal surface to change, thereby affecting the resonance conditions of the surface plasmon waves. By detecting parameters such as the angle and intensity of the reflected light, slight changes in the resonance conditions can be obtained, and then the interaction between the biological molecules immobilized on the surface of the metal film of the SPR chip and the molecules to be tested in the solution can be inferred, that is, the binding kinetics (binding rate, dissociation rate) and affinity information can be detected.

[0005] SPR technology can accurately measure the affinity of molecules and their kinetic parameters without chemically labeling the molecules, thus avoiding the potential impact of labeling on the molecular structure and activity. This makes SPR a core tool in drug development for screening and verifying candidate drugs with high affinity to target molecules.

[0006] SPR technology is mainly used to detect the following substances and interactions, such as interactions between biomolecules, interactions between biomolecules and other substances, interactions between cells or viruses and proteins, etc. In addition, the role of SPR technology in drug development is mainly reflected in the confirmation and verification of drug targets, drug screening, drug affinity and kinetic characterization, drug optimization and selection of lead compounds, drug toxicity prediction, drug quality control, etc.

[0007] However, the underlying principle of SPR technology has determined that this technology can only characterize the interactions between biomolecules on a two-dimensional plane without labeling, with high sensitivity and in real time, and cannot detect the interactions between biomolecules on the cell surface with a three-dimensional topological structure. In other words, there may be major loopholes in the evaluation criteria of SPR detection, which cannot accurately characterize the interactions between biomolecules on the cell surface and correctly evaluate them. For example, in SPR detection, the candidate drug molecules to be tested and the target protein may show a slower binding rate and a faster dissociation rate on the flat SPR metal film surface. The traditional SPR interpretation of the results will be considered to be poor efficacy, but in the real in vivo microenvironment, that is, on the three-dimensional cell surface with a specific topological structure, the candidate drug molecules to be tested and the target protein may show extremely strong affinity.

[0008] Therefore, SPR technology has a major limitation in drug screening, that is, although SPR provides accurate kinetic data in detecting molecular interactions, it ignores the complexity of the three-dimensional biological environment, which may lead to deviations in its efficacy prediction. Summary of the invention

[0009] The main purpose of the present invention is to provide a micro-electric biochip with a cell-like three-dimensional topological structure, which can simulate the cell surface topological structure and be used to characterize the interaction between biomolecules to overcome the shortcomings of the prior art.

[0010] Another object of the present invention is to provide a method for preparing the micro-electric biochip having a cell-like three-dimensional topological structure and its application.

[0011] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes: The embodiments of the present invention provide a micro-electric biochip with a cell-like three-dimensional topological structure for use in the fields of characterizing the interaction force between two biological molecules, characterizing the interaction force between biological molecules and other substances, characterizing the interaction force between cells or viruses and proteins, and drug research and development.

[0012] The embodiment of the present invention further provides a micro-electric biochip having a cell-like three-dimensional topological structure, which includes: The undulating three-dimensional cell-like topology; A metal conductive layer disposed on the surface of the cell-like three-dimensional topological structure; At least one first binding sample is solidified on the surface of the metal conductive layer having a cell-like three-dimensional topological structure, and the first binding sample can specifically bind to or dissociate from the second binding sample.

[0013] The embodiment of the present invention also provides a method for preparing a micro-electric biochip having a cell-like three-dimensional topological structure, which comprises: Through photolithography, deposition and etching techniques, a cell-like three-dimensional topological structure with uneven surfaces is prepared. Depositing a metal conductive layer on the surface of the cell-like three-dimensional topological structure to form a metal conductive layer having a cell-like three-dimensional topological structure; At least one first binding sample is solidified on the surface of the metal conductive layer having the cell-like three-dimensional topological structure.

[0014] The embodiment of the present invention further provides a sample detection system, which includes: The aforementioned micro-electric biochip with a cell-like three-dimensional topological structure; An electrical connection component, electrically connected to the micro-electric biochip having a cell-like three-dimensional topological structure, at least for real-time monitoring of changes in electrical properties of the micro-electric biochip; The microfluidic channel is correspondingly arranged above the detection area of ​​the micro-electric biochip with the cell-like three-dimensional topological structure, and is at least used to pass the microfluid containing the second binding sample to be detected.

