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

Abstract

The invention belongs to the technical field of biosensing and microelectronics, and relates to a preparation method and application of an extended gate field effect transistor for thrombin instant detection. The expanded gate field effect transistor is modified by the Prussian blue / platinum nanoparticle composite material, the electrochemical sensing performance of the field effect transistor is greatly improved, and the expanded gate field effect transistor prepared based on the composite material shows excellent anti-interference capability and recognition capability. The expanded gate field effect transistor has the advantages of being low in cost, easy to replace and the like, the preparation process is simple and controllable, large-scale and productization production is easy to achieve, the expanded gate field effect transistor based on the Prussian blue / platinum nanoparticles can achieve rapid recognition and instant detection of thrombin, the blank of thrombin detection products in the market at present is filled, and the application prospect is wide. And the detection efficiency of the blood coagulation index is greatly improved.

Description

Technical Field

[0001] The invention belongs to the field of biosensor and microelectronic technology, and relates to a preparation method and application of an extended gate field effect transistor for real-time detection of thrombin. Background Art

[0002] Thrombin is a serine protease commonly found in mammals. It mainly plays a role in the complex reaction between anticoagulants and procoagulants when vascular endothelial cells are damaged, and plays a vital role in the entire coagulation process. In addition to controlling hemostasis, wound healing, inflammation and tissue adhesion, thrombin also plays an important role in tumor growth and metastasis. When the concentration of thrombin is low, it promotes tumor growth, and when the concentration is high, it inhibits tumor growth and even produces apoptotic effects. In addition, abnormal levels of thrombin in the human body may lead to thrombotic diseases or 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 of related disease risks and therapeutic intervention can be achieved. Researchers have developed various quantitative detection technologies for thrombin concentrations, such as enzyme, fluorescence, immunity and surface plasmon resonance. However, in practical applications, chromatography / mass spectrometry-based methods and antibody-based immunoassays (such as enzyme-linked immunosorbent assay) are still the most commonly used diagnostic methods for thrombin detection. Although these detection technologies have high sensitivity, they require bulky equipment, high time costs and expensive labeling substances during actual detection, which has obvious limitations. Summary of the invention

[0004] Aiming at the problems existing in traditional thrombin detection, the present invention proposes a novel preparation method and application of an extended gate field effect transistor for real-time thrombin detection.

[0005] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: A method for preparing an extended gate field effect transistor for real-time detection of thrombin, the specific steps are as follows.

[0006] (1) Preparation of Prussian blue / platinum nanoparticle composites A transition metal salt solution and a transition metal potassium cyanide solution are mixed and stirred for a synthesis reaction. After the reaction is completed, the mixture is centrifuged and dried to obtain Prussian blue nanoparticles. The Prussian blue nanoparticles are dispersed in a solvent to obtain a dispersion A. Then, a platinum source solution and an auxiliary agent solution are added to perform a high-temperature reduction reaction. After the high-temperature reduction reaction is completed, the mixture is centrifuged, and the obtained solid is washed and dried to obtain a Prussian blue / platinum nanoparticle composite material.

[0007] (2) Extended-gate field-effect transistor based on Prussian blue / platinum nanoparticle composites The Prussian blue / platinum nanoparticle composite material is dispersed in water or ethanol to obtain dispersion B, a connection aid is drop-coated on the surface of the sensing layer of the extended gate field effect transistor and allowed to stand and dry once, and then dispersion B is continued to be drop-coated and allowed to stand and dry a second time.

[0008] (3) Fabrication of extended-gate field-effect transistor for thrombin recognition The thrombin-specific DNA probe solution is then dripped onto the sensing layer of the extended gate field effect transistor, reacted at low temperature, and rinsed after the reaction is completed, thereby obtaining an extended gate field effect transistor for thrombin recognition.

[0009] Preferably, in step (1), the transition metal salt solution and the transition metal potassium cyanide solution are mixed at a concentration of 70-80 mM, the transition metal salt is any one of FeCl2, Co(NO3)2, and CuSO4, the transition metal potassium cyanide is potassium ferrocyanide or potassium ferrocyanide, the synthesis reaction time is at least 6 hours, and the synthesis reaction temperature is room temperature.

