Biremote gate organic field effect transistor biosensor and preparation method and application thereof

Abstract

The invention discloses a double-remote-grid organic field effect transistor biosensor and a preparation method and application thereof. The sensor sequentially comprises double remote grids and a pair of insulated gate type semiconductor field effect transistors connected with the double remote grids respectively. Wherein the dual-remote grid electrode comprises two electrode substrates with the same size, and each substrate comprises a semiconductor layer and an insulating layer arranged on the surface of the semiconductor layer; the organic semiconductor layer is arranged on the surface of the substrate; the probe layer is arranged on the surface of the organic semiconductor layer; and the reference electrode is arranged above the probe layer and is immersed in a test liquid sample. Wherein the source electrode and the drain electrode of the insulated gate type semiconductor field effect transistor are respectively in short circuit, and the grid electrode of the insulated gate type semiconductor field effect transistor is respectively connected with the two remote grid electrode substrates. The transistor biosensor provided by the invention can be used for rapid biological analysis, and is reliable in detection and stable in signal.

Description

Technical Field

[0001] The invention relates to a dual remote gate organic field effect transistor biosensor and a preparation method and application thereof, belonging to the field of biosensors. Background Art

[0002] Rapid detection of disease markers in clinical practice and real-time monitoring of biological pollutants in environmental analysis are fundamental to timely and effective response measures, so the development of rapid on-site biosensing methods is very important and critical. Traditional large-scale instrument analysis is costly, complex, and time-consuming. Among portable sensing detection methods, field-effect transistor sensors have advantages over traditional optical and electrochemical sensors in terms of low power consumption, fast response speed, miniaturization, and integration. They are a new type of sensor device that can realize rapid on-site chemical and biological analysis.

[0003] At present, field effect transistors are mainly used for sensing analysis and rely on the sensitivity of one-dimensional or two-dimensional semiconductor structures between the source and drain. However, the uniformity of low-dimensional semiconductor material formation is difficult to ensure, and there is a lack of controllable functionalization strategies that give semiconductor materials selectivity, which limits the mass preparation of field effect transistor sensors and their application in the detection of a variety of biological molecules. Organic field effect transistors have advantages in the uniform formation of semiconductor films, and the rich functional groups of organic polymer semiconductor branches give transistors the possibility of more functionalized specific probe groups, making them ideal chemical and biological sensing platforms. However, the direct film formation between the source and drain still has the problem of structural differences between devices that are difficult to achieve commercialization, and there is a lack of specific probe groups or controllable functionalization methods for detecting biological molecules. It is necessary to redesign the structure of organic transistors, including gate structure and sensing response logic, selection of probe group structure and functionalization methods, and conversion methods of electrical signals, in order to provide a new organic field effect transistor sensor structure and analysis strategy with uniform structure, stable signal, controllable functionalization, fast and reliable analysis, so that field effect transistor sensors can be practically applied in biological sensing analysis. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dual remote gate organic field effect transistor biosensor and its preparation method and application, so as to realize rapid, stable, reliable, highly sensitive and specific detection of biological molecules on a sensing platform based on a transistor structure.

[0005] Technical solution: To solve the above technical problems, the present invention provides a dual remote gate organic field effect transistor biosensor, which has the structural characteristics of comprising: a substrate, including a semiconductor layer and an insulating layer arranged on the surface of the semiconductor layer; an organic semiconductor layer, arranged on the surface of the substrate; a probe layer, arranged on the surface of the organic semiconductor layer; a reference electrode, arranged above the probe layer and immersed in the test liquid; a pair of insulated gate semiconductor field effect transistors; wherein the substrate, the organic semiconductor layer, the probe layer, and the reference electrode constitute a remote gate, and the sensor comprises two remote gate structures; wherein the two sources of the pair of insulated gate semiconductor field effect transistors are connected and the two drains are connected; wherein the two remote gates are respectively connected to the two gates of the pair of insulated gate semiconductor field effect transistors; wherein the two reference electrodes are short-circuited as the sensor gate signal terminal, and the source and drain of the insulated gate semiconductor field effect transistor serve as the source and drain signal terminals of the sensor.

[0006] Wherein, the gate of the insulated gate type semiconductor field effect transistor is connected to the semiconductor layer of the remote gate substrate and the reference electrode respectively.

