High-precision full-quantitative tear marker detection chip and method for dry eye diagnosis

Abstract

The invention belongs to the technical field of precision medicine and novel diagnosis, and particularly relates to a high-precision full-quantitative tear marker detection chip and method for dry eye diagnosis. Cantilever structures are arranged on the upper portion and the lower portion in the base respectively. A driving electrode and a sensing electrode are deposited on the right side of the base respectively. The output electrode plate and the reaction electrode plate are connected through a metal wire, an ion sensitive membrane or a reference electrode is arranged on the reaction electrode plate, a modified electrode is deposited outside the fan-shaped sensitive area, and an antibody modified layer is arranged on the modified electrode; according to the invention, innovative design and temperature compensation design of a multi-parameter sensing structure are realized through large-adsorption-area and high-Q-value plane resonant symmetrical design, and a synergistic mechanism between system response and design parameters of the tear detection chip is deeply researched, so that high-precision simultaneous detection of multiple tear biomarkers is realized; therefore, the purpose of auxiliary diagnosis and treatment of the dry eye patient is achieved quickly.

Description

Technical Field

[0001] This invention belongs to the fields of precision medicine and novel diagnostic technology, specifically relating to a high-precision fully quantitative tear biomarker detection chip and method for dry eye diagnosis. Background Technology

[0002] Precision medicine plays a crucial role in personalized testing, disease prevention, improving medical efficiency, and advancing medical progress. It optimizes the allocation of medical resources and represents a significant direction for future medical development. Taking dry eye disease as an example, it is a common eye condition caused by a vicious cycle of tear film instability and hyperosmolarity, leading to ocular surface inflammation, damage, and abnormal nerve sensation. With the widespread use of electronic devices, changes in lifestyle, and increased environmental pollution, the prevalence of dry eye disease has further increased, affecting approximately 5% to 30% of the global population. Mild dry eye can cause visual impairment, while severe cases can lead to permanent blindness. Traditional dry eye diagnosis relies primarily on symptom inquiry, ocular surface examination, and semi-quantitative tear analysis using fluorescein chromatography. These methods are highly subjective and lack quantitative evaluation, making it difficult to accurately determine the diagnostic criteria and severity of dry eye. In recent years, the role of ocular surface inflammation in the pathogenesis of dry eye disease has gained increasing attention. Matrix metalloproteinase-9 (MMP-9) and sodium / potassium ions, as tear components closely related to ocular inflammation, have been proven to be the gold standard for dry eye diagnosis. Some existing commercially available products, such as the MMP-9 rapid diagnostic kit commonly used by clinicians in North America to determine whether a patient's tears are positive, are not yet available in China and can only perform qualitative detection of a single target with low accuracy. In addition, existing methods have high equipment requirements and complex operating procedures, which cannot meet the needs of rapid, home-based self-diagnosis. Summary of the Invention

[0003] To overcome the aforementioned challenges and achieve technological localization, and addressing the issue that existing commercial dry eye diagnostic products cannot perform high-precision, fully quantitative, and multi-parameter tear film detection, this invention provides a high-precision, fully quantitative tear film biomarker detection chip for dry eye diagnosis. This chip combines micro-nano resonance with electrochemical detection technology, resulting in a high-precision, fully quantitative tear film biomarker detection chip. Through a large adsorption area and a high-Q-value planar resonant symmetrical design, it achieves innovative multi-parameter sensing structure design and temperature compensation design. Furthermore, it delves into the synergistic mechanism between the tear film detection chip system response and design parameters, enabling high-precision simultaneous detection of multiple tear film biomarkers, thereby achieving the goal of rapidly assisting in the diagnosis and treatment of dry eye patients.

[0004] A high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis includes a base 1, a driving electrode 2, a reactive electrode 3, a sensing electrode 4, a piezoelectric layer 5, a modified electrode 6, an antibody modified layer 7, an ion-sensitive membrane 8, a reference electrode 9, and a cantilever structure 10. The base 1 has cantilever structures 10 located at the top and bottom, respectively. The right side of the cantilever structure 10 is connected to the base 1. The driving electrode 2 and the sensing electrode 4 are deposited at the front and rear ends of the top and bottom surfaces on the right side of the base 1, respectively.

[0005] The cantilever structure 10 includes a driving cantilever 1001, a supporting cantilever 1002, a sensing cantilever 1003, and a fan-shaped sensitive area 1004. The driving cantilever 1001, the supporting cantilever 1002, and the sensing cantilever 1003 are respectively arranged from front to back on the non-curved surface on the right side of the fan-shaped sensitive area 1004, and the right ends of the three are all connected to the base 1. Piezoelectric layers 5 are respectively provided on the outer surfaces of the right sides of the driving cantilever 1001 and the sensing cantilever 1003. The piezoelectric layer 5 on the driving cantilever 1001 is connected to the corresponding driving electrode 2, and the piezoelectric layer 5 on the sensing cantilever 1003 is connected to the corresponding sensing electrode 4.

[0006] The reaction electrode 3 includes an output electrode plate 301 deposited on the top and bottom right side of the base 1, metal wires 302 deposited on the outer surfaces of the upper and lower support cantilever 1002, and reaction electrode plates 303 deposited on the outer surfaces of the inner rings of the upper and lower fan-shaped sensitive areas 1004. The output electrode plate 301 and the corresponding reaction electrode plate 303 are connected by metal wires 302. An ion-sensitive membrane 8 is provided on the upper reaction electrode plate 303, and a reference electrode 9 is provided on the lower reaction electrode plate 303. Meanwhile, modification electrodes 6 are deposited on the outer surfaces of the outer rings of the upper and lower fan-shaped sensitive areas 1004, and antibody modification layers 7 are provided on the modification electrodes 6.

[0007] The base 1 is a quadrangular prism with a square base. The quadrangular prism has a rectangular through hole inside. Cantilever structures 10 are provided at the upper and lower ends of the rectangular through hole. The right side of the driving cantilever 1001, the supporting cantilever 1002 and the sensing cantilever 1003 are all connected to the inner wall of the base 1. A gap is left between the left side of the fan-shaped sensitive area 1004 and the inner wall of the base 1, so that the fan-shaped sensitive area 1004 does not contact the inner wall of the base 1.

[0008] The output electrode plate 301 is deposited on the base 1 between the driving electrode 2 and the sensing electrode 4.

[0009] The substrate 1 comprises two symmetrically arranged layers, each of which includes a silicon dioxide layer 101 and an SOI layer 102 arranged sequentially from the outside to the inside. The two SOI layers 102 are bonded together by silicon-silicon thermocompression bonding.

[0010] The driving electrode 2 and the sensing electrode 4 have the same structure, both divided into inner and outer parts. The inner part includes a square part and a long strip part, with the end of the long strip part disposed on the corresponding piezoelectric layer 5. The outer part includes a square part, a short strip part, and a long strip part, with the square part and the long strip part connected together by the short strip part. The short strip part and the long strip part have an angle of 90 degrees, and the end of the long strip part is disposed on the corresponding piezoelectric layer 5.

[0011] The cantilever structure 10 and the base 1 are an integral part.

