Potentiometric OECT biosensor and preparation method and application thereof

Abstract

The invention discloses a potentiometric OECT biosensor as well as a preparation method and application thereof, and belongs to the technical field of organic electrochemical sensing. The potentiometric OECT biosensor comprises a device module and a sensing module, the device module comprises a substrate, and a source electrode, a grid electrode and a first floating gate are arranged on the substrate; a semiconductor channel is arranged on the source electrode, and a drain electrode is arranged on the semiconductor channel; a grid control grid is arranged on the grid; a hydrogel electrolyte layer is arranged at the tops of the substrate, the drain electrode, the grid-control grid electrode and the first floating gate; the sensing module adopts an electrode plate; the electrode plate comprises a gold electrode, an Ag / AgCl electrode and a carbon electrode; the gold electrode serves as a sensing grid electrode, an SAM connecting layer, targeted protein molecules and a shielding layer are arranged on the gold electrode, and when the biosensor works, an electrolyte layer is arranged at the top of the sensing module, and the Ag / AgCl electrode serves as a second floating grid; the first floating gate and the second floating gate are connected through a wire. The potentiometric OECT biosensor is high in detection sensitivity and high in response speed, and can meet clinical requirements on rapid and accurate detection.

Description

Technical Field

[0001] The present invention relates to the field of organic electrochemical sensing technology, and in particular to a potential-type OECT biosensor and a preparation method and application thereof. Background Art

[0002] Organic electrochemical transistor (OECT) technology has received widespread attention and rapid development in the field of biosensors in recent years. The OECT channel is composed of organic semiconductor materials and has excellent stability in liquid environments, good signal conversion and amplification capabilities, and outstanding biocompatibility, making it show great potential in biomarker detection. Compared with traditional sensors, OECTs can monitor pathogens and biomarkers in body fluids in real time under conditions of low power consumption and high sensitivity, providing accurate physiological and pathological status information. Its efficient electrochemical properties enable the sensor to amplify tiny signal changes, enhance detection sensitivity, and operate stably for a long time in a biological environment. In addition, the low cost and ease of use of OECT technology give it broad application prospects in personalized health monitoring, early disease diagnosis and precision treatment. As the technology continues to improve, OECT-based biosensors are expected to become an important tool in the future medical testing field, promoting the development of health management and disease diagnosis and treatment.

[0003] However, when traditional OECTs are used to detect biomarkers in complex biological samples (such as serum and whole blood), they often face problems such as high device detection limits, unstable operation caused by gate electrode polarization interference, interface contamination, and poor portability of the test device, which limits their promotion and application in real-world scenarios. Therefore, how to ensure that OECT devices still have high amplification capabilities and operational stability under the condition of using polarizable functional gate electrodes, and have good anti-fouling ability in complex biological samples, and eliminate background noise interference caused by nonspecific adsorption, is the core difficulty of current research in this direction. Summary of the Invention

[0004] In order to solve the problems of conventional OECT biosensors such as high detection limit, unstable operation caused by gate electrode polarization interference, interface contamination, and poor portability of testing devices, the purpose of the present invention is to provide a potentiometric OECT biosensor and its preparation method and application.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a potential-type OECT biosensor, comprising a device module and a sensor module;

[0007] The device module includes a substrate, a source electrode, a gate electrode and a first floating gate are arranged on the substrate; a semiconductor channel is arranged on the source electrode, a drain electrode is arranged on the semiconductor channel, and a gate-controlled gate electrode is arranged on the gate electrode; a hydrogel electrolyte layer is arranged on top of the substrate, the drain electrode, the gate-controlled gate electrode and the first floating gate;

[0008] The sensing module uses an electrode sheet; the electrode sheet includes a gold electrode, an Ag / AgCl electrode, and a carbon electrode; the Ag / AgCl electrode serves as a second floating gate; the gold electrode serves as a sensing gate, on which a SAM connecting layer, a targeting protein molecule, and a shielding layer are provided; when the potentiometric OECT biosensor is operating, an electrolyte layer is provided on top of the electrode sheet, the second floating gate, the targeting protein molecule, and the shielding layer;

[0009] The first floating gate and the second floating gate are connected via a wire;

[0010] A drain voltage is applied to the loop consisting of the source, semiconductor channel and drain;

[0011] A gate voltage is applied to a loop consisting of a source electrode, a gate-controlled gate, a first floating gate, a second floating gate, a hydrogel electrolyte layer and a sensing gate.

[0012] The source electrode, gate electrode, drain electrode and first floating gate are all made of gold;

[0013] The gate-controlled grid is made of Ag / AgCl paste.

[0014] The semiconductor channel is a p-type semiconductor channel, and the semiconductor channel is made of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene].

[0015] The hydrogel electrolyte layer is made of a hydrogel electrolyte, which includes gelatin, poly(sodium p-styrene sulfonate), D-sorbitol, glycerol, sodium chloride and water; the mass ratio of the gelatin, poly(sodium p-styrene sulfonate), sorbitol, glycerol, sodium chloride and water is 5:5:0.25:0.25:0.1:15.

[0016] The SAM connecting layer is prepared by using a SAM modification solution, which is an ethanol solution of mercapto-tetraethylene glycol-propionic acid.