[0007] Preferably, the substrate material uses highly doped silicon and silicon dioxide as the semiconductor base layer and the insulating dielectric layer respectively.

[0008] Preferably, the organic semiconductor layer is made of polythiophene with carboxyl groups as side chains.

[0009] Preferably, the probe layer uses aptamer nucleic acid chains of the biological molecules to be detected.

[0010] Preferably, the probe layer and the semiconductor layer are covalently connected via probe layer nucleic acid aptamers and organic semiconductor branches.

[0011] Preferably, the semiconductor material of the insulated gate semiconductor field effect transistor is a metal oxide semiconductor.

[0012] Preferably, the reference electrode is a silver / silver chloride electrode.

[0013] The present invention also provides a method for preparing a dual remote gate organic field effect transistor biosensor, which is characterized by comprising the following steps:

[0014] 1. Make substrates of exactly the same size, and use isopropyl alcohol ultrasonic cleaning, concentrated sulfuric acid / hydrogen peroxide mixed solution immersion cleaning, ultrapure water rinsing, nitrogen drying, and ultraviolet ozone cleaning in sequence;

[0015] 2. Prepare an organic semiconductor organic solution, and form a uniform organic semiconductor layer on the surface of the substrate by spin coating and constant temperature heat treatment;

[0016] 3. Activate the branched carboxyl groups of the organic semiconductor layer with 1-ethyl-(3-dimethylaminopropyl)carbodiimide, treat the organic semiconductor layer with N-hydroxysuccinimide, and then hinge with the 5'-terminal amino-modified aptamer nucleic acid with an amide bond to form a covalently modified probe layer;

[0017] 4. Lead out the insulated gate semiconductor field effect transistor, short-circuit the source, short-circuit the drain, and short-circuit the reference electrode of the double remote gate, and connect them to the signal analysis equipment as the source, drain and gate of the field effect transistor sensor to control the input signal and capture the output signal.

[0018] The present invention also provides the application of the dual remote gate organic field effect transistor biosensor in VEGF detection.

[0019] Wherein, the concentration of VEGF is 10 -13 -10 -4 g / mL.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] 1. According to the dual remote gate organic field effect transistor biosensor and its preparation method involved in the present invention, the organic semiconductor sensitive film is arranged on the surface of the complete substrate, which can avoid the difficulty of uniformly forming the semiconductor film between the source and drain electrodes; 2. The dual remote gate structure can eliminate the random errors caused by accidental factors in the sensing and analysis process of different sensitive films through the circuit design of the field effect transistor; 3. The stability of the device structure and the stability of the response can be further improved by selecting and optimizing the probe group structure; 4. The circuit signal conversion structure of the coupled insulated gate field effect transistor can give the sensor a stable and reliable analysis signal; 5. The structural and performance characteristics of this sensor can improve the device repeatability and signal stability reliability of the field effect transistor as a biosensor, and to a certain extent, can realize the practical application and commercialization of transistor sensor bioanalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic structural diagram of a dual remote gate organic field effect transistor biosensor in an embodiment of the present invention;

[0023] Figure 2 is a chemical structure diagram of the organic semiconductor PT-COOH in an embodiment of the present invention;

[0024] Figure 3 The transistor sensor with VEGF aptamer as the probe layer in Example 1 of the present invention was subjected to different concentrations of VEGF solution (0 g / mL, 10 -13 g / mL,10 -12 g / mL,10 -11 g / mL,10-10 g / mL,10 -9 g / mL,10 -8 g / mL,10 -7 g / mL,10 - 6 g / mL,10 -5 g / mL,10 -4 g / mL, from low to high according to the arrow direction) transfer characteristic curve and threshold voltage change diagram;

[0025] Figure 4 The transistor sensor in Example 2 of the present invention, in which the VEGF aptamer and its complementary nucleic acid chain are used as the probe layer, is exposed to different concentrations of VEGF solution (0 g / mL, 10 -13 g / mL,10 -12 g / mL,10 -11 g / mL,10 -10 g / mL,10 -9 g / mL,10 -8 g / mL,10 -7 g / mL,10 -6 g / mL,10 -5 g / mL,10 -4 g / mL, from low to high according to the arrow direction) transfer characteristic curve and threshold voltage change diagram;

[0026] Figure 5 is a graph showing the function correlation between the sensor response signal and the VEGF concentration in the sample in an embodiment of the present invention;

