P-type organic electrochemical transistor biosensor and preparation method and application thereof

Abstract

The invention belongs to the technical field of organic electrochemical transistors, and discloses a p-type organic electrochemical transistor biosensor and a preparation method and application thereof.The sensor comprises a first substrate and a second substrate, a source electrode is arranged on one side of the first substrate, a polymer semiconductor channel is formed in the source electrode, a drain electrode is arranged on the polymer semiconductor channel, and the drain electrode is arranged on the second substrate; a first floating gate is arranged on the other side of the first substrate, and a first electrolyte layer covers the first substrate, the drain electrode and the first floating gate; a grid electrode is arranged on one side of the second substrate, a grid electrode modification layer is arranged on the grid electrode, a second floating gate is arranged on the other side of the second substrate, and the second substrate, the grid electrode modification layer and the second floating gate are covered with a second electrolyte layer; the first floating gate and the second floating gate are connected through a wire. According to the invention, the detection on the H1N1 virus marker with ultra-low concentration (10 to 20 mol / L) is realized, and the detection on the H1N1 virus marker with the concentration of 10 to 17 mol / L in ultra-short time is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of organic electrochemical transistors, and in particular relates to a p-type organic electrochemical transistor biosensor and a preparation method and application thereof. Background Art

[0002] With the improvement of social living standards and the continuous advancement of medical technology, people are paying more and more attention to health. Real-time detection using biomarkers has become an effective means to obtain information such as individual health status, disease development and treatment effects. Pathogen markers play an important role in the life process, and changes in their expression levels often reflect physiological or pathological changes in the body. By monitoring changes in pathogen levels in body fluids, medical personnel can more accurately assess the disease state, develop personalized treatment plans, and provide patients with more precise medical services. This application based on pathogen markers brings new possibilities for disease management and health monitoring, helps to improve the level of medical diagnosis and treatment, and provides more comprehensive attention and care for individual health.

[0003] In recent years, with the development of organic semiconductor materials and the optimization of processing technology, high-performance organic electrochemical transistors (OECTs) have been widely studied and developed rapidly. Thanks to their excellent working stability, signal conversion and amplification functions in the liquid phase, and good biocompatibility, OECT-based biosensors have shown great potential in the application of rapid and sensitive biomarker detection. However, the intrinsic content of pathogen biomarkers is low, and there are a large number of interfering substances such as ions and molecules in human body fluids (blood, sweat, saliva, etc.). There is an urgent need to build a type of biosensor with high stability, strong anti-interference ability, and high sensitivity. Summary of the invention

[0004] In view of the above-mentioned problems in the prior art, the purpose of the present invention is to provide a p-type organic electrochemical transistor biosensor and a preparation method and application. The p-type organic electrochemical transistor sensor can operate under positive voltage, avoiding the problem of small and unstable voltage modulation range of the device under negative voltage, and has the advantages of fast, sensitive and stable detection.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: A p-type organic electrochemical transistor biosensor comprises a first substrate and a second substrate; A source electrode is arranged on one side of the first substrate, a polymer semiconductor channel is arranged on the source electrode, a drain electrode is arranged on the polymer semiconductor channel, a first floating gate is arranged on the other side of the first substrate, and a first electrolyte layer is arranged on the first substrate, the drain electrode and the first floating gate; A gate is arranged on one side of the second substrate, a gate modification layer is arranged on the gate, a second floating gate is arranged on the other side of the second substrate, and a second electrolyte layer is arranged on the second substrate, the gate modification layer and the second floating gate; the first floating gate and the second floating gate are connected by a wire; A drain voltage is applied to a loop consisting of a source electrode, a polymer semiconductor channel and a drain electrode, and the source electrode is grounded; Applying a gate voltage to a loop consisting of a source electrode, a first floating gate, a second floating gate, a wire, a first electrolyte layer, a second electrolyte layer, a gate and a gate modification layer; The gate modification layer is a gate modification layer containing a target probe molecule layer of a target object to be detected.

[0006] Furthermore, the material of the first substrate and the second substrate is silicon oxide; The source electrode, the drain electrode and the gate electrode are made of Au.

[0007] Furthermore, the first electrolyte layer is prepared from a NaCl aqueous solution, and the second electrolyte layer is prepared from a PBS buffer solution.

[0008] Further, the material of the polymer semiconductor channel is poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene]; The first floating gate and the second floating gate are made of Ag / AgCl slurry.

[0009] Furthermore, the thickness of the source electrode is 60-70 nm; the thickness of the drain electrode is 60-70 nm; the thickness of the polymer semiconductor channel is 130-150 nm; the thickness of the gate electrode is 60-70 nm; the thickness of the gate modification layer is 5-10 nm; and the thickness of the first floating gate and the second floating gate is 5-10 μm.

[0010] A method for preparing a p-type organic electrochemical transistor biosensor comprises the following steps: Depositing Cr on the first substrate to form a Cr layer, then depositing Au on the Cr layer to form a source electrode, depositing Cr on the second substrate to form a Cr layer, then depositing Au on the Cr layer to form a gate electrode; Spin coating a chloroform solution of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the surface of the source electrode to obtain a polymer semiconductor channel; Au is deposited on the surface of the polymer semiconductor channel to form a drain electrode; Disposing a first floating gate on the first substrate and a second floating gate on the second substrate; A PBS buffer solution containing a target analyte probe is applied to the gate surface and allowed to stand to form a target probe molecular layer for the target analyte; a 6-mercapto-1-hexanol solution is dripped onto the gate surface to cover the gate surface not covered by the target analyte target probe molecular layer, and allowed to stand to form a 6-mercapto-1-hexanol blocking layer; the target probe molecular layer for the target analyte and the 6-mercapto-1-hexanol blocking layer form a gate modification layer.

[0011] A first electrolyte layer is arranged on the first substrate, the drain and the first floating gate, a second electrolyte layer is arranged on the second substrate, the gate modification layer and the second floating gate, and the first floating gate and the second floating gate are connected through a wire to obtain a p-type organic electrochemical transistor biosensor.