[0010] Preferably, the Prussian blue nanoparticle dispersion solvent is water or ethylene glycol, and the concentration of dispersion A is 6-8 mg / mL; the platinum source is any one of chloroplatinic acid, potassium chloroplatinate, and sodium chloroplatinate, the auxiliary agent is ascorbic acid or glucose, the concentration of the platinum source solution is 3-5 mM, and the concentration of the auxiliary agent solution is 5-10 mM; the volume ratio of dispersion A: platinum source solution: auxiliary agent solution is 2:1:1; the high-temperature reduction reaction temperature is 90-120°C, and the high-temperature reduction reaction time is 8-12h; the cleaning solvent is any one of water, ethanol, and acetone, the drying temperature is 60-80°C, the drying atmosphere is nitrogen, and the drying time is 6-12 hours.

[0011] Preferably, the sensing layer material of the extended gate field effect transistor in step (2) is any one of tantalum oxide, silicon nitride, and hafnium oxide, the linking aid is any one of cysteine, mercaptosilane, and aminosilane, the amount of the linking aid added is 1-5 μL, the concentration of dispersion B is 6-8 mg / mL, the amount of dispersion B added is 1-5 μL, and the first static drying time and the second static drying time are both at least 12 hours.

[0012] Preferably, in step (3), the concentration of the thrombin-specific DNA probe solution is 10-20 μM, the drop-coating amount is 1-5 μL, the reaction temperature at low temperature is 0°C, and the reaction time is 6-12 hours. After the reaction is completed, the unbound DNA probe is removed and rinsed, and can be used directly after drying at low temperature, or stored at 0°C for later use.

[0013] The present invention proposes the application of the extended gate field effect transistor prepared by the above method in real-time detection of thrombin.

[0014] Since the extended gate field effect transistor obtained by the present invention is very small, it can be applied to a handheld thrombin detection device. The extended gate field effect transistor obtained by the above preparation method can show excellent stability and high sensitivity in real-time thrombin detection, and the detection limit is extremely low.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are: 1. The present invention obtains a high-performance extended gate field effect transistor based on a high-performance Prussian blue / platinum nanoparticle composite material, which exhibits excellent electrochemical biosensing performance.

[0016] 2. The extended gate field effect transistor prepared by the present invention can realize high-sensitivity detection of thrombin with an extremely low detection limit; and has the advantages of low sample consumption, easy batch production, low cost and easy replacement. It can realize real-time and rapid monitoring of thrombin when combined with an external detection circuit, filling the gap in the current thrombin real-time detection products on the market and having high clinical application value.

[0017] 3. The extended gate field effect transistor can still specifically recognize thrombin in an environment with complex components, and the transistor is small in size, which is convenient for miniaturized integration of detection equipment, such as application in handheld devices to facilitate detection operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a scanning electron microscope image of the Prussian blue / platinum nanoparticles prepared in Example 1.

[0019] Figure 2 This is a detection circuit diagram for testing extended gate field effect transistors.

[0020] Figure 3 This is the detection signal curve of the extended gate field effect transistor for thrombin.

[0021] Figure 4 This is the calibration curve of thrombin detection concentration.

[0022] Figure 5 This is a graph showing the thrombin repeatability test results of an extended gate field effect transistor.

[0023] Figure 6 This is a graph showing the thrombin specificity test results of an extended gate field effect transistor. DETAILED DESCRIPTION

[0024] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with 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.

[0025] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0026] Example 1 This embodiment provides a method for preparing an extended gate field effect transistor for real-time detection of thrombin, the steps are as follows: (1) 100 mL of 70 mM FeCl2 solution was mixed with 100 mL of 70 mM potassium ferrocyanide solution, and the mixture was stirred at room temperature for 10 hours at a stirring rate of 300 rpm. After the reaction, the mixture was centrifuged at 8000 rpm for 5 minutes. The supernatant was discarded after centrifugation, and the solid at the bottom of the centrifuge tube was taken out and dried in an oven at 80°C for 24 hours to obtain Prussian blue nanoparticles.