[0027] Figure 6 1 and 2. The figures are a real-time change of the threshold voltage of the transistor sensor when the VEGF concentration in the sample to be tested on the remote gate surface changes in the embodiment of the present invention (a) and a comparative bar graph of the response signals of the transistor sensor in the embodiment of the present invention to the same concentration of sodium chloride, potassium chloride, urea, glucose, triglyceride, albumin, immunoglobulin G, VEGF165, VEGF121 and a mixture of all the above samples (b). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0029] Reference Figure 1 , a schematic structural diagram of a dual remote gate organic field effect transistor biosensor in an embodiment of the present invention, a dual remote gate organic field effect transistor biosensor provided by the present invention comprises a substrate 1, an organic semiconductor layer 2, a probe layer 3, a reference electrode 4, an insulated gate semiconductor field effect transistor 5, a source 6, a drain 7 and a gate 8.

[0030] A substrate 1, comprising a semiconductor layer and an insulating layer arranged on the surface of the semiconductor layer; wherein the material of the semiconductor layer of the substrate 1 is a doped conductive material such as doped silicon, defective graphene, etc.; the material of the insulating layer of the substrate 1 is a non-conductive material such as silicon dioxide, insulating glass, etc.;

[0031] The organic semiconductor layer 2 is arranged on the upper surface of the insulating layer of the substrate 1; wherein the material of the organic semiconductor layer 2 is a polymer having a conjugated structure along the main chain structure direction, such as polythiophene; the polymer molecular side chain of the organic semiconductor layer 2 has a functionalized structure, such as a carboxyl group, etc.;

[0032] The probe layer 3 is arranged on the surface of the organic semiconductor layer 2; wherein the probe layer 3 adopts a receptor that specifically binds to the molecule to be detected according to the structure of the molecule to be detected, and is covalently connected to the upper surface of the semiconductor layer 2 through chemical modification; the receptor that specifically binds to the molecule to be detected is the aptamer nucleic acid chain and its derivative structure of the molecule to be detected, and the terminal modified functional group, such as amino group, is covalently cross-linked with the polymer side chain of the semiconductor layer 2 through chemical reaction;

[0033] A reference electrode 4 is arranged above the probe layer 3, the surface of the reference electrode maintains a suitable spatial distance from the probe layer, and the surface of the reference electrode is immersed in the test liquid;

[0034] The substrate 1, the organic semiconductor layer 2, the probe layer 3, and the reference electrode 4 form a remote gate. The sensor includes two remote gate structures. The size, model, material, and preparation process of the substrate 1, the organic semiconductor layer 2, the probe layer 3, and the reference electrode 4 of the two remote gates are completely consistent.

[0035] The insulated gate semiconductor field effect transistor 5 is a pair of metal oxide field effect transistors, and any two identical metal oxide field effect transistors in a commercial monolithic integrated circuit can be selected;

[0036] The gates of the two metal oxide field effect transistors are respectively connected to the substrates 1 of the two remote gates by wires;

[0037] The source electrode 6 is arranged at the extension end of the wire after the two sources of the metal oxide field effect transistors are short-circuited;

[0038] The drain electrode 7 is arranged at the extension end of the wire after the drain electrodes of the two metal oxide field effect transistors are short-circuited;

[0039] The grid 8 is arranged at the extension end of the wire after the reference electrodes 4 of the two remote grids are short-circuited.

[0040] Embodiment 1:

[0041] In this embodiment, the substrate 1 includes a highly doped silicon wafer and a 300 nm thick silicon dioxide layer on its surface, and the organic semiconductor layer 2 is as follows: Figure 2The structure shown is poly[3-(3-carboxypropyl)thiophene-2,5-diyl] (RIEKE, source: Rieke metals (Catalog #: 4030)), i.e. PT-COOH, and the probe layer 3 is the aptamer nucleic acid chain VEap of the analyte vascular endothelial growth factor VEGF, and the 5' end is modified with an amino group, the sequence is NH2-5'-TGTGGGGGGGACGGGCCGGGTAGA-3'(NH2-VEap). In this embodiment, the organic semiconductor layer 2 is formed by spin coating of an organic dispersion, and the probe layer 3 is modified by an amidation reaction. The reference electrode 4 is an Ag / AgCl reference electrode, the electrode material is Ag / AgCl, the reference electrolyte is a 3 mol / L concentration of KCl solution, the liquid junction is made of ceramic, and the size is 3.8 mm in diameter.