[0012] Furthermore, a first floating gate is provided on the first substrate, and a second floating gate is provided on the second substrate, comprising the following steps: Ag / AgCl slurry is coated on the first substrate, Ag / AgCl slurry is coated on the second substrate, and then annealing is performed to form a first floating gate on the first substrate and a second floating gate on the second substrate.

[0013] Furthermore, annealing was performed under inert gas at a temperature of 100 °C for 30 min.

[0014] Application of a p-type organic electrochemical transistor biosensor in biomarker detection.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: In order to meet the high sensitivity and high selectivity detection requirements of OECT sensors, the present invention utilizes the interaction between the target analyte and the probe molecule to modulate the electrical properties of the device and achieve a high-sensitivity sensing response. The setting of the first floating gate and the second floating gate enables the p-type organic electrochemical transistor biosensor to always operate under optimal conditions and will not be contaminated by the analyte. It can be reused and the testing cost is reduced. The experimental results show that the p-type organic electrochemical transistor biosensor has good cyclic operation stability and sensitivity to voltage changes, and can complete 10 amol / L (10 -17 mol / L) detection of H1N1 virus markers, with a minimum detection limit as low as 10 zmol / L (10 - 20 mol / L) level, and the current response has a good linear relationship with the detection concentration, showing the advantages of high sensitivity and rapid detection, and showing extraordinary potential in the field of instant on-site diagnosis.

[0016] Furthermore, the present invention selects poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] as the channel material and selects a vertical device structure, so that the prepared p-type organic electrochemical transistor biosensor has an ultra-high transconductance value and signal amplification capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic structural diagram of a p-type organic electrochemical transistor biosensor of the present invention; Figure 2 is a current variation diagram of an OECT long-term cycle test when Au is used as a floating gate material in Example 1 of the present invention; Figure 3 This is a current variation diagram of an OECT long-term cycle test when Ag / AgCl is used as a floating gate material in Example 1 of the present invention; Figure 4 The actual gate voltage map is read out through the second floating gate when a positive gate voltage (0~+0.6 V) is applied in Embodiment 1 of the present invention; Figure 5 The actual gate voltage map is read out through the second floating gate when a negative gate voltage (0 to -0.6 V) is applied in Embodiment 1 of the present invention; Figure 6 The gate voltage applied during the feasibility verification of the effective gate voltage test method in Example 1 of the present invention is V g = 0 V when the voltage readout value of the second floating gate; Figure 7 The gate voltage applied during the feasibility verification of the effective gate voltage test method in Example 1 of the present invention is V g = 0.3 V when the voltage readout value of the second floating gate; Figure 8 To set the gate voltage initially V g = 0.3 V, the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig. 9 The voltage readout value of the second floating gate and the polymer semiconductor channel current value of the device under the same setting conditions after standing in the air for 1 hour; Fig.10 To set the gate voltage V g = 0 V, the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig.11 To set the gate voltage V g = 0.05 V, the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig.12 This is a schematic diagram of a gate with a gate modification layer of a p-type organic electrochemical transistor biosensor in Example 1 of the present invention; Fig.13 Schematic diagram of the gate after the gate modification layer is specifically combined with the H1N1 virus marker; Fig.14 for Fig.12 Schematic diagram of ion doping in the polymer semiconductor channel in the case shown in; Fig.15 for Fig.13 Schematic diagram of ion doping in the polymer semiconductor channel in the case shown in; Fig.16 To set the gate voltage for the p-type organic electrochemical transistor biosensor in Example 2 of the present invention V g = 0.3 V, the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig.17 After the gate of the modified target probe molecule was incubated in PBS buffer for 1 h, the original electrical test parameter settings were maintained, and the second floating gate voltage readout value and polymer semiconductor channel current value were plotted; Fig.18 The gate of the modified target probe molecule is at a concentration of 10 -20 After incubation in 1 mol / L H1N1 PBS buffer for 1 h, the original electrical test parameter settings were maintained, and the second floating gate voltage readout value and polymer semiconductor channel current value were plotted; Fig.19 for Fig.16 The transfer curve test data of the device module before the effective gate voltage test; Fig. 20 for Fig.17 The transfer curve test data of the device module before the effective gate voltage test; Fig.21 for Fig.18 The transfer curve test data of the device module before the effective gate voltage test; Fig. 22 for Fig.16 The baseline current obtained from Fig.17 The noise current obtained from Fig.18 Schematic diagram of comparison of binding response current magnitude obtained in ; Fig.23 To set the gate voltage for the p-type organic electrochemical transistor biosensor in Example 2 of the present invention V g = 0.3 V, the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig.24The second floating gate voltage readout value and the polymer semiconductor channel current value graph are obtained after the gate of the modified targeting probe molecule in Example 2 is incubated in PBS buffer for 5 minutes and the original electrical test parameter settings are maintained; Fig.25 The gate of the modified targeting probe molecule in Example 2 is at a concentration of 10 -17 After incubation in mol / L H1N1 PBS buffer for 5 min, the original electrical test parameter settings were maintained, and the second floating gate voltage readout value and polymer semiconductor channel current value were plotted; Fig.26 for Fig.23 The transfer curve test data of the device module before the effective gate voltage test; Fig. 27 for Fig.24 The transfer curve test data of the device module before the effective gate voltage test; Fig.28 for Fig.25 The transfer curve test data of the device module before the effective gate voltage test; Fig.29 for Fig.23 The baseline current obtained from Fig.24 The noise current obtained from Fig.25 Schematic diagram of comparison of binding response current magnitude obtained in ; Fig.30 It is a fitting curve of the current response change obtained for different concentrations of the target analyte in the detection of H1N1 virus in Example 2 of the present invention after the targeting probe molecule and the target analyte are combined for 5 minutes; Fig.31 To set the gate voltage for the p-type organic electrochemical transistor biosensor in Example 4 of the present invention V g = 0.3 V when the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig.32 After the gate of the modified target probe molecule was incubated in PBS buffer for 5 min, the original electrical test parameter settings were maintained, and the second floating gate voltage readout value and polymer semiconductor channel current value were obtained; Fig.33 The gate of the modified target probe molecule is at a concentration of 10 -17 After incubation in 5 mol / L H1N1 PBS buffer for 5 min, the original electrical test parameter settings were maintained, and the second floating gate voltage readout value and polymer semiconductor channel current value were obtained; Fig.34 for Fig.31 The transfer curve test data of the device module before the effective gate voltage test; Fig.35 for Fig.32 The transfer curve test data of the device module before the effective gate voltage test; Fig.36 for Fig.33 The transfer curve test data of the device module before the effective gate voltage test; Fig.37 for Fig.31 The baseline current obtained from Fig.32 The noise current obtained from Fig.33 Schematic diagram of comparison of binding response current magnitude obtained in ; Fig.38 To set the gate voltage for the p-type organic electrochemical transistor biosensor in Example 6 of the present invention V g = 0.3 V, the voltage readout value of the second floating gate and the polymer semiconductor channel current value; Fig.39 The gate electrode of the modified targeting probe molecule of Example 6 is incubated in PBS buffer for 5 min, and the original electrical test parameter settings are maintained, and the obtained second floating gate voltage readout value and polymer semiconductor channel current value are plotted; Fig.40 The gate of the modified targeting probe molecule of Example 6 is at a concentration of 10 -17 After incubation in mol / L H1N1 PBS buffer for 5 min, the original electrical test parameter settings were maintained, and the second floating gate voltage readout value and polymer semiconductor channel current value were plotted; Fig.41 for Fig.38 The transfer curve test data of the device module before the effective gate voltage test; Fig.42 for Fig.39 The transfer curve test data of the device module before the effective gate voltage test; Fig.43 for Fig.40 The transfer curve test data of the device module before the effective gate voltage test; Fig.44 for Fig.38 The baseline current obtained from Fig.39 The noise current obtained from Fig.40 Schematic diagram of comparison of binding response current magnitude obtained in ; In the figure, 1 is the first substrate, 2 is the source, 3 is the polymer semiconductor channel, 4 is the drain, 5 is the gate, 6 is the first electrolyte layer, 7 is the second electrolyte layer, 8 is the gate modification layer, 9 is the first floating gate, 10 is the wire, 11 is the second substrate, and 12 is the second floating gate. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0019] The PBS buffer solution in the present invention is a standard PBS buffer solution with a pH of 7.2-7.4 and a concentration of 10 mmol / L.