[0012] A method for using a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis is described below:

[0013] Step one involves calibrating the detection chip and obtaining the sensing curve, which includes the following:

[0014] Step 1.1: Place the detection chip in a tear environment containing matrix metalloproteinases at known concentrations of 0, 2, 4, 6, 8, and 10 ng / mL, respectively. Drive the chip by applying a driving signal through the driving electrode 2. Connect the oscilloscope to the sensing electrode 4 and obtain the amplitude-frequency curves of the detection chip under different concentrations of matrix metalloproteinases through the oscilloscope. The resonant peak point of the amplitude-frequency curve is the resonant frequency of the chip at the corresponding concentration of matrix metalloproteinases. Plot a curve with the resonant frequency difference as the ordinate and the matrix metalloproteinase concentration as the abscissa. This is the sensing curve. Fit the relationship expression between the frequency difference and the concentration through the sensing curve, where the resonant frequency difference is the resonant frequency of the chip at each non-zero concentration of matrix metalloproteinases minus the initial resonant frequency of the chip at zero concentration of matrix metalloproteinases.

[0015] Step 2: Place the calibrated detection chip in the tear fluid environment to be tested. At this time, the antibody-modified layer 7 and the ion-sensitive membrane 8 are used to detect matrix metalloproteinases and sodium / potassium ions in the tear fluid, respectively.

[0016] Step 3: Measure the initial potential difference E0 between the two output electrode plates 301 using a high-precision voltmeter.

[0017] Step 4: Drive the chip by applying a drive signal through the drive electrode 2, measure the potential difference E between the two output electrode plates 301 using a voltmeter, and obtain the resonant frequency of the detection chip using an oscilloscope.

[0018] Step 5: Calculate the sodium / potassium ion concentration using the potential difference E value from Step 4. Then, using the resonant frequency measured in Step 4, determine the frequency difference before and after tear detection according to Step 1. Finally, calculate the matrix metalloproteinase concentration at this point using the sensor curve fitting expression calibrated in Step 2. The sodium / potassium ion concentration is calculated using the following formula:

[0019]

[0020] In the formula, R is the gas constant, T is the tear temperature, z is the charge number of the target ion, F is the Faraday constant, and a is the concentration of the target ion.

[0021] A method for preparing and surface-modifying a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis, comprising the following:

[0022] Step a, a 500 nm silicon dioxide layer 101 is formed on the upper surface of the thin silicon layer 102a of the SOI layer 102 by an oxidation growth method;

[0023] Step b: Etching is performed on the silicon dioxide layer 101 in the corresponding region of the piezoelectric layer 5 using reactive ion etching technology to form an opening for ion implantation to form the piezoelectric layer 5;

[0024] Step c: Deep silicon etching technology is used to etch the structure corresponding to the cantilever structure 10 on the base silicon layer 102c, and then BOE process is used to etch the structure corresponding to the cantilever structure 10 on the insulating layer 102b, so as to obtain the whole suspended structure of the base 1 and the area where the cantilever structure 10 is located.

[0025] Step d: Repeat steps a, b, and c above to obtain two identical bases 1 and the entire suspended structure in the area where the cantilever structure 10 is located. Then, use the Si-Si hot-press bonding process to bond the remaining silicon base layer 102c of the two bases 1 to obtain the main structure of the detection chip.

[0026] Step e: Boron ions are implanted into the regions corresponding to the piezoelectric layer 5 at the roots of the driving cantilever 1001 and sensing cantilever 1003 through the openings formed in step b using an ion implantation process. After annealing at 950°C, the piezoelectric layer 5 is obtained. Then, a 20nm Cr layer and a 300nm Au layer are deposited on the silicon dioxide layer 101 at the positions corresponding to the driving electrode 2, the reactive electrode 3, the sensing electrode 4, the modification electrode 6, and the reference electrode 9, respectively, using a sputtering stripping process, to obtain the driving electrode 2, the reactive electrode 3, the sensing electrode 4, the modification electrode 6, and the reference electrode 9.

[0027] Step f: Reactive ion etching is used again to etch the silicon dioxide layer 101 to the thin silicon layer 102a of the non-cantilever structure area on the entire suspended structure where the substrate 1 and the cantilever structure 10 are located. Then, deep silicon etching is used to etch the thin silicon layer 102a to the insulating layer 102b of the non-cantilever structure area of ​​the entire suspended structure to release the cantilever structure 10. Thus, the main structure of the chip, substrate 1, driving electrode 2, reactive electrode 3, sensing electrode 4, piezoelectric layer 5, modification electrode 6, reference electrode 9 and cantilever structure 10, are obtained. Finally, a single chip is obtained by laser slicing.

[0028] Step g: An ion-sensitive membrane 8 and a reference electrode 9 are formed on the corresponding reaction electrode plate 303 by electrochemical method; and an antibody-modified layer 7 is formed on the modified electrode 6 by self-assembly method.

[0029] To achieve accurate detection of sodium / potassium ions in tears, an ion-sensitive membrane 8 and a reference electrode 9 are fabricated on a well-formed reaction electrode plate 303. The electrochemical process for preparing the sodium / potassium ion-sensitive membrane is as follows:

[0030] i. A PVB reference solution was prepared by dissolving 79.1 mg PVB and 50 mg NaCl in 1 mL of methanol;

[0031] ii. The reaction electrode plate 303 located below is deposited in the Ag electroplating solution under a constant current of 1 mA / cm2 for 30 min. Then, the reaction electrode plate 303 is placed in 0.1 mol / L FeCl3 and reacted for 60 s to obtain an Ag / AgCl layer on the reaction electrode plate 303. Then, 2 μL of PVB reference solution prepared in step i is drop-cast onto the Ag / AgCl layer to form the reference electrode 9.

[0032] iii. Pretreatment of the upper reaction electrode plate 303: at a current density of 2 mA / cm² 2 Under certain conditions, the upper reaction electrode plate 303 is placed in an electroplating solution composed of a mixture of 0.01M 3,4-ethylenedioxythiophene and 0.1M sodium poly-4-styrene sulfonate. When the transferred charge reaches 20mC, a conductive polymer working electrode is formed on the surface of the reaction electrode plate 303 through electrochemical polymerization. Subsequently, the reaction electrode plate 303 is rinsed clean with deionized water and dried to obtain Na. + and K + Working electrode;

[0033] iv.Na + Preparation of the sensitive membrane: 1 mg of Na ion carrier X-4-tert-butylcalix[4]arene-tetraethyl acetate, 0.55 mg of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 33 mg of polyvinyl chloride, and 65.45 mg of sodium sebacic acid diacid dioate were dissolved and mixed evenly in 660 μL of tetrahydrofuran to obtain Na + Selective membrane mixtures;

[0034] vK + Preparation of the sensitive membrane: 2 mg of valinemycin, 0.5 mg of sodium tetraphenylborate, 32.75 mg of PVC, and 64.75 mg of dioctyl sebacate were dissolved and mixed thoroughly in 350 μL of cyclohexanone solution to obtain K. + Selective membrane mixtures;

[0035] vi. Prepare 4 μL of Na + Or K + The selective membrane mixture is coated onto the top reactive electrode plate 303 and dried at room temperature to obtain the desired corresponding ion-sensitive membrane 8.

[0036] To achieve specific detection of tear biomarkers, the steps for fabricating antibody-modified layer 7 using a self-assembly method are as follows:

[0037] Step 1: Cleaning: Ultrasonic cleaning of the surface of electrode 6 with deionized water for 30 minutes, followed by drying with nitrogen gas;

[0038] Step 2: Formation of self-assembled monolayer: A self-assembled monolayer is formed by immersing the modified electrode 6 in an ethanol solution of 10 mM 11-mercaptoundecenoic acid.