[0042] The method for preparing the dual remote gate organic field effect transistor biosensor of this embodiment includes the following steps:

[0043] Step 1, cutting a single-sided oxidized highly doped silicon wafer into substrates 1 of completely uniform size of 1.5 cm×1.5 cm, sequentially using isopropyl alcohol ultrasonic cleaning for 15 minutes, concentrated sulfuric acid (98%) / hydrogen peroxide (10%) (1:1) mixed solution for 1 hour, ultrapure water rinsing 3 times, nitrogen drying, and ultraviolet ozone cleaning for 30 minutes;

[0044] Step 2, dissolving PT-COOH in dimethylformamide to prepare a 20 mg / mL organic dispersion, adsorbing the substrate 1 on a spin coater, coating at a speed of 1600 RPM for 5 minutes, and then standing on a 60° C. hot stage for 12 hours to form a uniform organic semiconductor layer 2 on the surface of the substrate 1;

[0045] Step 3, using 3 mg / mL concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to cover the branched carboxyl groups of the organic semiconductor layer 2, and then adding 1 mg / mL of N-hydroxysuccinimide (NHS) to cover the organic semiconductor layer 2, and after 20 minutes, covering with 3 μM concentration of NH2-VEap solution and incubating for 6 hours, NH2-VEap and the carboxyl groups of the organic semiconductor layer are connected by amide bonds to form a covalently modified probe layer 3;

[0046] Step 4, short-circuit the source electrodes 6 of the two metal oxide semiconductor field effect transistors 5 in the insulated gate semiconductor field effect transistor 5 with a wire to serve as the transistor sensor source; short-circuit the drain electrodes 7 of the two metal oxide semiconductor field effect transistors with a wire to serve as the transistor sensor drain; connect the gate electrodes 8 of the two metal oxide semiconductor field effect transistors with a wire respectively, and connect the reference electrode 4 of the dual remote gate with a wire to serve as the transistor sensor gate; thereby forming a complete dual remote gate organic field effect transistor biosensor as a VEGF analysis platform.

[0047] After the preparation is completed, the source electrode 6, the drain electrode 7, and the gate electrode 8 are connected to the Keithley 2400 semiconductor analyzer respectively, and the electrical signal curve in this embodiment is obtained by inputting and outputting electrical signals at these three terminals. During all electrical tests, the area between the probe layer surface and the reference electrode is filled with the sample of the solution to be tested.

[0048] Then, the test solution on the double remote gate surface of the organic field effect transistor sensor of this embodiment was sequentially increased from a blank 0.05×PBS buffer solution to a VEGF concentration of 10 -13 g / mL,10 -12 g / mL,10 -11 g / mL,10 -10 g / mL,10 -9 g / mL,10 -8 g / mL,10 -7 g / mL,10 -6 g / mL,10 -5 g / mL,10 -4 g / mL, and the transfer characteristic curve and threshold change of the transistor sensor were measured. The solution background was 0.05×PBS buffer solution, and the source-drain input voltage was kept at 50mV.

[0049] Figure 3 When the double remote gate surface of the sensor made in this embodiment is the VEGF solution with the above-mentioned concentration gradient, the transfer characteristic curve of the transistor sensor and its threshold voltage change from low to high VEGF concentration, indicating that the threshold voltage increases with the increase of VEGF concentration.

[0050] Then, the relative change of the threshold voltage is used as the response signal value to describe the relationship curve between the sensor response of this embodiment and the VEGF concentration; the relative change of the threshold voltage is used as the response signal value to describe the relationship curve between the sensor without the probe layer and the VEGF concentration, and its maximum noise value is used as the minimum identification signal for quantitatively determining the detection limit of the sensor of this embodiment;

[0051] Figure 5 The relationship curve between the response signal value of the sensor of this embodiment to VEGF and the VEGF concentration is presented. With the help of the response of the sensor without the probe layer, it is obtained that the detection limit of the sensor of this embodiment to VEGF is 40pg / mL.

[0052] Subsequently, the transfer characteristic curve of the transistor sensor of this embodiment in the process of analyzing the VEGF content in the solution is dynamically monitored. The transfer characteristic curve is measured once every 30 seconds and a threshold voltage value is output. The threshold voltage is output in real time while changing the sample to be tested on the surface of the dual remote gate. The samples are blank PBS buffer solution, VEGF solutions with concentrations from low to high, and blank PBS buffer solution in turn, so as to observe the real-time monitoring capability and reusability of the sensor.