[0017] In the targeting protein molecule and the shielding layer, the targeting protein molecule is prepared by using a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide and an antibody solution; the shielding layer is prepared by using an ethanolamine solution and a bovine serum albumin solution;

[0018] The electrolyte layer is prepared by using PBS buffer solution.

[0019] The present invention also provides a method for preparing the above-mentioned potentiometric OECT biosensor, comprising:

[0020] Cr is deposited on the substrate by vacuum evaporation to form a Cr layer, and Au is deposited on the Cr layer to form a source electrode, a gate electrode and a first floating gate;

[0021] A semiconductor channel was obtained by spin coating a chloroform solution of [thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the source electrode;

[0022] Depositing gold on the surface of the semiconductor channel to form a drain;

[0023] Coating Ag / AgCl slurry on the gate and annealing in an inert atmosphere to form a gate-controlled gate;

[0024] Covering the substrate, drain electrode, gate control gate and first floating gate with a hydrogel electrolyte to form a hydrogel electrolyte layer, and then covering the surface of the hydrogel electrolyte layer with ultraviolet curing glue and curing with ultraviolet light to produce a device module;

[0025] The sensing module uses an electrode sheet; the electrode sheet includes a gold electrode, an Ag / AgCl electrode, and a carbon electrode; the Ag / AgCl electrode serves as the second floating gate, and the gold electrode serves as the sensing gate. The sensing gate is subjected to cyclic voltammetry scanning, and then the pre-treated sensing gate is immersed in a SAM modification solution to obtain a SAM connection layer;

[0026] The sensing gate with the SAM connecting layer is placed in a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide for reaction activation, an antibody solution is added dropwise to the surface of the activated sensing gate to react and obtain a targeting protein molecule, the sensing gate is then treated with an ethanolamine solution, and the surface of the sensing gate is subsequently sealed with a bovine serum albumin solution to obtain the targeting protein molecule and a shielding layer, thereby preparing a sensing module;

[0027] The device module is connected to the sensor module to obtain a potential-type OECT biosensor.

[0028] The semiconductor channel is obtained by spin coating a chloroform solution of [thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the source electrode, specifically:

[0029] Spin coating a chloroform solution of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on a substrate containing a source electrode, a gate electrode and a first floating gate to obtain a semiconductor channel film;

[0030] Masking the area other than the top of the source electrode on the substrate containing the semiconductor channel film, and depositing Au on the surface of the semiconductor channel film on the source electrode to form a drain electrode;

[0031] The drain electrode is used as a mask, and the substrate containing the drain electrode is etched by reactive ion etching to form a semiconductor channel.

[0032] The molar concentration of the SAM modification solution is 1 mM ethanol solution of mercapto-tetraethylene glycol-propionic acid; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in the mixed solution is 20 mg / mL, and the concentration of N-hydroxysuccinimide is 10 mg / mL; the concentration of the antibody solution is 20 μg / mL; the concentration of the ethanolamine solution is 1 M, and the pH is adjusted to 8 with concentrated hydrochloric acid during the blocking process; the concentration of the bovine serum albumin solution is 10 mg / mL.

[0033] The present invention also provides the use of the above-mentioned potential-type OECT biosensor in biomarker detection.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] The potential-type OECT biosensor provided by the present invention separates the sensing module from the device module by introducing a floating gate structure; at the same time, a new gate degree of freedom is additionally introduced, so that the gate has both the functions of a reference electrode and a counter electrode; the functions of the reference electrode and the counter electrode based on a single gate are separated into two independent electrodes, namely a sensing gate for realizing a bio-recognition function and a gate-controlled gate for realizing a channel control function. The present invention combines the open-circuit potential test method of a two-electrode system in an electrochemical workstation to achieve a state close to an open circuit between the sensing gate and the source (extremely high impedance), and almost no current flows between the sensing gate and the source, thereby avoiding polarization of the sensing gate surface, thereby protecting the integrity and stability of the functional probe. The potential-type OECT biosensor and the circuit module are combined to form a portable monitoring system. Compared with the complex wiring method and large size when the source meter is used in conjunction with the electrochemical workstation, the plug-and-play working mode and compact size of the portable detection system reduce the operating threshold and improve the detection efficiency. The portable monitoring system with the potential-type OECT biosensor has a large linear response range and high sensitivity during detection.

[0036] Furthermore, the gate-controlled gate serves as the driving end that actually applies the gate voltage and regulates the channel state. It can use a non-polarizable Ag / AgCl electrode with excellent stability to maintain a stable electrochemical environment and achieve efficient amplification of the source-drain current.

[0037] Furthermore, the SAM connecting layer uses SH-(PEG)4-COOH molecules with more anti-fouling properties, which significantly inhibits nonspecific adsorption and improves the interface-specific recognition efficiency.

