Field effect transistor biosensor and preparation method thereof

Abstract

The invention relates to the field of biosensors, in particular to a field effect transistor biosensor and a preparation method thereof. The field effect transistor biosensor comprises a field effect transistor chip, a surface modification layer and a biological probe layer, the field effect transistor chip comprises a semiconductor channel layer, and the surface modification layer is located on the surface of the semiconductor channel layer; the biological probe layer comprises a double-end aptamer, the 5'end and the 3 'end of the double-end aptamer both have modification groups-SH, and the double-end aptamer is connected with the surface modification layer through the modification groups-SH. According to the biosensor, the double-end-SH modified aptamer is adopted, the distance between a target molecule binding site and the surface of a semiconductor can be remarkably reduced, and the Debye shielding effect can be effectively overcome, so that the sensitivity of the biosensor is greatly improved, the detection limit of a device is reduced, and the stability is improved.

Description

Technical Field

[0001] This invention relates to the field of biosensors, and more specifically, to a field-effect transistor biosensor and its fabrication method. Background Technology

[0002] Detecting low concentrations of disease biomarkers is crucial for early medical diagnosis. PCR technology, as the mainstream molecular diagnostics method, utilizes the principle of DNA double-strand replication to amplify specific DNA fragments in large quantities outside the organism. However, the PCR amplification process is time-consuming, involves numerous steps, and requires expensive equipment and highly specialized laboratories due to its high level of human interaction. Therefore, PCR is far from meeting people's needs due to numerous limitations in high-throughput screening, point-of-care testing, and implementation in primary hospitals. Thus, the demand for rapid, effective, cost-effective, and easy-to-use detection methods remains increasingly urgent.

[0003] Over the past few decades, nanobiosensors have demonstrated significant achievements and superiority in various biomolecule detection applications. Among existing biosensors, field-effect transistor (FET) biosensors, due to their label-free and non-amplified advantages, meet the demands for rapid, economical, and convenient use, and are considered an ideal choice for next-generation medical diagnostic platforms. FETs possess advantages such as reliable readout of small signals, direct gate-to-test solution contact, and low operating gate voltage, resulting in excellent performance including high sensitivity and fast response speed. FET-based biosensors (Bio-FETs) show even broader application prospects in detecting biological targets, especially disease-related biomarkers (such as nucleic acids, characteristic proteins, and metabolism).

[0004] However, the exceptionally complex solution environments of real-world samples (such as serum and urine) and the unprecedented accuracy requirements for detecting even the smallest residual disease molecules still pose significant challenges to biofield-effect transistors (BFETs). Therefore, improving the sensitivity of transistor sensor devices has become a hot topic in this field. To improve device sensitivity, the Debye shielding effect is a fundamental limitation that BFET sensors cannot avoid. The Debye length is defined by the detectable range of the channel surface. Within the Debye length, specific binding generates an equivalent gate voltage effect on the semiconductor surface, shifting the transistor's threshold voltage and causing a change in drain current, thus converting the target concentration change into an observable electrical signal. Outside the Debye length, the charge of molecules cannot be effectively sensed. Therefore, overcoming the Debye shielding effect is key to improving device sensitivity.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One objective of this invention is to provide a field-effect transistor biosensor that can effectively overcome the Debye shielding effect, significantly improve the sensitivity of the biosensor, and reduce the detection limit of the device.

[0007] Another objective of this invention is to provide a method for fabricating a field-effect transistor biosensor that is simple and easy to implement. The field-effect transistor biosensor obtained through the coordinated steps can overcome the Debye shielding effect, improve the sensitivity of the biosensor, and reduce the detection limit.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: A field-effect transistor (FET) biosensor includes a FET chip, a surface modification layer, and a bioprobe layer; the FET chip includes a semiconductor channel layer, and the surface modification layer is located on the surface of the semiconductor channel layer; the bioprobe layer includes a bipolar aptamer, the 5' end and 3' end of the bipolar aptamer both having a modifying group -SH, and the bipolar aptamer is connected to the surface modification layer through the modifying group -SH.

[0009] In some embodiments, the probes in the bioprobe layer have a U-shaped structure.

[0010] In some embodiments, the bi-terminal aptamers in the bioprobe layer are used to complementarily pair with the target molecule to form a binding compound, and the vertical distance between the binding compound and the semiconductor channel layer is less than the Debye length.

[0011] In some embodiments, the surface modification layer includes an aminosilane layer, wherein the -NH2 of the aminosilane layer is linked to the -SH modification group of the dual-terminal aptamer via a bifunctional crosslinking agent.

[0012] In some embodiments, the surface modification layer includes an aminosilane layer comprising 3-aminopropyltriethoxysilane; In some embodiments, the bifunctional crosslinking agent in the surface modification layer comprises sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide.

[0013] In some implementations, the semiconductor channel layer comprises IGZO.