[0039] Step 3: Carboxyl activation: The modified electrode 6 treated above was immersed in a mixed solution of 400 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 100 mM N-hydroxysuccinimide for 2 hours to activate the carboxyl groups in the MUA.

[0040] Step 4: Antibody conjugation: The modified electrode 6 after step 3 is immersed in MMP-9 antibody solutions of different concentrations and continuously flowed for 2 hours, so that the antibody is conjugated to the modified electrode 6 to form an antibody-modified layer 7.

[0041] Step 5: Further fixation of the antibody: Immerse the antibody-modified layer 7 obtained in step 4 in a 0.05% glutaraldehyde solution to fix the antibody-modified layer 7 and prevent the antibody layer from peeling off.

[0042] Step 6: Surface passivation: The antibody-modified layer 7 obtained in step 5 was surface passivated in a 1 mg / mL casein solution for 30 min.

[0043] The beneficial effects of this invention are:

[0044] This invention employs an in-plane resonance method, which greatly reduces the damping effect of the silicon microcantilever structure in the fluid, thereby improving the quality factor of the tear resonance detection chip from the perspective of resonance mechanism.

[0045] This invention innovatively combines micro-nano resonance and electrochemical principles to replace the traditional semi-quantitative fluorescence detection principle, enabling accurate and rapid detection of multiple tear film biomarkers such as inflammatory factors MMP-9, Na+, and K+. The chip has advantages such as high-precision multi-biomarker detection, full quantification, rapid reusability, etc.

[0046] This invention requires simple, small-sized, and low-cost instruments, which can provide a more accurate and effective means for the real-time tear detection and home self-diagnosis of hundreds of millions of dry eye patients. It can also enhance the self-protection capability of high-end precision medical detection chips. This invention will effectively promote the development of real-time tear detection technology and has great social significance and value.

[0047] In summary, this invention introduces an atomically-resolution resonance mechanism into a biosensor element and designs two symmetrically distributed resonance structures. This enables high-precision, fully quantitative detection of multiple ions in tears simultaneously. Through the full quantitative detection of multiple biomarkers, data analysis and comparison, and integration with dry eye diagnostic mechanisms, the health status of a patient's eyes can be rapidly assessed, providing doctors with a more comprehensive diagnostic basis. After experimentation, optimization, and mature application, dry eye examination techniques, diagnostic procedures, and standards suitable for clinical practice in China can be proposed, improving the current level of clinical diagnosis and treatment of dry eye. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the content of the embodiments of the present invention and these drawings without creative effort.

[0049] Figure 1 This is a schematic diagram of the tear biomarker detection chip structure according to an embodiment of the present invention;

[0050] Figure 2 This is a top view of the tear biomarker detection chip structure according to an embodiment of the present invention;

[0051] Figure 3 This is a front view of the tear biomarker detection chip structure according to an embodiment of the present invention;

[0052] Figure 4 These are the first 1-6 modal diagrams of the tear biomarker detection chip according to an embodiment of the present invention;

[0053] Figure 5 This is a flowchart illustrating the fabrication process of the tear biomarker detection chip according to an embodiment of the present invention.

[0054] Figure 6 This is a schematic diagram of MMP-9 antibody modification according to an embodiment of the present invention.

[0055] Figure 7 This is a diagram illustrating the ion detection mechanism of the tear biomarker detection chip according to an embodiment of the present invention. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.

[0057] Example 1

[0058] A high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis includes a base 1, a driving electrode 2, a reactive electrode 3, a sensing electrode 4, a piezoelectric layer 5, a modified electrode 6, an antibody modified layer 7, an ion-sensitive membrane 8, a reference electrode 9, and a cantilever structure 10. The base 1 has a cantilever structure 10 located at the top and bottom, respectively. The right side of the cantilever structure 10 is connected to the base 1. The driving electrode 2 and the sensing electrode 4 are deposited at the front and rear ends of the top and bottom surfaces on the right side of the base 1, respectively.

[0059] The cantilever structure 10 includes a driving cantilever 1001, a supporting cantilever 1002, a sensing cantilever 1003, and a fan-shaped sensitive area 1004 located at the ends of the three cantilever arms. The driving cantilever 1001, the supporting cantilever 1002, and the sensing cantilever 1003 are respectively arranged from front to back on the non-curved surface on the right side of the fan-shaped sensitive area 1004, and the right ends of the three are all connected to the base 1. Piezoelectric layers 5 are respectively provided on the outer surfaces of the right sides of the driving cantilever 1001 and the sensing cantilever 1003. The piezoelectric layer 5 on the driving cantilever 1001 is connected to the corresponding driving electrode 2, and the piezoelectric layer 5 on the sensing cantilever 1003 is connected to the corresponding sensing electrode 4.

[0060] Specifically: piezoelectric layers 5 are respectively provided on the top surface of the right side of the drive cantilever 1001 and sensing cantilever 1003 of the cantilever structure 10 located above the interior of the base 1; piezoelectric layers 5 are respectively provided on the bottom surface of the right side of the drive cantilever 1001 and sensing cantilever 1003 of the cantilever structure 10 located below the interior of the base 1; the piezoelectric layer 5 on the upper drive cantilever 1001 is connected to the drive electrode 2 located on the top surface of the base 1, and the end of the drive electrode 2 is located on the piezoelectric layer 5; the piezoelectric layer 5 on the lower drive cantilever 1001 is connected to the drive electrode 2 located on the bottom surface of the base 1; the piezoelectric layer 5 on the upper sensing cantilever 1003 is connected to the sensing electrode 4 located on the top surface of the base 1; the piezoelectric layer 5 on the lower sensing cantilever 1003 is connected to the sensing electrode 4 located on the bottom surface of the base 1, and the end of the sensing electrode 4 is located on the piezoelectric layer 5.

[0061] The reaction electrode 3 is an ion detection reaction electrode, including an output electrode plate 301 deposited on the top and bottom right side of the base 1, metal wires 302 deposited on the outer surfaces of the upper and lower support cantilever 1002, and reaction electrode plates 303 deposited on the outer surfaces of the inner rings of the upper and lower fan-shaped sensitive areas 1004. The output electrode plate 301 and the corresponding reaction electrode plate 303 are connected by metal wires 302. An ion-sensitive membrane 8 is provided on the upper reaction electrode plate 303, and a reference electrode 9 is provided on the lower reaction electrode plate 303. Meanwhile, modification electrodes 6 are deposited on the outer surfaces of the outer rings of the upper and lower fan-shaped sensitive areas 1004, and antibody modification layers 7 are provided on the modification electrodes 6.

[0062] The base 1 is a quadrangular prism with a square base. The quadrangular prism has a rectangular through hole inside. Cantilever structures 10 are provided at the upper and lower ends of the rectangular through hole. The right side of the driving cantilever 1001, the supporting cantilever 1002 and the sensing cantilever 1003 are all connected to the inner wall of the base 1. A gap is left between the left side of the fan-shaped sensitive area 1004 and the inner wall of the base 1, so that the fan-shaped sensitive area 1004 does not contact the inner wall of the base 1.

[0063] The output electrode plate 301 is deposited on the base 1 between the driving electrode 2 and the sensing electrode 4.

[0064] The substrate 1 comprises two symmetrically arranged layers, each including a silicon dioxide layer 101 and an SOI layer 102 arranged sequentially from the outside to the inside. The two SOI layers 102 are bonded together by silicon-silicon thermocompression bonding. The SOI layer 102 is a standard SOI wafer, which includes a thin silicon layer 102a, an insulating layer 102b, and a base silicon layer 102c fixed together sequentially from the outside to the inside.

