Disease marker high-throughput electrochemical sensing chip based on carbon nanotubes, preparation method and application

Abstract

The invention discloses a disease marker high-flux electrochemical sensing chip based on a carbon nano tube and a preparation method and application of the disease marker high-flux electrochemical sensing chip. A chip body specifically comprises three polyimide substrate layers which are laminated mutually, and a plurality of parallel marker detection modules are arranged on the first polyimide substrate layer; each marker detection module comprises a plurality of detection units, and each detection unit at least comprises one baseline detection unit for calibration and a plurality of actual detection units; electric signal amplification units and micron-sized detector arrays are implanted into the two sides of each baseline detection unit for calibration and the two sides of each actual detection unit; the single-walled carbon nanotube is coated with an antibody combined with the marker; and an electric signal transmission unit is implanted between the polyimide substrate layer II and the polyimide substrate layer III. The carbon nanotube working groups with different marker antibodies are loaded on the chip body so as to construct a detection module of corresponding indexes, and the results are collected for medical index analysis, so that intelligent management of a subject is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensor chip preparation, and more specifically to a high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes, a preparation method and an application thereof. Background Art

[0002] In the process of tumor development, immunosensors are widely used in clinical work as an important non-invasive examination method. One of the methods widely used in clinical laboratories to detect tumor markers is enzyme-linked immunosorbent assay. However, some disadvantages of traditional enzyme-linked immunosorbent assay, such as time-consuming, poor accuracy, and difficulty in automation, limit its application in rapid decision-making on the spot. Electrochemical sensing methods have the characteristics of fast analysis speed, easy operation, moderate cost, easy miniaturization, and real-time continuous monitoring. They have now become one of the important technologies in the field of analytical chemistry. Carbon-based nanocomposites have unique advantages such as small size, high specific surface area, easy functionalization, excellent biocompatibility and stability. Based on the unique advantages of carbon-based nanocomposites and electrochemical sensing, as well as the introduction of signal amplification methods, new electrochemical biosensors based on carbon-based nanocomposites are designed and constructed for high-sensitivity and high-specificity detection of disease-related biomarkers such as nucleic acids, small biomolecules or proteins, laying a certain foundation for the development of nano-integrated devices, rapid and accurate clinical diagnosis, and the realization of personalized medicine.

[0003] In recent years, a lot of research has been done on the application of carbon nanomaterials in biosensors, and breakthrough progress has been made. As one of the important forms, carbon nanotubes have attracted widespread attention from researchers in the field of analysis. Carbon nanotubes have good electrical conductivity, large specific surface area and high mechanical strength, which enables them to play an important material function in the field of biosensors. Because of its nanoscale structure close to or even smaller than that of protein molecules, it is expected to be constructed into an electrochemical sensing interface with excellent performance and applied in the field of bioanalysis. It still has good electrical conductivity when combined with more biological molecules. These novel special properties enable single-arm carbon nanotubes to play an important role in the miniaturization of analytical instruments and equipment. Whether it is constructed on the electrode surface or as a material for probe molecules in the detection process, single-walled carbon nanotubes are undoubtedly nanomaterials that many researchers are interested in in recent years. In terms of electrode construction, single-walled carbon nanotubes are constructed on the electrode surface by assembly, electrodeposition, covalent coupling, etc., which undoubtedly increases the effective area of ​​the electrode on the one hand, and more importantly, enhances the material transport and electrical conductivity of the electrode surface. In addition, since the surface of functionalized single-walled carbon nanotubes has abundant sites for modification, constructing enzyme molecules, antigens or antibodies on the surface of carbon nanotubes shows better application prospects and effectively enhances the electrode signal.

[0004] Therefore, how to enhance the electrical signal of the electrochemical chip and specifically capture and detect multiple markers in the body is an urgent problem that those skilled in the art need to solve. Summary of the invention

[0005] In view of this, the present invention is proposed.

[0006] One of the purposes of the present invention is to provide a high-throughput electrochemical sensor chip.

[0007] A second object of the present invention is to provide a method for preparing a high-throughput electrochemical sensor chip.