[0038] Furthermore, the electrolyte layer uses a hydrogel electrolyte. Hydrogel has good water retention and can maintain a uniform electrolyte concentration during long-term testing, effectively avoiding concentration fluctuations caused by liquid volatilization, and can significantly improve the stability and reproducibility of the device response. At the same time, the hydrogel electrolyte facilitates the overall packaging and integration of the OECT device, making it more suitable for subsequent portable detection systems, and providing a structural foundation for the engineering implementation of OECT sensors in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the structure of the potentiometric OECT biosensor of the present invention;

[0040] Figure 2 Schematic diagram of the structure of the sensor module of the present invention;

[0041] Figure 3 Schematic diagram of the circuit of an existing OECT biosensor;

[0042] Figure 4 A schematic circuit diagram of the potentiometric OECT biosensor provided by the present invention;

[0043] Figure 5 This is a diagram showing the results of CV cleaning of electrodes during the preparation of the sensor module in Example 1;

[0044] Figure 6 This is the result diagram of electrode potential change during the operation of traditional OECT;

[0045] Figure 7 The electrode potential changes during the operation of the potentiometric OECT biosensor prepared in Example 1;

[0046] Figure 8 A comparison chart of transfer curve test results of a conventional OECT biosensor and the potentiometric OECT biosensor prepared in Example 1;

[0047] Figure 9 This is a current fluctuation diagram generated when the electrolyte of Comparative Example 1 is an aqueous electrolyte test;

[0048] Figure 10 The current fluctuation diagram generated when the electrolyte of Example 1 is a hydrogel electrolyte test; wherein (a) is the current fluctuation diagram at a scan rate of 0.04 V / s, and (b) is the 1-h stability diagram at a scan rate of 0.04 V / s;

[0049] Figure 11 This is a photo of a portable detection platform composed of a potentiometric OECT biosensor prepared according to Example 1;

[0050] Figure 12 Performance comparison of a portable detection system composed of the potentiometric OECT biosensor prepared in Example 1 and used in conjunction with an electrochemical workstation / source meter; (a) is a transfer curve comparison, and (b) is a current-time curve comparison.

[0051] Figure 13 This is a graph showing clinical sample test results of a portable detection system composed of the potentiometric OECT biosensor prepared in Example 1;

[0052] Among them, 1. substrate; 2. source; 3. semiconductor channel; 4. drain; 5. gate; 6. gate-controlled gate; 7. first floating gate; 8. hydrogel electrolyte layer; 9. screen-printed electrode sheet; 10. sensing gate; 11. second floating gate; 12. electrolyte layer; 13. SAM connecting layer; 14. targeting protein molecules and shielding layer. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0054] like Figure 1 As shown, the present invention provides a potential-type OECT biosensor, including a device module and a sensing module;

[0055] The device module includes a substrate 1, on which a source electrode 2, a gate electrode 5 and a first floating gate 7 are arranged; a semiconductor channel 3 is arranged on the source electrode 2, a drain electrode 4 is arranged on the semiconductor channel 3, and a gate-controlled gate 6 is arranged on the gate 5; a hydrogel electrolyte layer 8 is arranged on top of the substrate 1, the drain electrode 4, the gate-controlled gate 6 and the first floating gate 7;

[0056] like Figure 2 As shown, the sensing module uses an electrode sheet 9; the electrode sheet 9 includes a gold electrode, an Ag / AgCl electrode, and a carbon electrode; the Ag / AgCl electrode serves as a second floating gate 11; the gold electrode serves as a sensing gate 10, and a self-assembled monomolecule (SAM) connecting layer 13, a targeting protein molecule, and a shielding layer 14 are provided on the sensing gate 10; when the potential-type OECT biosensor is working, an electrolyte layer 12 is provided on top of the electrode sheet 9, the second floating gate 11, the targeting protein molecule, and the shielding layer 14;

[0057] The first floating gate 7 and the second floating gate 11 are connected via a wire;

[0058] A drain voltage is applied to the loop consisting of the source 2, the semiconductor channel 3 and the drain 4;

[0059] A gate voltage is applied to a loop consisting of the source electrode 2 , the gate control gate 6 , the first floating gate 7 , the second floating gate 11 , the hydrogel electrolyte layer 8 and the sensing gate 10 .

[0060] The source electrode 2, gate electrode 5, drain electrode 4 and first floating gate 7 are all made of gold (Au);

[0061] The gate control grid 6 is made of Ag / AgCl paste.

[0062] The semiconductor channel 3 is a p-type semiconductor channel, and is made of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] (P(g2T-T)).

[0063] The hydrogel electrolyte layer 8 is made of a hydrogel electrolyte, which includes gelatin, poly (sodium p-styrene sulfonate), D-sorbitol, glycerol, sodium chloride and water; the mass ratio of gelatin, poly (sodium p-styrene sulfonate), sorbitol, glycerol, sodium chloride and water is 5:5:0.25:0.25:0.1:15.

[0064] The SAM connecting layer 13 is made of a SAM modification solution, which is an ethanol solution of mercapto-tetraethylene glycol-propionic acid.

[0065] In the targeting protein molecule and the shielding layer 14, the targeting protein molecule is prepared using a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide and an antibody solution; the shielding layer is prepared using an ethanolamine solution and a bovine serum albumin solution; and the electrolyte layer 12 is prepared using a PBS buffer solution.