[0027] (2) Add the above-mentioned Prussian blue nanoparticles to deionized water to prepare a dispersion with a concentration of 6 mg / mL; take 20 mL of the dispersion, add 10 mL of a 3 mM chloroplatinic acid solution and 10 mL of a 5 mM ascorbic acid solution, and perform reduction reaction at 90°C for 12 hours. The obtained product is centrifuged at 3000 rpm for 5 minutes, and the supernatant is poured out after centrifugation. The solid obtained by centrifugation is washed twice with deionized water, and then dried at 60°C in a nitrogen atmosphere for 12 hours to obtain a Prussian blue / platinum nanoparticle composite material, such as Figure 1 .

[0028] (3) An extended gate field effect transistor with a sensing layer of tantalum oxide was selected (the other structures and preparation processes of the transistors used in this embodiment and other embodiments adopt the method disclosed in Example 1 of CN 114660157A), 4 μL of cysteine ​​(Merck) was dripped on the surface of the sensing layer as a connecting agent and allowed to stand and dry at room temperature for 12 hours. Then, the Prussian blue / platinum nanoparticles obtained in step (2) were added to deionized water to prepare a dispersion solution with a concentration of 6 mg / mL, and 5 μL was taken and dripped on the surface of the connecting agent of the sensing layer, and allowed to stand and dry at room temperature for 12 hours to obtain a Prussian blue / platinum nanoparticle composite modified field effect transistor.

[0029] (4) The DNA probe lyophilized powder (Shanghai Sangon Biotechnology Co., Ltd., DNA sequence: AGT CCG TGGTAG GGC AGG TTG GGG TGA CT) was dissolved in TE buffer (Shanghai MacLean Biochemical Technology Co., Ltd., pH 7.0) to prepare a 100 μM DNA probe solution, which was further diluted to 12 μM with deionized water to obtain a DNA probe dilution solution. 4 μL of the DNA probe dilution solution was added dropwise to the surface of the composite material of the extended gate field effect transistor sensing layer after drying, and the mixture was placed in a 0°C environment for reaction for 6 hours. The extended gate field effect transistor was then taken out, and the unbound DNA probe on the surface of the sensing layer was rinsed off with deionized water. The mixture was then placed in a 0°C environment for drying for 6 hours to obtain an extended gate field effect transistor for specific detection of thrombin.

[0030] Example 2 The conditions in this embodiment and subsequent embodiments that are not specifically described are consistent with those in embodiment 1. This embodiment provides a method for preparing an extended gate field effect transistor for real-time detection of thrombin, comprising the following steps.

[0031] (1) Mix 100 mL of 75 mM Co(NO3)2 solution and 100 mL of 75 mM potassium ferrocyanide solution, stir at room temperature for 12 hours at a stirring rate of 300 rpm. After the reaction, centrifuge at 8000 rpm for 5 minutes and then dry in an oven at 90°C for 24 hours to obtain Prussian blue nanoparticles.

[0032] (2) Prussian blue nanoparticles were added to ethylene glycol to prepare a dispersion with a concentration of 7 mg / mL; 20 mL of the dispersion was taken and 10 mL of 4 mM potassium chloroplatinate solution and 10 mL of 7 mM glucose solution were added, and the reduction reaction was carried out at 100°C for 10 hours. The obtained product was centrifuged at 3000 rpm for 6 minutes, washed twice with deionized water after centrifugation, and then dried at 70°C in a nitrogen atmosphere for 9 hours to obtain a Prussian blue / platinum nanoparticle composite material.

