Preparation method and application of extended gate field effect transistor for thrombin instant detection

Abstract

The application belongs to the field of biosensing and microelectronic technology, and relates to a preparation method and application of an extended gate field effect transistor for instant detection of thrombin. The extended gate field effect transistor is modified by a Prussian blue / platinum nanoparticle composite material, which greatly improves the electrochemical sensing performance of the field effect transistor. The extended gate field effect transistor prepared based on the composite material exhibits excellent anti-interference ability and recognition ability. The extended gate field effect transistor has the advantages of low cost and easy replacement, and the preparation process is simple and controllable, and easy to realize large-scale and productized production. The extended gate field effect transistor based on the Prussian blue / platinum nanoparticle can realize rapid recognition and instant detection of thrombin, fill the blank of thrombin detection products on the market, and greatly improve the detection efficiency of coagulation indicators.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biosensing and microelectronics technology, and relates to a preparation method and application of an extended gate field effect transistor for thrombin real-time detection. BACKGROUND

[0002] Thrombin is a ubiquitous serine protease in mammals, which mainly plays a role in the complex reaction between anticoagulants and procoagulants when the vascular endothelial cells are damaged, and plays a crucial role in the whole coagulation process. In addition to controlling hemostasis, wound healing, inflammation and tissue adhesion, thrombin also plays an important role in tumor growth and metastasis. Thrombin promotes tumor growth at low concentrations, inhibits tumor growth at high concentrations, and even produces an apoptotic effect. In addition, an abnormal level of human thrombin content may cause thrombotic diseases and even death. Thrombin has also been found to be a potential biomarker to assist in the detection of diseases such as systemic lupus erythematosus, hemophilia, kidney disease, and influenza virus.

[0003] Through the detection of thrombin, the purpose of early diagnosis and treatment intervention of related diseases can be achieved. Researchers have developed various quantitative detection techniques for thrombin concentration, such as enzyme, fluorescence, immunity, and surface plasmon resonance. However, in practical applications, methods based on chromatography / mass spectrometry and antibody-based immunoassays (such as enzyme-linked immunosorbent assay) are still the most commonly used thrombin detection and diagnosis methods. Although these detection techniques have high sensitivity, they require cumbersome equipment, high time cost and expensive labeling materials, which have obvious limitations. SUMMARY

[0004] The application proposes a new preparation method and application of an extended gate field effect transistor for thrombin real-time detection to solve the problems in traditional thrombin detection.

[0005] To achieve the above purpose, the application is implemented by using the following technical solutions:

[0006] A preparation method of an extended gate field effect transistor for thrombin real-time detection is provided, and the specific steps are as follows.

[0007] (1) Preparation of Prussian blue / platinum nanoparticle composite material

[0008] The transition metal salt solution and the potassium cyanide solution of the transition metal are mixed and stirred to perform a synthesis reaction. After the reaction is completed, centrifugation and drying are performed to obtain Prussian blue nanoparticles. The Prussian blue nanoparticles are dispersed in a solvent to obtain a dispersion liquid A. Then, a platinum source solution and an additive solution are added to perform a high-temperature reduction reaction. After the high-temperature reduction reaction is completed, centrifugation is performed, and the obtained solid is washed and dried to obtain a Prussian blue / platinum nanoparticle composite material.

[0009] (2) Extended-gate field effect transistor based on Prussian blue / platinum nanoparticle composite

[0010] The Prussian blue / platinum nanoparticle composite is dispersed in water or ethanol to obtain dispersion liquid B, a connecting aid is dropped on the surface of the sensing layer of the extended-gate field effect transistor and is dried once, then the dispersion liquid B is continuously dropped, and is dried twice.

[0011] (3) Preparation of extended-gate field effect transistor for thrombin recognition

[0012] The thrombin-specific DNA probe solution is continuously dropped on the sensing layer of the extended-gate field effect transistor, and is reacted at low temperature, then is washed after the reaction is completed, to obtain the extended-gate field effect transistor for thrombin recognition.

