A sensor chip for detecting the binding kinetics between biomolecules, a preparation method thereof, and an application thereof

Abstract

The present invention provides a sensor chip for detecting the binding kinetics between biomolecules, a preparation method thereof and an application thereof, relating to the technical field of sensor chips. The sensor chip for detecting the binding kinetics between biomolecules of the present invention comprises: a control substrate and a sensing substrate. The top end of the sensing substrate is bonded to the bottom end of the control substrate. A first liquid accumulation groove covered by a reaction groove is dug at the top end of the sensing substrate. A structural film or a sensitive film is erected above the first liquid accumulation groove. A local modification area is arranged at the top end of the sensitive film. Four cantilever beams are circumferentially and uniformly distributed on the side walls of both the structural film and the sensitive film and are fixedly connected to the inner wall of the first liquid accumulation groove. A piezoresistor is arranged at the top end of each cantilever beam. Every two adjacent piezoresistors above the same first liquid accumulation groove are connected by leads to form a Wheatstone bridge. Each lead is connected to a pad. The sensor chip prepared by the present invention has high sensitivity, high resolution, small temperature drift, low cost, and can realize miniaturized detection.

Description

Technical Field

[0001] The present invention relates to the technical field of sensor chips, and in particular, to a sensor chip for detecting the binding kinetics between biomolecules, a preparation method thereof, and an application thereof. Background Art

[0002] The interactions between biomolecules such as proteins, DNA, and small molecules are the basis for maintaining life activities. By studying the binding characteristics of these molecules, the mechanisms of important biological processes such as cell signal transduction, enzyme catalysis, and transcriptional regulation can be revealed. The detection of the binding kinetics between biomolecules has important scientific and practical significance in the fields of biology, drug development, disease treatment, etc.

[0003] The kinetic measurement methods for biomolecular interactions mainly include fluorescence method, gravimetric method, and optical method. Among them, the fluorescence method is used to detect by directly binding the analyte, and is especially suitable for the binding evaluation of unconventional materials such as cell surfaces. However, this method requires the use of labeled probes to modify the ligand, which is not only time-consuming and laborious, but may also interfere with the natural binding behavior of the target biomolecule, thus affecting the measurement accuracy. In contrast, the gravimetric method and the optical method adopt the non-labeled detection principle, avoiding the potential interference caused by probe modification. However, these two types of methods have high requirements for the size of the target molecule, and the detection sensitivity for low molecular weight substances is insufficient. It should be noted that such technologies generally have the problem of equipment dependence in the application level - large-scale precision instruments need to be equipped, resulting in limited detection throughput and high R & D costs. These technical bottlenecks jointly restrict their application and popularization in conventional laboratories and high-throughput screening scenarios.

[0004] In recent years, more and more sensors based on micro-nano processing technology have been applied to the research of biomolecular interactions. Among them, the biosensor based on surface stress is a common label-free biosensing technology. This biosensor based on surface stress uses the binding energy of chemical bonds between biomolecules to test the analyte. When biomolecules interact with the recognition elements (such as antibodies, enzymes, DNA, etc.) on the sensor surface, it will cause stress changes on the sensor surface. This stress change can be detected and quantified by the small deformation of the sensor structure or the change of physical properties such as capacitance and resistance. By monitoring the changes of these physical parameters, qualitative and quantitative analysis of biomolecules can be achieved. The relevant research mainly focuses on two structures: microcantilever type or microfilm type. They can be made into an array structure with parallel arrangement to realize the simultaneous detection of multiple independent sensing units with different surface functionalizations with high sensitivity. The patent with the application number 202010327183.3 discloses a design method of a hollow micro-nano composite beam for biochemical molecule detection. The design structure proposed by this method includes a beam body and a hollow microchannel for biochemical reactions. The beam is composed of two structural materials: a silicon or silicon compound substrate and a flexible film. Between the flexible film layer and the silicon or silicon compound substrate layer, there are a liquid inlet, a liquid outlet, a liquid inlet microchannel, a liquid outlet microchannel, and a reaction pool for the sample to be tested. The reaction pool is located in the mass-sensitive area of the beam. This method proposes a "liquid reaction-vacuum detection" method, adsorbing the analyte inside the microchannel and placing the whole structure in a vacuum environment. This detection method can effectively isolate the reaction environment from the detection environment. By detecting the frequency change caused by the reaction of the analyte, high-sensitivity, in-situ real-time rapid detection of biochemical molecules can be completed, and it can be widely applied to engineering fields such as medicine and chemical engineering. However, this method requires an additional vacuum pumping instrument to perform vacuum pumping operations to obtain a vacuum environment, so its detection cost rises sharply.