[0008] The third object of the present invention is to provide the use of the electrochemical sensor chip prepared as above.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] On one hand, the present invention provides a high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes, comprising a chip body, wherein the chip body comprises a polyimide base layer 1, a polyimide base layer 2 and a polyimide base layer 3 laminated on each other, wherein a plurality of parallel marker detection modules are arranged on the polyimide base layer 1, each of the marker detection modules comprises a plurality of detection units, and each of the detection units comprises at least one calibration baseline detection unit and a plurality of actual detection units;

[0011] Each of the calibration baseline detection unit and the actual detection unit is implanted with an electrical signal amplification unit and a micrometer-level detector array for capturing signals on both sides; each of the calibration baseline detection unit and the actual detection unit is coated with a single-walled carbon nanotube that binds to a marker antibody;

[0012] An electrical signal transmission unit is implanted between the second polyimide base layer and the third polyimide base layer, and each actual detection unit is commonly connected to an electrical signal transmission unit.

[0013] In a preferred embodiment, the micron-scale detector array for capturing signals is a JPS-TP3050-SMT silicon photomultiplier tube; the electrical signal amplification unit is a common-phase circuit amplifier; and the electrical signal transmission unit is a converter nRF52832.

[0014] In a preferred embodiment, the electrochemical sensor chip also includes a micro energy storage power supply unit; the micro energy storage power supply unit is arranged on a side of the polyimide base layer 3 away from the electrical signal transmission unit, and the chip body, the micro energy storage power supply unit, and the micron-scale detector array for capturing signals are bonded with biocompatible materials.

[0015] In a preferred embodiment, the chip has a single detection time of ≤30 min, a specificity of >90%, a sensitivity of >90%, and no less than 3 types of signals detected in a single detection.

[0016] In a preferred embodiment, the markers include CEA, PSA, HE4 and hCG, and the detection limit indicators of the electrochemical sensor chip are: CEA<60fM / mL, PSA<0.6ng / mL, HE4<30pM / L, hCG<1pM / L.

[0017] The second aspect of the present invention provides a method for preparing the electrochemical sensor chip, the process comprising:

[0018] Step 1: Add single-walled carbon nanotubes to an aqueous solution containing a surfactant, disperse them ultrasonically, remove the undispersed carbon nanotubes by centrifugation, and obtain a carbon nanotube solution. Then, mix the dispersed carbon nanotube solution with mixed acid, react at 300°C for 15 minutes, and centrifuge at 16,000 rpm for 10 minutes to obtain a carbon nanotube precipitate. Resuspend the carbon nanotubes with 1 mL of PBS, add 1 mL of a mixed solution of 1 mg / mL EDC and 1 mL of 2 mg / mL NHS, react for 4 hours, and centrifuge at 16,000 rpm for 10 minutes, filter, and wash to obtain a functionalized carbon nanotube solution; drip or spray the functionalized carbon nanotube solution onto the surface of the chip body, and perform heat treatment or plasma treatment to firmly bond the carbon nanotubes to the chip body to obtain a sensor chip.

[0019] Step 2: Optimize the sensor chip with the marker to be detected, couple the marker antibody with the carbon nanotubes on the sensor chip, and at the same time, introduce a blocking agent or competitive inhibitor to inhibit nonspecific binding and improve the specific binding between the sensor chip and the target molecule to obtain a sensor chip coupled with the antibody;

[0020] Step 3: Assemble the signal-capturing micrometer-scale detector array, the electrical signal amplification unit, the electrical signal transmission unit and the micro energy storage power supply unit onto the antibody-coupled sensor chip, and then encapsulate the entire chip with biocompatible materials to obtain an electrochemical sensor chip.

[0021] In a preferred embodiment, in step 1, the ultrasonic dispersion time is 30 minutes; the mixed acid is a mixed solution of nitric acid solution and sulfuric acid solution, wherein the concentration of the nitric acid solution is 65%, the concentration of the sulfuric acid solution is 98%, and the volume ratio of the two solutions is 1:1.

[0022] In a preferred embodiment, in step three, the biocompatible material includes PLGA or PDMS.

[0023] The third aspect of the present invention provides the use of the electrochemical sensor chip, which includes preparing a sensor for detecting disease markers in a living body.