[0014] In some embodiments, the field-effect transistor chip further includes: a substrate layer, a bottom gate layer, a top gate layer, an Al2O3 layer, a SiO2 layer, a source electrode, a drain electrode, a passivation layer, an electrode layer, and a via; the bottom gate layer is located on a portion of the surface of the substrate layer; the Al2O3 layer is located on the surface of the substrate layer and the surface of the bottom gate layer, and the SiO2 layer is located on the surface of the Al2O3 layer; the via penetrates through the Al2O3 layer and the SiO2 layer; the top gate layer is located in the via and is in contact with the substrate layer; the electrode layer is disposed on the surface of the top gate layer; the semiconductor channel layer is located on a portion of the surface of the SiO2 layer; the source electrode and the drain electrode are each independently located on a portion of the surface of the semiconductor channel layer and the SiO2 layer; the passivation layer is located on the surface of the source electrode, the drain electrode, the semiconductor channel layer, and the SiO2 layer. The fabrication method of the field-effect transistor biosensor as described above includes the following steps: Obtain a field-effect transistor chip.

[0015] A surface modification layer is prepared on the surface of the field-effect transistor chip.

[0016] Under the action of a bifunctional crosslinking agent, the bi-terminal aptamer is anchored to the surface of the surface-modified layer to form a biological probe layer.

[0017] In some embodiments, the method for obtaining the field-effect transistor chip specifically includes: A bottom gate material is deposited on a substrate to form a bottom gate layer; an Al2O3 layer is deposited on the surface of the substrate and the bottom gate layer, and a SiO2 layer is deposited on the surface of the Al2O3 layer; a semiconductor channel material is grown on the surface of the SiO2 layer to form a semiconductor channel layer; a via is fabricated through the SiO2 layer and the Al2O3 layer; a composite layer of Ti and Au is deposited on a portion of the surface of the semiconductor channel layer, a portion of the surface of the SiO2 layer, and the via to form a source, drain, and top gate layer; a passivation material is coated on a portion of the surface of the semiconductor channel layer, the source, the drain, and a portion of the surface of the SiO2 layer to form a passivation layer; and an electrode layer is fabricated on the surface of the top gate layer. In some embodiments, preparing a surface modification layer on the surface of the field-effect transistor chip specifically includes: placing the field-effect transistor chip in an alcohol solution of aminosilane and performing a first incubation treatment; treating the field-effect transistor chip after the first incubation treatment with an alcohol solvent, and then performing a first drying treatment to obtain an aminosilane-modified field-effect transistor chip; placing the aminosilane-modified field-effect transistor chip in a PBS solution of a bifunctional crosslinking agent and performing a second incubation treatment, and then performing PBS solution washing and a second drying treatment to obtain a field-effect transistor chip with a surface modification layer.

[0018] In some embodiments, the volume fraction of aminosilane in the alcoholic solution of the aminosilane is 3% to 7%.

[0019] In some embodiments, the temperature of the first incubation treatment is 20~30°C, the rotation speed of the first incubation treatment is 150~250 r / min, and the time of the first incubation treatment is 0.5~2 h.

[0020] In some embodiments, the alcohol solvent treatment includes washing and soaking.

[0021] In some embodiments, the drying process includes nitrogen blowing and drying, wherein the drying temperature is 100-130°C and the drying time is 10-60 minutes.

[0022] In some embodiments, the concentration of the bifunctional cross-linking agent in the PBS solution is 0.8~1.2 mg / mL, the concentration of the PBS solution is 8~12 mmol / L, and the pH is 7.2~7.6.

[0023] In some embodiments, the rotation speed of the second incubation treatment is 150~200 r / min, and the incubation time is 1~3 h.

[0024] In some embodiments, the PBS solution used for washing has a concentration of 8-12 mmol / L and a pH of 7.2-7.6.

[0025] In some embodiments, the second drying process uses nitrogen gas to dry the product.

[0026] In some embodiments, the preparation of the biological probe layer specifically includes: mixing a PBS solution of reducing agent and a double-ended aptamer powder and then performing a third incubation treatment to obtain a mixed system; adding the mixed system dropwise to the surface modification layer region of the field-effect transistor chip and then performing a fourth incubation treatment to obtain the biological probe layer.

[0027] In some embodiments, the reducing agent in the PBS solution comprises tri-(2-formylethyl)phosphonic acid hydrochloride, the concentration of the reducing agent is 8-12 mmol / L, the concentration of the PBS solution is 8-12 mmol / L, and the pH of the PBS solution is 7.2-7.6.

[0028] In some embodiments, the third incubation treatment lasts for 0.5 to 1.5 hours.

[0029] In some embodiments, the concentration of the biterminal aptamer powder is 8~12 μmol / mL.