[0005] Therefore, there is a need to provide a sensitive and effective method for directly detecting biomolecular binding with electrical signals without using expensive additional equipment. Summary of the Invention

[0006] The problem solved by the present invention is to provide a sensor chip for detecting the binding kinetics between biomolecules, its preparation method and application. The sensor chip has high sensitivity, high resolution, small temperature drift, low cost, and can realize miniaturized detection.

[0007] The first aspect of the present invention provides a sensor chip for detecting the binding kinetics between biomolecules, including:

[0008] A control substrate, on which at least one sample inlet and at least one sample outlet are provided. A reaction tank and two microfluidic channels communicating with the reaction tank are dug at the bottom end of the control substrate. The reaction tank is communicated with the sample inlet and the sample outlet respectively through the two microfluidic channels.

[0009] A sensing substrate, the top end of the sensing substrate is bonded to the bottom end of the control substrate. At least two first liquid accumulation grooves covered by the reaction tank are dug at the top end of the sensing substrate. Above the two first liquid accumulation grooves, a structural film and a sensitive film are respectively erected. A local modification area is provided at the top end of the sensitive film. Four cantilever beams are circumferentially and uniformly distributed on the side walls of the structural film and the sensitive film and are fixedly connected to the inner wall of the first liquid accumulation groove through the four cantilever beams. A piezoresistor is provided at the top end of each cantilever beam. Every two adjacent piezoresistors above the same first liquid accumulation groove are connected by leads to form a Wheatstone bridge. Each lead is connected to a pad.

[0010] Compared with the prior art, the sensor chip for detecting the binding kinetics between biomolecules of the present invention has the following advantages:

[0011] In the present invention, independent piezoresistors are arranged on each cantilever beam, and adjacent piezoresistors are electrically connected by leads to form a Wheatstone bridge with self-compensation benefits. A local modification area is provided at the top end of the sensitive film to form a stress concentration area. And an independent reaction tank and microfluidic channels are integrated at the bottom end of the control substrate. By covering the reaction tank over each first liquid accumulation groove to form a closed reaction chamber, it ensures the directional capture and localized control of the target molecule binding model when the sensor chip is used; and the fluid loop formed by the sample inlet, microfluidic channel, reaction tank, and sample outlet supports dynamic sample circulation and real-time flushing, which can not only maintain the stability of the reaction environment, avoid cross-contamination, but also realize the automatic switching of multi-step detection processes. While significantly improving the detection throughput, it also takes into account the efficient utilization of trace samples and the consistent regulation of detection conditions.

[0012] Furthermore, in the present invention, a thin-film type cantilever beam array is selected. By taking the differential processing of the measurement results of the sensitive film with a locally specific reaction biosensitive layer and the structural film without a specific reaction biosensitive layer, the measurement error caused by the non-specific adsorption problem is effectively reduced.

[0013] In a possible implementation manner, each lead includes two sub-leads with one end connected to each other. Each sub-lead includes a heavily doped lead and a metal lead connected to the heavily doped lead. Every two adjacent piezoresistors above the same first liquid accumulation groove are sequentially connected through the heavily doped lead and the metal lead.

[0014] Compared with the prior art, after adopting the above technical solution, the heavily doped lead is used as the first connection segment, and the metal lead is used as the second connection segment. Due to the characteristic of the heavily doped lead doped with a high concentration of impurity elements, the number of carriers that can be transmitted is significantly increased, and the conductivity can be improved.

[0015] In a possible implementation manner, a second liquid accumulation groove is dug at the top ends of the structural thin film and the sensitive thin film, a through hole is provided in the middle of the local modification area, and the through hole communicates with the second liquid accumulation groove on the sensitive thin film.

[0016] In a possible implementation manner, four first liquid accumulation grooves covered by the reaction groove are dug at the top end of the sensing substrate, a structural thin film is erected above one of the first liquid accumulation grooves, and sensitive thin films are erected above the remaining three first liquid accumulation grooves.

[0017] Compared with the prior art, after adopting the above technical solution, the reference value is tested through the structural thin film in a single area, and the measured value is tested through the sensitive thin films in three areas. Compared with the setting of the sensitive thin film in a single area, the accuracy of the measured value can be further improved, and the error influence caused by the change of a single parameter can be reduced.