[0066] The preparation method of the above-mentioned potentiometric OECT biosensor comprises the following steps:

[0067] Device module preparation:

[0068] Step 1: Using a vacuum evaporation method to perform mask deposition, a Cr layer is deposited on a clean substrate 1 as an adhesion layer to enhance the bonding strength between gold and the substrate 1. An Au layer is deposited on the Cr layer to form a source electrode 2, a gate electrode 5, and a first floating gate 7.

[0069] Step 2: Spin-coating a P(g2T-T) chloroform solution on the surface of the substrate with the source electrode 2, the gate electrode 5, and the first floating gate 7 obtained in step 1 to form a semiconductor channel film, wherein the concentration of the P(g2T-T) chloroform solution is 20 mg / mL;

[0070] Step 3: The surface of the substrate 1 containing the semiconductor channel film obtained in step 2 is masked, and the area other than the top of the source electrode 2 on the substrate 1 containing the semiconductor channel film is masked, and an Au layer is vacuum evaporated on the surface of the semiconductor channel film on the source electrode 2 as the drain electrode 4;

[0071] Step 4: Using the drain electrode 4 as a mask, the semiconductor channel film is etched by reactive ion etching to obtain a semiconductor channel 3, which is a p-type semiconductor channel; removing excess P(g2T-T) can avoid ineffective doping; the etching gas in the reactive ion etching method is an inert gas;

[0072] Step 5: Ag / AgCl slurry is coated on the surface of the gate 5 obtained in step 1, and then thermally annealed in an inert gas atmosphere to obtain a gate-controlled gate 6;

[0073] Step 6: Cover the substrate 1, the drain electrode 4, the gate-controlled gate 6 and the top of the first floating gate 7 with a hydrogel electrolyte to form a hydrogel electrolyte layer 8, and then evenly cover the surface of the hydrogel electrolyte layer with ultraviolet curing glue and use ultraviolet light curing to obtain a device module; the hydrogel electrolyte includes gelatin, poly (sodium p-styrene sulfonate), sorbitol, glycerol, sodium chloride and deionized water; gelatin, poly (sodium p-styrene sulfonate), sorbitol, glycerol, sodium chloride and deionized water are in a mass ratio of 5:5:0.25:0.25:0.1:15.

[0074] Preparation of sensor module:

[0075] The sensing module uses an electrode sheet 9, which includes a gold electrode, an Ag / AgCl electrode, and a carbon electrode. The Ag / AgCl electrode serves as the second floating gate 11; the gold electrode serves as the sensing gate 10. To construct an Au electrode biointerface with specific recognition capabilities, the gold electrode is modified as follows:

[0076] Step 1: Perform a cyclic voltammetry (CV) scan using the Ag / AgCl electrode on the electrode sheet as the reference electrode, the gold electrode as the working electrode, and the carbon electrode as the counter electrode. Place the gold electrode in an H2SO4 solution and perform a cyclic voltammetry (CV) scan. The CV scan voltage range is set to -0.2 to 1.2 V. Immediately rinse the gold electrode with anhydrous ethanol for later use. CV scanning of the Au electrode can etch the surface, effectively stripping away surface impurities, restoring the electrode's clean active surface and stabilizing its electrochemical properties to improve subsequent functional group binding efficiency.

[0077] Step 2: Immerse the pretreated Au electrode in a SAM modification solution and allow to stand overnight in the dark at room temperature under nitrogen protection (to avoid oxidation) to obtain a SAM connecting layer 13; the SAM modification solution is an ethanol solution of thiol-tetraethylene glycol-propionic acid, and the molar concentration of the SAM modification solution is 1 mM; in this step, the Au electrode is immersed in the SAM modification solution to react to form an ordered monolayer with carboxyl functional groups, which provides active sites for subsequent antibody coupling;

[0078] Step 3: The Au electrode with the SAM modified layer 13 obtained in step 2 is taken out and the surface is repeatedly washed with deionized water; then, a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is used to activate the carboxyl groups on the surface of the SAM connecting layer 13, and the reaction is carried out at room temperature. After activation, the surface of the Au electrode is repeatedly washed with PBS buffer, and an antibody solution is added dropwise to the activated Au electrode surface for incubation to obtain a targeting protein molecule; wherein the antibody solution is a PBS solution containing an antibody, and the concentration of EDC in the mixed solution of EDC and NHS is 20 mg / mL, and the concentration of NHS is 10 mg / mL; the concentration of the PBS solution containing the antibody is 20 μg / mL; the mixed solution of EDC and NHS is used to convert the carboxyl groups on the surface of the SAM modified layer into active ester intermediates to promote the subsequent coupling reaction of amine molecules; the PBS solution of the antibody is added dropwise to the surface of the activated Au electrode to form a stable covalent bond between the amino group in the antibody and the active ester on the Au surface;

[0079] Step 4: Rinse the gold electrode surface obtained in step 3 with PBS buffer, and then treat the gold electrode with ethanolamine solution. The concentration of the ethanolamine solution is 1M. During the blocking process, concentrated hydrochloric acid is added dropwise to gradually adjust the pH to 8; the gold electrode is treated with ethanolamine solution to block the residual unreacted active ester to prevent the occurrence of nonspecific reactions during subsequent detection; further, rinse the gold electrode surface with PBS buffer again, and use 10 mg / mL bovine serum albumin solution (BSA) solution to block the gold electrode surface, and let it stand at room temperature to fill nonspecific adsorption sites and enhance interface stability; finally, the targeting protein molecule and the shielding layer 14 are formed, and the electrode after treatment is the sensing module.