[0065] The driving electrode 2 and the sensing electrode 4 have the same structure, both divided into inner and outer parts. The inner part includes a square part and a long strip part, with the end of the long strip part disposed on the corresponding piezoelectric layer 5. The outer part includes a square part, a short strip part, and a long strip part, with the square part and the long strip part connected together by the short strip part. The short strip part and the long strip part have an angle of 90 degrees, and the end of the long strip part is disposed on the corresponding piezoelectric layer 5.

[0066] The piezoelectric layer 5 is obtained by ion implantation and is used for driving the cantilever structure 10 and detecting the output signal.

[0067] The antibody-modified layer 7 is a specific modified antibody, prepared according to the target biomolecule to be detected. It forms gold-thiol bonds through self-assembly technology and achieves MMP-9 antibody modification on the modified electrode 6.

[0068] The ion-sensitive membrane 8 is a selective ion detection membrane, which is prepared according to the target ion to be detected. It consists of a target ion detection membrane and a solid electrolyte layer, and is deposited on the reaction electrode 303 by deposition technology.

[0069] The reference electrode 9 is a reference electrode that is matched with the ion-sensitive membrane 8. It is symmetrically distributed on the cantilever structure 10 with the ion-sensitive membrane 8 to provide a stable potential reference to ensure the accuracy and reliability of the measurement.

[0070] The cantilever structure 10 and the base 1 are an integral part.

[0071] A method for using a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis is described below:

[0072] Step one involves calibrating the detection chip and obtaining the sensing curve, which includes the following:

[0073] Step 1.1: Place the detection chip in a tear fluid environment containing matrix metalloproteinases at known concentrations of 0, 2, 4, 6, 8, and 10 ng / mL, respectively. Drive the chip by applying a driving signal through the driving electrode 2. Connect an oscilloscope to the sensing electrode 4 and obtain the amplitude-frequency curves of the detection chip at different concentrations of matrix metalloproteinases using the oscilloscope. The resonant peak point of the amplitude-frequency curve is the resonant frequency of the chip at the corresponding concentration of matrix metalloproteinases (i.e., the resonant frequency of the cantilever structure 10). Plot a curve with the resonant frequency difference as the ordinate and the matrix metalloproteinase concentration as the abscissa; this is the sensing curve. Fit the relationship between the frequency difference and the concentration using this sensing curve, where the resonant frequency difference is the resonant frequency f of the chip at each non-zero concentration of matrix metalloproteinases. r - The initial resonant frequency f0 of the chip at 0 concentration of matrix metalloproteinase;

[0074] Step 2: Place the calibrated detection chip in the tear fluid environment to be tested. At this time, the antibody-modified layer 7 and the ion-sensitive membrane 8 are used to detect matrix metalloproteinases (MMP-9) and sodium / potassium ions in the tear fluid, respectively.

[0075] Step 3: Measure the initial potential difference E0 between the two output electrode plates 301 using a high-precision voltmeter.

[0076] Step 4: Drive the chip by applying a drive signal through the drive electrode 2, measure the potential difference E between the two output electrode plates 301 using a high-precision voltmeter, and obtain the resonant frequency of the detection chip using an oscilloscope.

[0077] Step 5: Calculate the sodium / potassium ion concentration using the potential difference E value from Step 4. Using the resonant frequency measured in Step 4, determine the frequency difference before and after tear detection according to Step 1. Then, calculate the matrix metalloproteinase-9 concentration at this point using the sensor curve fitting expression calibrated in Step 2. The sodium / potassium ion concentration is calculated using the following formula:

[0078]

[0079] In the formula, E is the potential difference between the two output electrode plates 301 when detecting tears, E0 is the initial potential difference between the two output electrode plates 301, R is the gas constant, T is the tear temperature, and z is the target ion (Na+). + K + The charge number of the target ion (Na₂) is given by F, where F is the Faraday constant and a is the target ion (Na₂). + K + The concentration of ).

[0080] Step 6: Compare the concentrations of matrix metalloproteinase-9 (MMP-9) and target ion concentrations with medical data from healthy individuals (Ion content in tear fluid of healthy individuals: MMP-9: 0.5-5 ng / mL; Sodium ions: 90–144 mmol / L; Potassium ions: 15–25 mmol / L). If any of the above ions or some of the ions are significantly higher or lower than healthy levels, the individual is identified as a potential patient with dry eye syndrome or requires further medical diagnosis and treatment depending on the clinical situation.

[0081] The principle of this invention: According to classical vibration theory, the resonant frequency of a system is related to its equivalent stiffness and equivalent mass. The basic form of the free vibration frequency f0 of the cantilever structure 10 is as follows:

[0082]

[0083] Where k eff It is the effective stiffness of the cantilever structure 10, m eff It is the effective mass of the cantilever structure 10;

[0084] When the cantilever structure 10 vibrates in a viscous fluid, the fluid impedes its motion. The effect exerted by the fluid environment on the cantilever structure 10 is mainly a damping force proportional to the resonant velocity of the cantilever structure 10. This effect greatly influences the dynamic response of the cantilever structure 10, such as... Figure 4 As shown, using the second-order vibration mode as the working mode can greatly reduce the damping force in the liquid environment. The resonant frequency of the arm beam structure 10 is expressed as f. r :

[0085]

[0086] Where Q is the quality factor, L is the effective length of the cantilever structure 10, and g2 is the inertial parameter determined by the fluid.

[0087] In tear detection environments, the Q value of a resonant sensor depends on the system's energy loss. Since the medium for tear detection is a liquid, energy loss due to liquid viscosity damping is dominant. In this case, the expression for the Q value is as follows:

[0088]

[0089] Where g i , (i = 1, 2) are parameters that depend on the viscosity of the tear being measured, and the expressions for g1 and g2 are as follows:

[0090] g1=πηR e Γ i (R e )

[0091]

[0092] Where η is the viscosity of the fluid, R e It is the Reynolds number, Γ r and Γ i These are the real and imaginary parts of the dimensionless fluid dynamics equations, respectively; the Reynolds number is expressed as follows:

[0093]

[0094] Where, ρ f Where is the fluid density, and b is the effective width of the cantilever structure 10; in the equation, Lg1 / m eff It depends on the dissipation of the fluid (viscous loss), while Lg2 / m eff Related to the added mass of the cantilever structure; when the cantilever structure 10 vibrates, it drags the surrounding fluid, thus the mass of the dragged fluid is equivalent to the added mass of the cantilever structure 10; in most cases, the viscous loss term Lg1 / m eff The value is higher than that of the fluid mass term Lg2 / m eff The value is at least three orders of magnitude larger; therefore, the energy loss caused by fluid dissipation dominates the energy loss of the resonator and basically determines the Q value.

[0095] The detection mechanism of the ion-sensitive membrane 8 is achieved through an ion exchange mechanism, which involves the exchange of target ions within the ion-sensitive membrane 8 with target ions in the tear fluid being tested. According to the Nernst equation, when the ion-sensitive membrane 8 exchanges ions with the target ions in the tear fluid being tested, a potential difference related to the concentration of the target ions in the tear fluid is generated on the two output electrode plates 301. The magnitude of this potential difference is logarithmically related to the concentration of the target ions in the solution, and the specific conversion relationship is as follows:

[0096]

[0097] In the formula, E is the potential difference between the two output electrode plates 301 when detecting tears, E0 is the initial potential difference between the two output electrode plates 301, R is the gas constant, T is the temperature, and z is the target ion (Na+). + K + The charge number of the target ion (Na₂) is given by F, where F is the Faraday constant and a is the target ion (Na₂). + K + The concentration of ).