[0018] Another object of the present invention is to provide a method for preparing a sensor chip, including the following steps:

[0019] S1. Select an SOI substrate including a silicon support layer, a buried oxide layer, and a silicon device layer from bottom to top;

[0020] S2. Perform photolithography, development, and dry etching processes on the SOI substrate in sequence to obtain grooves on the surfaces of the silicon device layer and the buried oxide layer;

[0021] S3. Grow a first SiO2 layer on the surfaces of the grooves and the silicon device layer;

[0022] S4. After performing photolithography and development processes in sequence, obtain piezoresistors through light doping treatment with boron ions, and then obtain heavily doped leads through heavy doping treatment with boron ions;

[0023] S5. Deposit a second SiO2 layer on the top surface and the bottom surface respectively;

[0024] S6. After performing photolithography, development, and dry etching processes in sequence, obtain lead holes at the ends of the heavily doped leads;

[0025] After performing sputtering metal treatment, photolithography, development, and etching processes in sequence, obtain metal leads and pads, and make the metal leads form ohmic contacts with the heavily doped leads;

[0026] S7. Selectively sputter at least one metal layer on the top surface, and obtain a sensitive thin film through a lift-off method;

[0027] S8. Deposit a third SiO2 layer and a silicon nitride layer on the top surface in sequence, and then perform photolithography and development processes to obtain a metal passivation layer;

[0028] S9. After performing photolithography, development, and dry etching processes on the top surface in sequence, expose the silicon support layer and obtain through-holes in the local modification area to obtain a second liquid accumulation tank;

[0029] S10. Perform anisotropic wet etching. After obtaining the first liquid accumulation tank and the cantilever beam, obtain a sensing substrate;

[0030] S11. Use a flexible film to fabricate a control substrate;

[0031] S12. Bond the control substrate and the sensing substrate to obtain a sensor chip.

[0032] Compared with the prior art, this preparation method realizes the high-precision manufacturing of sensor chips through the collaborative integration of an SOI substrate and multiple processes: based on the etching and doping processes of the SOI heterostructure, the nanoscale morphology and piezoresistive characteristics of the cantilever beam are precisely controlled to ensure the matching of mechanical and electrical properties; multi-step oxidation and passivation layer deposition strengthen the interface stability and biocompatibility; the combination of wet etching and bonding processes, while ensuring the integrity of the suspended structure, seamlessly integrates the microfluidic functional module, taking into account the process repeatability and the reliable forming of complex three-dimensional structures, providing an efficient and controllable technical path for the batch preparation of high-performance biosensing chips; moreover, the preparation process provided by the present invention all adopts conventional and mature MEMS processing techniques, and this method has low cost and high reliability, and can achieve large-scale batch manufacturing.

[0033] In a possible implementation manner, in the step S1, in the SOI substrate, the thickness of the silicon device layer is 2.9 - 3.1 μm, the thickness of the buried oxide layer is 0.9 - 1.1 μm, and the thickness of the silicon support layer is 690 - 710 μm.

[0034] Compared with the prior art, the present invention realizes the collaborative optimization of the mechanical characteristics of the device and the process feasibility by precisely matching the thickness ratios of the functional layers of the SOI substrate: the thickness of the silicon device layer ensures that the cantilever beam has both high-sensitivity flexure and fatigue resistance strength, the thickness of the buried oxide layer balances the etching accuracy and the interface stress buffering effect, and the thickness of the silicon support layer provides sufficient mechanical stability for the subsequent release of the suspended structure. The three cooperate to ensure the structural integrity and process compatibility of the chip in nanoscale deformation sensing and microfluidic integration, laying a foundation for high-yield manufacturing.

[0035] In a possible implementation, in step S7, the material of the metal layer is a mixture of gold and chromium or a mixture of gold and titanium.

[0036] Compared with the prior art, the present invention takes into account both the interfacial bonding force and the biocompatibility of biological functions through the design of a composite metal layer: the mixed use of gold and chromium or titanium not only utilizes the high adhesion of chromium / titanium to ensure the firm bonding of the metal layer to the silicon substrate, but also provides a stable and low-background-interference active interface for subsequent biomolecule modification through the chemical inertness of gold. At the same time, the electrical signal transmission efficiency and the anti-environmental corrosion ability are optimized, enabling the sensing interface to maintain long-term functional stability in a complex biochemical environment.

[0037] In a possible implementation, in step S8, the thickness of the third SiO2 layer is 195 - 205 nm, and the thickness of the silicon nitride layer is 95 - 105 nm.