[0015] Accordingly, an embodiment of the present invention further provides a sample detection method, which includes: Passing the microfluid containing the second binding sample to be tested into the detection area of ​​the micro-electric biochip with a cell-like three-dimensional topological structure in the aforementioned sample detection system, specifically binding or dissociating with the first binding sample solidified on the surface of the metal conductive layer, causing changes in the electrical properties of the micro-electric biochip; The electrical connection component is used to monitor the changes in the electrical properties of the micro-electric biochip in real time, and to determine whether the second binding sample to be tested has specifically bound or dissociated with the first binding sample, thereby achieving sample detection.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention uses a purely electrical detection principle (no longer based on optical imaging technology) to introduce a cell-like three-dimensional topological structure on the surface of a biochip, and more realistically restores the biological microenvironment (such as the cell surface) where biological molecules interact with each other. It not only comprehensively improves the anti-interference ability of the detection system (environmental factors, such as vibration, will have a great impact on the light-based SPR system), but also solves the limitation that traditional SPR can only characterize the interaction between biological molecules on a two-dimensional plane. It can be applied to the characterization of the interaction force between two biological molecules, the characterization of the interaction force between biological molecules and other substances, the characterization of the interaction force between cells or viruses and proteins, and drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the accompanying drawings of the present invention will be briefly introduced below. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0018] Figure 1A This is a schematic diagram of unactivated free platelets; Figure 1B is a schematic diagram of platelets in the adherent state after activation; Figure 2 It is a quantitative relationship diagram between the expression level of PS and the specific binding of Lactadherin and Annexin V to it; Figure 3A This is a schematic diagram of PS expression on the platelet surface under mild stimulation; Figure 3B This is a schematic diagram of PS expression on the platelet surface under strong stimulation; Figure 4A and Figure 4B It is a schematic structural diagram of a micro-electric biochip having a cell-like three-dimensional topological structure in a typical embodiment of the present invention; Figure 5A and Figure 5B is a schematic structural diagram of another micro-electric biochip having a cell-like three-dimensional topological structure in a typical embodiment of the present invention; Figure 6 It is a schematic diagram of the structure and preparation process of a split micro-electric biochip with a cell-like three-dimensional topological structure and a detection system in a typical embodiment of the present invention; Figure 7 It is a schematic diagram of the structure and preparation process of an integrated micro-electric biochip with a cell-like three-dimensional topological structure and a detection system in another typical embodiment of the present invention; Figure 8 It is a schematic diagram of solidifying biomolecules on the surface of a metal layer of a chip by contact transfer in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0019] In view of the limitation that traditional SPR can only characterize the interactions between biomolecules on a two-dimensional plane, the inventors of this case, after long-term research and practice, were able to propose the technical solution of the present invention, which mainly provides a micro-electric biochip that can simulate the cell surface topological structure and is used to characterize the interactions between biomolecules.

[0020] The inventors of this case used conventional Annexin V and Lactadherin as detection proteins to perform localization and quantitative analysis on the expression of phosphatidylserine (PS) on the surface of activated platelets.

[0021] 1. When a blood vessel is damaged, platelets are rapidly activated. Figure 1A The oval spherical shape (free without activation) in the blood quickly adheres to the damaged bleeding site and changes into Figure 1B The disc shape in the middle (adhesive state after activation) is like a flattened egg (with a hill-like protrusion in the center).

[0022] 2. Activated platelets will express PS protein on the cell membrane surface.

[0023] 3. It is generally believed in the industry that the expression of PS on the cell membrane surface of activated / apoptotic cells is relatively random and the distribution is irregular.

[0024] 4. Both Annexin V and Lactadherin can bind specifically to PS.

[0025] 5. If Figure 2 The figure shows the quantitative relationship between the expression level of PS and the specific binding of Lactadherin and Annexin V to PS. When the PS expression is 1-5%, only Lactadherin can specifically bind to PS; when the PS expression exceeds 6%, both Lactaherin and Annexin V can specifically bind to PS.

[0026] The inventors of this case used different doses of activators to activate platelets, such as Figure 3A As shown in the figure, under slight stimulation, the PS expression of platelets is mainly concentrated in the junction of the central bulge and the flat area (a circle of valleys), and the expression amount is relatively low (less than 6%), among which the green fluorescence is Lactadherin and the red fluorescence is Annexin V. Therefore, the inventors of this case saw that only Lactadherin coupled with green fluorescence had specific binding with it; Figure 3B As shown in the figure, under strong stimulation, the PS expression of platelets is mainly concentrated in the central bulge and the junction of the central bulge and the flat area, and the expression level is relatively high (greater than 6%), so it can be seen that Lactadherin coupled with green fluorescence and Annexin V coupled with red fluorescence both specifically bind to it. Figure 3B It also allowed the inventor of this case to see the following new discovery that broke the conventional rules for the first time: Although both Lactadherin and Annexin V can specifically bind to PS, Lactadherin only binds specifically to PS expressed on the cell membrane with a concave curvature (the junction of the central bulge and the flat area, i.e., the circle of the valley); while Annexin V only binds specifically to PS expressed on the cell membrane with a convex curvature (the central bulge, i.e., the top of the hill).

[0027] This shows that, in addition to the influence of the concentration of conventional molecules on the specific binding between biomolecules, the surface where the specific binding occurs, its curvature, and the three-dimensional topological structure will also have a significant impact on the specific binding between biomolecules. This also means that SPR technology has a major limitation in drug screening: although SPR provides accurate kinetic data in detecting molecular interactions, it ignores the complexity of the three-dimensional biological environment, which may lead to deviations in its efficacy prediction.

[0028] The present invention introduces a cell-like three-dimensional topological structure on the surface of the biochip through the principle of pure electrical detection, which more realistically restores the biological microenvironment (such as the cell surface) where biological molecules interact with each other, and comprehensively improves the anti-interference ability of the detection system (environmental factors, such as vibration, will have a great impact on the light-based SPR system).

[0029] The technical solution, its implementation process and principle will be further explained as follows.

[0030] As one aspect of the technical solution of the present invention, it involves the application of a micro-electric biochip with a cell-like three-dimensional topological structure in the fields of characterizing the interaction force between two biological molecules, characterizing the interaction force between biological molecules and other substances, characterizing the interaction force between cells or viruses and proteins, and drug research and development.