[0098] This invention provides a method for preparing and surface-modifying a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis, comprising the following process steps (e.g.) Figure 5 , Figure 6 ):

[0099] Step a, a 500 nm silicon dioxide layer 101 is formed on the upper surface of the thin silicon layer 102a of the SOI layer 102 by an oxidation growth method;

[0100] Step b: Etching is performed on the silicon dioxide layer 101 in the corresponding region of the piezoelectric layer 5 using reactive ion etching technology to form an opening for ion implantation to form the piezoelectric layer 5;

[0101] The region corresponding to the piezoelectric layer 5 and the dicing region on the silicon dioxide layer 101 obtained in step a are etched to the thin silicon layer 102a by reactive ion etching to obtain an opening on the piezoelectric layer 5 for ion implantation.

[0102] Step c: Deep silicon etching technology is used to etch the structure corresponding to the cantilever structure 10 on the base silicon layer 102c, that is, the part of the base silicon layer 102c that does not belong to the cantilever structure 10 is etched away. Then, the BOE process is used to etch the structure corresponding to the cantilever structure 10 on the insulating layer 102b, that is, the excess part of the cantilever structure 10 on the insulating layer 102b is etched away, resulting in the entire suspended structure of the base 1 and the area where the cantilever structure 10 is located. The cantilever structure 10 includes a driving cantilever 1001, a supporting cantilever 1002, a sensing cantilever 1003, and a fan-shaped sensitive area 1004 located at the ends of the three cantilevers.

[0103] Step d: Repeat steps a, b, and c above to obtain two identical bases 1 and the entire suspended structure in the area where the cantilever structure 10 is located. Then, use the Si-Si hot-press bonding process to bond the remaining silicon base layer 102c of the two bases 1 to obtain the main structure of the detection chip.

[0104] In step e, boron ions are implanted into the regions corresponding to the piezoelectric layer 5 at the roots of the driving cantilever 1001 and the sensing cantilever 1003 through the openings formed in step b using an ion implantation process. After annealing at 950°C, the piezoelectric layer 5 is obtained. Then, a 20nm Cr layer and a 300nm Au layer are deposited on the silicon dioxide layer 101 at the positions corresponding to the driving electrode 2, the reactive electrode 3, the sensing electrode 4, the modification electrode 6, and the reference electrode 9, respectively, using a sputtering stripping process (the 20nm Cr layer and the 300nm Au layer are deposited sequentially using a magnetron sputtering process, followed by immersion in an acetone solution for stripping). This yields the driving electrode 2 for driving the chip, the reactive electrode 3 for ion detection, and the sensing electrode 4, the modification electrode 6, and the reference electrode 9 for output signal detection.

[0105] Step f: Reactive ion etching is used again to etch the silicon dioxide layer 101 to the thin silicon layer 102a of the non-cantilever structure area of ​​the entire suspended structure in the region where the substrate 1 and the cantilever structure 10 are located. Then, deep silicon etching is used to etch the thin silicon layer 102a to the insulating layer 102b of the non-cantilever structure area of ​​the entire suspended structure in the region where the substrate 1 and the cantilever structure 10 are located to release the cantilever structure 10 (that is, to etch away the part of the entire region where the cantilever structure 10 is located that does not belong to the cantilever structure 1010). Thus, the main structure of the chip, substrate 1, driving electrode 2, reactive electrode 3, sensing electrode 4, piezoelectric layer 5, modification electrode 6, reference electrode 9 and cantilever structure 10, are obtained. Finally, a single chip is obtained by laser slicing.

[0106] Step g: An ion-sensitive membrane 8 and a reference electrode 9 are formed on the corresponding reaction electrode plate 303 by electrochemical method; and an antibody-modified layer 7 is formed on the modified electrode 6 by self-assembly method.

[0107] To achieve accurate detection of sodium / potassium ions in tears, an ion-sensitive membrane 8 and a reference electrode 9 are fabricated on a well-formed reaction electrode plate 303. The electrochemical process for preparing the sodium / potassium ion-sensitive membrane is as follows:

[0108] i. A PVB reference solution was prepared by dissolving 79.1 mg PVB and 50 mg NaCl in 1 mL of methanol;

[0109] ii. The reaction electrode plate 303 located below is deposited in the Ag electroplating solution under a constant current of 1 mA / cm2 for 30 min. Then, the reaction electrode plate 303 is placed in 0.1 mol / L FeCl3 and reacted for 60 s to obtain an Ag / AgCl layer on the reaction electrode plate 303. Then, 2 μL of PVB reference solution prepared in step i is drop-cast onto the Ag / AgCl layer to form Ag / AgCl / PVB reference electrode 9.

[0110] iii. Pretreatment of the upper reaction electrode plate 303: at a current density of 2 mA / cm² 2 Under certain conditions, the upper reaction electrode plate 303 is placed in an electroplating solution composed of a mixture of 0.01M 3,4-ethylenedioxythiophene (EDOT) and 0.1M sodium poly(4-styrene sulfonate) (NaPSS). When the transferred charge reaches 20mC, a conductive polymer working electrode is formed on the surface of the reaction electrode plate 303 through electrochemical polymerization. Subsequently, the reaction electrode plate 303 is rinsed clean with deionized water and dried to obtain Na… + and K + Working electrode;

[0111] iv.Na + Preparation of the sensitive membrane: 1 mg of Na ion carrier X-4-tert-butylcalix[4]arene-tetraacetic acid tetraethyl ester, 0.55 mg of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), 33 mg of polyvinyl chloride (PVC), and 65.45 mg of sodium sebacic acid diacid dioate were dissolved and mixed evenly in 660 μL of tetrahydrofuran to obtain Na + Selective membrane mixtures;

[0112] vK + Preparation of the sensitive membrane: 2 mg of valinemycin, 0.5 mg of sodium tetraphenylborate (NaTPB), 32.75 mg of PVC, and 64.75 mg of dioctyl sebacate (DOS) were dissolved and mixed thoroughly in 350 μL of cyclohexanone solution to obtain K. + Selective membrane mixtures;

[0113] vi. Prepare 4 μL of Na + Or K + The selective membrane mixture is coated onto the top reactive electrode plate 303 and dried at room temperature to obtain the desired corresponding ion-sensitive membrane 8.

[0114] To achieve specific detection of tear biomarkers, the steps for fabricating antibody-modified layer 7 using a self-assembly method are as follows:

[0115] Step 1: Cleaning: Ultrasonic cleaning of the surface of electrode 6 with deionized water for 30 minutes, followed by drying with nitrogen gas;

[0116] Step 2: Formation of self-assembled monolayer: A self-assembled monolayer is formed by immersing the modified electrode 6 in an ethanol solution of 10 mM 11-mercaptoundecenoic acid (MUA). This process provides a basis for further chemical reactions by forming stable gold-sulfur bonds through the binding of thiol groups in MUA with the gold surface. The incubation time is 12 hours or more to ensure that the monolayer fully covers the sensor surface.

[0117] Step 3: Carboxyl activation: The modified electrode 6 treated above was immersed in a mixed solution of 400 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS) for 2 hours to activate the carboxyl groups in the MUA; EDC / NHS converts the carboxyl groups into highly reactive intermediates, enabling them to covalently bind to the amino groups in the antibody.