[0038] Compared with the prior art, the present invention realizes a dual optimization between mechanical protection and interfacial functions by precisely regulating the thickness ratio of the passivation layer: the third SiO2 layer provides flexible stress buffering and electrical insulation isolation, while the silicon nitride layer endows a highly chemically inert surface barrier. The two cooperate to form a gradient protection system, which not only effectively resists the erosion of biological fluids and ion penetration, but also avoids the rigid constraint of the over-thick coating on the micro-nano deformation of the cantilever beam, ensuring the stability and signal fidelity of the sensing interface during long-term biochemical reactions.

[0039] In a possible implementation, the specific operation of step S11 is as follows:

[0040] S11. Take a single-sided polished single-crystalline silicon wafer, and obtain a silicon wafer mold through photolithography and development processes in sequence;

[0041] Prepare a mixture containing a flexible film material and a curing agent, and place the silicon wafer mold in the mixture until it cures. Then, through demolding, slicing, and punching processes in sequence, obtain a control substrate with a sample inlet, a sample outlet, a reaction chamber, and a microchannel.

[0042] Compared with the prior art, the present invention realizes high-precision structure replication and biocompatibility guarantee in the preparation of microfluidic control components through the combination of a single-crystalline silicon mold and a flexible material replication process: the photolithographic forming of the silicon mold ensures the strict controllability of the dimensions of the reaction chamber and the flow channel, forming a spatial match with the cantilever beam unit of the sensing component; the pouring and curing process of the flexible material endows the control component with excellent deformation adaptability, which not only simplifies the processing difficulty of complex three-dimensional channels, but also maintains the intrinsic activity of the biological reaction environment through the chemically inert surface characteristics, providing an efficient solution for the high-reliability packaging and fluid control of the chip.

[0043] The third aspect of the present invention provides an application of a sensor chip in detecting the binding kinetics between biomolecules. This method has the characteristics of being simple, rapid, low-cost, and reusable, and can be widely used for measuring the binding equilibrium constants and kinetic parameters of various types of molecules. Description of the Drawings

[0044] Figure 1 It is a schematic diagram of the explosion structure of the present invention;

[0045] Figure 2 It is a schematic diagram of the structure at the top of the sensing substrate of the present invention;

[0046] Figure 3 It is a schematic diagram of the structure at the bottom of the control substrate of the present invention;

[0047] Figure 4 It is a schematic diagram of the local structure in the middle of the top of the sensing substrate of the present invention;

[0048] Figure 5 It is a partial schematic diagram of one of the first liquid accumulation tank areas of the present invention;

[0049] Figure 6 It is a process flow chart for preparing the sensing substrate of the present invention;

[0050] Figure 7 It is a cross-sectional view of the sensor chip of the present invention;

[0051] Figure 8 It is one of the SEM scanning electron microscope images of the sensor chip of the present invention;

[0052] Figure 9 It is another SEM scanning electron microscope image of the sensor chip of the present invention;

[0053] Figure 10 It is yet another SEM scanning electron microscope image of the sensor chip of the present invention;

[0054] Figure 11 It is the SEM scanning electron microscope image of the sensor chips with different geometric shapes prepared by the present invention;

[0055] Description of the Reference Numerals:

[0056] 1. Control substrate; 11. Sampling inlet; 12. Sampling outlet; 13. Reaction tank; 14. Microchannel; 2. Sensing substrate; 21. First liquid accumulation tank; 22. Structural film; 23. Sensitive film; 24. Cantilever beam; 25. Piezoresistor; 26. Lead; 261. Heavily doped lead; 262. Metal lead; 27. Pad; 28. Second liquid accumulation tank; 3. Local modification area. Detailed Embodiments

[0057] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following provides a detailed description of specific embodiments of the present invention. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0058] It should be noted that the endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0059] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, for the purpose of clarification or convenient reference, terms with conventional understood meanings are defined herein. Such definitions herein should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments follows the protocols and parameters given by the manufacturers.