[0031] Furthermore, the drug development includes areas such as drug target confirmation and validation, drug screening, drug affinity and kinetic characterization, drug toxicity testing or drug quality control.

[0032] Specifically, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention can be used to characterize the interaction force between two biomolecules, for example: - Protein-protein: For example, studying the binding between proteins in cell signaling pathways, such as the interaction between Ras protein and other related proteins, to understand physiological processes such as cell proliferation and differentiation; -Protein-DNA: It can detect the binding of transcription factors to DNA, helping to understand the transcriptional regulation mechanism of genes, which is of great significance for studying gene expression and genetic information transmission; -Protein-small molecules: In drug development, detect the binding of drug small molecules to protein targets, determine the affinity and kinetic properties of drugs, and assist in drug screening and optimization; - Antibody-antigen: It can be used for antibody screening, identification and affinity determination. In disease diagnosis and immunotherapy research, it helps to screen out high-affinity antibodies and improve detection and treatment effects; -Receptor-ligand: Detecting the binding of neurotransmitter receptors and corresponding ligands is of great value for studying the pathogenesis of neural signal transduction and related diseases.

[0033] Specifically, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention can be used to characterize the interaction force between biomolecules and other substances, for example: -Protein-polysaccharide: Studying the interaction between proteins and polysaccharides in the body is helpful for understanding the formation of extracellular matrix, immune regulation and other processes; -Protein-Nanomaterials: Detecting the binding of proteins to nanomaterials provides a basis for the development of nanomedicine and biosensors, such as studying the interaction between nanodrug carriers and proteins to optimize drug delivery systems; -Lipid membrane-protein: It can explore the binding of lipids and proteins on the cell membrane, which helps to understand the structure and function of the cell membrane, as well as the mechanism of related diseases, such as the interaction between β-amyloid protein and cell membrane lipids in Alzheimer's disease.

[0034] Specifically, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention can be used to characterize the interaction between cells or viruses and proteins, for example: -Cell-Protein: Detecting the binding of cell surface receptors and proteins is of great significance for studying cell recognition, adhesion and other processes, such as the interaction between immune cells and surface proteins of antigen-presenting cells.

[0035] -Virus-Protein: The binding of viral proteins to host cell proteins can be studied to help reveal the virus's invasion mechanism and pathogenic mechanism, such as the binding of Ebola virus glycoprotein to receptors in host cells.

[0036] Furthermore, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention can also be used in drug research and development, which is mainly reflected in the following aspects: 1. Confirmation and validation of drug targets Potential target proteins can be fixed on the surface of the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention, and then small drug molecules can be allowed to interact with it, and binding signals can be detected to determine whether there is specific binding between the two, thereby verifying the effectiveness of the target and providing a basis for subsequent drug design. For example, when confirming tumor-related targets, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention can be used to screen compounds that bind to it to verify the feasibility of the target; 2. Drug Screening It is possible to quickly and efficiently screen the binding ability of a large number of drug candidate compounds to the target. The target molecules are fixed on the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention, and different compounds are allowed to flow through in sequence. The binding process is monitored in real time, and candidate compounds with high affinity are screened out based on data such as binding constants. This can be used to construct compound library screening, improve screening efficiency and accuracy, and accelerate the drug discovery process; 3. Drug affinity and kinetic characterization The affinity, binding rate constant, dissociation rate constant and other kinetic parameters between drugs and targets can be accurately measured to help gain a deeper understanding of the drug's mechanism of action and efficacy. For example, for drugs for treating cardiovascular diseases, the kinetic characteristics of the drugs and targets can be studied through the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention, so as to optimize the drug structure and improve the efficacy. 4. Drug optimization and lead compound selection After the Lead compound is transformed and modified, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention is used to quickly compare the binding properties of different derivatives with the target, and the compound with the best comprehensive performance is selected as a candidate drug for preclinical research, thereby improving the success rate of research and development; 5. Prediction of drug toxicity Proteins related to drug toxicity are fixed on the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention to detect the binding of drugs to these proteins, predict potential toxicity risks, optimize drug structures or adjust research and development directions in advance, and reduce the risk of failure in late drug research and development; 6. Drug quality control In the production of biological drugs, the micro-electric biochip with a cell-like three-dimensional topological structure of the present invention can detect quality indicators such as product activity and immunogenicity, such as detecting the binding activity of monoclonal antibody products with target receptors, and monitoring the non-specific binding of impurity proteins to drugs during the production process of biological drugs, thereby ensuring the quality and safety of drugs.

[0037] Specifically, the micro-electric biochip with a cell-like three-dimensional topological structure involved in the present invention includes: The undulating three-dimensional cell-like topology; A metal conductive layer disposed on the surface of the cell-like three-dimensional topological structure; At least one first binding sample is solidified on the surface of the metal conductive layer having a cell-like three-dimensional topological structure, and the first binding sample can specifically bind to or dissociate from the second binding sample.

[0038] In some embodiments, the cell-like three-dimensional topological structure includes a plurality of concave-convex units, each of which has a diameter of 1 mm to 1000 mm and a height of -50 mm to +50 mm.

[0039] In some embodiments, the first combined sample may be solidified at a peak end or a valley end of the concave-convex unit.