[0118] Step 4: Antibody conjugation: Immerse the modified electrode 6 after step 3 into MMP-9 antibody solutions of different concentrations (0.5, 1, 10 and 20 ug / mL) and keep it flowing for 2 hours (the antibody solution should be kept flowing for 2 hours to ensure more complete antibody conjugation), so that the antibody is conjugated to the modified electrode 6 to form the antibody modified layer 7.

[0119] Step 5: Further fixation of the antibody: Immerse the antibody-modified layer 7 obtained in step 4 in a 0.05% glutaraldehyde solution to fix the antibody-modified layer 7 and prevent the antibody layer from peeling off.

[0120] Step 6: Surface passivation: The antibody-modified layer 7 obtained in step 5 is surface passivated in a 1 mg / mL casein solution for 30 min; the purpose is to reduce non-specific adsorption and improve the sensitivity and specificity of detection.

[0121] Example 2

[0122] Please see Figure 1 , Figure 2 and Figure 3 The embodiments of the present invention include:

[0123] A high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis includes a base 1, a driving electrode 2, a reactive electrode 3, a sensing electrode 4, a piezoelectric layer 5, a modifying electrode 6, an antibody-modified layer 7, an ion-sensitive membrane 8, a reference electrode 9, a cantilever structure 10, and a fan-shaped sensitive region 11. One end of the cantilever structure 10 is disposed on the base 1, and the fan-shaped sensitive region 11 is connected to the base 1 through the cantilever structure 10. The driving electrode 2 and the sensing electrode 4 are deposited on the base 1, the reactive electrode 3 is deposited on the base 1, the cantilever structure 10, and the fan-shaped sensitive region 11, and the modifying electrode 6 is deposited on the fan-shaped sensitive region 11.

[0124] The substrate 1 consists of a total of 8 layers, including two symmetrically distributed silicon dioxide layers 101 and SOI layers 102. The SOI layer 102 consists of a thin silicon layer 102a, an insulating layer 102b, and a base silicon layer 102c. Two adjacent SOI layers are bonded together by silicon-silicon thermocompression bonding.

[0125] The reaction electrode 3 is an ion detection reaction electrode, including an output electrode plate 301 deposited on the base 1, a metal wire 302 deposited on the support cantilever 1003, and a reaction electrode plate 303 deposited on the fan-shaped sensitive area 11, wherein the output electrode plate 301 and the reaction electrode plate 303 are connected by the metal wire 302.

[0126] The piezoelectric layer 5 is obtained by ion implantation and is used for driving the cantilever structure 10 and detecting the output signal.

[0127] The antibody modification layer 7 is a specific modified antibody, which is selected from the target biomolecule to be detected. It forms gold-thiol bonds through self-assembly technology and realizes the antibody modification of MMP-9 on the modification electrode 6.

[0128] The ion-sensitive membrane 8 is a selective ion detection membrane. The target ion to be detected is preferably obtained. It is composed of a target ion detection membrane and a solid electrolyte layer and is deposited on the reaction electrode 303 by deposition technology.

[0129] The reference electrode 9 is a reference electrode that is matched with the ion-sensitive membrane 8, and is used to provide a stable potential reference to ensure the accuracy and reliability of the measurement.

[0130] The cantilever structure 10, the fan-shaped sensitive area 11, and the base 1 are integrated into one piece.

[0131] The cantilever structure 10 consists of a driving cantilever 1001, a supporting cantilever 1002, and a sensing cantilever 1003, wherein a piezoelectric layer 5 is provided at one end of the driving cantilever 1001 and the sensing cantilever 1003 near the base 1.

[0132] This invention provides a method for preparing and surface-modifying a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis, comprising the following process steps:

[0133] Step a, deposit a 500 nm silicon dioxide layer 101 on the surface of the thin silicon layer 102a by oxidation growth method;

[0134] Step b involves etching the silicon dioxide layer 101 to the thin silicon layer 102a using reactive ion etching technology to obtain openings, scribe lines, and alignment marks for ion implantation to form the piezoelectric layer 5.

[0135] Step c: The base silicon layer 102c is etched to the insulating layer 102b using deep silicon etching technology, and then the back cavity is etched to the thin silicon layer 102a using BOE process.

[0136] Step d: The base silicon layer 102c of the device obtained in the above steps is bonded using a Si-Si hot-press bonding process to obtain the main chip structure.

[0137] Step e: A piezoelectric layer 5 is formed at the root of the driving cantilever 1001 and the sensing cantilever 1003 using an ion implantation process; then, a driving electrode 2 for driving the chip, a reaction electrode 3 for ion detection, and a sensing electrode 4 for output signal detection are obtained by a sputtering stripping process.

[0138] Step f: The silicon dioxide layer 101 is etched to the thin silicon layer 102a using reactive ion etching technology, and then the thin silicon layer 102a is etched to the insulating layer 102b using deep silicon etching technology to release the cantilever structure to obtain the main structure base 1, cantilever structure 10 and fan-shaped sensitive area 11 of the chip. Finally, the chip is cut and separated by laser slicing technology to obtain a single chip.

[0139] Step g: An ion-sensitive membrane 8 and a reference electrode 9 are formed on two symmetrical reaction electrode plates 303 by electrochemical method; an antibody-modified layer 7 is formed on the modified electrode 6 by self-assembly method.

[0140] To achieve accurate detection of sodium and potassium ions in tears, an ion-sensitive membrane and a reference electrode are fabricated on a well-formed reaction electrode plate. The fabrication process of the sodium / potassium ion-sensitive membrane is as follows:

[0141] i. A PVB reference solution was prepared by dissolving 79.1 mg PVB and 50 mg NaCl in 1 mL of methanol.

[0142] ii. The reaction electrode plate 303 on one side is deposited in the Ag electroplating solution under a constant current of 1 mA / cm2 for 30 min, then reacted with 0.1 mol / L FeCl3 for 60 s, and then 2 μL of PVB reference solution is drop-cast onto the formed Ag / AgCl layer to form the Ag / AgCl / PVB reference electrode 9.

[0143] iii.Na + and K + Working electrode preparation: Under the conditions of 20mC polymerization charge and constant current of 2mA / cm2, the working electrode is formed on the reaction electrode plate 303 on the other side in an electroplating solution of 0.01M 3,4-ethylenedioxythiophene (EDOT) and 0.1M sodium poly4-styrenesulfonate (NaPSS).

[0144] iv.Na +Preparation of the sensitive membrane: Na ion carrier X (1 mg), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB) (0.55 mg), polyvinyl chloride (PVC) (33 mg), and sodium sebacic acid dioic acid dioic acid (65.45 mg) were mixed in 660 μL of tetrahydrofuran to prepare Na + Selective membrane mixture

[0145] vK + Preparation of the sensitive membrane: A 350 μL solution of cyclohexanone containing valamicin (2 mg), sodium tetraphenylborate (NaTPB) (0.5 mg), PVC (32.75 mg), and DOS (64.75 mg) was used to prepare the K-membrane. + Selective membrane mixture

[0146] vi. Prepare 4 μL of Na + Or K + A selective membrane mixture is coated onto the reactive electrode plate 303 on the other side to form an ion-sensitive membrane 8;

[0147] To achieve specific detection of tear biomarkers, this invention also provides a method for surface antibody modification using self-assembly technology:

[0148] Step 1: Cleaning: Use deionized water to ultrasonically clean the surface of the chip-modified electrode 6 for 30 minutes, and then dry it with nitrogen.