[0013] As preferred, in step (1), the mixing concentration of the transition metal salt solution and the potassium cyanide solution of transition metal is 70-80 mM, the transition metal salt is any one of FeCl2, Co(NO3)2, and CuSO4, the potassium cyanide of transition metal is potassium ferricyanide or potassium ferrocyanide, the synthesis reaction time is at least 6 h, and the synthesis reaction temperature is room temperature.

[0014] As preferred, the dispersion solvent of the Prussian blue nanoparticle is water or ethylene glycol, and the concentration of the dispersion liquid A is 6-8 mg / mL; the platinum source is any one of chloroplatinic acid, potassium chloroplatinate, and sodium chloroplatinate, the aid is ascorbic acid or glucose, the concentration of the platinum source solution is 3-5 mM, and the concentration of the aid solution is 5-10 mM; the volume ratio of the dispersion liquid A, the platinum source solution, and the aid solution is 2:1:1; the high-temperature reduction reaction temperature is 90-120℃, the high-temperature reduction reaction time is 8-12 h; the cleaning solvent is any one of water, ethanol, and acetone, the drying temperature is 60-80℃, the drying atmosphere is nitrogen, and the drying time is 6-12 hours.

[0015] As preferred, in step (2), the sensing layer material of the extended-gate field effect transistor is any one of tantalum oxide, silicon nitride, and hafnium oxide, the connecting aid is any one of cysteine, mercaptosilane, and aminosilane, the dropping amount of the connecting aid is 1-5 μL, the concentration of the dispersion liquid B is 6-8 mg / mL, and the dropping amount of the dispersion liquid B is 1-5 μL; the drying time of the first static drying and the second static drying is at least 12 h.

[0016] As preferred, in step (3), the concentration of the thrombin-specific DNA probe solution is 10-20 μM, and the dropping amount is 1-5 μL; the reaction temperature at low temperature is 0℃, and the reaction time is 6-12 h. After the reaction is completed, the unbound DNA probe is taken out and washed, and is dried at low temperature and can be directly used or stored at 0℃ for standby use.

[0017] The application provides application of the extended gate field effect transistor prepared by the method in real-time detection of thrombin.

[0018] The extended gate field effect transistor obtained by the application has a small volume, and can be applied to a handheld thrombin detection device.

[0019] Compared with the prior art, the application has the advantages and positive effects that:

[0020] 1. The application obtains a high-performance extended gate field effect transistor based on a high-performance Prussian blue / platinum nanoparticle composite material, and the extended gate field effect transistor has excellent electrochemical biosensing performance.

[0021] 2. The extended gate field effect transistor prepared by the application can realize high-sensitivity detection of thrombin, has an extremely low detection limit, has the advantages of small sample consumption, easy batch manufacturing, low cost and easy replacement, and can realize real-time and rapid monitoring of thrombin in combination with an external detection circuit, fills the blank of real-time thrombin detection products on the market, and has high clinical application value.

[0022] 3. The extended gate field effect transistor can specifically recognize thrombin in a complex environment, and has a small volume, so that the detection device can be miniaturized and integrated, for example, applied to a handheld device, and convenient for detection operation. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A scanning electron microscope image of the Prussian blue / platinum nanoparticles prepared in Example 1.

[0024] Figure 2 A detection circuit diagram for testing the extended gate field effect transistor.

[0025] Figure 3 A detection signal curve of the extended gate field effect transistor for thrombin.

[0026] Figure 4 A calibration curve diagram of the thrombin detection concentration.

[0027] Figure 5 A thrombin repeatability test result diagram of the extended gate field effect transistor.

[0028] Figure 6 A thrombin specificity test result diagram of the extended gate field effect transistor. DETAILED DESCRIPTION

[0029] In order to enable the above-mentioned objects, features and advantages of the present application to be more clearly understood, the present application will be further described below with reference to specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0030] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and therefore the present application is not limited to the specific embodiments disclosed in the following description.