[0020] See also Figure 1 A p-type organic electrochemical transistor biosensor of the present invention comprises a first substrate 1 and a second substrate 11; a source electrode 2 is arranged on one side of the first substrate 1, a polymer semiconductor channel 3 is arranged on the source electrode 2, a drain electrode 4 is arranged on the polymer semiconductor channel 3, a first floating gate 9 is arranged on the other side of the first substrate 1, the source electrode 2 and the first floating gate 9 are arranged at intervals, and a first electrolyte layer 6 is covered on the first substrate 1, the drain electrode 4 and the first floating gate 9; a gate electrode 5 is arranged on one side of the second substrate 11, a gate modification layer 8 is arranged on the gate electrode 5, a second floating gate 12 is arranged on the other side of the second substrate 11, the gate electrode 5 and the second floating gate 12 are arranged at intervals, and a second electrolyte layer 7 is covered on the second substrate 11, the gate modification layer 8 and the second floating gate 12; the first floating gate 9 and the second floating gate 12 are connected by a wire 10.

[0021] The first substrate 1 and the second substrate 11 are made of silicon oxide.

[0022] The source electrode 2 , the drain electrode 4 and the gate electrode 5 are made of Au.

[0023] The first electrolyte layer 6 is a NaCl aqueous solution, and the second electrolyte layer 7 is a PBS buffer solution.

[0024] The material of the polymer semiconductor channel 3 is poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] (CAS No. 1974310-62-7, referred to as P(g2T-T)).

[0025] The gate modification layer 8 is a target probe molecule layer of the target object to be detected and randomly staggeredly combined on the surface of the gate 5 and a 6-mercapto-1-hexanol blocking layer.

[0026] The first floating gate 9 and the second floating gate 12 are electrodes made of Ag / AgCl slurry.

[0027] The wire 10 is a copper wire.

[0028] The source electrode 2 has a length of 250-300 μm, a width of 95-105 μm, and a thickness of 60-70 nm; the drain electrode 4 has a length of 1200-1400 μm, a width of 95-105 μm, and a thickness of 60-70 nm; the polymer semiconductor channel 3 has a length of 100-110 μm, a width of 100-110 μm, and a thickness of 130-150 nm; the gate electrode 5 has a length of 9-10 mm, a width of 5-6 mm, and a thickness of 60-70 nm; the gate modification layer 8 has a length of 9-10 mm, a width of 5-6 mm, and a thickness of 5-10 nm; the first floating gate 9 and the second floating gate 12 have a length of 11-13 mm, a width of 6-7 mm, and a thickness of 5-10 μm.

[0029] The method for preparing the p-type organic electrochemical transistor biosensor as described above comprises the following steps: Metal Cr (chromium) is deposited on a clean first substrate 1 by vacuum thermal evaporation through a mask, and the evaporation rate is set to 0.2 Å / s to form a Cr layer to improve the bonding strength between Au (gold) and the first substrate 1, and then metal Au is deposited on the Cr layer by vacuum thermal evaporation through a mask, and the evaporation rate is set to 1 Å / s~1.5 Å / s to form a source 2; metal Cr (chromium) is deposited on a clean second substrate 11 by vacuum thermal evaporation through a mask, and the evaporation rate is set to 0.2 Å / s to form a Cr layer to improve the bonding strength between Au and the second substrate 11, and then metal Au is deposited on the Cr layer by vacuum thermal evaporation through a mask, and the evaporation rate is set to 1 Å / s~1.5 Å / s to form a gate 5.

[0030] A chloroform solution of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] (molecular weight 20000) was spin coated on the surface of the source electrode 2 to obtain a polymer semiconductor channel 3; wherein, the concentration of the chloroform solution of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] was 20 mg / ml, the spin coating speed was 5000 r / min, the spin coating time was 25 s~30 s, the volume was 25 μL, and the thickness of the polymer semiconductor channel 3 was 130 nm~150 nm.