[0080] The principle of the potentiometric OECT biosensor provided in this scheme is as follows: In the conventional OECT biosensor structure, the gate electrode not only performs the function of signal acquisition, but also serves as the control terminal for applying gate voltage and injecting ions into the channel. Due to the existence of ion conduction paths in the electrolyte, the gate inevitably undergoes redox reactions or changes in double-layer capacitance under working conditions, resulting in irreversible shifts in its potential (polarization). This polarization will directly affect the voltage distribution and device stability, causing drift and distortion of the output signal. At the same time, in order to achieve specific recognition, the surface of the gate electrode is often modified with biological molecules such as nucleic acid probes, antibodies, and aptamers. These molecules usually have complex chemical structures and are environmentally sensitive. Under the action of the operating voltage, the redox reactions or changes in double-layer capacitance occurring on the gate electrode will destroy the probe structure, reduce binding efficiency, shorten the device life, and may cause problems such as nonspecific adsorption and uneven response. Therefore, how to construct an OECT structure that can achieve highly sensitive signal transduction while avoiding functional electrode polarization and molecular probe damage is the key to achieving high-performance potentiometric biosensing.

[0081] like Figure 3 The functions of different electrodes in the ion and electron circuits are given in the following. GS ), the source 2 acts as a counter electrode and reference electrode, the gate 5 applies a voltage to the doped channel compared to the source 2 (reference function), and the source collects the gate current (counter electrode function). In the electronic circuit (V DS ), while source 2 functions similarly, serving as a reference for the voltage applied to drain 4 and recording the channel current. The gate electrode, crucial for sensing, functions solely as a working electrode in the circuit, while source 2 possesses two degrees of freedom (acting as both a reference electrode and a counter electrode). To achieve conduction in the OECT device, the gate and source 2 functions can be interchanged simply by changing the polarity of the applied voltage.

[0082] The present invention introduces a new gate degree of freedom, which enables the gate to have both the functions of reference electrode and counter electrode. Furthermore, the functions of reference electrode and counter electrode based on a single gate are separated into two independent electrodes, namely sensing gate 10 (SensingGate, G S ) and the gate-controlled gate 6 (GatingGate, G G). In addition, the present invention introduces a test method for the open-circuit potential of a two-electrode system in an electrochemical workstation to achieve a state close to a disconnection between the sensing gate 10 and the source 2 (extremely high impedance). The advantage of this arrangement is that there is almost no current flowing between the sensing gate 10 and the source 2, avoiding polarization of the surface of the sensing gate 10, thereby protecting the integrity and stability of the functional probe. The gate-controlled gate 6 serves as the driving end that actually applies the gate voltage and regulates the channel state. It can use a non-polarizable Ag / AgCl electrode with excellent stability to maintain a stable electrochemical environment and achieve efficient amplification of the source-leakage current. Finally, a floating gate structure is introduced to separate the sensing module from the device module. The circuit structure and function are shown in the figure below. Figure 4 shown.

[0083] Example 1

[0084] This example describes a method for preparing a potentiometric OECT biosensor. The specific preparation steps include:

[0085] Device module preparation:

[0086] Step 1: The substrate 1 is a rigid glass substrate, which is ultrasonically cleaned in detergent water, ultrapure water, acetone, and isopropyl alcohol for 30 minutes.

[0087] Step 2: Use an air gun to blow clean the surface of the substrate 1, and then place the substrate 1 in a UV ozone cleaning machine for 15 minutes to improve the wettability and cleanliness of the substrate surface;

[0088] Step 3: Use vacuum evaporation method to perform mask deposition on the cleaned substrate 1. A 2 nm thick Cr layer was deposited as an adhesion layer, followed by A 60nm thick Au layer is deposited on the Cr layer at a speed of 100 nm to form a source electrode 2, a gate electrode 5 and a first floating gate 7. The source electrode 2 has a length of 250 μm and a width of 95 μm, the gate electrode 5 has a length of 9 mm and a width of 5 mm; the first floating gate 7 has a total length of 7.65 mm, a head length of 2 mm and a width of 2.5 mm; and a tail width of 0.5 mm.

[0089] Step 4: P(g2T-T) was dissolved in chloroform and stirred at 50°C and 600 rpm for 15 h to obtain a 20 mg / mL P(g2T-T) chloroform solution; the substrate 1 with the source 2, gate 5 and first floating gate 7 obtained in step 3 was treated with UVO for 15 min to optimize the work function of the electrode and improve the wettability of the substrate as a whole. The substrate 1 was then accelerated to 5000 rpm using a spin coater, and 25 μL of the 20 mg / mL P(g2T-T) chloroform solution was then pipetted using a pipette and dropped onto the surface of the high-speed rotating substrate to form a semiconductor channel film with a channel length of 100 μm, a width of 110 μm and a thickness of 140 nm.