[0031] Embodiment 1

[0032] The present embodiment provides a preparation method of an extended gate field effect transistor for thrombin instant detection, and the steps are as follows:

[0033] (1) Mix 100 mL of FeCl2 solution with a concentration of 70 mM with 100 mL of potassium ferricyanide solution with a concentration of 70 mM, stir at room temperature for 10 hours at a stirring rate of 300 rpm, centrifuge at 8000 rpm for 5 min after the reaction is completed, pour off the supernatant after centrifugation, then take out the solid at the bottom of the centrifuge tube and dry it in a 80°C oven for 24 hours, to obtain Prussian blue nanoparticles.

[0034] (2) Add the above-mentioned Prussian blue nanoparticles to deionized water to prepare a dispersion solution with a concentration of 6 mg / mL; take 20 mL of the dispersion solution, and add 10 mL of chloroplatinic acid solution with a concentration of 3 mM and 10 mL of ascorbic acid solution with a concentration of 5 mM, and reduce at 90°C for 12 hours. Centrifuge the obtained product at 3000 rpm for 5 min, pour off the supernatant after centrifugation, and then wash the solid obtained by centrifugation with deionized water twice, and then dry it at 60°C under a nitrogen atmosphere for 12 hours to obtain Prussian blue / platinum nanoparticle composite material, as shown in Figure 1 .

[0035] (3) Select an extended gate field effect transistor with a sensing layer of tantalum oxide (the other structures and preparation processes of the transistor used in the present embodiment and other embodiments use the method disclosed in CN 114660157A embodiment 1), and drop 4 μL of cysteine (Merck) as a connecting aid on the surface of the sensing layer and dry it at room temperature for 12 hours. Then add the Prussian blue / platinum nanoparticles obtained in step (2) to deionized water to prepare a dispersion solution with a concentration of 6 mg / mL, take 5 μL and continue to drop on the surface of the connecting aid of the sensing layer, and dry it at room temperature for 12 hours to obtain a Prussian blue / platinum nanoparticle composite material modified field effect transistor.

[0036] (4) The DNA probe freeze-dried powder (Shanghai Biotech Engineering Co., Ltd., DNA sequence: AGT CCG TGGTAG GGC AGG TTG GGG TGA CT) is dissolved in TE buffer (Shanghai Maikelin Biochemical Technology Co., Ltd., pH 7.0) to prepare a 100 μM DNA probe solution, which is further diluted with deionized water to 12 μM to obtain a DNA probe diluent. 4 μL of the DNA probe diluent is added dropwise to the surface of the composite material of the dried extended gate field effect transistor sensing layer, and the reaction is carried out at 0°C for 6 hours. Then the extended gate field effect transistor is taken out, and the unbound DNA probe on the sensing layer is washed away with deionized water. The extended gate field effect transistor is continuously dried at 0°C for 6 hours to obtain an extended gate field effect transistor for specific detection of thrombin.

[0037] Example 2

[0038] In this and subsequent examples, the conditions not specifically stated are consistent with those of Example 1. This example provides a preparation method of an extended gate field effect transistor for real-time detection of thrombin, comprising the following steps.

[0039] (1) Mix 100 mL of Co(NO3)2 solution with a concentration of 75 mM with 100 mL of potassium ferricyanide solution with a concentration of 75 mM, stir at room temperature for 12 hours at a stirring rate of 300 rpm, centrifuge at 8000 rpm for 5 min after the reaction is completed, and then dry at a constant temperature of 90°C for 24 hours in an oven to obtain Prussian blue nanoparticles.