[0024] It can be seen from the above technical solution that compared with the prior art, the present invention loads a carbon nanotube working group with different marker antibodies on a high-throughput electrochemical sensor chip to construct a detection module for the corresponding indicator. Each independent detection module is responsible for data collection of the corresponding unit and translating the chemical signal into a digital signal, cascading amplifying the collected data and transmitting it to the terminal for processing, collecting the results and performing medical indicator analysis, thereby realizing intelligent management of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The accompanying drawing is a schematic diagram of the carbon nanotube-based disease marker high-throughput electrochemical sensor chip of the present invention.

[0027] Figure 2 The attached figure is a physical picture of the carbon nanotube-based disease marker high-throughput electrochemical sensor chip of the present invention.

[0028] Figure 3 The attached figure shows the morphology characterization of the single-walled carbon nanotubes in the present invention by scanning electron microscopy.

[0029] Figure 4 The attached figure shows the morphology characterization of the single-walled carbon nanotubes in the present invention by transmission electron microscopy.

[0030] Figure 5 The attached figure is a scanning electron microscope surface morphology characterization of the single-walled carbon nanotubes sprayed in the chip flow channel in the present invention.

[0031] Figure 6 The attached figure is a transmission electron microscope surface morphology characterization of the single-walled carbon nanotubes sprayed in the chip flow channel of the present invention.

[0032] Figure 7 The attached figure shows the detection signal captured by the sensor chip in the present invention.

[0033] Figure 8 The attached figure shows the detection signal captured by the sensor chip after signal amplification in the present invention.

[0034] Fig. 9 The attached figure is an interface diagram of the signal transmission module in the present invention.

[0035] Fig.10The attached figure is a verification of the specificity and sensitivity of the high-throughput electrochemical sensor chip for carbon nanotube-based disease markers of the present invention. The low, medium and high concentrations of the test objects noted in the figure are as follows: CEA = 30fM / mL, CEA = 60fM / mL, CEA = 90fM / mL; PSA = 0.2ng / mL, PSA = 0.6ng / mL, PSA = 1.0ng / mL; HE4 = 10pM / L, HE4 = 30pM / L, HE4 = 60pM / L; hCG = 0.5pM / L, hCG = 1pM / L, hCG = 1.5pM / L.

[0036] Fig.11 The attached figure is a graph showing the chip's anti-interference performance test results.

[0037] Fig.12 The attached figure is a schematic diagram of the accuracy of the chip in detecting different types of samples. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The electrochemical detector micrometer detector array translates the detection results of different markers by the high-throughput chip, and the signal amplification unit cascades the detection results, and finally transmits the signal wirelessly to the downstream terminal to realize data integration and mobilization, ensuring the stable output of the signal of the electrochemical sensor chip under complex conditions.

[0040] Since the chip is packaged with biocompatible materials, it is suitable for patch-type chips and skin-mounted testing. There is no need to pre-process the test samples, which ensures the high fidelity and real-time output of the test results of the sensor chip. The sensor chip captures the object to be tested using electrochemical immunoassay technology to achieve strong specific detection of the object to be tested. The amplifier ADA4530 in the sensor chip is used to amplify the signal of the carbon nanotube detected by the silicon photomultiplier tube JPS-TN3050-SMT, thereby achieving the initial amplification of the conversion of biological signals in the body into electrochemical signals, which can make the detection sensitivity reach the fM level and achieve high-sensitivity detection of the object to be tested; the chip has built-in partition operation units for different objects to be tested to achieve repeatable detection of the object to be tested.

[0041] The present invention uses CEA, PSA, HE4 and hCG as biosafety factor indicators to optimize the performance of the sensor chip. Monoclonal antibodies or nano antibodies are used to react with corresponding biosafety factors to improve the specificity of recognition. Through chemical modification (click chemistry) or biological modification (covalent coupling), the affinity sites of the carbon nanotube surface and the antibody are regulated to enhance the specific mutual recognition with the target molecule. Blockers or competitive inhibitors are introduced to inhibit nonspecific binding and improve the specific binding of the sensor chip to the target molecule. In addition, due to the use of patch-type sensor chips, the target molecule does not require any pre-treatment operations such as extraction and purification, thereby ensuring the fidelity of the output results.