[0040] In some embodiments, the metal conductive layer includes a gold (Au) layer or other biocompatible conductive materials, but is not limited thereto.

[0041] Furthermore, the thickness of the metal conductive layer is 5 nm to 10 mm.

[0042] In some embodiments, the types of the first combined sample and the second combined sample are determined according to the objects to be detected and characterized in the aforementioned different application scenarios.

[0043] In some preferred embodiments, the structure of the micro-electric biochip with a cell-like three-dimensional topological structure may specifically include: a silicon substrate with a cell-like three-dimensional topological structure, an insulating layer, and a metal conductive layer stacked in sequence from bottom to top, wherein at least one first binding sample is solidified on the surface of the metal conductive layer. When the first binding sample is solidified at the valley end position of the concave-convex unit, the structure is as follows Figure 4A As shown, when located at the peak end, the structure is as follows Figure 4B shown.

[0044] In some other preferred embodiments, the structure of the micro-electric biochip with a cell-like three-dimensional topological structure may specifically include: an insulating layer, a silicon substrate, an insulating layer, a first metal conductive layer, a photoresist layer with a cell-like three-dimensional topological structure, and a second metal conductive layer with a cell-like three-dimensional topological structure, which are stacked in sequence from bottom to top, and at least one first binding sample is solidified on the surface of the second metal conductive layer; and at least part of the first metal conductive layer extends out of the setting area of ​​the photoresist layer with a cell-like three-dimensional topological structure. When the first binding sample is solidified at the valley end position of the concave-convex unit, the structure is as follows Figure 5A As shown, when located at the peak end, the structure is as follows Figure 5B Furthermore, in this micro-electric biochip structure, the insulating layer, the silicon substrate, and the first metal conductive layer are flat layered structures.

[0045] Furthermore, the material of the insulating layer may include silicon nitride, silicon oxide, etc., but is not limited thereto.

[0046] Furthermore, the thickness of the insulating layer is 10 nm to 10 mm.

[0047] As another aspect of the technical solution of the present invention, a method for preparing a micro-electric biochip having a cell-like three-dimensional topological structure includes: Through photolithography, deposition and etching techniques, a cell-like three-dimensional topological structure with uneven surfaces is prepared. Depositing a metal conductive layer on the surface of the cell-like three-dimensional topological structure to form a metal conductive layer having a cell-like three-dimensional topological structure; At least one first binding sample is solidified on the surface of the metal conductive layer having the cell-like three-dimensional topological structure.

[0048] In some embodiments, having Figure 4A or Figure 4B The method for preparing the micro-electric biochip of the structure shown specifically includes: Spin-coating photoresist on the surface of the silicon substrate, performing photolithography by grayscale ultraviolet photolithography technology, and removing the photoresist to form a photoresist with a cell-like three-dimensional topological structure, and then performing deep reactive ion etching to obtain a silicon substrate with a cell-like three-dimensional topological structure; Depositing an insulating layer on the surface of the silicon substrate having a cell-like three-dimensional topological structure; A metal conductive layer is deposited on the surface of the insulating layer, and then the first combined sample is solidified on the surface of the metal conductive layer having a cell-like three-dimensional topological structure by contact transfer.

[0049] In the preparation method, the insulating layer is formed by adopting low-pressure chemical vapor deposition technology.

[0050] In the preparation method, the metal conductive layer is deposited on the surface of the insulating layer by using an electron beam evaporation technique with a shadow mask.

[0051] In other embodiments, the Figure 5A or Figure 5B The method for preparing the micro-electric biochip of the structure shown specifically includes: Depositing insulating layers on both sides of the silicon substrate; Depositing a first metal conductive layer on the surface of one of the insulating layers; Spin-coating photoresist on the surface of the first metal conductive layer, performing photolithography using grayscale ultraviolet photolithography technology, and removing the photoresist to form a photoresist layer having a cell-like three-dimensional topological structure; A second metal conductive layer is deposited on the surface of the photoresist layer having the cell-like three-dimensional topological structure, and then the first combined sample is solidified on the surface of the second metal conductive layer having the cell-like three-dimensional topological structure by contact transfer. Preferably, the first combined sample is transferred to a specific area, i.e., a peak end or a valley end of the concave-convex unit, by contact transfer.

[0052] In the preparation method, the deposition methods of the insulating layer and the first metal conductive layer are as described above.

[0053] Furthermore, the preparation method comprises: using electron beam evaporation technology with a shadow mask to deposit and form the second metal conductive layer on the surface of the photoresist layer having the cell-like three-dimensional topological structure.

[0054] In summary, the present invention constructs a concave and convex topological structure with a diameter ranging from 1 mm to 1000 mm and a height ranging from -50 mm to +50 mm on the metal layer of the micro-electric biochip through advanced micro-nano processing technologies such as grayscale ultraviolet lithography (i.e., giving different exposure doses to the photoresist at different positions, and utilizing the incomplete development of the photoresist under low-dose exposure to obtain a continuous exposure method of lithographic three-dimensional structure), low-pressure chemical vapor deposition (LPCVD), and deep reactive ion etching (DRIE), thereby forming a non-planar three-dimensional spatial effect similar to that after cells attach to the wall.