[0033] (3) An extended gate field effect transistor with a sensing layer of hafnium oxide was selected, and 3 μL of mercaptosilane linking agent (Merck) was dripped on the surface of the sensing layer and allowed to stand and dry at room temperature for 14 h. Then, the Prussian blue / platinum nanoparticles obtained in step (2) were added to ethanol to prepare a dispersion solution with a concentration of 7 mg / mL, and 4 μL was dripped on the surface of the linking agent of the sensing layer and allowed to stand and dry at room temperature for 12 hours to obtain a Prussian blue / platinum nanoparticle composite modified field effect transistor.

[0034] (4) Dissolve the DNA probe freeze-dried powder in TE buffer to obtain a 100 μM DNA probe solution. Further dilute it to 15 μM with deionized water. Take 3 μL of the DNA probe solution diluted with deionized water and continue to drip it on the surface of the composite material of the extended gate field effect transistor sensing layer, place it in a 0°C environment to react for 9 hours, then take out the extended gate field effect transistor, rinse off the unbound DNA probe on the surface of the sensing layer with deionized water, and continue to dry it at 0°C for 6 hours to obtain an extended gate field effect transistor for thrombin specific detection.

[0035] Example 3 This embodiment provides a method for preparing an extended gate field effect transistor for real-time detection of thrombin, comprising the following steps.

[0036] (1) 100 mL of 80 mM CuSO4 solution was mixed with 100 mL of 80 mM potassium ferrocyanide solution, and the mixture was stirred at room temperature for 14 hours at a stirring rate of 300 rpm. After the reaction, the mixture was centrifuged at 8000 rpm for 5 minutes, and then dried in an oven at 80°C for 24 hours to obtain Prussian blue nanoparticles.

[0037] (2) Prussian blue nanoparticles were added to ethylene glycol to prepare a dispersion with a concentration of 8 mg / mL; 20 mL of the dispersion was taken and 10 mL of 5 mM sodium chloroplatinate solution and 10 mL of 9 mM glucose solution were added, and the reduction reaction was carried out at 120°C for 8 hours. The obtained product was centrifuged at 3000 rpm for 6 minutes, washed twice with deionized water after centrifugation, and then dried at 80°C in a nitrogen atmosphere for 7 hours to obtain a Prussian blue / platinum nanoparticle composite material.

[0038] (3) Select an extended gate field effect transistor with a sensing layer of silicon nitride, add 2 μL of aminosilane linker (Merck) on the surface of the sensing layer and let it stand and dry at room temperature for 14 hours. Then add the Prussian blue / platinum nanoparticles obtained in step (2) to ethanol to prepare a dispersion solution with a concentration of 8 mg / mL, take 2 μL and continue to drop it on the surface of the linker of the sensing layer, let it stand and dry at room temperature for 12 hours, and obtain a Prussian blue / platinum nanoparticle composite modified field effect transistor.

[0039] (4) Dissolve the DNA probe freeze-dried powder in TE buffer to obtain a 100 μM DNA probe solution. Further dilute it to 18 μM with deionized water. Take 2 μL of the DNA probe solution diluted with deionized water and continue to drip it on the surface of the composite material of the extended gate field effect transistor sensing layer, place it in a 0°C environment to react for 10 hours, then take out the extended gate field effect transistor, rinse off the unbound DNA probe on the surface of the sensing layer with deionized water, and continue to dry it at 0°C for 6 hours to obtain an extended gate field effect transistor for thrombin specific detection.

[0040] The extended gate field effect transistor prepared in each embodiment is used in combination with a detection circuit to test the performance 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. The layout of each module is as follows: Figure 2 As shown. 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 the Stm32rct6 chip, the signal acquisition module 3 is composed of the ADS1115 chip, the excitation power module 4 is composed of the DAC8562 chip, and the current amplifier module 5 is composed of the 100Ω current sensing resistor CRM2512-FX-1000ELF and the INA181A3IDBVT voltage amplifier. The extended gate field effect transistor interface module 6 is provided with three interfaces and is respectively connected to the drain, gate, and source of the transistor. 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 that drives 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.