[0090] Step 5: Place the sample obtained in step 4 into a mask, mask the area on the substrate 1 containing the semiconductor channel film except the top of the source electrode 2, and use vacuum evaporation to deposit a layer of semiconductor channel film on the surface of the source electrode 2. A 60 nm thick Au layer is deposited at a speed of , as the drain electrode 4, wherein the drain electrode 4 has a length of 1200 μm and a width of 95 μm;

[0091] Step 6: Using the drain electrode 4 as a mask, the semiconductor channel film is etched by reactive ion etching to obtain a semiconductor channel 3; the semiconductor channel 3 is a p-type semiconductor channel; the etching gas in the reactive ion etching method is argon, the flow rate is 15 sccm, the etching power is 25 W, and the etching time is 250 s;

[0092] Step 7: Ag / AgCl slurry is coated on the surface of the gate 5 obtained in step 3, and then the substrate 1 is placed in an inert gas atmosphere and thermally annealed at 100° C. for 30 minutes to obtain a gate-controlled gate 6;

[0093] Step 8: Gelatin, poly (sodium styrene sulfonate), sorbitol, glycerol, sodium chloride, and deionized water were mixed in a mass ratio of 5:5:0.25:0.25:0.1:15, stirred at 85°C until clear, and then poured into a mold while hot. After cooling, a hydrogel electrolyte was obtained; the hydrogel electrolyte was cut into 5×10mm 2 , ensuring that the hydrogel electrolyte layer 8 can be formed by simultaneously covering the substrate 1, the drain electrode 4, the gate-controlled gate 6 and the top of the first floating gate 7, and then evenly covering the surface of the hydrogel electrolyte layer 8 with ultraviolet curing glue, and then using ultraviolet lamp irradiation for 10 seconds to cure, completing the packaging of the device module to obtain the device module;

[0094] Preparation of sensor module:

[0095] The sensing module uses a screen-printed electrode sheet (manufacturer: Shenzhen Haoyang Technology Co., Ltd.). The screen-printed electrode sheet includes a gold electrode, an Ag / AgCl electrode, and a carbon electrode. The Ag / AgCl electrode serves as the second floating gate 11; the gold electrode serves as the sensing gate 10. To construct an Au electrode biointerface with specific recognition capabilities, the gold electrode is modified as follows:

[0096] Step 1: Place the Au electrode in 0.5M H2SO4 solution and perform cyclic voltammetry (CV) scanning. The CV scanning setting voltage range is 1V, the scanning rate is 0.1V / s, and the number of cycles is 5. The scanning results are as follows: Figure 5 As shown in Figure 2, after five cyclic CV scans, the electrode redox characteristics tend to be stable and are suitable for subsequent functionalization steps; the Au electrode is then immediately rinsed with anhydrous ethanol for later use;

[0097] Step 2: Add mercapto-tetraethylene glycol-propionic acid (SH-(PEG)4-COOH) to anhydrous ethanol to obtain a SAM modification solution, wherein the molar concentration of the SAM modification solution is 1 mM; immerse the pretreated Au electrode in the SAM modification solution and stand in the dark at 25° C. under nitrogen protection for 15 hours (to prevent oxidation) to obtain a SAM connecting layer 13;

[0098] Step 3: Dissolve 10 mg of NHS powder in 1 mL of deionized water. Use a pipette to draw 20 mg of EDC solution and add it to the NHS aqueous solution. Mix them evenly to obtain a mixed solution of 20 mg / mL EDC and 10 mg / mL NHS. Remove the Au electrode with the SAM connecting layer 13 obtained in step 2 and repeatedly rinse the surface with deionized water. Then, activate the carboxyl groups on the SAM-modified surface with a mixed solution of 20 mg / mL EDC and 10 mg / mL NHS and react at room temperature for 30 minutes.

[0099] Step 4: Dilute the purchased CA19-9 antibody solution to 20 μg / mL with PBS solution; repeatedly wash the Au electrode surface with PBS buffer, and add 20 μg / mL CA19-9 monoclonal antibody PBS solution to the activated Au electrode surface, incubate at 4°C for 15 hours, and obtain the targeted protein molecule after incubation.

[0100] Step 5: Rinse the gold electrode surface obtained in step 4 with PBS buffer, and then treat the gold electrode with a concentration of 1M for 10 minutes. During the sealing process, adjust the pH of the ethanolamine solution to 8 with concentrated hydrochloric acid; rinse the gold electrode surface with PBS buffer, and further, use 10 mg / mL BSA solution to seal the gold electrode surface. Let it stand at room temperature for 1 hour to obtain the targeted protein molecule and the shielding layer 14. The electrode after treatment is the sensing module; during the working process, the device module and the sensing model are connected to form a potential-type OECT biosensor for detection.

[0101] The circuit function of the potentiometric OECT biosensor prepared in Example 1 was tested:

[0102] The present invention has carried out the performance test of OECT device, such as Figure 6 and Figure 7 The actual operating potential change diagrams of the traditional OECT biosensor and the potentiometric OECT biosensor are shown respectively. The applied voltages of the two are equivalent to the OECT operating effect, both with a leakage bias of -0.4V and a triangle wave voltage scan of 0.4 to -0.2V. For the potentiometric OECT structure, G G The potential test is measured on the gate-controlled gate when the sensing gate is at a low potential and the bias current is 0A during device operation.