[0055] Furthermore, the concentration of the first binding sample solidified on the surface of the metal conductive layer having a cell-like three-dimensional topological structure of the present invention can be freely adjusted from 1% to 100% (simulating different concentrations of protein expression on the cell surface under different circumstances).

[0056] Specifically, if it is only necessary to consider whether the first binding sample and the second binding sample can specifically bind, that is, it is not necessary to have a detailed understanding of whether the substrate with a cell-like three-dimensional topological structure has an effect on the specific binding of the first binding sample and the second binding sample, the first binding sample can be simply and fully solidified to the surface of the metal conductive layer by immersion, and the solidification method can be through the covalent connection of a self-assembled monolayer (SAM) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS). The specific operation steps are: (1) anchoring of SAM on an inert surface, (2) subsequent surface activation by EDC / NHS to form an active layer, and (3) covalent connection of the first binding sample. In another scheme, biological molecules or linker molecules with thiol ends (such as thiol compounds containing active functional groups that can react with biological molecules) can be first adsorbed onto the surface of the metal conductive layer to form a tightly arranged monolayer (self-assembled monolayer, SAMs); then, their terminal hydroxyl groups can be used to specifically bind to the carboxyl or amino groups on the biological molecules that need to be cured on the surface of the metal conductive layer, thereby achieving the curing of the biological molecules on the surface of the metal conductive layer. The basic principle is also to use molecules containing thiol (-SH) groups to form strong Au-S bonds with the surface of the metal conductive layer (such as Au).

[0057] Furthermore, the cross-linking agent used in the above curing method can be disulfide bis (succinimidyl propionate, DSP), the disulfide linkage contained in it can be rapidly chemically adsorbed to the surface of the metal conductive layer, and the active NHS groups at both ends can specifically bind to the functional groups of the protein (such as the primary amine groups of human fibronectin), thereby achieving the curing of the protein on the surface of the metal conductive layer.

[0058] If one needs to understand in detail how the substrate with a cell-like three-dimensional topological structure affects the specific binding of the first binding sample and the second binding sample, that is, whether the different curvatures on the substrate will affect the specific binding between the two samples, it is necessary to transfer the first binding sample to a specific area on the surface of the metal conductive layer, that is, the peak end or valley end of the concave-convex unit, through contact transfer.

[0059] As another aspect of the technical solution of the present invention, a sample detection system (for example, a detection system for characterizing interactions between biomolecules) is provided, comprising: The aforementioned micro-electric biochip with a cell-like three-dimensional topological structure; An electrical connection component, electrically connected to the micro-electric biochip having a cell-like three-dimensional topological structure, at least for real-time monitoring of changes in electrical properties of the micro-electric biochip; The microfluidic channel is correspondingly arranged above the detection area of ​​the micro-electric biochip with the cell-like three-dimensional topological structure, and is at least used to pass the microfluid containing the second binding sample to be detected.

[0060] Furthermore, the selected Figure 4A or Figure 4B In the case of the micro-electric biochip of the structure shown, a lower electrode plate is also provided below the micro-electric biochip, which is electrically connected to the electrical connection component, and can also be called a "split chip structure".

[0061] Further, Figure 5A or Figure 5B The micro-electric biochip of the structure shown can also be called an "integrated chip structure".

[0062] In summary, the present invention connects the micro-electric biochip that has undergone bioactivation treatment / protein modification with a high-precision LCR bridge that can monitor the changes in the electrical properties of the micro-electric biochip in real time, and then connects it to a microfluidic system to form a detection system that can be used to characterize the interaction between biomolecules.

[0063] Accordingly, another aspect of the technical solution of the present invention further relates to a sample detection method (for example, a drug screening method), which comprises: Passing the microfluid containing the second binding sample to be tested into the detection area of ​​the micro-electric biochip with a cell-like three-dimensional topological structure in the aforementioned sample detection system, specifically binding or dissociating with the first binding sample solidified on the surface of the metal conductive layer, causing changes in the electrical properties of the micro-electric biochip; The electrical connection components are used to monitor the changes in the electrical properties of the micro-electric biochip in real time to determine whether the second binding sample to be tested specifically binds or dissociates with the first binding sample, thereby achieving sample detection, such as drug screening.

[0064] Specifically, when the microfluid containing the second binding sample to be tested flows at a uniform speed through the detection area of ​​the micro-electric biochip, if it specifically binds / dissociates with the target protein immobilized in the detection area (on the surface of the gold conductive layer with a cell-like three-dimensional topological structure), the electrical properties (resistance, impedance, capacitance, etc.) of the micro-electric biochip will change accordingly. By monitoring the dynamic changes of its electrical properties through a high-precision LCR bridge, it is possible to infer the specific binding / dissociation of the second binding sample to be tested with the target protein immobilized on the chip surface, thereby more accurately characterizing the interaction between biological molecules.

[0065] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention. In addition, unless otherwise specified, all kinds of raw materials, reagents, and equipment used in the following embodiments can be obtained from market channels, and the magnetron sputtering, photolithography, dry etching, wet etching, etc. used therein can be implemented in a manner known in the art.

[0066] Example 1 In this embodiment, the structure of a micro-electric biochip with a cell-like three-dimensional topological structure includes: a silicon wafer with a cell-like three-dimensional topological structure, an insulating layer (silicon nitride or silicon oxide), and a metal conductive layer (Au layer) stacked in sequence from bottom to top, and biological molecules (such as target proteins) are solidified on the surface of the Au layer.