[0060] See Figures 1-5 , an embodiment of the present application discloses a sensor chip for detecting the binding kinetics between biomolecules, including a control substrate 1 and a sensing substrate 2 bonded to each other. The control substrate 1 covers the top of the sensing substrate 2. Among them, at least one injection port 11 and one sampling port 12 are provided on the control substrate 1. In this embodiment, preferably two injection ports 11 are provided. A microchannel 14 and a reaction tank 13 are provided at the bottom of the control substrate 1. The two injection ports 11 are respectively connected to the reaction tank 13 through a microchannel 14, and the sampling port 12 is connected to the reaction tank 13 through a microchannel 14; at least two first liquid accumulation grooves 21 are formed in the middle of the top of the sensing substrate 2. Both of the two first liquid accumulation grooves 21 are covered by the reaction tank 13. A structural film 22 and a sensitive film 23 are respectively erected above the two first liquid accumulation grooves 21. Among them, a local modification area 3 is provided at the top of the sensitive film 23. Four cantilever beams 24 are circumferentially and uniformly distributed on the side walls of the structural film 22 and the sensitive film 23 and are fixedly connected to the inner wall of the first liquid accumulation groove 21 through the four cantilever beams 24. A piezoresistor 25 is provided at the top of each cantilever beam 24. Every two adjacent piezoresistors 25 above the same first liquid accumulation groove 21 are connected by a lead 26 to form a Wheatstone bridge. Each lead 26 is connected to a pad 27, and the input and output of electrical signals are realized through the pad 27.

[0061] In the embodiment of the present application, in order to further improve the detection effect, four first liquid accumulation grooves 21 are opened in the middle of the top of the sensing substrate 2. The four first liquid accumulation grooves 21 are arranged in a 2x2 array. A structural film 22 and a sensitive film 23 are arranged above the four first liquid accumulation grooves 21. Among them, the sensitive film 23 is arranged above any three first liquid accumulation grooves 21, and the structural film 22 is arranged above the remaining one first liquid accumulation groove 21. Local modification areas 3 are provided at the tops of the sensitive films 23.

[0062] In the embodiment of the present application, the width of the microchannel 14 is 300μm - 500μm, and the diameters of the sample inlet 11 and the sample outlet 12 are both 1mm.

[0063] In the embodiment of the present application, the cross-sections of the structural film 22 and the sensitive film 23 are both annular octagons, and the thickness is 2μm - 4μm. The width of the cantilever beam 24 is 80μm - 140μm. The bottom end of the cantilever beam 24 is at the same horizontal height as the top of the structural film 22 or the sensitive film 23, that is, there is a height difference between the cantilever beam 24 and the local modification area 3, so as to form a stress concentration area on the sensitive film 23. Local modification areas 3 are provided on each sensitive film 23, and the surface of the local modification area 3 is used to modify biorecognition molecules.

[0064] In the embodiment of the present application, each lead 26 includes two sub-leads. Each sub-lead includes a heavily doped lead 261 and a metal lead 262 connected to the heavily doped lead 261. Every two adjacent piezoresistors 25 above the same first liquid accumulation groove 21 are sequentially connected through the heavily doped lead 261 and the metal lead 262. Second liquid accumulation grooves 28 are dug at the tops of the structural film 22 and the sensitive film 23. A through hole is provided in the middle of the local modification area 3, and the through hole communicates with the second liquid accumulation groove 28 on the sensitive film 23.

[0065] As Figure 6 and Figure 7 shown, the specific embodiment of the present invention also provides a preparation method of a sensor chip. The preparation method specifically includes the following steps:

[0066] S1. An SOI substrate with a (100) crystal plane is used. The SOI substrate includes a silicon support layer, a buried oxide layer, and a silicon device layer from bottom to top.

[0067] S2. The silicon device layer is sequentially subjected to photolithography, development, and dry etching processes to obtain grooves on the surfaces of the silicon device layer and the buried oxide layer.

[0068] S3. A first SiO2 layer is grown by thermal oxidation on the grooves obtained in step S2 and the surface of the silicon device layer.

[0069] S4. The structure obtained in step S3 is successively subjected to photolithography, development, then boron ion light doping treatment to form piezoresistors 25, and then boron ion heavy doping treatment to form heavily doped leads 261;

[0070] S5. Deposit a second SiO2 layer on the top and bottom surfaces of the structure obtained in step S4;

[0071] S6. On the basis of the structure obtained in step S5, successively through photolithography, development, and dry etching treatment, form lead holes at the ends of the heavily doped leads 261;

[0072] Then successively through sputtering metal treatment, photolithography, development, and etching treatment, obtain metal leads 262 and pads 27, where the metal leads 262 and the heavily doped leads 261 form an ohmic contact through high-temperature boron ion diffusion treatment and annealing treatment;

[0073] S7. Selectively sputter at least one metal layer on the top surface of the structure obtained in step S6, and form the sensitive film 23 by the lift-off method;

[0074] S8. Deposit a third SiO2 layer and a silicon nitride layer on the top surface of the structure obtained in step S7 in sequence, and then through photolithography and development, form a metal passivation layer;