[0041] During the detection process, the main control module 2 drives the excitation power supply module 4 to output a scanning voltage of 0-2V and a step length of 0.05V / S between the source and drain of the extended gate field effect transistor, and outputs a fixed voltage of 1V between the gate and the source to drive the extended gate field effect transistor to work normally. After the thrombin in the test solution preferentially and specifically binds to the DNA aptamer probe fixed on the surface of the field effect transistor sensing layer modified by the Prussian blue / platinum nanoparticle composite material, a more stable ligand is formed. The signal response generated in this process causes the induced potential on the surface of the sensing layer to change, which in turn affects the step-by-step distribution of carriers in the channel of the extended gate field effect transistor, and ultimately causes the current (source-drain current) at both ends of the source and drain of the extended gate field effect transistor to change. The source-drain current passes through the current-sense resistor of the current amplifier module 5, and the voltage at both ends of the current-sense resistor is amplified by the INA181A3IDBVT voltage amplifier and then output to the ADS1115 chip of the signal acquisition module 3 for analog-to-digital conversion. The current value at both ends of the source and drain (i.e., the source-drain current V DS ), and transmits the response signal to the main control module through I2C communication. The main control module receives, stores and transmits it.

[0042] 1. Extended Gate Field Effect Transistor Performance Test First, add 100 μL of PBS buffer solution (5000 mL of deionized water plus 32.275 g of potassium chloride, 40.8275 g of dihydrogen phosphate, 45.643 g of dihydrogen phosphate, and 10 g of potassium sorbate; the same below) to the detection pool, immerse the extended gate in the PBS buffer solution, connect the gate, drain, and source electrode contacts of the extended gate field effect transistor to the corresponding electrode wires on the detection circuit interface module, and use the excitation power module to set the extended gate field effect transistor source-drain voltage to 0-2V, with a step size of 0.05V, and the gate-source voltage to 1V; the detection curve when the thrombin concentration is 0 pM can be obtained, which is the baseline. After the baseline test is completed, the detection conditions remain unchanged, and the detection system is changed: pour out the PBS buffer solution in the detection pool, and then replace it with 100 μL of thrombin concentrations of 1, 10, 100, 500, 1000, 5000, and 10000 pM detection solutions for detection in turn. Among them, thrombin was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, stored at -20 degrees Celsius). When testing samples, the lyophilized thrombin powder can be diluted with PBS buffer, and each sample must 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 As shown, 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.

[0043] 2. Repeatability test According to the steps in Example 1, 6 extended gate field effect transistors for specific detection of thrombin are repeatedly prepared, and the prepared extended gate field effect transistors are matched with the detection circuit to carry out stability test of thrombin detection. The prepared extended gate field effect transistors are numbered from 1 to 6 according to the preparation order. First, the extended gate of the field effect transistor numbered 1 is immersed in the detection system, 100 μL of the test solution with a thrombin concentration of 1 pM is added, the source-drain voltage of the extended gate field effect transistor is set to 0-2V, and the gate-source voltage is set to 1V; the detection curve of the field effect transistor numbered 1 when the thrombin concentration is 1 pM can be obtained. After the test is completed, the extended gate field effect transistor numbered 2 is replaced, and 100 μL of the test solution with a thrombin concentration of 1 pM is added again for detection, and the above operation is repeated. The repeatability test results of the extended gate field effect transistor are as follows. Figure 5 As shown, the signal difference between each extended gate field effect transistor is small, indicating that it has good signal reproducibility.

[0044] 3. Stability The extended gate field effect transistor was stored in a 4°C environment for 30 days. After testing, its response signal was 92% of the initial signal, indicating that the extended gate field effect transistor has excellent stability.