[0040] (2) The Prussian blue nanoparticles are added to ethylene glycol to prepare a dispersion solution with a concentration of 7 mg / mL; 20 mL of the dispersion solution is taken, and 10 mL of potassium chloroplatinate solution with a concentration of 4 mM and 10 mL of glucose solution with a concentration of 7 mM are added, and the reduction reaction is carried out at 100°C for 10 hours. The obtained product is centrifuged at 3000 rpm for 6 min, washed twice with deionized water after centrifugation, and then dried at 70°C under a nitrogen atmosphere for 9 hours to obtain Prussian blue / platinum nanoparticle composite material.

[0041] (3) Select an extended gate field effect transistor with a hafnium oxide sensing layer, and add 3 μL of mercaptosilane connecting aid (Merck) dropwise to the surface of the sensing layer and dry at room temperature for 14 hours. Then the Prussian blue / platinum nanoparticles obtained in step (2) are added to ethanol to prepare a dispersion solution with a concentration of 7 mg / mL, and 4 μL is further added dropwise to the surface of the connecting aid on the sensing layer, and dried at room temperature for 12 hours to obtain a Prussian blue / platinum nanoparticle composite material modified field effect transistor.

[0042] (4) The DNA probe freeze-dried powder is dissolved in TE buffer to obtain a 100 μΜ DNA probe solution. Further dilution with deionized water is performed to obtain a 15 μΜ DNA probe solution. 3 μL of the deionized water-diluted DNA probe solution is added dropwise to the surface of the composite material of the extended gate field effect transistor sensing layer, and the reaction is performed at 0 °C for 9 hours. Then, the extended gate field effect transistor is taken out, and the unbound DNA probe on the sensing layer surface is washed away with deionized water. The extended gate field effect transistor is dried at 0 °C for 6 hours to obtain the extended gate field effect transistor for specific thrombin detection.

[0043] Example 3

[0044] The embodiment provides a preparation method of an extended gate field effect transistor for real-time thrombin detection, including the following steps.

[0045] (1) 100 mL of a CuSO4 solution with a concentration of 80 mM is mixed with 100 mL of a potassium ferrocyanide solution with a concentration of 80 mM. The mixture is stirred at room temperature for 14 hours at a stirring rate of 300 rpm. After the reaction is completed, the mixture is centrifuged at 8000 rpm for 5 min. Then, the mixture is dried in an oven at 80 °C for 24 hours to obtain Prussian blue nanoparticles.

[0046] (2) The Prussian blue nanoparticles are added to ethylene glycol to prepare a dispersion solution with a concentration of 8 mg / mL. 20 mL of the dispersion solution is taken, and 10 mL of a sodium chloroplatinate solution with a concentration of 5 mM and 10 mL of a glucose solution with a concentration of 9 mM are added. The mixture is reduced at 120 °C for 8 hours. The obtained product is centrifuged at 3000 rpm for 6 min. After centrifugation, the product is washed twice with deionized water, and then dried at 80 °C under a nitrogen atmosphere for 7 hours to obtain Prussian blue / platinum nanoparticle composite material.

[0047] (3) An extended gate field effect transistor with a sensing layer of silicon nitride is selected. 2 μL of an amino silane connecting aid (Merck) is added dropwise to the surface of the sensing layer, and the mixture is dried at room temperature for 14 hours. Then, the Prussian blue / platinum nanoparticles obtained in step (2) are added to ethanol to prepare a dispersion solution with a concentration of 8 mg / mL. 2 μL of the dispersion solution is added dropwise to the surface of the connecting aid on the sensing layer, and the mixture is dried at room temperature for 12 hours to obtain a Prussian blue / platinum nanoparticle composite material modified field effect transistor.

[0048] (4) The DNA probe freeze-dried powder is dissolved in TE buffer to obtain a 100 mM DNA probe solution. Further dilute it to 18 mM with deionized water. Take 2 μL of the DNA probe solution diluted with deionized water and continue to drop on the surface of the composite material of the extended gate field effect transistor sensing layer, and react in a 0°C environment for 10 hours. Then take out the extended gate field effect transistor, wash off the unbound DNA probe on the sensing layer surface with deionized water, and continue to dry at 0°C for 6 h to obtain the extended gate field effect transistor for specific detection of thrombin.