[0067] The preparation process of the micro-electric biochip with this structure is as follows Figure 6 As shown: Spin coating photoresist on a silicon wafer, performing photolithography using grayscale ultraviolet photolithography technology, and removing the photoresist to form a photoresist having a cell-like three-dimensional topological structure, and then performing deep reactive ion etching to obtain a silicon wafer having a cell-like three-dimensional topological structure; Depositing silicon nitride or silicon oxide as an insulating layer on the surface of the silicon wafer having the cell-like three-dimensional topological structure by LPCVD; The surface of the insulating layer is plated with gold using electron beam evaporation technology with a shadow mask, and then contact transfer is performed to solidify the biological molecules (such as target proteins) on the surface of the gold layer. When the target protein is specifically connected to the valley end of the concave-convex unit, the structure can be referred to Figure 4A As shown, when located at the peak position, the structure can be seen Figure 4B Specifically, considering the cell-like three-dimensional topological structure on the metal conductive layer surface of the micro-electric biochip, contact transfer (such as Figure 8 As shown in the figure, the ECM will only be transferred from the PDMS Stamp to the top of the micro-pillar, and will not appear on the side wall or bottom of the micro-pillar).

[0068] The micro-electric biochip that has undergone bioactivation treatment / protein modification is combined with the lower electrode plate, connected to a high-precision LCR bridge that can monitor the changes in the electrical properties of the micro-electric biochip in real time, and then connected to the microfluidic channel shell for assembly, forming a detection system that can be used to characterize the interactions between biomolecules.

[0069] Example 2 In this embodiment, the structure of a micro-electric biochip with a cell-like three-dimensional topological structure includes: silicon nitride or silicon oxide (as an insulating layer), a silicon wafer, silicon nitride or silicon oxide (as an insulating layer), a first Au layer, a photoresist layer with a cell-like three-dimensional topological structure, and a second Au layer with a cell-like three-dimensional topological structure, which are stacked in sequence from bottom to top, and biological molecules (such as target proteins) are solidified on the surface of the second Au layer with a cell-like three-dimensional topological structure; the first Au layer partially extends out of the setting area of ​​the photoresist layer.

[0070] The preparation process of the micro-electric biochip with this structure is as follows Figure 7 As shown: Silicon nitride or silicon oxide (as an insulating layer) is deposited on the upper and lower surfaces of a silicon wafer by LPCVD, gold is plated on the surface of the insulating layer by electron beam evaporation technology, and then photoresist is spin-coated, photolithography is performed by grayscale ultraviolet lithography technology, and the photoresist is removed to form a photoresist with a cell-like three-dimensional topological structure; Au is deposited on the surface of the photoresist layer with a cell-like three-dimensional topological structure by electron beam evaporation technology with a shadow mask, and then contact transfer is performed to solidify biological molecules (such as target proteins) on the surface of the Au layer with a cell-like three-dimensional topological structure. When the target protein is solidified at the valley end of the concave-convex unit, the structure can be referred to Figure 5A As shown, when located at the peak position, the structure can be seen Figure 5B shown.

[0071] The micro-electric biochip that has undergone bioactivation treatment / protein modification is connected to a high-precision LCR bridge that can monitor the changes in the electrical properties of the micro-electric biochip in real time, and then connected to the microfluidic channel shell for assembly, forming a detection system that can be used to characterize the interactions between biomolecules.

[0072] Application Example 1 If it is only necessary to consider whether the first binding sample and the second binding sample can specifically bind to each other, that is, there is no need to understand in detail whether the substrate with a cell-like three-dimensional topological structure has an effect on the specific binding of the first binding sample and the second binding sample, the first binding sample can be simply and fully solidified onto the surface of the metal conductive layer by immersion.

[0073] The basic process of screening is as follows: - Sample preparation: Identify the binding sample pairs that need to be characterized for interaction, such as antigen and antibody, protein and small molecule drugs, etc. Purify and quantify the first binding sample (ligand) to be fixed on the surface of the micro-electric biochip and the second binding sample as the analyte to ensure that their purity and concentration meet the experimental requirements. At the same time, prepare appropriate buffers for diluting samples, maintaining the pH and ionic strength of the experimental system, etc.

[0074] - Chip pretreatment: Install the micro-electric biochip that has not been treated with biological activation into the supporting instrument, and then use specific cleaning and buffer to clean the chip surface to remove possible impurities and pollutants to ensure the cleanliness and activity of the chip surface. Some chips also need pretreatment, such as chemical modification to make their surface have specific functional groups to better fix the first binding sample.

[0075] Specifically, the first binding sample (ligand) is fixed: the ligand molecule is dissolved in a suitable buffer at an appropriate concentration, and the ligand solution is injected into the chip surface by a syringe pump or manual injection, so that the ligand molecule is covalently bound to the chip surface or specifically adsorbed and fixed on the chip. During the fixation process, it is necessary to control parameters such as the concentration of the ligand, injection flow rate and time to ensure that the ligand can be fixed on the chip surface evenly and stably, while avoiding excessive fixation that leads to waste of chip active sites or increase in non-specific binding.