[0103] Under this operating condition, the present invention carried out transfer curve test of 5 cycle scans, and the results are as follows Figure 8 The results show that although the gate function is split into two independent electrodes, it does not affect the electrical performance of the device. The transfer curves of the two cycles remain highly consistent, thus successfully completing the principle verification of the potential-type OECT device.

[0104] In summary, the potential-type OECT biosensor effectively overcomes the problems of gate polarization, potential drift, and probe molecule damage that exist in traditional OECT biosensing by introducing a design that decouples the sensing gate from the gate-controlled gate. During the detection process, the sensing gate operates at an open-circuit potential, avoiding electrochemical perturbations and significantly improving the device's cyclic and repeated operation stability. At the same time, while maintaining the high gain and low power consumption advantages of OECT, this structure achieves non-perturbation recognition of the target signal, providing a better solution for building a highly sensitive, long-lasting and stable biosensing platform.

[0105] Comparative Example 1

[0106] The same as Example 1, except that the electrolyte layer uses NaCl aqueous solution as the channel electrolyte.

[0107] The potential-type OECT biosensors of Example 1 and Comparative Example 1 were tested; Figure 9 As shown in FIG, when NaCl aqueous solution is used as the channel electrolyte, the device output current is easily disturbed by the fluctuation of electrolyte concentration, resulting in unstable signal response and affecting the repeatability and reliability of the detection results. In Example 1, the electrolyte of the electrolyte layer is replaced by NaCl hydrogel instead of NaCl aqueous solution. Figure 10 As shown in (a), when the voltage scan rate is increased to 0.04 V / s, the potentiometric OECT biosensor can achieve stable output and reversible doping behavior; under this condition, a long-term stability test of up to 1 hour was carried out, and the results are shown in Figure 10 As shown in (b), I-I0 = 23.8 ± 19.8 μA, similar to the results obtained in NaCl aqueous solution. Therefore, replacing the electrolyte with NaCl hydrogel effectively avoids concentration fluctuations caused by liquid volatilization, improves the stability and repeatability of the device during testing, and facilitates device packaging and integration into a portable platform.

[0108] Example 2

[0109] like Figure 11 As shown, the device module, sensor module, and circuit module prepared in Example 1 are combined to form a portable detection system. Specifically, the circuit module uses an STM32F as the main control chip. The adapter board above the main PCB board is the device module prepared in Example 1. The OECT device module is connected to the adapter PCB via wire bonding, and the main PCB is then connected to the adapter board using reserved pin headers. The sensor module is located on the side of the main PCB and is designed with a pluggable interface for easy replacement. The entire circuit board measures 8cm × 5cm, making it compact and portable.

[0110] First, to verify the functional consistency of the portable detection system when used in conjunction with the source meter / electrochemical workstation, a -0.4V voltage bias and a -0.4 to 0.2V triangle wave voltage sweep were set for both test modes, and the same device module was tested. Figure 12 (a) and (b) show the transfer curves and their corresponding current-time curves obtained from five cyclic scans using different devices, respectively. The test results show that the transfer curves obtained using the designed PCB closely match those obtained using a source-meter instrument (SMU) / electrochemical workstation (WS) and exhibit high stability during operation. The current-time curves also demonstrate a high degree of similarity between the PCB and SMU / electrochemical workstation tests across the entire range.

[0111] After verifying the functionality of the portable detection system, it was used to test clinical samples from four patients. The test process was consistent with the combined use of a source meter / electrochemical workstation. The test results are as follows: Figure 13 As shown, the goodness of fit of the linear detection reached 0.99, and the linear response range covered three orders of magnitude, including the diagnostic critical concentration value of 37U / mL indicated by the National Health Commission and 1000U / mL indicating critically ill patients, indicating that the portable detection system has high sensitivity and is suitable for quantitative detection of CA19-9 in multiple scenarios. In addition, the rapid incubation time of 10 minutes allows the entire test process to be completed within half an hour. Therefore, a portable biological detection system with rapid and highly sensitive detection capabilities was finally realized, laying a solid foundation for its practical application in scenarios such as early screening and on-site diagnosis of diseases.

[0112] The above description is merely 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 replacements, and these modifications and replacements are also within the scope of protection covered by the claims.

Claims

1. A potentiometric OECT biosensor, characterized in that: Including device module and sensor module; The device module comprises a substrate (1), a source electrode (2), a gate electrode (5) and a first floating gate (7) are arranged on the substrate (1); a semiconductor channel (3) is arranged on the source electrode (2), a drain electrode (4) is arranged on the semiconductor channel (3), and a gate-controlled gate electrode (6) is arranged on the gate electrode (5); and a hydrogel electrolyte layer (8) is arranged on top of the substrate (1), the drain electrode (4), the gate-controlled gate electrode (6) and the first floating gate (7); The sensing module adopts an electrode sheet (9); the electrode sheet (9) includes a gold electrode, an Ag / AgCl electrode and a carbon electrode; the Ag / AgCl electrode serves as a second floating gate (11); the gold electrode serves as a sensing gate (10), and a SAM connecting layer (13), a targeting protein molecule and a shielding layer (14) are arranged on the sensing gate (10); when the potential-type OECT biosensor is working, an electrolyte layer (12) is arranged on top of the electrode sheet (9), the second floating gate (11), the targeting protein molecule and the shielding layer (14); The first floating gate (7) and the second floating gate (11) are connected via a wire; A drain voltage is applied to a loop consisting of a source electrode (2), a semiconductor channel (3) and a drain electrode (4); A gate voltage is applied to a loop consisting of a source electrode (2), a gate-controlled gate (6), a first floating gate (7), a second floating gate (11), a hydrogel electrolyte layer (8) and a sensing gate (10).