[0049] The extended gate field effect transistors prepared in each example are matched with a detection circuit for performance test of thrombin. The detection circuit is composed of a power module 1, a main control module 2, a signal acquisition module 3, an excitation power module 4, a current amplification module 5, and an extended gate field effect transistor interface module 6, and each module is arranged as shown in Figure 2 The power module 1 converts the 5V lithium battery output voltage into a 3.3V available voltage source through the LDO chip ME6211C33M5G, which is used to power the entire circuit module. The main control module 2 is composed of the minimum system of Stm32rct6 chip, the signal acquisition module 3 is composed of ADS1115 chip, the excitation power module 4 is composed of DAC8562 chip, the current amplification module 5 is composed of 100Ω current sensing resistor CRM2512-FX-1000ELF and INA181A3IDBVT voltage amplifier. The extended gate field effect transistor interface module 6 is provided with three interfaces and is connected with the transistor drain, gate and source respectively. The main control module 2 establishes communication with the excitation power module 4 through the SPI communication interface, and drives the DAC8562 chip to generate an excitation signal source for driving the extended gate field effect transistor to work. The main control module 2 establishes communication with the signal acquisition module 3 through the I2C communication interface, and drives the ADS1115 chip to perform digital-to-analog conversion on the detection signal.

[0050] During the detection process, the main control module 2 drives the excitation power supply module 4 to output a scanning voltage of 0-2V between the source and drain of the extended gate field effect transistor, with a step size of 0.05V / S, and outputs a fixed voltage of 1V between the gate and source, to drive the extended gate field effect transistor to work normally. After the thrombin in the to-be-detected solution and the DNA aptamer probe fixed on the surface of the field effect tube sensing layer of the prussian blue / platinum nanoparticle composite material are preferentially and specifically combined, a more stable ligand is formed, a signal response is generated in this process, the sensing layer surface is changed, and then the step of the carrier in the channel of the extended gate field effect transistor is affected, and finally the current between the source and drain of the extended gate field effect transistor (source-drain current) is changed. The source-drain current passes through the current detection resistance of the current amplification module 5, is amplified by the INA181A3IDBVT voltage amplifier between the current detection resistance, and is then output to the ADS1115 chip of the signal acquisition module 3 for analog-to-digital conversion. The MCU is operated to obtain the current value between the source and drain (i.e., the source-drain current V DS ), and the response signal is transmitted to the main control module through I2C communication. The main control module receives, saves and transmits the response signal.

[0051] 1. Performance test of the extended gate field effect transistor

[0052] First, 100μL of PBS buffer solution (5000mL of deionized water, 32.275g of potassium chloride, 40.8275g of monopotassium phosphate, 45.643g of dipotassium hydrogen phosphate, and 10g of potassium sorbate; the same below) is added to the detection pool, the extended gate is immersed in the PBS buffer solution, the gate, drain and source three electrode contacts of the extended gate field effect transistor are connected to the corresponding electrode lines on the detection circuit interface module, the source-drain voltage of the extended gate field effect transistor is set to 0-2V with a step size of 0.05V by using the excitation power supply module, and the gate-source voltage is set to 1V; the detection curve when the concentration of thrombin is 0pM is obtained, that is, the baseline. After the baseline test is completed, the detection conditions remain unchanged, and the detection system is replaced: the PBS buffer solution in the detection pool is poured out, and then 100μL of detection solution with a thrombin concentration of 1, 10, 100, 500, 1000, 5000 and 10000pM is sequentially detected. The thrombin is purchased from Sigma Aldrich Trading Co., Ltd. (Shanghai, frozen at minus 20 degrees Celsius). The thrombin lyophilized powder can be diluted with PBS buffer solution for sample detection, and each sample needs to be prepared and used immediately. The detection signal curve of the extended gate field effect transistor for different concentrations of thrombin is shown in Figure 3 and Figure 4 The results show that the linear detection range of the extended gate field effect transistor obtained in Example 1 for thrombin can reach 1-10000pM, the linearity R² value is 95.64%, and the response time is 4 min.