[0045] 4. Anti-interference detection The prepared extended gate field effect transistor was matched with the detection circuit for thrombin anti-interference test. First, the extended gate was immersed in the detection system, 120μL of thrombin (Tob) solution with a concentration of 1pM was added, the source-drain voltage of the extended gate field effect transistor was set to 0-2V, and the gate-source voltage was set to 1V; the detection curve when the thrombin concentration was 1pM was obtained. After the test, the detection system was changed, and 120μL of immunoglobulin G (lgG), bovine serum albumin (BSA), prostate-specific antigen (PSA), cardiac troponin T (cTnT), and lysozyme (Lzm) solution diluted to 1μM with PBS buffer were added respectively. The detection system was changed after each test, and finally the following two samples were mixed for detection: Sample 1: 20μL PBS buffer with a concentration of 1pM Tob; Sample 2: 20μL PBS buffer (Mix) with a concentration of 1μM Lzm, lgG, BSA, PSA, and cTnT. Among them, BSA and Lzm were purchased from Shanghai Shenggong Biotechnology Co., Ltd., IgG, PSA, and cTnT were purchased from Abcam, and all were stored in a refrigerated state (2-8°C).

[0046] The test results of the extended gate field effect transistor anti-interference performance are as follows: Figure 6As shown. The extended gate field effect transistor also exhibits excellent anti-interference ability and can still maintain a specific signal response to thrombin in a detection system with a concentration of 1000 times that of the interferent. The above results all indicate that the extended gate field effect transistor has excellent thrombin recognition ability and real system application potential.

[0047] 5. Real sample testing 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 Drum Tower Hospital). Each time 120 μL of serum was added to the detection system, and the extended gate field effect transistor prepared in Example 1 was used with the detection circuit to test the performance of thrombin. Figure 4 The corresponding relationship between the detection signal change and the serum thrombin content established in the calculation was used. The results obtained by using the Abcam thrombin activity kit (ab197006) were used to calculate the deviation. The obtained detection results are shown in Table 1.

[0048] Table 1 Test results of thrombin in real serum samples From the results in Table 1, the field effect transistor prepared by the present invention can realize rapid and accurate detection of thrombin in real serum, with a low detection limit and a wide detection range, which is consistent with the detection results of the commercial kit. In addition, the extended gate field effect transistor of the present invention is small in size and the required inspection 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.

[0049] Comparative Example 1 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 Prussian blue / platinum nanoparticles, and the other conditions remain unchanged. After testing, the obtained extended gate field effect transistor has no obvious signal response to thrombin.

[0050] Comparative Example 2 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 other conditions remain unchanged. After testing, the obtained extended gate field effect transistor has no obvious signal response to thrombin.

[0051] Comparative Example 3 The difference between this comparative example and Example 1 is that a single Prussian blue and a single platinum nanoparticle are mixed to directly modify the extended gate of the field effect transistor instead of using Prussian blue / platinum nanoparticles, and the other conditions remain unchanged. After testing, the linear detection range of the obtained extended gate field effect transistor for thrombin is 10-200pM, and the response time is 5min. Compared with the detection performance of the field effect transistor in Example 1, it is significantly reduced, which fully demonstrates the superiority of the Prussian blue / platinum composite material.

[0052] From the detection performance comparison of the field effect transistor obtained in the above-mentioned embodiment, it can be found that nano materials play a vital 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, due to the weak change of the response signal generated by the recognition process, conventional electrochemical biosensors are difficult to meet the rapid detection of low-concentration thrombin. Field effect transistors can have excellent sensitivity and resolution to low current signals, but are subject to the general effect of current field effect transistor surface modification technology, and nano materials are difficult to be compatible with field effect transistors, resulting in the existing reported field effect transistors and other detection means being difficult to accurately and quickly determine the content of thrombin in whole blood.