[0031] Metal Au is deposited on the surface of the polymer semiconductor channel 3 by vacuum thermal evaporation through a mask, and the evaporation rate is set to 1Å / s~1.5Å / s to form a drain 4; The poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the first substrate 1 except the polymer semiconductor channel 3 between the source 2 and the drain 4 is removed by reactive ion etching; specifically, the drain 4 is used as a mask for etching, the etching gas is argon, the flow rate is 15 sccm, the etching power is 25 W, and the etching time is 250 s.

[0032] Ag / AgCl slurry (Ag / AgCl slurry is manufactured by Shanghai Julong Electronic Technology Co., Ltd. and is named JLL20) is coated on the first substrate 1 and the second substrate 11 respectively using a mask. The coated first substrate 1 and the second substrate 11 are heated at 100° C. for 30 min in an inert gas environment for annealing to obtain a first floating gate 9 and a second floating gate 12 . The first floating gate 9 is located at one end of the first substrate 1 , and the second floating gate 12 is located at the other end of the second substrate 11 .

[0033] A PDMS (Polydimethylsiloxane) groove is used to surround the gate 5, and a PBS (Phosphate Buffered Saline) buffer solution added with a target analyte probe molecule (the target analyte targeted probe molecule in the present invention is an H1N1 molecule) is dripped on the surface of the gate 5 surrounded by the PDMS groove, and the mixture is allowed to stand for 15 hours at 4°C for modification to form a targeted probe molecule layer of the target analyte; the surface of the gate 5 bounded with the targeted probe molecule layer is washed with PBS buffer, and then 1 mmol / L MCH (6-Mercapto-1-hexanol) solution is dripped on the targeted probe molecule layer of the target analyte to fill the blank area where the targeted probe molecule layer of the target analyte is not bound to the gate 5, and the mixture is allowed to stand at room temperature for 1 hour to form a 6-mercapto-1-hexanol blocking layer; finally, the surface of the gate 5 is washed with PBS buffer, and the targeted probe molecule layer of the target analyte and the 6-mercapto-1-hexanol blocking layer form a gate modification layer 8.

[0034] A first electrolyte is dripped onto the first substrate 1, the drain 4 and the first floating gate 9 to form a first electrolyte layer 6, and a second electrolyte is dripped onto the second substrate 11, the gate modification layer 8 and the second floating gate 12 to form a second electrolyte layer 7. The first floating gate 9 and the second floating gate 12 are connected by a wire 10 to obtain a p-type organic electrochemical transistor biosensor.

[0035] The application of the p-type organic electrochemical transistor biosensor as described above is to detect pathogen biomarkers using an effective gate voltage test method. The present invention uses a vertical structure OECT device and optimizes its performance to achieve an ultra-high transconductance of 438 mS. By setting the first floating gate 9 and the second floating gate 12, the gate control capability of the gate 5 is greatly increased, and by using an effective gate voltage test method, while ensuring the stability of the device, an ultra-sensitive small voltage offset response is achieved, laying a foundation for the subsequent realization of ultra-low concentration pathogen detection. The present invention can achieve 10 -20 mol / L H1N1 marker detection and complete 10 - 17 mol / L H1N1 marker detection, the current response and the concentration of the detected substance have a good linear change relationship. The p-type organic electrochemical transistor biosensor of the present invention has a simple preparation method and has broad application prospects in the field of rapid and portable detection of trace biomarkers.

[0036] The present invention introduces a floating gate structure to separate the test part from the device part, thereby preventing the p-type organic electrochemical transistor from being contaminated by the sample of the object to be tested, ensuring that the p-type organic electrochemical transistor biosensor is in the optimal test condition, and adopts an effective gate voltage test method to enable the p-type organic electrochemical transistor sensor to operate under a positive voltage, avoiding the problem of a small and unstable voltage modulation range for the device under a negative voltage, thereby achieving rapid, sensitive and stable detection.

[0037] Example 1 This embodiment relates to a method for preparing a p-type organic electrochemical transistor biosensor, and the specific steps are as follows: Step 1, cleaning the rigid silicon oxide substrate: placing the substrate in detergent water, ultrapure water, acetone and isopropanol for ultrasonic cleaning for 30 minutes.

[0038] Step 2: Use an air gun to blow clean the surface of the substrate, and then place it in a UV ozone cleaner for 15 minutes to improve the wettability of the substrate surface.

[0039] Step 3, depositing on the cleaned first substrate 1 by vacuum evaporation through a mask, first depositing a 2 nm thick Cr layer as an adhesion layer at 0.2 Å / s to enhance the bonding force between gold and the substrate, and then depositing a 60 nm thick Au layer at a rate of 1 Å / s~1.5 Å / s to form a source 2; depositing on the cleaned second substrate 11 by vacuum evaporation through a mask, first depositing a 2 nm thick Cr layer as an adhesion layer at 0.2 Å / s to enhance the bonding force between gold and the substrate, and then depositing a 60 nm thick Au layer at a rate of 1 Å / s~1.5 Å / s to form a gate 5; wherein the length of the source 2 is 250 μm and the width is 95 μm, and the length of the gate 5 is 9 mm and the width is 5 mm.

[0040] Step 4, adding poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] (molecular weight of 20000) into chloroform and stirring for 15 h. After the poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] is fully dissolved, a 20 mg / ml poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] chloroform solution is obtained. After accelerating the first substrate 1 to 5000 rpm, 25 μL of the poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] chloroform solution is added dropwise onto the first substrate 1, and the solvent is completely evaporated in a vacuum environment to form a polymer semiconductor channel 3. The length of the polymer semiconductor channel 3 is 100 μm, width is 110 μm and thickness is 130 nm.

[0041] Step 5, depositing a 60 nm thick Au layer as the drain electrode 4 on the surface of the polymer semiconductor channel 3 by vacuum evaporation through a mask at a rate of 1 Å / s to 1.5 Å / s; wherein the drain electrode 4 has a length of 1200 μm and a width of 95 μm.

[0042] Step 6, using the drain 4 as a mask, patterning the polymer semiconductor channel 3 by reactive ion etching (RIE) to remove excess poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] to avoid ineffective doping; the etching gas in the reactive ion etching method is inert gas argon, the flow rate is 15 sccm, the etching power is 25 W, and the etching time is 250 s.