[0075] S9. On the top surface of the structure obtained in step S8, successively through photolithography, development, and dry etching treatment, etch away the upper first SiO2 layer and second SiO2 layer, expose the silicon support layer of the SOI substrate and form through holes in the local modification area 3 to form the second liquid accumulation groove 28;

[0076] S10. Perform anisotropic wet etching on the structure obtained in step S9 to obtain the first liquid accumulation groove 21 and the cantilever beam 24, and complete the fabrication of the sensing substrate 2;

[0077] S11. Fabricate an inlet 11, an outlet 12, a reaction tank 13, and a microchannel 14 on a flexible film with biocompatibility to obtain a control substrate 1;

[0078] S12. Adopt a chemical bonding method to bond the control substrate 1 and the sensing substrate 2 to obtain a sensor chip.

[0079] In the embodiment of the present application, in step S1, in the SOI substrate, the thickness of the silicon device layer is 2.9 - 3.1 μm, the thickness of the buried oxide layer is 0.9 - 1.1 μm, and the thickness of the silicon support layer is 690 - 710 μm.

[0080] In the embodiment of the present application, in step S7, the material of the metal layer is a mixture of gold and chromium or a mixture of gold and titanium.

[0081] In the embodiment of the present application, in step S8, the thickness of the third SiO2 layer is 195 - 205 nm, and the thickness of the silicon nitride layer is 95 - 105 nm.

[0082] In the embodiment of the present application, the specific operation of step S11 is as follows: S11. Take a single-sided polished single-crystalline silicon wafer, and obtain a silicon wafer mold through photolithography and development processes in sequence.

[0083] Prepare a mixed solution containing a flexible film material and a curing agent, and place the silicon wafer mold in the mixed solution until it cures. Then, through demolding, slicing, and punching processes in sequence, obtain a control substrate 1 having a sample inlet 11, a sample outlet 12, a reaction tank 13, and a microchannel 14.

[0084] Specifically, in the preparation process of the present invention, processes such as photolithography, development, dry etching, and thermal oxidation growth for volume are all in accordance with the prior art. More specifically, they can be selected from the following parameters and adjusted according to the actual preparation process.

[0085] Photolithography: Uniformly coat a photoresist on the surface of the structure through a spin coater (thickness 0.5 - 2 μm). After pre-baking (90 - 120 °C, 1 - 5 minutes) for curing, use an ultraviolet light source (such as 193 nm DUV) to expose through a mask (dose 100 - 300 mJ / cm²), and then perform post-baking (110 - 130 °C, 1 - 5 minutes) to enhance the pattern stability. Finally, form a high-resolution (0.1 - 0.5 μm) photoresist pattern to provide an accurate mask for subsequent etching.

[0086] Development: Use a TMAH solution (positive photoresist) or a special developer (negative photoresist) to remove the photoresist in the unexposed / exposed areas, control the development time (30 - 120 seconds) and temperature (20 - 25 °C), and use ultrasonic assistance to improve the uniformity, precisely retain the pattern edge (roughness < 1 nm), avoid undercut effect, and ensure high consistency with the mask pattern.

[0087] Dry etching: Use reactive ion etching (RIE) or inductively coupled plasma (ICP - RIE), with an SF6 / Cl2 mixed gas (ratio 50:10 sccm) as the main etchant, supplemented by O2 for depolymerization and He for cooling. Set the radio frequency power (50 - 300 W), bias voltage (-50 to -500 V), and low pressure (1 - 100 mTorr). Transfer the photoresist pattern to the silicon and buried oxide layer through anisotropic etching (aspect ratio 10:1 - 50:1), and control the etching depth (such as 500 nm) through endpoint detection (OES monitoring of the SiF characteristic peak). At the same time, optimize the selectivity (Si / PR reaches 10:1 - 100:1) to avoid damage to the buried oxide layer.

[0088] Thermal oxidation growth: The steps for thermal oxidation growth of the first SiO2 layer include: First, remove the contaminants on the silicon wafer surface through the RCA cleaning method and dry it; Subsequently, select dry oxygen (800 - 1000 °C, O2), wet oxygen (700 - 900 °C, with H2O participation), or steam oxidation (directly introduce steam), and control the temperature, time, and gas flow rate (such as the O2 flow rate of dry oxygen being 1 - 10 slm) to adjust the thickness (10 - 500 nm); After growth, repair the lattice defects through rapid thermal annealing (900 - 1100 °C), and finally obtain a dense oxide layer with a low interface state density (<1×10 10 cm -2 eV -1 ), and high uniformity (±3%).