[0149] Step 2: Formation of a self-assembled monolayer: A self-assembled monolayer is formed by immersing the chip-modified electrode 6 in an ethanol solution of 10 mM 11-mercaptoundecenoic acid (MUA). This process provides a basis for further chemical reactions by forming stable gold-sulfur bonds through the binding of thiol groups in MUA to the gold surface. The incubation time is 12 hours or more to ensure that the monolayer fully covers the sensor surface.

[0150] Step 3: Carboxyl activation: Next, the carboxyl groups in the MUA are activated by immersing the chip-modified electrode 6 in a mixed solution of 400 mM EDC and 100 mM NHS for 2 hours. EDC / NHS converts the carboxyl groups into highly reactive intermediates, enabling them to covalently bind to the amino groups in the antibody.

[0151] Step 4: Antibody conjugation: Immerse the chip-modified electrode 6 in MMP-9 antibody solutions of different concentrations (0.5, 1, 10 and 20 ug / mL) and allow them to flow continuously for 2 hours to conjugate the antibody to the chip-modified electrode 6 to form an antibody-modified layer 7.

[0152] Step 5: Further fixation of antibodies: Antibodies are fixed using 0.05% glutaraldehyde. Glutaraldehyde further stabilizes antibody adsorption by forming covalent bonds, preventing the antibody layer from peeling off, while not affecting antibody activity.

[0153] Step 6: Surface passivation: The surface of antibody-modified layer 7 is passivated using a 1 mg / mL casein solution; the purpose is to reduce non-specific adsorption and improve the sensitivity and specificity of detection; casein is a commonly used passivating agent that can effectively fill the non-functionalized areas on the sensor surface and prevent non-specific binding of other proteins.

[0154] The mechanism and steps of a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis are as follows:

[0155] When the detection chip of this embodiment is placed in the tear fluid environment to be tested, for the detection of MMP-9, the MMP-9 antibody modified on the antibody-modified layer 7 will specifically recognize the inflammatory factor MMP-9 present in the target tear fluid. After MMP-9 is adsorbed onto the antibody-modified layer 7, the resonant frequency of the detection chip will shift. The content of MMP-9 factor in the tear fluid is calculated by the frequency shift output by the sensing electrode 4. For the detection of Na+, the Na+ content in the tear fluid is calculated by the change in the output point position of the symmetrical output electrode plates 301 on both sides. + / K + Concentration, replacing the ion-sensitive membrane 8 can achieve the control of Na + / K + Specific detection of multiple ions.

[0156] Based on the initial signal acquisition output, a symmetrical structure can be used to realize temperature acquisition and temperature compensation design, reducing the measurement error caused by tear temperature changes during the test.

[0157] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the scope of protection of the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, any person skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention within the scope of the technology disclosed in the present invention. These simple modifications are all within the scope of protection of the present invention.

[0158] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0159] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A high-precision, fully quantitative tear film biomarker detection chip for dry eye diagnosis, characterized in that, The device includes a base (1), a driving electrode (2), a reaction electrode (3), a sensing electrode (4), a piezoelectric layer (5), a modification electrode (6), an antibody modification layer (7), an ion-sensitive membrane (8), a reference electrode (9), and a cantilever structure (10). The base (1) is provided with cantilever structures (10) at the top and bottom. The right side of the cantilever structure (10) is connected to the base (1). The driving electrode (2) and the sensing electrode (4) are deposited at the front and rear ends of the top and bottom surfaces on the right side of the base (1), respectively. The cantilever structure (10) includes a driving cantilever (1001), a supporting cantilever (1002), a sensing cantilever (1003), and a fan-shaped sensitive area (1004). The driving cantilever (1001), the supporting cantilever (1002), and the sensing cantilever (1003) are respectively arranged on the non-curved surface on the right side of the fan-shaped sensitive area (1004) from front to back, and the right ends of the three are all connected to the base (1). Piezoelectric layers (5) are respectively provided on the outer surface of the right side of the driving cantilever (1001) and the sensing cantilever (1003). The piezoelectric layer (5) on the driving cantilever (1001) is connected to the corresponding driving electrode (2), and the piezoelectric layer (5) on the sensing cantilever (1003) is connected to the corresponding sensing electrode (4).

2. The high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 1, characterized in that, The reaction electrode (3) includes an output electrode plate (301) deposited on the top and bottom right side of the base (1), metal wires (302) deposited on the outer surface of the upper and lower support cantilever (1002), and reaction electrode plates (303) deposited on the outer surface of the inner ring of the upper and lower fan-shaped sensitive areas (1004). The output electrode plate (301) and the corresponding reaction electrode plate (303) are connected by metal wires (302). An ion-sensitive membrane (8) is provided on the upper reaction electrode plate (303), and a reference electrode (9) is provided on the lower reaction electrode plate (303). Meanwhile, a modification electrode (6) is deposited on the outer surface of the outer ring of the upper and lower fan-shaped sensitive areas (1004), and an antibody modification layer (7) is provided on the modification electrode (6).

3. The high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 1, characterized in that, The base (1) is a quadrangular prism with a square bottom surface. The quadrangular prism has a rectangular through hole inside. The upper and lower ends of the rectangular through hole are respectively provided with cantilever structures (10). The right side of the driving cantilever (1001), the supporting cantilever (1002) and the sensing cantilever (1003) are all connected to the inner wall of the base (1). A gap is left between the left side of the fan-shaped sensitive area (1004) and the inner wall of the base (1), so that the fan-shaped sensitive area (1004) does not contact the inner wall of the base (1).

4. A high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 2, characterized in that, The output electrode plate (301) is deposited on the base (1) between the driving electrode (2) and the sensing electrode (4).

5. A high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 1, characterized in that, The base (1) includes two symmetrically arranged layers, each of which includes a silicon dioxide layer (101) and an SOI layer (102) arranged sequentially from the outside to the inside. The two SOI layers (102) are bonded together by silicon-silicon thermocompression bonding.

6. A high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 1, characterized in that, The driving electrode (2) and the sensing electrode (4) have the same structure, both divided into inner and outer parts. The inner part includes a square part and a long strip part, with the end of the long strip part set on the corresponding piezoelectric layer (5). The outer part includes a square part, a short strip part and a long strip part, with the square part and the long strip part connected together by the short strip part. The angle between the short strip part and the long strip part is 90 degrees, and the end of the long strip part is set on the corresponding piezoelectric layer (5).