[0043] Step 7, using a pre-customized mask to coat Ag / AgCl slurry on the first substrate 1, then placing the first substrate 1 in an inert gas atmosphere and thermally annealing it at 100°C for 30 min to form a first floating gate 9, and using a pre-customized mask to coat Ag / AgCl slurry on the second substrate 11, then placing the second substrate 11 in an inert gas atmosphere and thermally annealing it at 100°C for 30 min to form a second floating gate 12, wherein the first floating gate 9 and the second floating gate 12 are 11 mm long, 6 mm wide, and 5 μm thick.

[0044] Step 8, add 10 μL of 1 mmol / L tris(2-carboxyethyl)phosphine (TCEP) aqueous solution to 100 μL of 1 μmol / L PBS buffer solution of the target analyte targeting probe molecule to obtain a mixed solution. The purpose of adding tris(2-carboxyethyl)phosphine (TCEP) aqueous solution is to open the disulfide bond of the target analyte targeting probe molecule to better bind to the surface of the gate 5. The modified area is fixed by the PDMS groove, the mixed solution is dripped into the PDMS groove, and the second substrate 11 is placed at 4°C for 15 hours for modification to form a target analyte targeting probe molecule layer.

[0045] Step 9, rinse the modified surface of the gate 5 with PBS buffer, blow the surface dry with an ear-cleaning bulb, and drop 100 μL of 1 mmol / L MCH solution into the PDMS groove in step 8, and let it stand at room temperature for 1 hour to form a 6-mercapto-1-hexanol blocking layer. The purpose of standing for 1 hour is to allow the MCH solution to cover the surface of the gate 5 that is not covered by the target probe molecule layer of the target analyte, and to arrange the probe molecules regularly to prevent nonspecific adsorption on the surface of the gate 5. Subsequently, the surface of the gate 5 is rinsed with PBS buffer to form a gate modification layer 8, which has a length of 9 mm, a width of 5 mm, and a thickness of 5 nm.

[0046] Step 10, drip 0.1 mol / L NaCl aqueous solution on the first substrate 1, the polymer semiconductor channel 3 and the first floating gate 9 as the first electrolyte layer 6; drip PBS buffer solution on the second substrate 11, the gate modification layer 8 and the second floating gate 12 as the second electrolyte layer 7, use the copper wire 10 to connect the first floating gate 9 and the second floating gate 12 to complete the preparation of the p-type organic electrochemical transistor biosensor.

[0047] Among them, the first substrate 1, the source 2, the polymer semiconductor channel 3, the drain 4, the first electrolyte layer 6 and the first floating gate 9 are used as an organic electrochemical transistor (OECT) device module, the second substrate 11, the gate 5, the gate modification layer 8, the second electrolyte layer 7 and the second floating gate 12 are used as a sensing module, and the OECT device module is used to amplify the signal sensed by the sensing module. The two modules together constitute a p-type organic electrochemical transistor biosensor. The drain voltage ( V DS ), the source electrode 2 is grounded. A gate voltage ( V GS ), source 2 is grounded.

[0048] The performance improvement mechanism of the p-type organic electrochemical transistor biosensor of this embodiment is as follows: 1) Influence of floating gate material on device performance: First, the performance of the OECT device module is optimized. The source 2, the first floating gate 9 and the first electrolyte layer 6 form a loop, and the gate voltage is applied to the first floating gate 9, and the source 2 is grounded; the source 2, the polymer semiconductor channel 3 and the drain 4 form a loop, and the drain voltage is applied to the drain 4, and the source 2 is grounded. First, the polarizable electrode Au is used as the electrode material of the first floating gate 9 to test the long-term stability of the device module, such as Figure 2 As shown in FIG. 1 , the maximum on-state current of the device module changes randomly with the increase of the number of scans during the triangular wave cyclic scanning process. This poor cyclic stability will cause problems such as excessive noise and baseline drift in the subsequent device module test. Subsequently, the non-polarizable electrode Ag / AgCl is used as the electrode material of the first floating gate 9 to perform a cyclic stability test, as shown in FIG. Figure 3 As shown, the device module has good cycle stability, and the device module performance is almost not attenuated within the test time of 3000 s, which meets the test requirements of the sensor for performance stability. Therefore, it is speculated that the test stability is poor when Au is used as the gate material because it is a polarized electrode. During the test, the surface generates charge accumulation, which makes the electrolyte and the gate interface capacitance constantly change, thereby affecting the potential at the polymer semiconductor channel, and the source-drain current constantly drifts from the perspective of device electrical performance. However, the gate modification layer 8 can only be combined with Au, so the present invention uses Ag / AgCl slurry as a floating gate to read the surface potential change of the gate 5, thereby realizing the conduction of the device module, which can greatly improve the stability of the device.

[0049] 2) Effect of different gate voltages on device performance: The second floating gate 12 reads the change in the surface potential of the gate 5. The test results are as follows: Figure 4 and Figure 5 As shown, when a positive voltage is applied to the surface of the gate 5, the readout voltage of the second floating gate 12 is linearly related to the applied triangular wave voltage, but when a negative voltage is applied to the surface of the gate 5, the readout voltage has a large loss compared to the applied voltage, and the voltage modulation range is very small. As the number of scanning cycles increases, the charge further accumulates on the surface of the gate 5, and the readout voltage of the second floating gate 12 decreases due to the increase in the surface capacitance of the gate 5. It can be seen that in the PBS solution environment, the adsorption and desorption process of anions on the surface of the gate 5 under a positive gate voltage is reversible, but the adsorption and desorption process of cations under a negative gate voltage is basically irreversible, resulting in a small range of actual gate voltage changes and a weak modulation effect on the device current. Therefore, during testing, it is necessary to apply a positive gate voltage to the gate 5 to ensure that the device can produce an obvious current response to a small voltage change, that is, it has high sensitivity.