[0053] 2. Reproducibility test

[0054] Six extended gate field effect transistors for thrombin specific test were prepared according to the steps in Example 1, and stability test of thrombin detection was carried out by using the prepared extended gate field effect transistors and detection circuit. The prepared extended gate field effect transistors were numbered 1 to 6 according to the preparation sequence. First, the extended gate of the field effect transistor numbered 1 was immersed in the detection system, 100 μL of 1 pM thrombin test solution was added, the source-drain voltage of the extended gate field effect transistor was set to 0-2 V, and the gate-source voltage was set to 1 V; the detection curve of the field effect transistor numbered 1 at 1 pM thrombin concentration was obtained. After the test was completed, the extended gate field effect transistor numbered 2 was replaced, and 100 μL of 1 pM thrombin test solution was added again for detection, and the above operation was repeated. The reproducibility test results of the extended gate field effect transistor are shown in Table 1. Figure 5 As shown in Table 1, the signal difference between each extended gate field effect transistor is small, indicating that it has good signal reproducibility.

[0055] 3. Stability

[0056] The prepared extended gate field effect transistor was stored in a 4°C environment for 30 days, and the response signal was 92% of the initial signal, indicating that the extended gate field effect transistor has excellent stability.

[0057] 4. Anti-interference test

[0058] The prepared extended gate field effect transistor was used for thrombin anti-interference test by using the detection circuit. First, the extended gate was immersed in the detection system, 120 μL of 1 pM thrombin (Tob) test solution was added, the source-drain voltage of the extended gate field effect transistor was set to 0-2 V, and the gate-source voltage was set to 1 V; the detection curve at 1 pM thrombin concentration was obtained. After the test was completed, the detection system was replaced, and 120 μL of immunoglobulin G (IgG), bovine serum albumin (BSA), prostate specific antigen (PSA), cardiac troponin T (cTnT), lysozyme (Lzm) test solution diluted to 1 μM with PBS buffer was added, respectively. The detection system was replaced after each test, and finally the following two samples were mixed for detection: sample one: 20 μL of 1 pM Tob PBS buffer; sample two: 20 μL of 1 μM Lzm, IgG, BSA, PSA, cTnT mixed PBS buffer (Mix). Among them, BSA and Lzm were purchased from Shanghai Sangon Biological Engineering Co., Ltd., IgG, PSA, and cTnT were purchased from Abeam company, and the storage method was cold storage (2-8°C).

[0059] The anti-interference test results of the extended gate field effect transistor are shown in Table 2.Figure 6 The extended gate field effect transistor also showed excellent anti-interference ability, and still maintained specific signal response to thrombin in a detection system with 1000-fold concentration of interferents. The above results all indicate that the extended gate field effect transistor has excellent thrombin recognition ability and real system application potential.

[0060] 5. Real sample detection

[0061] Based on the above results, the extended gate field effect transistor was applied to the detection of thrombin content in real serum (real serum samples were provided by Nanjing Gulou Hospital). 120 μL of serum was added to the detection system each time, and the extended gate field effect transistor prepared in Example 1 was used to test the performance of thrombin in combination with the detection circuit. The corresponding relationship between the change of the detection signal and the content of serum thrombin established by the method of Example 4 was used for calculation. The results obtained by using the thrombin activity kit (ab197006) of Abeam Company were used for calculation deviation. The detection results are shown in Table 1. Figure 4

[0062] Table 1. Test results of thrombin in real serum samples

[0063]

[0064] From the results in Table 1, it can be seen that the field effect transistor prepared by the present application can realize rapid and accurate detection of thrombin in real serum, has a low detection limit and a wide detection range, and is consistent with the detection results of the commercial kit. In addition, the extended gate field effect transistor of the present application has a small size, and the required detection circuit structure is relatively simple, which provides a new perspective and solution for the miniaturization of the thrombin detector, thereby bringing more possibilities for the miniaturization of the thrombin detector.