[0053] Figure 6 a shows that the sensor of this embodiment is covered with a blank PBS buffer solution on the double remote gate surface, 10 -11 g / mL, 10 -9 g / mL, 10 -7 g / mL, 10 -5 The real-time changes in the threshold voltage during the process of adding 100 g / mL VEGF solution and re-covering with a blank PBS buffer solution show that the sensor prepared in this example has a rapid and sensitive response capability to VEGF.

[0054] The sensor of this embodiment was then used to analyze other common molecules in body fluid samples, including inorganic salts, small molecular organic matter, common proteins, two VEGFs and their mixtures. The rate of change of the threshold voltage relative to the blank sample was used as the sensitivity. The selectivity of the sensor detection and the ability to detect VEGF in mixed samples were observed through the sensitivity intensity.

[0055] Figure 6 b shows a comparison chart of the sensitivity of the sensor of this embodiment to 1 ng / mL sodium chloride, potassium chloride, urea, glucose, triglyceride, albumin, immunoglobulin G, VEGF165, VEGF121 and mixed solution samples of all the above samples, showing the selective detection ability of the sensor of this embodiment to VEGF121 and the ability to detect VEGF121 in complex biological samples.

[0056] Embodiment 2:

[0057] In this embodiment, the substrate 1 includes a highly doped silicon wafer and a 300 nm thick silicon dioxide layer on its surface, and the organic semiconductor layer 2 is as follows: Figure 2PT-COOH shown, the probe layer 3 is the aptamer nucleic acid chain VEap and its complementary chain CS of the analyte VEGF, the 5' end of VEap is modified with an amino group, the sequence is NH2-5'-TGTGGGGGGGACGGGCCGGGTAGA-3'(NH2-VEap), and the CS sequence is 3'-CCACCTGCCCGG-5'. In this embodiment, the organic semiconductor layer 2 is formed by spin coating of an organic dispersion, and the probe layer 3 is modified by an amidation reaction. The reference electrode 4 is an Ag / AgCl electrode, the electrode material is Ag / AgCl, the reference electrolyte is a 3 mol / L concentration of KCl solution, the liquid junction is made of ceramic, and the size is 3.8 mm in diameter.

[0058] The method for preparing the dual remote gate organic field effect transistor biosensor of this embodiment includes the following steps:

[0059] Step 1, cutting a single-sided oxidized highly doped silicon wafer into substrates 1 of completely uniform size of 1.5 cm×1.5 cm, sequentially using isopropyl alcohol ultrasonic cleaning for 15 minutes, soaking and cleaning with a mixture of concentrated sulfuric acid (98%) / hydrogen peroxide (10%) (volume ratio 1:1) for 1 hour, rinsing with ultrapure water 3 times, blowing dry with nitrogen, and ultraviolet ozone cleaning for 30 minutes;

[0060] Step 2, dissolving PT-COOH in dimethylformamide to prepare a 20 mg / mL organic dispersion, adsorbing the substrate 1 on a spin coater, coating at a speed of 1600 RPM for 5 minutes, and then standing on a 60° C. hot stage for 12 hours to form a uniform organic semiconductor layer 2 on the surface of the substrate 1;

[0061] Step 3, using 3 mg / mL concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to cover the branched carboxyl groups of the organic semiconductor layer 2, and then adding 1 mg / mL of N-hydroxysuccinimide (NHS) to cover the organic semiconductor layer 2, 6 μM H2N-VEap and 6 μM CS solution were mixed 1:1 on a shaker for 20 minutes and allowed to stand for more than 2 hours to obtain a H2N-VEap / CS solution, and then the NH2-VEap / CS solution was covered and incubated for 6 hours, and the NH2-VEap / CS and the organic semiconductor carboxyl groups were connected by amide bonds to form a covalently modified probe layer 3;

[0062] Step 4, short-circuit the source electrodes 6 of the two metal oxide semiconductor field effect transistors in the insulated gate semiconductor field effect transistor 5 with a wire to serve as the transistor sensor source; short-circuit the drain electrodes 7 of the two metal oxide semiconductor field effect transistors with a wire to serve as the transistor sensor drain; connect the gate electrodes 8 of the two metal oxide semiconductor field effect transistors with a wire respectively, and connect the reference electrode of the dual remote gate with a wire to serve as the transistor sensor gate; thereby forming a complete dual remote gate organic field effect transistor biosensor for analyzing VEGF.