[0050] 3) Influence of effective gate voltage test on device performance: Based on the above-mentioned influence of applying different gate voltages on device performance, the present invention proposes to change the traditional transfer curve test method of applying constant negative drain voltage and scanning negative gate voltage to applying a constant 0.1 V positive gate voltage to the gate, and applying a 0-0.6 V triangle wave voltage scan to the drain 4. In this process, when the drain 4 voltage is greater than 0.1 V, the gate potential is less than the drain 4 potential. At this time, a positive electric field pointing from the drain 4 to the gate is generated, and the anions move toward the channel under the action of the electric field force, realizing the doping of the p-type channel under positive voltage.

[0051] The feasibility of the effective gate voltage test method is analyzed below to determine the modulation range and stability of the applied voltage. Figure 6 and Figure 7 As shown in the figure, when a positive voltage is applied, the actual gate voltage is linear and stable, indicating that this effective gate voltage test method is feasible in principle. The sensing detection principle is that after the object to be tested specifically binds to the probe, the electrolyte and the gate interface capacitance C G Therefore, before being formally applied to actual pathogen detection, it is necessary to test the stability of the device in this mode and its sensitivity to gate voltage changes.

[0052] like Figure 8 , Fig. 9 , Fig.10 and Fig.11 As shown, the initial leakage current I d = 9.27 mA after one hour of standing I d =9.37 mA, the change is about 1%, which meets the requirements of device test stability. Then, the specific binding process at the gate is simulated, such as Fig.10 and Fig.11 As shown in the figure, the gate voltage is changed from 0 V to 0.05 V. I d The voltage change of 50 mV from 10.21 mA to 9.27 mA caused a current response of about 1 mA, which proved the feasibility of OECT (Organic Electrochemical Transistor) devices to detect pathogen markers using an effective gate voltage test method.

[0053] Based on the above test results, it can be seen that the setting of the floating gate structure greatly improves the stability of the device for long-term operation, avoiding problems such as excessive noise and baseline drift in subsequent sensor tests. When Au is used as the gate electrode material, its polarization characteristics lead to ion adsorption and accumulation on the surface during the test, and this process is reversible only under positive gate voltage. Therefore, a new effective gate voltage test method is proposed. By reasonably setting the bias gate voltage and the drain voltage range of the triangular wave scan, under the drive of the electric field force, anions are doped into the channel and coupled out holes to achieve the conduction of the device. Under this test method, the device performance only shifted by 1% under the static condition of 1 h, and a current response change of nearly 1 mA was generated for a voltage offset of 50 mV, which greatly confirmed the feasibility and superiority of this new test method.

[0054] Example 2 This embodiment relates to the application of a p-type organic electrochemical transistor biosensor; the p-type organic electrochemical transistor biosensor can detect trace pathogen markers (such as H1N1 virus RNA), and the specific method is as follows: Step 1: Prepare 10 portions of the same mixture with a concentration gradient of 10 -12 mol / L~10 -21 mol / L H1N1 RNA in PBS.

[0055] Step 2, a PDMS groove for testing is placed on the surface of the gate 5 of the p-type organic electrochemical transistor biosensor prepared in Example 1, and 250 μL of PBS buffer solution is added to the PDMS groove with a pipette, a triangular wave scanning voltage of 0.2 V to -0.4 V is applied to the first floating gate 9, and a bias voltage of -0.6 V is applied between the source 2 and the drain 4, and a conventional transfer curve test is performed; then a triangular wave scanning voltage of 0 to 0.6 V is applied between the source 2 and the drain 4, and a bias voltage of 0.3 V is applied to the gate to perform an effective gate voltage test, and 10 scanning cycles are repeated, and the maximum current average value of each cycle is taken as the baseline current.

[0056] Step 3, after the test is completed, the second substrate 11 with the gate 5 for the test is taken out, and its surface is rinsed with PBS buffer, and the surface of the gate 5 is blown dry with an ear-cleaning bulb, and then a PBS buffer solution is added to the gate modification layer 8 for incubation, and the time is consistent with the time required for specific binding in the subsequent step 4. After the incubation is completed, the surface is rinsed with PBS buffer, and the surface of the gate 5 is blown dry with an ear-cleaning bulb, and a PDMS groove for testing is placed on the surface of the gate 5, and 250 μL of PBS buffer solution is added to the PDMS groove with a pipette gun, and a triangular wave scanning voltage of 0.2 V to -0.4 V is applied to the first floating gate 9, and a bias voltage of -0.6 V is applied between the source 2 and the drain 4, and a conventional transfer curve test is performed; then a triangular wave scanning voltage of 0~0.6 V is applied between the source 2 and the drain 4, and a bias voltage of 0.3 V is applied to the gate 5, and an effective gate voltage test is performed, and 10 scanning cycles are repeated, and the maximum current average value of each cycle is taken as the noise current.

[0057] Step 4, after the test is completed, take out the second substrate 11, rinse its surface with PBS buffer, blow the surface of the gate 5 dry with an ear bulb, and then add a PBS solution of the test concentration on the gate modification layer 8 for incubation. The time can be adjusted according to the purpose of the test (for example, in this embodiment, the binding time required for the lowest detection limit is 1h and the binding time required for the fastest detection speed is 5min). After the incubation is completed, the surface is rinsed with PBS buffer, the gate surface is blown dry with an ear bulb, a PDMS groove for testing is placed on the gate surface, and 250 μL of PBS buffer solution is added to the PDMS groove with a pipette. A triangle wave scanning voltage of 0.2 V to -0.4 V is applied to the first floating gate 9, and a bias voltage of -0.6 V is applied between the source 2 and the drain 4 to perform a conventional transfer curve test; then a triangle wave scanning voltage of 0 to 0.6 V is applied between the source 2 and the drain 4, and a bias voltage of 0.3 V is applied to the gate 5 to perform an effective gate voltage test. Ten scanning cycles are repeated, and the average value of the maximum current in each cycle is taken as the response current.

[0058] The principle of the p-type organic electrochemical transistor biosensor for detecting H1N1 is as follows: Fig.12 , Fig.13 , Fig.14 and Fig.15 As shown, the specific RNA sequence of the H1N1 virus can specifically bind to the single-stranded DNA probe modified at gate 5. After binding, the ion transfer to the surface impedance of gate 5 increases, reducing the interface capacitance between the electrolyte and the gate. C G . Due to the electrolyte and channel capacitance C Chis a series relationship, so after specific binding occurs, the electrolyte and channel interface partial pressure V G,Ch As the effective gate voltage decreases, the number of cations driven by the electric field to enter the channel decreases, causing the source-drain current to change accordingly. V G,Ch The change in is proportional to the H1N1 concentration, so the change in source-drain current is also proportional to the H1N1 concentration.