[0065] Comparative Example 1

[0066] The difference between this comparative example and Example 1 is that Prussian blue is used to directly modify the extended gate of the field effect transistor, instead of using Prussian blue / platinum nanoparticles, and the rest of the conditions remain unchanged. It was detected that the obtained extended gate field effect transistor had no obvious signal response to thrombin.

[0067] Comparative Example 2

[0068] The difference between this comparative example and Example 1 is that a single platinum nanoparticle is used to directly modify the extended gate of the field effect transistor, instead of using Prussian blue / platinum nanoparticles, and the rest of the conditions remain unchanged. It was detected that the obtained extended gate field effect transistor had no obvious signal response to thrombin.

[0069] Comparative Example 3

[0070] ​The comparative example is different from example 1 in that a single Prussian blue and a single platinum nanoparticle are mixed and then directly used to modify the extended gate of the field effect transistor, instead of using Prussian blue / platinum nanoparticles, and the rest of the conditions remain unchanged. It is detected that the linear detection range of the obtained extended gate field effect transistor for thrombin is 10-200 pM, and the response time is 5 min. Compared with the detection performance of the field effect transistor in example 1, there is a significant decrease, which fully illustrates the superiority of the Prussian blue / platinum composite material.

[0071] It can be found from the comparison of the detection performance of the field effect transistor obtained from the above examples that the nanomaterial plays a crucial role in the grafting of DNA molecules and signal catalytic amplification in the thrombin detection process. For the thrombin detection process based on the DNA recognition strategy, the response signal change generated by the recognition process is weak, and the conventional electrochemical biosensor cannot meet the rapid detection of low-concentration thrombin. The field effect transistor has excellent sensitivity and resolution to low current signals, but is limited by the general effect of the current field effect transistor surface modification technology and the poor compatibility of the nanomaterial with the field effect transistor, which leads to the difficulty of accurately and rapidly determining the content of thrombin in whole blood by using the existing field effect transistor and other detection methods.

[0072] In order to solve the above problems, the application uses Prussian blue analog material as the research object, uses its excellent catalytic activity and pH sensitivity as the modification material of the extended gate of the field effect transistor; at the same time, in order to further improve the detection performance and ensure that the thrombin specific probe molecule can exist stably in the system, platinum nanoparticles are grown in situ on the surface of the Prussian blue analog to prepare a Prussian blue / platinum nanomaterial, and the high specific surface area and excellent biocompatibility of the composite nanoparticle structure are used to improve the detection performance of the field effect transistor. In the thrombin detection process, the DNA probe molecule fixed on the surface of the Prussian blue / platinum nanoparticle can quickly recognize thrombin, the change of its configuration triggers the catalytic action of Prussian blue, thereby causing the change of the surface potential of the gate, so that the field effect transistor generates a weak current response signal. In this composite structure, the synergistic effect of Prussian blue and platinum nanoparticles is used to realize the stable loading and large amount of loading of the thrombin specific probe and the amplification and transmission of the response signal, which greatly improves the detection performance and detection efficiency of thrombin.

[0073] The above is only a preferred embodiment of the application, and does not limit other forms of the application. Any person skilled in the art can use the disclosed technical content to make changes or modifications to equivalent embodiments applied to other fields, but any simple modification, equivalent change and modification made on the basis of the technical essence of the application to the above embodiments still belongs to the protection scope of the technical solution of the application.