[0063] After the preparation is completed, the source electrode 6, the drain electrode 7, and the gate electrode 8 are respectively connected to the Keithley 2400 semiconductor analyzer, and the electrical signal diagram in this embodiment is obtained by controlling the input signal of the source electrode 6, the drain electrode 7, and the gate electrode 8 and recording the output signal. During all electrical tests, the area between the probe layer surface and the reference electrode is filled with the sample of the solution to be tested.

[0064] Then, the test solution in which the double remote gate surface of the organic field effect transistor sensor of this embodiment is immersed is successively increased from the blank 0.05×PBS buffer solution to the VEGF concentration of 10 -13 g / mL,10 -12 g / mL,10 -11 g / mL,10 - 10 g / mL,10 -9 g / mL,10 -8 g / mL,10 -7 g / mL,10 -6 g / mL,10 -5 g / mL,10 -4 g / mL, and the transfer characteristic curve and threshold change of the transistor sensor were measured. The solution background was 0.05×PBS buffer solution, and the source-drain input voltage was set to 50mV.

[0065] Figure 4 When the double remote gate surface of the sensor made in this embodiment is the VEGF solution with the above-mentioned concentration gradient, the transfer characteristic curve of the transistor sensor and its threshold voltage change from low to high VEGF concentration, indicating that the threshold voltage decreases with the increase of VEGF concentration.

[0066] Then, the relative change of the threshold voltage is used as the response signal value to describe the relationship curve between the sensor response of this embodiment and the VEGF concentration; the relative change of the threshold voltage is used as the response signal value to describe the relationship curve between the sensor without the probe layer and the VEGF concentration, and its maximum noise value is used as the minimum identification signal for quantitatively determining the detection limit of the sensor of this embodiment;

[0067] Figure 5The relationship curve between the response signal value of the sensor of this embodiment to VEGF and the VEGF concentration is presented. With the help of the response of the sensor without the probe layer, it is obtained that the detection limit of the sensor of this embodiment to VEGF is 20pg / mL.

[0068] Subsequently, the transfer characteristic curve of the transistor sensor of this embodiment in the process of analyzing the VEGF content in the solution is dynamically monitored. The transfer characteristic curve is measured once every 30 seconds and a threshold voltage value is output. The threshold voltage is output in real time while changing the sample to be tested on the surface of the dual remote gate. The samples are blank PBS buffer solution, VEGF solutions with concentrations from low to high, and blank PBS buffer solution in turn, so as to observe the real-time monitoring capability and reusability of the sensor.

[0069] Figure 6 a shows that the sensor of this embodiment is covered with a blank PBS buffer solution on the double remote gate surface, 10 -11 g / mL, 10 -9 g / mL, 10 -7 g / mL, 10 -5 The real-time changes in the threshold voltage during the process of adding 100 g / mL VEGF solution and re-covering with a blank PBS buffer solution show that the sensor prepared in this example has a rapid and sensitive response capability to VEGF.

[0070] The sensor of this embodiment was then used to analyze other common molecules in body fluid samples, including inorganic salts, small molecular organic matter, common proteins, two VEGFs and their mixtures. The rate of change of the threshold voltage relative to the blank sample was used as the sensitivity. The selectivity of the sensor detection and the ability to detect VEGF in mixed samples were observed through the sensitivity intensity.

[0071] Figure 6 b shows a comparison chart of the sensitivity of the sensor of this embodiment to 1 ng / mL sodium chloride, potassium chloride, urea, glucose, triglyceride, albumin, immunoglobulin G, VEGF165, VEGF121 and mixed solution samples of all the above samples, showing the selective detection ability of the sensor of this embodiment to VEGF121 and the ability to detect VEGF121 in complex biological samples.

[0072] It can be seen from the above embodiments that the dual remote gate organic field effect transistor sensor modified with the detected biological molecule VEGF aptamer nucleic acid and its paired double-stranded structure has high sensitivity, rapid response and high specific response capability for the detection of VEGF.