[0059] To verify the ability of the p-type organic electrochemical transistor biosensor to detect low-concentration biomarkers, the p-type organic electrochemical transistor biosensor of Example 1 was used to perform ultra-low concentration (10 -20 mol / L) H1N1 test, the binding time of H1N1 marker and target probe molecule was set to 1 h. The data results are as follows Fig.16 , Fig.17 , Fig.18 , Fig.19 , Fig. 20 , Fig.21 and Fig. 22 Baseline current I d,基线 = 7.50±0.03 mA. Noise current I d,噪声 = 7.45 ± 0.02 mA. The offset of the second test data compared to the baseline is taken as the test noise, and the concentration corresponding to the three-fold response of the test noise is defined as the detection limit (LoD). In this case, the three-fold noise = 3( I d,基线 - I d,噪声 ) = 136.4 μA. Finally, the response current I d,响应 = 7.13±0.03mA. In summary, the current change Δ I d = I d,噪声 - I d,响应 = 7.45-7.13 = 329.8 μA>136.4 μA, the test is successful. At the same time, the transfer curve test is performed before the effective gate voltage test to determine whether the device performance has been degraded. Fig.19 , Fig. 20 and Fig.21 It can be seen that the performance of the p-type organic electrochemical transistor biosensor did not decay during the test, which means that 10 -20 mol / L H1N1 virus was successfully detected.

[0060] In actual applications, the lowest detection limit of the device is certainly important, but fast current detection in a short time is more suitable for real application scenarios. Therefore, the lowest detection limit that can be achieved with a binding time of 5 minutes was further explored. The H1N1 concentration used was 10 -17 mol / L, the data results are as follows Fig.23 , Fig.24 , Fig.25 , Fig.26 , Fig. 27 , Fig.28 and Fig.29 Baseline current I d,基线 = 4.44±0.01 mA. Noise current I d,噪声 = 4.42±0.01 mA, triple noise = 3( I d,基线 - I d,噪声 ) =51.9 μA, response current I d,响应 = 4.34±0.04 mA. In summary, the current change Δ I d = I d,噪声 - I d,响应 = 4.42-4.34= 80.9 μA>51.9 μA, the detection is successful. Fig.26 , Fig. 27 and Fig.28 It can be seen that the device performance did not decay during the test, which means that 10 -17 mol / L H1N1 virus was successfully detected within a binding time of 5 min.

[0061] In order to achieve quantitative analysis of H1N1 concentration in the samples to be tested, H1N1 variable concentration test was carried out in PBS buffer with a concentration range of 10 -16 mol / L changes to 10 -12 mol / L. Fig.30 It can be seen that the current change response has a good linear relationship with the H1N1 concentration, which shows that the device performance and the test method are superior and can realize the quantitative detection of H1N1.

[0062] Example 3 Same as Example 1, except that, in step 3, the length of the source electrode 2 is 300 μm, the width is 105 μm, and the thickness is 65 nm; the length of the gate electrode 5 is 10 mm, the width is 6 mm, and the thickness is 70 nm; In step 4, the length of the polymer semiconductor channel 3 is 100 μm, the width is 105 μm, and the thickness is 140 nm; In step 5, the drain electrode 4 has a length of 1400 μm, a width of 105 μm, and a thickness of 65 nm; In step 7, the first floating gate 9 and the second floating gate 12 have a length of 11 mm, a width of 6 mm, and a thickness of 10 μm; In step 9, the gate modification layer 8 has a length of 10 mm, a width of 6 mm, and a thickness of 8 nm.

[0063] Example 4 To verify the ability of the p-type organic electrochemical transistor biosensor to detect low-concentration biomarkers, the p-type organic electrochemical transistor biosensor of Example 3 was used to perform ultra-low concentration (10 -17 mol / L) H1N1 test, the binding time between H1N1 marker and targeted probe molecule was set to 5 min. The data results are as follows Fig.31 , Fig.32 , Fig.33 , Fig.34 , Fig.35 , Fig.36 and Fig.37 Baseline current I d,基线 = 4.82±0.11 mA. Noise current I d,噪声 = 4.83±0.08 mA. The offset of the second test data compared to the baseline is taken as the test noise, and the concentration corresponding to the three-fold response of the test noise is defined as the detection limit (LoD). In this case, the three-fold noise = 3( I d,基线 - I d,噪声 ) = 30 μA. Finally, the response current I d,响应 = 4.62±0.13 mA. In summary, the current change Δ I d = I d,噪声 - I d,响应 = 4.83-4.62 = 210 μA>30 μA, the test is successful. At the same time, the transfer curve test is performed before the effective gate voltage test to determine whether the device performance has been degraded. Fig.34 , Fig.35 and Fig.36 It can be seen that the performance of the p-type organic electrochemical transistor biosensor did not decay during the test, which means that 10 -20 mol / LH1N1 virus was successfully detected.

[0064] Example 5 Same as Example 1, except that, in step 3, the length of the source electrode 2 is 280 μm, the width is 100 μm, and the thickness is 70 nm; the length of the gate electrode 5 is 9 mm, the width is 5 mm, and the thickness is 65 nm; In step 4, the polymer semiconductor channel 3 has a length of 110 μm, a width of 100 μm, and a thickness of 150 nm; In step 5, the drain electrode 4 has a length of 1300 μm, a width of 100 μm, and a thickness of 70 nm; In step 7, the first floating gate 9 and the second floating gate 12 have a length of 13 mm, a width of 7 mm, and a thickness of 5 μm; In step 9, the gate modification layer 8 has a length of 9 mm, a width of 5 mm, and a thickness of 10 nm.