Claims

1. A method for fabricating an extended gate field effect transistor for thrombin instantaneous detection, characterized in that, The specific steps are as follows: (1) Preparation of Prussian blue / platinum nanoparticle composite material The transition metal salt solution and the transition metal potassium cyanide solution are mixed, stirring is performed for the synthesis reaction, centrifugation and drying are performed after the reaction is completed, and Prussian blue nanoparticles are obtained; the Prussian blue nanoparticles are dispersed in a solvent to obtain dispersion liquid A; then, a platinum source solution and an auxiliary solution are added, a high-temperature reduction reaction is performed, centrifugation is performed after the high-temperature reduction reaction is completed, and the obtained solid is washed and dried to obtain the Prussian blue / platinum nanoparticle composite material; (2) Preparation of extended gate field effect transistor based on the Prussian blue / platinum nanoparticle composite material The Prussian blue / platinum nanoparticle composite material is dispersed in water or ethanol to obtain dispersion liquid B, a connecting auxiliary is dropped on the surface of the sensing layer of the extended gate field effect transistor and is once statically dried, then the dispersion liquid B is continuously dropped, and twice static drying is performed; (3) Preparation of extended gate field effect transistor for thrombin recognition The thrombin-specific DNA probe solution is continuously dropped on the sensing layer of the extended gate field effect transistor, reaction is performed at low temperature, washing is performed after the reaction is completed, and low-temperature drying is performed, and the extended gate field effect transistor for thrombin recognition is obtained.

2. The method of claim 1, wherein the extended-gate field effect transistor for the instant detection of thrombin is prepared by the steps of: In step (1), the mixing concentration of the transition metal salt solution and the transition metal potassium cyanide solution is 70-80 mM, the transition metal salt is any one of FeCl2, Co(NO3)2 and CuSO4, the transition metal potassium cyanide is potassium ferricyanide or potassium ferrocyanide, the synthesis reaction time is at least 6 h, and the synthesis reaction temperature is room temperature.

3. The method of claim 1, wherein the extended-gate field effect transistor for the instant detection of thrombin is prepared by the steps of: In step (1), the dispersion solvent of the Prussian blue nanoparticles is water or ethylene glycol, and the concentration of the dispersion liquid A is 6-8 mg / mL; the platinum source is any one of chloroplatinic acid, potassium chloroplatinate and sodium chloroplatinate, the auxiliary is ascorbic acid or glucose, the concentration of the platinum source solution is 3-5 mM, and the concentration of the auxiliary solution is 5-10 mM; the volume ratio of the dispersion liquid A, the platinum source solution and the auxiliary solution is 2:1:1; the high-temperature reduction reaction temperature is 90-120℃, the high-temperature reduction reaction time is 8-12 h; the drying temperature is 60-80℃, the drying atmosphere is nitrogen, and the drying time is 6-12 h.

4. The method of claim 1, wherein the extended-gate field effect transistor for the instant detection of thrombin is prepared by the steps of: In step (2), the sensing layer material of the extended gate field effect transistor is any one of tantalum oxide, silicon nitride and hafnium oxide, the connecting auxiliary is any one of cysteine, mercaptosilane and aminosilane, the dropping amount of the connecting auxiliary is 1-5 μL, the concentration of the dispersion liquid B is 6-8 mg / mL, and the dropping amount of the dispersion liquid B is 1-5 μL; the once static drying and twice static drying time are both at least 12 h.

5. The method of claim 1, wherein the extended-gate field effect transistor for the instant detection of thrombin is prepared by the steps of: In step (3), the concentration of the thrombin-specific DNA probe solution is 10-20 μM, and the dropping amount is 1-5 μL; the reaction temperature at low temperature is 0℃, and the reaction time is 6-12 h.

6. Application of the extended gate field effect transistor prepared by the method of any one of claims 1-5 in thrombin instant detection.

7. Application of the extended gate field effect transistor prepared by the method of any one of claims 1-5 in a thrombin handheld detection device.

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