[0042] Example 1

[0043] Based on the chip body requirements, various chip bodies of 1cm*1cm, 2cm*2cm (16 holes / 24 holes) and other specifications are customized from SMIC. This embodiment takes the 1cm*1cm 16-hole chip body as an example. The chip body length*width*height=1cm*1cm*0.1cm, with 16 detection units built in, 4 detection units as 1 module, and each module is set with 1 baseline detection unit and 3 actual detection units to ensure the accuracy of the detection results.

[0044] The preparation process of the electrochemical sensor chip is as follows:

[0045] Step 1: Add 100 mg of single-arm carbon nanotubes (SWCNTs) to 5 mL of an aqueous solution containing 1% SDS, dispersing evenly by ultrasonic treatment for 30 min, centrifuging at 16000 rpm for 10 min to remove undispersed carbon nanotubes to obtain a carbon nanotube solution; then mix the dispersed carbon nanotube solution with a mixture of nitric acid and sulfuric acid, react at 300 ° C for 15 min, centrifuge at 16000 rpm for 10 min to obtain a carbon nanotube precipitate; resuspend the carbon nanotubes with 1 mL of PBS, then add 1 mL of a 1 mg / mL EDC and 1 mL of a 2 mg / mL NHS mixture to react for 4 h, introduce a carboxyl (-COOH) functional group after reacting with EDC and NHS, centrifuge at 16000 rpm for 10 min, remove excess acid by filtering and washing with deionized water, and obtain a carboxyl-activated functionalized carbon nanotube solution. The functionalized carbon nanotube solution is dripped or sprayed onto the chip surface. The carbon nanotubes are firmly bonded to the chip through heat treatment or plasma treatment, wherein the concentration of the nitric acid solution is 65%, the concentration of the sulfuric acid solution is 98%, and the volume ratio of the two solutions is 1:1.

[0046] Monoclonal antibodies against biomolecules CEA, PSA, HE4, and hCG were coupled to activated carbon nanotubes to form stable covalent bonds. Surface analysis techniques, such as scanning electron microscopy and transmission electron microscopy, were used to confirm the modification of the carbon nanotube surface and the binding effect of the antibodies. Antibody capture experiments were performed to verify the specificity and sensitivity of the carbon nanotube high-throughput chip for capturing target molecules.

[0047] Step 2:

[0048] 1) Design of micron-scale detector array circuit for capturing signals: Micron-scale detector arrays are implanted on both sides of the carbon nanotube substrate to sense the electrochemical signals of the carbon nanotubes after the sensor chip captures the object to be detected. The working electrode can detect the electrical signal at the input end. The stable potential of the reference electrode is designed to provide a baseline for detection to reduce the transmission of irrelevant signals and reduce signal noise. The small and low-priced JPS-TN3050-SMT silicon photomultiplier tube is selected as the electrical signal sensor. The silicon photomultiplier tube consists of multiple avalanche photodiode (APD) units in parallel. Electrons generate electrical pulses in the APD and accumulate to form current output, the size of which reflects the concentration of the object to be detected.

[0049] 2) Design of the circuit of the electric signal amplification unit: After the signal fluctuation of the carbon nanotube is detected, it needs to be amplified before it can be transmitted by the communication circuit. The low bias current and integrated operation common-mode circuit amplifier ADA4530 launched by ADI was selected. It is a fA (10-15A) input bias current operational amplifier with integrated protection buffer to adapt to the input of high impedance signal source and ensure high-precision and high-bandwidth output. Its gain formula is Av=1+Rf / Rin, which is used for signal amplification after receiving the electrical signal and can meet the use requirements.

[0050] 3) Design of the electrical signal transmission unit circuit: Select the converter nRF52832 launched by Nordic, use the Sigma-Delta analog-to-digital conversion formula, and collect the electrochemical signal amplified by the circuit amplifier ADA4530 at a settable interval frequency. Each sample value is converted to the closest discrete level. The converter nRF52832 is a Bluetooth wireless low-power microcontroller that supports multi-protocol SoC and uses a 32-bit Cortex TM -M4F CPU, with 512kB flash memory + 64kB RAM. The embedded 2.4GHz transceiver supports Bluetooth low energy, ANT and proprietary 2.4GHz. It can simultaneously meet the needs of using GPIO to control the switch of LED and using the low-power Bluetooth transmission protocol. The quantized discrete level will be converted into a binary digital signal and transmitted to the patient terminal through the Bluetooth module.