[0073] In summary, according to the dual remote gate organic field effect transistor biosensor and its preparation method involved in Example 1 and Example 2, because the organic semiconductor film of the prepared organic field effect transistor sensor is set on the surface of the complete substrate, the difficulty of uniformly forming the semiconductor film between the source and drain electrodes is avoided; because the dual remote gate structure is designed, the random error caused by accidental factors in the interface response process of different semiconductor sensitive films is eliminated; because the probe layer of nucleic acid aptamer and its complementary chain structure is involved, the stability of the device structure and the stability of the response are further improved; because the commercial insulated gate field effect transistor is coupled as a circuit signal conversion structure, the sensor is given a stable and reliable output signal. Therefore, the organic field effect transistor sensor of this embodiment has the characteristics of structure and performance, which can improve the device repeatability and signal stability reliability of the field effect transistor as a biosensor, and can realize the practical application and commercialization of transistor sensor bioanalysis to a certain extent.

[0074] The above-described embodiments are only preferred cases of the present invention and do not limit the protection scope of the present invention. It should be pointed out that modifications and improvements made without departing from the technical principles of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A dual remote gate organic field effect transistor biosensor, characterized in that: The invention comprises two identical remote gates and a pair of insulated gate semiconductor field effect transistors (5); the remote gate comprises a substrate (1), an organic semiconductor layer (2), a probe layer (3), and a reference electrode (4); the substrate (1) comprises a semiconductor layer and a double insulating layer arranged on the surface of the semiconductor layer; the surface of the double insulating layer of the substrate (1) is provided with an organic semiconductor layer (2); the surface of the organic semiconductor layer (2) is provided with a probe layer (3); a reference electrode (4) for immersion in a test liquid is arranged above the probe layer (3); and the gate (8) of the insulated gate semiconductor field effect transistor (5) is connected to the remote gate substrate (1) and the reference electrode (4) respectively.

2. The dual remote gate organic field effect transistor biosensor according to claim 1, characterized in that: The source electrodes (6) of the two insulated gate semiconductor field effect transistors (5) are connected to each other, and the two drain electrodes (7) of the two insulated gate semiconductor field effect transistors (5) are connected to each other.

3. The dual remote gate organic field effect transistor biosensor according to claim 1, characterized in that: The semiconductor layer of the substrate (1) is made of highly doped silicon, and the double insulating layer material of the substrate (1) is made of silicon dioxide.

4. The dual remote gate organic field effect transistor biosensor according to claim 1, characterized in that: The organic semiconductor layer (2) uses polythiophene whose side chains are carboxyl groups.

5. The dual remote gate organic field effect transistor biosensor according to claim 1, characterized in that: The detection group used in the probe layer (3) is the aptamer nucleic acid chain of the detected biological molecule.

6. The dual remote gate organic field effect transistor biosensor according to claim 5, characterized in that: The 5' end of the aptamer nucleic acid chain is functionalized with an amino group and is covalently connected to the organic semiconductor layer (2).

7. The dual remote gate organic field effect transistor biosensor according to claim 1, characterized in that: The reference electrode (4) is short-circuited and serves as a sensor gate signal terminal, and the source and drain of the insulated gate semiconductor field effect transistor serve as the source and drain signal terminals of the sensor.

8. A method for preparing a dual remote gate organic field effect transistor biosensor according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step 1, preparing an organic semiconductor organic solution, and forming a uniform organic semiconductor layer on the surface of a substrate by spin coating and constant temperature heat treatment; the substrate is cleaned and includes a semiconductor layer and a double insulating layer arranged on the surface of the semiconductor layer; Step 2, using 1-ethyl-(3-dimethylaminopropyl)carbodiimide to activate the branched carboxyl groups of the organic semiconductor layer, then treating the organic semiconductor layer with N-hydroxysuccinimide, and then hinged with the 5'-terminal amino-modified aptamer nucleic acid with an amide bond to form a covalently modified probe layer; Step 3, lead out the insulated gate semiconductor field effect transistor short-circuited source, short-circuited drain, double remote gate short-circuited reference electrode, and use them as the source, drain and gate of the field effect transistor sensor to connect to the signal analysis equipment to control the input signal and capture the output signal.

9. Use of the dual remote gate organic field effect transistor biosensor according to any one of claims 1 to 7 in VEGF detection.

10. The use according to claim 9, characterized in that: The concentration of VEGF is 10 -13 -10 -4 g / mL.

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