7. A method of using the high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis as described in any one of claims 1-6, characterized in that, The details are as follows: Step one involves calibrating the detection chip and obtaining the sensing curve, which includes the following: Step 1.1: Place the detection chip in a tear environment containing matrix metalloproteinases at known concentrations of 0, 2, 4, 6, 8, and 10 ng / mL. Drive the chip by applying a driving signal through the driving electrode (2). Connect the oscilloscope to the sensing electrode (4). Obtain the amplitude-frequency curves of the detection chip under different concentrations of matrix metalloproteinases through the oscilloscope. The resonant peak point of the amplitude-frequency curve is the resonant frequency of the chip at the corresponding concentration of matrix metalloproteinases. Plot a curve with the resonant frequency difference as the vertical axis and the matrix metalloproteinase concentration as the horizontal axis. This is the sensing curve. Fit the relationship expression between the frequency difference and the concentration through the sensing curve. The resonant frequency difference is the resonant frequency of the chip at each non-zero concentration of matrix metalloproteinases - the initial resonant frequency of the chip at zero concentration of matrix metalloproteinases. Step 2: Place the calibrated detection chip in the tear fluid environment to be tested. At this time, the antibody-modified layer (7) and the ion-sensitive membrane (8) are used to detect matrix metalloproteinases and sodium / potassium ions in the tear fluid, respectively. Step 3: Measure the initial potential difference E0 between the two output electrode plates (301) using a high-precision voltmeter; Step 4: Drive the chip by applying a drive signal through the drive electrode (2), measure the potential difference E between the two output electrode plates (301) by a voltmeter, and obtain the resonant frequency of the detection chip by an oscilloscope. Step 5: Calculate the sodium / potassium ion concentration using the potential difference E value from Step 4. Then, using the resonant frequency measured in Step 4, determine the frequency difference before and after tear detection according to Step 1. Finally, calculate the matrix metalloproteinase concentration at this point using the sensor curve fitting expression calibrated in Step 2. The sodium / potassium ion concentration is calculated using the following formula: In the formula, R is the gas constant, T is the tear temperature, z is the charge number of the target ion, F is the Faraday constant, and a is the concentration of the target ion.

8. A method for preparing a high-precision, fully quantitative tear biomarker detection chip for dry eye diagnosis as described in any one of claims 1-6, characterized in that, Includes the following: Step a, a 500 nm silicon dioxide layer (101) is formed on the upper surface of the thin silicon layer (102a) of the SOI layer (102) by oxidation growth method; Step b, etching is performed on the silicon dioxide layer (101) in the corresponding piezoelectric layer (5) region by reactive ion etching technology to form an opening for ion implantation to form the piezoelectric layer (5); Step c: Deep silicon etching technology is used to etch the structure corresponding to the cantilever structure (10) on the base silicon layer (102c), and then the BOE process is used to etch the structure corresponding to the cantilever structure (10) on the insulating layer (102b) to obtain the entire suspended structure of the base (1) and the area where the cantilever structure (10) is located. Step d: Repeat steps a, b, and c above to obtain two identical bases (1) and the entire suspended structure in the area where the cantilever structure (10) is located. Then, use the Si-Si hot-press bonding process to bond the remaining base silicon layer (102c) of the two bases (1) to obtain the main structure of the detection chip. In step e, boron ions are implanted into the region corresponding to the piezoelectric layer (5) at the root of the driving cantilever (1001) and sensing cantilever (1003) through the opening formed in step b using an ion implantation process. Then, the piezoelectric layer (5) is obtained by annealing at 950°C. Then, a 20nm Cr layer and a 300nm Au layer are deposited on the silicon dioxide layer (101) at the positions corresponding to the driving electrode (2), the reactive electrode (3), the sensing electrode (4), the modification electrode (6), and the reference electrode (9) respectively by a sputtering stripping process, so as to obtain the driving electrode (2), the reactive electrode (3), the sensing electrode (4), the modification electrode (6), and the reference electrode (9). Step f: Reactive ion etching is used again to etch the silicon dioxide layer (101) to the thin silicon layer (102a) of the non-cantilever structure area on the entire suspended structure where the base (1) and cantilever structure (10) are located. Then, deep silicon etching is used to etch the thin silicon layer (102a) to the insulating layer (102b) of the non-cantilever structure area of ​​the entire suspended structure to release the cantilever structure (10). Thus, the main structure base (1), driving electrode (2), reactive electrode (3), sensing electrode (4), piezoelectric layer (5), modification electrode (6), reference electrode (9) and cantilever structure (10) of the chip are obtained. Finally, a single chip is obtained by laser slicing. In step g, an ion-sensitive membrane (8) and a reference electrode (9) are formed on the corresponding reaction electrode plate (303) by electrochemical method; and an antibody-modified layer (7) is formed on the modified electrode (6) by self-assembly method.

9. The method for preparing the high-precision fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 8, characterized in that, The process for preparing an ion-sensitive membrane (8) and a reference electrode (9) on a well-formed reaction electrode plate (303) and for preparing a sodium / potassium ion-sensitive membrane by electrochemical method is as follows: i. A PVB reference solution was prepared by dissolving 79.1 mg PVB and 50 mg NaCl in 1 mL of methanol; ii. The reaction electrode plate (303) located below is deposited in the Ag electroplating solution under a constant current of 1 mA / cm2 for 30 min. Then, the reaction electrode plate (303) is placed in 0.1 mol / L FeCl3 and reacted for 60 s to obtain an Ag / AgCl layer on the reaction electrode plate (303). Then, 2 μL of PVB reference solution prepared in step i is drop-cast onto the Ag / AgCl layer to form a reference electrode (9). iii. Pretreatment of the upper reaction electrode plate (303): at a current density of 2 mA / cm² 2 Under certain conditions, the upper reaction electrode plate (303) is placed in an electroplating solution composed of 0.01M 3,4-ethylenedioxythiophene and 0.1M sodium poly(4-styrenesulfonate). When the transferred charge reaches 20mC, a conductive polymer working electrode is formed on the surface of the reaction electrode plate (303) through electrochemical polymerization. Subsequently, the reaction electrode plate (303) is rinsed clean with deionized water and dried to obtain Na. + and K + Working electrode; iv.Na + Preparation of the sensitive membrane: 1 mg of Na ion carrier X-4-tert-butylcalix[4]arene-tetraethyl acetate, 0.55 mg of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 33 mg of polyvinyl chloride, and 65.45 mg of sodium sebacic acid diacid dioate were dissolved and mixed evenly in 660 μL of tetrahydrofuran to obtain Na + Selective membrane mixtures; vK + Preparation of the sensitive membrane: 2 mg of valinemycin, 0.5 mg of sodium tetraphenylborate, 32.75 mg of PVC, and 64.75 mg of dioctyl sebacate were dissolved and mixed thoroughly in 350 μL of cyclohexanone solution to obtain K. + Selective membrane mixtures; vi. Prepare 4 μL of Na + Or K + The selective membrane mixture is coated onto the top reactive electrode plate (303) and dried at room temperature to obtain the desired corresponding ion-sensitive membrane (8).

10. The method for preparing the high-precision fully quantitative tear biomarker detection chip for dry eye diagnosis according to claim 8, characterized in that, The preparation of the antibody-modified layer (7) using the self-assembly method is as follows: Step 1: Cleaning: Ultrasonic cleaning of the surface of the modified electrode (6) with deionized water for 30 minutes, followed by drying with nitrogen gas; Step 2: Formation of self-assembled monolayer: A self-assembled monolayer is formed by immersing the modified electrode (6) in an ethanol solution of 10 mM 11-mercaptoundecenoic acid. Step 3: Carboxyl activation: The modified electrode (6) treated above was immersed in a mixed solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride with a concentration of 400 mM and N-hydroxysuccinimide with a concentration of 100 mM for 2 hours to activate the carboxyl groups in the MUA. Step 4: Antibody coupling: The modified electrode (6) after step 3 is immersed in MMP-9 antibody solutions of different concentrations and continuously flowed for 2 hours, so that the antibody is coupled to the modified electrode (6) to form an antibody modified layer (7). Step 5: Further fixation of antibody: Immerse the antibody-modified layer (7) obtained in step 4 in a 0.05% glutaraldehyde solution to fix the antibody-modified layer (7) and prevent the antibody layer from peeling off; Step 6: Surface passivation: The antibody-modified layer (7) obtained in step 5 was surface passivated in a 1 mg / mL casein solution for 30 min.

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