[0051] 4) Design of micro energy storage power supply unit: We customized lead zirconate titanate (PZT) with high piezoelectric coefficient from SMIC to make a piezoelectric battery with a cantilever beam structure. The battery will generate electricity when subjected to pressure and vibration. The PZT surface is covered with electrodes to the greatest extent to collect charges, and the charges are stored in a high-energy-density supercapacitor made of graphene to ensure the long-term battery life of the micro-implanted sensor chip.

[0052] The above-mentioned capture signal micrometer-level detector array, electrical signal amplification unit, electrical signal transmission unit and micro energy storage power supply unit are fixed in series on the chip to receive the chemical signals received by the carbon nanotubes.

[0053] Step 3: Optimize the specificity, sensitivity and repeatability of different biosafety factors, and optimize the performance of the sensor chip using CEA, PSA, HE4 and hCG as biosafety factor indicators. Use monoclonal antibodies or nanoantibodies to react with the corresponding biosafety factors to improve the specificity of recognition. Through biological modification (covalent coupling), regulate the affinity sites of the carbon nanotube surface and the antibody to enhance the specific mutual recognition with the target molecule. Introduce a blocking agent or competitive inhibitor bovine serum albumin (BSA) to inhibit nonspecific binding and improve the specific binding of the sensor chip to the target molecule.

[0054] Step 4: Use PDMS for packaging. The schematic diagram of the prepared electrochemical sensor chip is as follows: Figure 1 As shown, the actual picture is shown Figure 2 .

[0055] The electrochemical sensor chip prepared in this embodiment has a single detection time of ≤30min, a specificity of >90%, a sensitivity of >90%, and a single detection signal of no less than 3. The detection limits for each marker are: CEA <60fM / mL, PSA <0.6ng / mL, HE4 <30pM / L, and hCG <1pM / L.

[0056] Example 2 Experimental evaluation of the function of a high-throughput biosafety factor chip based on a carbon nanotube

[0057] First, the single-walled carbon nanotubes of Example 1 were dispersed in double distilled water to obtain a single-walled carbon nanotube suspension. The double distilled water solution containing the single-walled carbon nanotubes was dropped on a glass slide. After the water evaporated, the single-walled carbon nanotubes were characterized by scanning electron microscopy and transmission electron microscopy. The single-walled carbon nanotubes had uniform particle size and good dispersion. Figure 3 and Figure 4 Then the carboxyl-modified single-walled carbon nanotubes in Example 1 were sprayed on the chip surface, and the dispersion and spray thickness of the single-walled carbon nanotubes were characterized by scanning electron microscopy and transmission electron microscopy. It can be seen that the single-walled carbon nanotubes are uniformly dispersed in the chip flow channel. Figure 5 and Figure 6.

[0058] Example 3 evaluates the functions of the micrometer-scale detector array circuit, signal amplification unit and signal transmission unit for capturing signals in the present invention.

[0059] The JPS-TN3050-SMT silicon photomultiplier tube is used as an electrical signal sensor. The silicon photomultiplier tube is composed of multiple avalanche photodiode (APD) units connected in parallel. Electrons generate electrical pulses in the APD and accumulate to form current output, the size of which reflects the concentration of the object to be tested. The results are shown in Figure 7 The ADA4530 fA (10-15A) input bias current operational amplifier is used to amplify the signal after receiving the electrical signal, which can meet the use requirements. The results are shown in Figure 8 The nRF52832 collects the electrochemical signal amplified by the circuit amplifier ADA4530. The nRF52832 can simultaneously control the switch of the LED using GPIO and use the low-power Bluetooth transmission protocol. The quantized discrete level will be converted into a binary digital signal and transmitted to the patient terminal through the Bluetooth module. The results are shown in Fig. 9 .