[0076] - Binding analysis: A solution containing a certain concentration of the second binding sample, i.e., the analyte, is injected into the chip surface at a certain flow rate to interact with the ligand molecules fixed on the chip. During the analyte injection process, the electrical signal detection module in the instrument monitors the dynamic changes of the electrical properties (such as capacitance) of the micro-electric biochip in real time and records them as a sensorgram of electrical signal vs. time. By analyzing the dynamic change curve of the electrical signal in the sensorgram over time, the kinetic information of the binding of the analyte to the ligand can be obtained, such as the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD).

[0077] - Chip regeneration: After completing a binding analysis, the chip needs to be regenerated to remove the analyte molecules bound to the chip surface so that the chip can be reused. Select a suitable regeneration solution, such as a high-concentration salt solution, acidic or alkaline solution, etc., and inject the regeneration solution into the chip surface at a certain flow rate through a syringe pump to dissociate the complex between the analyte and the ligand, thereby restoring the activity of the chip. During the regeneration process, it is necessary to pay attention to parameters such as the concentration, flow rate and action time of the regeneration solution to avoid excessive regeneration that causes the ligand molecules on the chip surface to fall off or the activity of the chip to decrease.

[0078] Application Example 2 If one needs to understand in detail how the substrate with a cell-like three-dimensional topological structure affects the specific binding of the first binding sample and the second binding sample, that is, whether the different curvatures on the substrate will affect the specific binding between the two samples, it is necessary to transfer the first binding sample to a specific area on the surface of the metal conductive layer through contact transfer.

[0079] The basic process of screening is as follows: - The first binding sample (ligand) is adsorbed onto a stamp made of soft silicone material such as PDMS with a matching cell-like three-dimensional topological structure: the ligand molecules are dissolved in a suitable buffer at an appropriate concentration and then adsorbed onto the surface of the PDMS stamp by soaking.

[0080] The surface of the micro-electric biochip is treated with ozone or plasma to change the hydrophilicity / hydrophobicity of the surface of the metal conductive layer of the micro-electric biochip.

[0081] The PDMS stamp is precisely positioned with the micro-electric biochip with a cell-like three-dimensional topological structure.

[0082] The PDMS stamp is gently placed on the surface of the micro-electric biochip, that is, the first binding sample (ligand) solidified on the surface of the PDMS stamp is transferred to a specific area of ​​the metal conductive layer of the micro-electric biochip, such as the peak end or valley end of the concave-convex unit, by contact transfer.

[0083] - Chip pretreatment: Install the micro-electric biochip that has completed the bioactivation treatment (i.e. the first binding sample / ligand has been solidified on the chip) into the supporting instrument, and then clean the chip surface with a specific cleaning and buffer solution to remove possible impurities and contaminants to ensure the cleanliness and activity of the chip surface.

[0084] - Binding analysis: A solution containing a certain concentration of the second binding sample, i.e., the analyte, is injected into the chip surface at a certain flow rate to interact with the ligand molecules fixed on the chip. During the analyte injection process, the electrical signal detection module in the instrument monitors the dynamic changes of the electrical properties (such as capacitance) of the micro-electric biochip in real time and records them as a sensorgram of electrical signal vs. time. By analyzing the dynamic change curve of the electrical signal in the sensorgram over time, the kinetic information of the binding of the analyte to the ligand can be obtained, such as the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD).

[0085] - Chip regeneration: After completing a binding analysis, the chip needs to be regenerated to remove the analyte molecules bound to the chip surface so that the chip can be reused. Select a suitable regeneration solution, such as a high-concentration salt solution, acidic or alkaline solution, etc., and inject the regeneration solution into the chip surface at a certain flow rate through a syringe pump to dissociate the complex between the analyte and the ligand, thereby restoring the activity of the chip. During the regeneration process, it is necessary to pay attention to parameters such as the concentration, flow rate and action time of the regeneration solution to avoid excessive regeneration that causes the ligand molecules on the chip surface to fall off or the activity of the chip to decrease.

[0086] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention.

Claims

1. Application of micro-electric biochips with cell-like three-dimensional topological structures in the characterization of the interaction between two biomolecules, the interaction between biomolecules and other substances, the interaction between cells or viruses and proteins, and in the field of drug research and development; The micro-electric biochip with a cell-like three-dimensional topological structure comprises: The undulating three-dimensional cell-like topology; A metal conductive layer disposed on the surface of the cell-like three-dimensional topological structure; Solidify at least one first binding sample on the surface of the metal conductive layer having a cell-like three-dimensional topological structure, wherein the first binding sample can specifically bind to or dissociate from the second binding sample; Preferably, the drug development includes confirmation and validation of drug targets, drug screening, drug affinity and kinetic characterization, drug optimization and selection of lead compounds, drug toxicity prediction or drug quality control.

2. A micro-electric biochip with a cell-like three-dimensional topological structure, characterized in that: include: The undulating three-dimensional cell-like topology; A metal conductive layer disposed on the surface of the cell-like three-dimensional topological structure; At least one first binding sample is solidified on the surface of the metal conductive layer having a cell-like three-dimensional topological structure, and the first binding sample can specifically bind to or dissociate from the second binding sample.