2. A potentiometric OECT biosensor according to claim 1, characterized in that: The source electrode (2), gate electrode (5), drain electrode (4) and first floating gate (7) are all made of gold; The gate-controlled grid (6) is made of Ag / AgCl slurry.

3. The potentiometric OECT biosensor according to claim 1, wherein: The semiconductor channel (3) is a p-type semiconductor channel, and the semiconductor channel (3) is made of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene].

4. The potentiometric OECT biosensor according to claim 1, wherein: The hydrogel electrolyte layer (8) is made of a hydrogel electrolyte, which comprises gelatin, poly(sodium p-styrene sulfonate), D-sorbitol, glycerol, sodium chloride and water; the mass ratio of the gelatin, poly(sodium p-styrene sulfonate), sorbitol, glycerol, sodium chloride and water is 5:5:0.25:0.25:0.1:

15.

5. The potentiometric OECT biosensor according to claim 1, characterized in that: The SAM connecting layer (13) is made of a SAM modification solution, which is an ethanol solution of mercapto-tetraethylene glycol-propionic acid.

6. The potentiometric OECT biosensor according to claim 1, characterized in that: In the targeting protein molecule and the shielding layer (14), the targeting protein molecule is prepared by using a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide and an antibody solution; the shielding layer is prepared by using an ethanolamine solution and a bovine serum albumin solution; The electrolyte layer (12) is made of PBS buffer solution.

7. A method for preparing a potentiometric OECT biosensor according to claim 1, characterized in that: include: Cr is deposited on a substrate (1) by a vacuum evaporation method to form a Cr layer, and Au is deposited on the Cr layer to form a source electrode (2), a gate electrode (5) and a first floating gate (7); A semiconductor channel (3) is obtained by spin coating a chloroform solution of [thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the source electrode (2); Depositing gold on the surface of the semiconductor channel (3) to form a drain electrode (4); Coating Ag / AgCl slurry on the gate (5) and annealing in an inert atmosphere to form a gate-controlled gate (6); Covering the substrate (1), the drain electrode (4), the gate-controlled gate (6) and the first floating gate (7) with a hydrogel electrolyte to form a hydrogel electrolyte layer (8), then covering the surface of the hydrogel electrolyte layer (8) with ultraviolet curing glue and curing with ultraviolet light to produce a device module; The sensing module adopts an electrode sheet (9); the electrode sheet (9) includes a gold electrode, an Ag / AgCl electrode and a carbon electrode; the Ag / AgCl electrode serves as a second floating gate (11), the gold electrode serves as a sensing gate (10), the sensing gate (10) is subjected to a cyclic voltammetry scanning process, and then the pre-treated sensing gate (10) is immersed in a SAM modification solution to obtain a SAM connection layer (13); The sensing gate (10) with the SAM connecting layer (13) is placed in a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide for reaction activation, an antibody solution is added dropwise to the surface of the activated sensing gate (10) to react and obtain a targeting protein molecule, the sensing gate (10) is then treated with an ethanolamine solution, and the surface of the sensing gate (10) is then sealed with a bovine serum albumin solution to obtain a targeting protein molecule and a shielding layer (14), thereby obtaining a sensing module; The device module is connected to the sensor module to obtain a potential-type OECT biosensor.

8. A method for preparing a potentiometric OECT biosensor according to claim 7, characterized in that: The semiconductor channel (3) is obtained by spin coating a chloroform solution of [thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on the source (2), specifically: A semiconductor channel film is obtained by spin coating a chloroform solution of poly[thiophene-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2,2'-bithiophene] on a substrate (1) containing a source electrode (2), a gate electrode (5) and a first floating gate (7); Masking the area other than the top of the source electrode (2) on the substrate (1) containing the semiconductor channel film, and depositing Au on the surface of the semiconductor channel film on the source electrode (2) to form a drain electrode (4); The drain electrode (4) is used as a mask, and a substrate (1) containing the drain electrode (4) is etched by reactive ion etching to form a semiconductor channel.

9. A method for preparing a potentiometric OECT biosensor according to claim 7, characterized in that: The molar concentration of the SAM modification solution is 1 mM ethanol solution of mercapto-tetraethylene glycol-propionic acid; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in the mixed solution is 20 mg / mL, and the concentration of N-hydroxysuccinimide is 10 mg / mL; the concentration of the antibody solution is 20 μg / mL; the concentration of the ethanolamine solution is 1 M, and the pH is adjusted to 8 with concentrated hydrochloric acid during the blocking process; the concentration of the bovine serum albumin solution is 10 mg / mL.

10. Use of the potentiometric OECT biosensor according to any one of claims 1 to 6 in biomarker detection.

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