[0053] In order to solve the above problems, the present invention adopts Prussian blue analog material as research object, utilizes its excellent catalytic activity and pH sensitivity, uses it as the modification material of field effect transistor extension gate;Meanwhile, in order to further improve detection performance, and ensure that thrombin specific probe molecule can stably exist in the system, in situ growth platinum nanoparticles on the surface of Prussian blue analog, prepare Prussian blue / platinum nanomaterial, utilize the high specific surface area and excellent biocompatibility of its composite nanoparticle structure to improve the detection performance of field effect transistor.In thrombin detection process, the DNA probe molecule fixed on the surface of Prussian blue / platinum nanoparticle can quickly identify thrombin, and the change of its configuration triggers the catalytic action of Prussian blue, thereby causing the gate surface potential to change, so that the field effect transistor produces a weak current response signal.In this composite structure, utilizing the synergistic effect of Prussian blue and platinum nanoparticles, the stability of thrombin specific probe and a large amount of load and the amplification and transmission of response signal are realized, greatly improving the detection performance and detection efficiency of thrombin.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing an extended gate field effect transistor for real-time detection of thrombin, characterized in that: The specific steps are as follows: (1) Preparation of Prussian blue / platinum nanoparticle composites A transition metal salt solution and a transition metal potassium cyanide solution are mixed and stirred to perform a synthesis reaction. After the reaction is completed, the mixture is centrifuged and dried to obtain Prussian blue nanoparticles; the Prussian blue nanoparticles are dispersed in a solvent to obtain a dispersion A; a platinum source solution and an auxiliary agent solution are then added to perform a high-temperature reduction reaction. After the high-temperature reduction reaction is completed, the mixture is centrifuged, and the obtained solid is washed and dried to obtain a Prussian blue / platinum nanoparticle composite material; (2) Fabrication of extended gate field effect transistor based on Prussian blue / platinum nanoparticle composites The Prussian blue / platinum nanoparticle composite material is dispersed in water or ethanol to obtain a dispersion B, a connection aid is drop-coated on the surface of the sensing layer of the extended gate field effect transistor and allowed to stand and dry once, and then the dispersion B is continued to be dropped and allowed to stand and dry for a second time; (3) Fabrication of extended-gate field-effect transistor for thrombin recognition The thrombin-specific DNA probe solution is then dripped onto the sensing layer of the extended gate field effect transistor, reacted at low temperature, rinsed after the reaction is completed, and dried at low temperature to obtain an extended gate field effect transistor for thrombin recognition.

2. The method for preparing an extended gate field effect transistor for real-time detection of thrombin according to claim 1, characterized in that: In step (1), the transition metal salt solution and the transition metal potassium cyanide solution are mixed at a concentration of 70-80 mM, the transition metal salt is any one of FeCl2, Co(NO3)2, and CuSO4, the transition metal potassium cyanide is potassium ferrocyanide or potassium ferrocyanide, the synthesis reaction time is at least 6 hours, and the synthesis reaction temperature is room temperature.

3. The method for preparing an extended gate field effect transistor for real-time detection of thrombin according to claim 1, characterized in that: In step (1), the Prussian blue nanoparticle dispersion solvent is water or ethylene glycol, and the concentration of dispersion A is 6-8 mg / mL; the platinum source is any one of chloroplatinic acid, potassium chloroplatinate, and sodium chloroplatinate, the auxiliary agent is ascorbic acid or glucose, the concentration of the platinum source solution is 3-5 mM, and the concentration of the auxiliary agent solution is 5-10 mM; the volume ratio of dispersion A: platinum source solution: auxiliary agent solution is 2:1:1; the high-temperature reduction reaction temperature is 90-120°C, and the high-temperature reduction reaction time is 8-12h; the drying temperature is 60-80°C, the drying atmosphere is nitrogen, and the drying time is 6-12h.

4. The method for preparing an extended gate field effect transistor for real-time detection of thrombin according to claim 1, characterized in that: 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 agent is any one of cysteine, mercaptosilane, and aminosilane, the amount of the connecting agent added is 1-5 μL, the concentration of dispersion B is 6-8 mg / mL, the amount of dispersion B added is 1-5 μL, and the first and second standing drying times are both at least 12 hours.

5. The method for preparing an extended gate field effect transistor for real-time detection of thrombin according to claim 1, characterized in that: In step (3), the concentration of the thrombin-specific DNA probe solution is 10-20 μM, and the drop-coating amount is 1-5 μL; the reaction temperature at low temperature is 0° C., and the reaction time is 6-12 h.

6. Use of the extended gate field effect transistor prepared by any one of the methods of claims 1 to 5 in real-time detection of thrombin.

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

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