[0060] Example 4 Experimental evaluation of the specificity and sensitivity of the electrochemical sensor chip of the present invention in detecting different markers CEA, PSA, HE4 and hCG

[0061] Monoclonal antibodies are used to react with corresponding biosafety factors to improve the specificity of recognition. Through covalent coupling, the affinity sites of the carbon nanotube surface and the antibody are regulated to enhance the specific mutual recognition with the target molecule.

[0062] Taking CEA as an example, the specificity and sensitivity optimization experiment of the electrochemical sensor chip of the present invention is described in detail below.

[0063] Select 1mg / mL CEA monoclonal antibody, use the covalent cross-linking of amino and carboxyl groups to couple the CEA monoclonal antibody to the flow channel surface for specific recognition of CEA. Use a syringe to drip a drop of the following samples: CEA = 30fM / mL, CEA = 60fM / mL, CEA = 90fM / mL; PSA = 0.2ng / mL, PSA = 0.6ng / mL, PSA = 1.0ng / mL; HE4 = 10pM / L, HE4 = 30pM / L, HE4 = 60pM / L; hCG = 0.5pM / L, hCG = 1pM / L, hCG = 1.5pM / L. The results are shown in the figure. Fig.10The electrochemical sensor chip responded to CEA, but not to PSA, HE4 and hCG, indicating that the detection chip has specific detection performance. In addition, the detection sensitivity of the detection chip to CEA is CEA = 60fM / mL, which meets the gold standard for CEA detection, indicating that the detection chip has a high sensitivity.

[0064] Example 5 Chip Anti-interference Performance Test

[0065] The performance was tested by differential pulse voltammetry using an electrochemical workstation. At the same time, the anti-interference ability of the sensing performance was tested in the presence of different ion concentrations, different pH values ​​and different interfering substances to verify the stability.

[0066] Taking CEA as an example, the anti-interference performance test of the electrochemical sensor chip of the present invention is described in detail below.

[0067] The detection chip detects CEA standard, serum and in vivo implant samples. It can be seen that there is no significant difference in the test results of the three test samples. The standard, serum and in vivo implant samples expose the single-walled carbon nanotubes of the chip to different ion concentrations, different pH and different anti-interference substances. For example, compared with the CEA standard, serum and in vivo implant samples have more protein interference. Compared with the standard and serum tests, it has the anti-interference performance of the in vivo implant test method. It can be used as an auxiliary clinical detection method. The results are shown in Fig.11 .

[0068] Example 6 Chip Detection Performance Test

[0069] Preliminary preparation: ① System design and testing: Complete system design, prototype production, and conduct preliminary testing and verification in a laboratory environment. ② Ethical approval and informed consent: Submit the test plan to the hospital ethics committee, obtain approval, and ensure that all participating patients sign the informed consent form. Clinical implementation: ① Personnel screening: Appropriate test personnel were selected based on the patients visiting the Department of Thoracic Surgery to ensure the representativeness and diversity of the sample. ② Personnel grouping: The patients participating in the test were randomly divided into two groups, one group was implanted with the actual sensor chip, and the other group was implanted with a placebo chip (without function). ③ Concealment of test subjects: Ensure that medical staff and patients are unaware of the chip type. Test implementation: ① Implantation surgery: Experienced thoracic surgeons perform chip implantation surgery in a strict sterile environment to ensure standardized operation and patient safety. ② Postoperative monitoring: Use wireless data transmission modules to collect patient data in real time, record the health status and test results of each patient, and upload them to the cloud server for storage and analysis; conduct regular patient follow-up, collect postoperative recovery data, and make adjustments and optimizations as needed. Data Analysis:① Blind data analysis: Use statistical analysis methods and machine learning algorithms to analyze the data of the sensor chip and the placebo group to evaluate the detection accuracy and reliability of the sensor chip. ② Comparison after unblinding: Compare the two groups of data after unblinding to verify the effectiveness and advantages of the sensor chip.

[0070] According to the grouping, the accuracy of the chip in detecting different types of samples was observed. The results are shown in Fig.12 :The detection chip detects the standard products, serum and in vivo implant samples of CEA, PSA, HE4 and hCG respectively. It can be seen that there is no significant difference in the test results of the three test samples. The detection accuracy of in vivo implant samples is >85%. Compared with the standard product and serum detection, it has the anti-interference performance of in vivo implant detection. It can be used as an auxiliary clinical detection method.