3. The micro-electric biochip with a cell-like three-dimensional topological structure according to claim 2, characterized in that: The cell-like three-dimensional topological structure includes a plurality of concave-convex units, each of which has a diameter of 1 mm to 1000 mm and a height of -50 mm to +50 mm; And / or, the metal conductive layer includes a gold layer; and / or, the thickness of the metal conductive layer is 5 nm to 10 mm; And / or, the first combined sample is solidified at a peak end or a valley end of the concavo-convex unit.

4. The micro-electric biochip with a cell-like three-dimensional topological structure according to claim 2, characterized in that: Specifically include: A silicon substrate, an insulating layer and a metal conductive layer having a cell-like three-dimensional topological structure are sequentially stacked, and at least one first binding sample is solidified on the surface of the metal conductive layer.

5. The micro-electric biochip with a cell-like three-dimensional topological structure according to claim 2, characterized in that: Specifically include: An insulating layer, a silicon substrate, an insulating layer, a first metal conductive layer, a photoresist layer having a cell-like three-dimensional topological structure, and a second metal conductive layer having a cell-like three-dimensional topological structure are sequentially stacked, wherein at least one first binding sample is solidified on a surface of the second metal conductive layer; and at least a portion of the first metal conductive layer extends out of a setting area of ​​the photoresist layer having a cell-like three-dimensional topological structure; Preferably, the insulating layer, the silicon substrate, and the first metal conductive layer are flat layered structures; And / or, the material of the insulating layer includes silicon nitride or silicon oxide; and / or, the thickness of the insulating layer is 10 nm to 10 mm.

6. The method for preparing a micro-electric biochip having a cell-like three-dimensional topological structure according to any one of claims 2 to 5, characterized in that: include: Through photolithography, deposition and etching techniques, a concave and convex cell-like three-dimensional topological structure was prepared; Depositing a metal conductive layer on the surface of the cell-like three-dimensional topological structure to form a metal conductive layer having a cell-like three-dimensional topological structure; At least one first binding sample is solidified on the surface of the metal conductive layer having the cell-like three-dimensional topological structure.

7. The preparation method according to claim 6, characterized in that: Specifically include: Spin-coating photoresist on the surface of the silicon substrate, performing photolithography by grayscale ultraviolet photolithography technology, and removing the photoresist to form a photoresist with a cell-like three-dimensional topological structure, and then performing deep reactive ion etching to obtain a silicon substrate with a cell-like three-dimensional topological structure; Depositing an insulating layer on the surface of the silicon substrate having a cell-like three-dimensional topological structure; Depositing a metal conductive layer on the surface of the insulating layer, and then solidifying the first combined sample on the surface of the metal conductive layer having a cell-like three-dimensional topological structure by contact transfer; Preferably, the preparation method comprises: forming the insulating layer by adopting low pressure chemical vapor deposition technology; Preferably, the preparation method comprises: using electron beam evaporation technology with a shadow mask to deposit a metal conductive layer on the surface of the insulating layer.

8. The preparation method according to claim 6, characterized in that: Specifically include: Depositing insulating layers on both sides of the silicon substrate; Depositing a first metal conductive layer on the surface of one of the insulating layers; Spin-coating photoresist on the surface of the first metal conductive layer, performing photolithography using grayscale ultraviolet photolithography technology, and removing the photoresist to form a photoresist layer having a cell-like three-dimensional topological structure; Depositing a second metal conductive layer on the surface of the photoresist layer having the cell-like three-dimensional topological structure, and then solidifying the first combined sample on the surface of the second metal conductive layer having the cell-like three-dimensional topological structure by contact transfer; Preferably, the preparation method comprises: forming the insulating layer by adopting low pressure chemical vapor deposition technology; Preferably, the preparation method comprises: using electron beam evaporation technology with a shadow mask to deposit the first metal conductive layer on the surface of the insulating layer; Preferably, the preparation method comprises: using electron beam evaporation technology with a shadow mask to deposit the second metal conductive layer on the surface of the photoresist layer having the cell-like three-dimensional topological structure; And / or, the concentration of the solidified first bonding sample is 1% to 100%.

9. A sample detection system, characterized in that: include: The micro-electric biochip with a cell-like three-dimensional topological structure according to any one of claims 2 to 5; An electrical connection component, electrically connected to the micro-electric biochip having a cell-like three-dimensional topological structure, at least for real-time monitoring of changes in electrical properties of the micro-electric biochip; A microfluidic channel, correspondingly arranged above the detection area of ​​the micro-electric biochip having a cell-like three-dimensional topological structure, and at least used to pass a microfluid containing a second binding sample to be detected; Preferably, a lower electrode plate is further provided below the micro-electric biochip having a cell-like three-dimensional topological structure and is electrically connected to the electrical connection component.

10. A sample detection method, characterized in that: include: Passing the microfluid containing the second binding sample to be tested into the detection area of ​​the micro-electric biochip with a cell-like three-dimensional topological structure in the sample detection system of claim 9, specifically binding or dissociating with the first binding sample solidified on the surface of the metal conductive layer, causing changes in the electrical properties of the micro-electric biochip; The electrical connection component is used to monitor the changes in the electrical properties of the micro-electric biochip in real time, and to determine whether the second binding sample to be tested has specifically bound or dissociated with the first binding sample, thereby achieving sample detection.

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