[0089] According to the above preparation method, the SEM scanning electron microscope image of the prepared sensor chip is as shown in Figures 8-10 ; Figure 11 It shows the SEM scanning electron microscope images of sensor chips with different geometries prepared by the inventor.

[0090] The present invention also provides a method for detecting the binding kinetics between biomolecules, including the following steps:

[0091] Step A1, fix the probe molecules on the surface of the local modification area 3 of the sensing substrate 2 through methods such as thiol self-assembly or physical adsorption;

[0092] Step A2, inject the buffer solution into the reaction tank 13 of the sensor chip through the injection port 11, and measure the initial baseline output voltage through the sensing substrate 2 V b ;

[0093] Step A3, inject the solution with the target molecule concentration of [A] into the reaction tank 13 through the injection port 11 for detection, so that the target molecule binds to the probe molecule, and obtain the binding curve of the output voltage changing with time during the binding process V m ( t ), where the relative change amount of the output voltage during the binding process is Δ V ( t ) = V m ( t ) - V b ;

[0094] Step A4, after reaching equilibrium during the binding process, obtain the maximum value of the output voltage signal response as Δ V max, inject the buffer solution into the reaction cell 13 through the injection port 11, start the dissociation of the target molecule and the probe molecule, and obtain the dissociation curve of the output voltage varying with time during the dissociation process. The minimum value of the output signal response during the dissociation process is Δ V min ;

[0095] Step A5: Perform kinetic fitting on the binding curve in Step A3 and the dissociation curve in Step A4 to obtain the binding rate constant k on and the dissociation rate constant k off , and calculate the equilibrium dissociation constant k D = k off / k on ;

[0096] Step A6: Verify the linear relationship between k obs and the concentration [A] of the target molecule through experiments with multiple concentrations of the target molecule.

[0097] In the embodiment of the present application, in Step A5, the calculation formula for performing kinetic fitting is as follows:

[0098] ;

[0099] ;

[0100] .

[0101] In the embodiment of the present application, when the biomolecular binding kinetics detection method is applied to the detection of antigen - antibody binding kinetic parameters, it is evolved into the following steps:

[0102] Step B1: Fix the anti - IgG antibody (concentration of 10 μg / mL) diluted with PBS buffer (pH 7.4) on the surface of the local modification area of the sensing substrate 2 by thiol self - assembly method, and block the non - specific binding sites with 1% BSA;

[0103] Step B2: Inject the PBS buffer into the reaction cell 13 of the sensor chip through the injection port 11, control the flow rate to be 5 μL / min. After the flow rate is stable, record the output voltage of the sensing substrate 2 V b , and take the average value for 5 minutes as the baseline signal;

[0104] Step B3: Inject the PBS solution containing the target antigen IgG at concentration gradients of 0.1 nM, 1 nM, and 10 nM into the reaction cell 13 through the injection port 11 for detection, enabling the anti-IgG antibody to bind to the target antigen IgG, and obtaining the binding curve of the output voltage varying with time during the binding process. V m ( t ) Calculate the relative change in the output voltage during the binding process as Δ V ( t ) = V m ( t ) - V b ;

[0105] Step B4: After reaching equilibrium during the binding process, record the maximum value of the output voltage signal response as Δ V max . Inject the PBS buffer solution into the reaction cell 13 through the injection port 11 to initiate the dissociation of the anti-IgG antibody from the target antigen IgG, record the dissociation curve of the output voltage varying with time during the dissociation process, and the minimum value of the output signal response during the dissociation process is Δ V min ;

[0106] Step B5: Perform kinetic fitting on the binding curve in Step 3 and the dissociation curve in Step 4 to obtain the binding rate constant k on and the dissociation rate constant k off , and calculate the equilibrium dissociation constant k D = k off / k on ;

[0107] Step B6: Repeat the above steps, and measure the corresponding k obs values at IgG concentrations of 0.1 nM and 1 nM, plot the linear relationship graph of the k obs value versus the IgG concentration, and calculate the error range therefrom.

[0108] As described above, it is only the specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in this application should be covered within the protection scope of this application. Therefore, the protection scope of this application shall be subject to the protection scope of the claims.