[0071] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0072] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the concept or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes, characterized in that: The chip body comprises a polyimide base layer 1, a polyimide base layer 2 and a polyimide base layer 3 which are laminated on each other, a plurality of parallel marker detection modules are arranged on the polyimide base layer 1, each of the marker detection modules comprises a plurality of detection units, and each of the detection units comprises at least one calibration baseline detection unit and a plurality of actual detection units; Each of the calibration baseline detection unit and the actual detection unit is implanted with an electrical signal amplification unit and a micrometer-level detector array for capturing signals on both sides; each of the calibration baseline detection unit and the actual detection unit is coated with a single-walled carbon nanotube that binds to a marker antibody; An electrical signal transmission unit is implanted between the second polyimide base layer and the third polyimide base layer, and each actual detection unit is commonly connected to an electrical signal transmission unit.

2. The high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes according to claim 1, characterized in that: The micron-scale detector array used to capture the signal is a JPS-TP3050-SMT silicon photomultiplier tube; The electrical signal amplification unit is a common-phase circuit amplifier; The electrical signal transmission unit is a converter nRF52832.

3. The high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes according to claim 1, characterized in that: It also includes a micro energy storage power supply unit; The micro energy storage power supply unit is arranged on a side of the polyimide substrate layer 3 away from the electrical signal transmission unit; The chip body, the micro energy storage power supply unit and the micrometer-level detector array for capturing signals are bonded with biocompatible materials.

4. The high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes according to claim 3, characterized in that: The electrochemical sensor chip has a single detection time of ≤30 min, a specificity of >90%, a sensitivity of >90%, and a single detection signal of no less than 3 types.

5. The high-throughput electrochemical sensor chip for disease markers based on carbon nanotubes according to claim 4, characterized in that: The markers include CEA, PSA, HE4 and hCG, and the detection limits of the electrochemical sensor chip for each marker are: CEA<60fM / mL, PSA<0.6ng / mL, HE4<30pM / L, hCG<1pM / L.

6. The method for preparing the electrochemical sensor chip according to any one of claims 1 to 5, characterized in that: The process includes: Step 1: add 100 mg of single-walled carbon nanotubes to an aqueous solution containing a surfactant, disperse by ultrasonication, remove undispersed carbon nanotubes by centrifugation, obtain a carbon nanotube solution, then mix the dispersed carbon nanotube solution with mixed acid, react at 300°C for 15 minutes, and centrifuge at 16000 rpm for 10 minutes to obtain a carbon nanotube precipitate; resuspend the carbon nanotubes with 1 mL of PBS, add 1 mL of a mixed solution of 1 mg / mL EDC and 1 mL of 2 mg / mL NHS to react for 4 hours, and centrifuge at 16000 rpm for 10 minutes, filter, and wash to obtain a functionalized carbon nanotube solution; drip or spray the functionalized carbon nanotube solution onto the surface of the chip body, perform heat treatment or plasma treatment to firmly combine the carbon nanotubes with the chip body, and obtain a sensor chip; Step 2: Optimize the sensor chip with the marker to be detected, and couple the marker antibody with the carbon nanotubes on the sensor chip, while introducing a blocking agent or a competitive inhibitor to obtain a sensor chip coupled with the antibody; Step 3: Assemble the signal-capturing micrometer-scale detector array, the electrical signal amplification unit, the electrical signal transmission unit and the micro energy storage power supply unit onto the antibody-coupled sensor chip, and then encapsulate the entire chip with biocompatible materials to obtain an electrochemical sensor chip.

7. The method according to claim 6, characterized in that In step 1, the ultrasonic dispersion time is 30 minutes; the mixed acid is a mixed solution of nitric acid solution and sulfuric acid solution; Among them, the concentration of nitric acid solution is 65%, the concentration of sulfuric acid solution is 98%, and the volume ratio of the two solutions is 1:

1.

8. The method according to claim 7, characterized in that In step three, the biocompatible material includes PLGA or PDMS.

9. Use of the electrochemical sensor chip according to any one of claims 1 to 5, comprising preparing a sensor for detecting disease markers in a living body.

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