[0065] Example 6 To verify the ability of the p-type organic electrochemical transistor biosensor to detect low-concentration biomarkers, the p-type organic electrochemical transistor biosensor of Example 5 was used to perform ultra-low concentration (10 -17 mol / L) H1N1 test, the binding time between H1N1 marker and targeted probe molecule was set to 5 min. The data results are as follows Fig.38 , Fig.39 , Fig.40 , Fig.41 , Fig.42 , Fig.43 and Fig.44 Baseline current I d,基线 = 6.68±0.03 mA. Noise current I d,噪声 = 6.73 ± 0.01 mA. The offset of the second test data compared to the baseline is taken as the test noise, and the concentration corresponding to the three-fold response of the test noise is defined as the detection limit (LoD). In this case, the three-fold noise = 3( I d,基线 - I d,噪声 ) = 150 μA. Finally, the response current I d,响应 = 6.56±0.01 mA. In summary, the current change Δ I d = I d,噪声 - I d,响应= 6.73-6.56 = 170.4 μA>150 μA, the test is successful. At the same time, the transfer curve test is performed before the effective gate voltage test to determine whether the device performance has been degraded. Fig.41 , Fig.42 and Fig.43 It can be seen that the performance of the p-type organic electrochemical transistor biosensor did not decay during the test, which means that 10 - 17 mol / L H1N1 virus was successfully detected.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to impose any limitation on the technical solution of the present invention. Those skilled in the art should understand that, without departing from the spirit and principles of the present invention, the technical solution can also be subjected to several simple modifications and substitutions, and these modifications and substitutions are also within the scope of protection covered by the claims.

Claims

1. A p-type organic electrochemical transistor biosensor, characterized in that: It comprises a first substrate (1) and a second substrate (11); A source electrode (2) is arranged on one side of a first substrate (1), a polymer semiconductor channel (3) is arranged on the source electrode (2), a drain electrode (4) is arranged on the polymer semiconductor channel (3), a first floating gate (9) is arranged on the other side of the first substrate (1), and a first electrolyte layer (6) is arranged on the first substrate (1), the drain electrode (4) and the first floating gate (9); A gate (5) is arranged on one side of the second substrate (11), a gate modification layer (8) is arranged on the gate (5), a second floating gate (12) is arranged on the other side of the second substrate (11), and a second electrolyte layer (7) is arranged on the second substrate (11), the gate modification layer (8) and the second floating gate (12); the first floating gate (9) and the second floating gate (12) are connected via a wire (10); A drain voltage is applied to a loop consisting of a source electrode (2), a polymer semiconductor channel (3) and a drain electrode (4), and the source electrode (2) is grounded; A gate voltage is applied to a loop consisting of a source electrode (2), a first floating gate (9), a second floating gate (12), a wire (10), a first electrolyte layer (6), a second electrolyte layer (7), a gate electrode (5) and a gate modification layer (8); The gate modification layer (8) is a gate modification layer containing a target probe molecule layer of a target analyte.

2. The p-type organic electrochemical transistor biosensor according to claim 1, characterized in that: The material of the first substrate (1) and the second substrate (11) is silicon oxide; The source electrode (2), the drain electrode (4) and the gate electrode (5) are made of Au.

3. The p-type organic electrochemical transistor biosensor according to claim 1, characterized in that: The first electrolyte layer (6) is prepared from a NaCl aqueous solution, and the second electrolyte layer (7) is prepared from a PBS buffer solution.

4. The p-type organic electrochemical transistor biosensor according to claim 1, characterized in that: The material of the polymer semiconductor channel (3) is poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene]; The first floating gate (9) and the second floating gate (12) are made of Ag / AgCl slurry.

5. The p-type organic electrochemical transistor biosensor according to claim 1, characterized in that: The thickness of the source electrode (2) is 60-70 nm; the thickness of the drain electrode (4) is 60-70 nm; the thickness of the polymer semiconductor channel (3) is 130-150 nm; the thickness of the gate electrode (5) is 60-70 nm; the thickness of the gate modification layer (8) is 5-10 nm; and the thickness of the first floating gate (9) and the second floating gate (12) is 5-10 μm.

6. A method for preparing a p-type organic electrochemical transistor biosensor according to any one of claims 1 to 5, characterized in that: The following steps are involved: Cr is deposited on a first substrate (1) to form a Cr layer, and then Au is deposited on the Cr layer to form a source electrode (2); Cr is deposited on a second substrate (11) to form a Cr layer, and then Au is deposited on the Cr layer to form a gate electrode (5); Spin-coat a chloroform solution of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the surface of the source electrode (2) to obtain a polymer semiconductor channel (3); Depositing Au on the surface of the polymer semiconductor channel (3) to form a drain electrode (4); A first floating gate (9) is arranged on a first substrate (1), and a second floating gate (12) is arranged on a second substrate (11); A PBS buffer solution containing a target analyte probe is applied to the surface of the gate (5) and allowed to stand to form a target probe molecule layer of the target analyte; a 6-mercapto-1-hexanol solution is added dropwise to the surface of the gate (5) to cover the surface of the gate (5) not covered by the target probe molecule layer of the target analyte, and allowed to stand to form a 6-mercapto-1-hexanol blocking layer; the target probe molecule layer of the target analyte and the 6-mercapto-1-hexanol blocking layer form a gate modification layer (8); A first electrolyte layer (6) is arranged on a first substrate (1), a drain electrode (4) and a first floating gate (9); a second electrolyte layer (7) is arranged on a second substrate (11), a gate modification layer (8) and a second floating gate (12); the first floating gate (9) and the second floating gate (12) are connected via a wire (10) to obtain a p-type organic electrochemical transistor biosensor.

7. The method for preparing a p-type organic electrochemical transistor biosensor according to claim 6, characterized in that: A first floating gate (9) is arranged on a first substrate (1), and a second floating gate (12) is arranged on a second substrate (11), comprising the following steps: Ag / AgCl slurry is coated on a first substrate (1), and Ag / AgCl slurry is coated on a second substrate (11), and then annealing is performed to form a first floating gate (9) on the first substrate (1), and a second floating gate (12) on the second substrate (11).

8. The method for preparing a p-type organic electrochemical transistor biosensor according to claim 7, characterized in that: The annealing was carried out under inert gas at a temperature of 100 °C for 30 min.

9. Use of the p-type organic electrochemical transistor biosensor according to any one of claims 1 to 5 in biomarker detection.

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