Claims

1. A sensor chip for detecting the binding kinetics between biomolecules, characterized in that, Comprising: A control substrate, on which at least one sample inlet and at least one sample outlet are formed. At the bottom end of the control substrate, a reaction tank and two microfluidic channels communicating with the reaction tank are dug. The reaction tank is communicated with the sample inlet and the sample outlet respectively through the two microfluidic channels; A sensing substrate, the top end of the sensing substrate is bonded to the bottom end of the control substrate. At least two first liquid accumulation grooves covered by the reaction tank are dug at the top end of the sensing substrate. Above the two first liquid accumulation grooves, a structural film and a sensitive film are respectively erected. A local modification area is arranged at the top end of the sensitive film. Four cantilever beams are circumferentially and evenly distributed on the side walls of the structural film and the sensitive film and are fixedly connected to the inner wall of the first liquid accumulation groove through the four cantilever beams. A piezoresistor is arranged at the top end of each cantilever beam. Every two adjacent piezoresistors above the same first liquid accumulation groove are connected by leads to form a Wheatstone bridge. Each lead is connected to a pad. Second liquid accumulation grooves are dug at the top ends of the structural film and the sensitive film. A through hole is arranged in the middle of the local modification area and the through hole communicates with the second liquid accumulation groove on the sensitive film.

2. The sensor chip for detecting the binding kinetics between biomolecules according to claim 1, wherein Each lead includes two sub-leads with one ends connected to each other. Each sub-lead includes a heavily doped lead and a metal lead connected to the heavily doped lead. Every two adjacent piezoresistors above the same first liquid accumulation groove are sequentially connected through the heavily doped lead and the metal lead.

3. The sensor chip for detecting the binding kinetics between biomolecules according to claim 1, characterized in that, Four first liquid accumulation grooves covered by the reaction tank are dug at the top end of the sensing substrate. Above one of the first liquid accumulation grooves, a structural film is erected. Above the remaining three first liquid accumulation grooves, a sensitive film is erected.

4. A method for preparing a sensor chip as described in claim 1, characterized in that, Including the following steps: S1. Select an SOI substrate including a silicon support layer, a buried oxide layer, and a silicon device layer from bottom to top; S2. Perform photolithography, development, and dry etching on the SOI substrate in sequence to obtain grooves on the surfaces of the silicon device layer and the buried oxide layer; S3. Grow a first SiO2 layer on the grooves and the surface of the silicon device layer; S4. After performing photolithography and development in sequence, obtain piezoresistors through boron ion light doping treatment, and then obtain heavily doped leads through boron ion heavy doping treatment; S5. Deposit second SiO2 layers on the top surface and the bottom surface respectively; S6. After performing photolithography, development, and dry etching in sequence, obtain lead holes at the ends of the heavily doped leads; After performing sputtering metal treatment, photolithography, development, and etching in sequence, obtain metal leads and pads, and make the metal leads form ohmic contacts with the heavily doped leads; S7. Selectively sputter at least one metal layer on the top surface, and obtain a sensitive thin film by the lift-off method; S8. Deposit a third SiO2 layer and a silicon nitride layer on the top surface in sequence, and then perform photolithography and development processes to obtain a metal passivation layer; S9. After performing photolithography, development, and dry etching processes on the top surface in sequence, expose the silicon support layer and obtain a through hole in the local modification area to obtain a second liquid accumulation groove; S10. Perform anisotropic wet etching. After obtaining the first liquid accumulation groove and the cantilever beam, obtain a sensing substrate; S11. Use a flexible film to fabricate a control substrate; S12. Bond the control substrate and the sensing substrate to obtain a sensor chip.

5. The preparation method according to claim 4, characterized in that, In the step S1, in the SOI substrate, the thickness of the silicon device layer is 2.9 - 3.1 μm, the thickness of the buried oxide layer is 0.9 - 1.1 μm, and the thickness of the silicon support layer is 690 - 710 μm.

6. The preparation method according to claim 4, characterized in that, In the step S7, the material of the metal layer is a mixture of gold and chromium or a mixture of gold and titanium.

7. The preparation method according to claim 4, characterized in that, In the step S8, the thickness of the third SiO2 layer is 195 - 205 nm, and the thickness of the silicon nitride layer is 95 - 105 nm.

8. The preparation method according to claim 4, characterized in that, The specific operation of the step S11 is as follows: S11. Take a single-sided polished monocrystalline silicon wafer, and obtain a silicon wafer mold through photolithography and development processes in sequence; Prepare a mixed solution containing a flexible film material and a curing agent, and place the silicon wafer mold in the mixed solution until it cures. Through demolding, slicing, and punching processes in sequence, obtain a control substrate with a sample inlet, a sample outlet, a reaction tank, and a microchannel.

9. Application of a sensor chip as described in any one of claims 1 - 3 in the detection of biomolecular binding kinetics.

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