[0030] In some embodiments, the temperature of the fourth incubation treatment is 3~8°C, and the time of the fourth incubation treatment is 8~15h.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The field-effect transistor biosensor of the present invention uses a dual-terminal -SH modified aptamer, which forms a stable connection with the semiconductor channel layer of the field-effect transistor chip under the action of the surface modification layer; the biosensor can significantly reduce the distance between the target molecule binding site and the semiconductor surface, effectively overcome the Debye shielding effect, thereby greatly improving the sensitivity of the biosensor, reducing the detection limit of the device, and improving stability.

[0032] (2) The preparation method of the field-effect transistor biosensor of the present invention is simple and easy to implement. Through the cooperation of each step, the distance between the target molecule binding site and the semiconductor surface can be reduced, thereby effectively overcoming the Debye shielding effect. This method can improve the sensitivity of the biosensor and reduce the detection limit of the device. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1a This is a schematic diagram of the structure of the biosensor in Example 1; Figure 1b This is a schematic diagram of the fabrication process of the biosensor in Example 1; Figure 2a This is a schematic diagram of the preparation process of the surface modification layer and the biological probe layer of the biosensor in Example 1; Figure 2b This is a schematic diagram of the preparation process of the surface modification layer and the biological probe layer of the biosensor in Comparative Example 1; Figure 3a The Id-Vg curves of the biosensor in Example 1 are shown when the leakage voltage is 0.005V-1V. Figure 3b The Id-Vd curves of the biosensor in Example 1 are shown when the gate voltage is 0.3V-0.9V. Figure 4 The Id-Vg curves of the biosensors of Example 1 and Comparative Example 1 during the surface modification process; Figure 5a This is an atomic force microscopy (AFM) image of the functionalized dual-ended probe from Example 1. Figure 5b An atomic force microscope image of a single-ended probe functionalized in Comparative Example 1; Figure 6a The biosensor of Example 1 is used at a concentration of 10 -16 Up to 10 -6 Id-Vg standard concentration gradient curve of NMP22 at g / mL; Figure 6b The biosensor for Comparative Example 1 was used at a concentration of 10... -16 Up to 10 -6 Id-Vg standard concentration gradient curve of NMP22 at g / mL; Figure 6c The figure shows a comparison of the real-time current response of the biosensors in Example 1 and Comparative Example 1; Figure 7a The image shows the Id-Time real-time response curves of the biosensor in Example 1 to different concentrations of DNA standards. Figure 7b This is a bar chart showing the drain current of the biosensor in Example 1 detecting DNA standards at different concentrations. Figure 7c This is a graph showing the Id / I0-Time test results of the biosensor in Example 1; Figure 7d The graph shows the current change response of the biosensor in Example 1 when detecting four different DNA standards.

[0035] Figure label: 1-Substrate layer, 2-Bottom gate layer, 3-Al2O3 layer, 4-SiO2 layer, 5-Semiconductor channel layer, 6-Top gate layer, 7-Drain, 8-Source, 9-Passivation layer, 10-Electrode layer, 11-Through hole, 12-Surface modification layer, 13-Biological probe layer. Detailed Implementation

[0036] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0037] According to one aspect of the present invention, the present invention relates to a field-effect transistor biosensor, comprising a field-effect transistor chip, a surface modification layer, and a bioprobe layer; the field-effect transistor chip includes a semiconductor channel layer, and the surface modification layer is located on the surface of the semiconductor channel layer; the bioprobe layer includes a bipolar aptamer, wherein the 5' end and 3' end of the bipolar aptamer each have a modifying group -SH, and the bipolar aptamer is connected to the surface modification layer through the modifying group -SH.

[0038] The field-effect transistor biosensor of the present invention employs a dual-ended -SH modified aptamer, which, under the action of a surface modification layer, forms a stable connection with the semiconductor channel layer of the field-effect transistor chip. This biosensor can significantly reduce the distance between the target molecule binding site and the semiconductor surface, effectively overcome the Debye shielding effect, thereby greatly improving the sensitivity of the biosensor, reducing the detection limit of the device, and improving stability. In some embodiments, the probe in the biological probe layer has a U-shaped structure. The dual-ended aptamer can be fixed at both ends, allowing the aptamer to form a U-shaped bent conformation.

[0039] In some embodiments, the bi-terminal aptamers in the bioprobe layer are used to complementarily pair with the target molecule to form a binding compound. The vertical distance between the binding compound and the semiconductor channel layer is less than the Debye length, thereby effectively overcoming the Debye shielding effect.

[0040] In some embodiments, the surface modification layer includes an aminosilane layer, wherein the -NH2 group of the aminosilane layer is linked to the -SH group of the dual-terminal aptamer via a bifunctional crosslinking agent. In some embodiments, the aminosilane layer comprises 3-aminopropyltriethoxysilane. In some embodiments, the bifunctional crosslinking agent comprises sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide (sulfo-SMCC). This invention introduces an appropriate amount of -NH2 onto the surface of the semiconductor channel layer through the aminosilane layer, thereby increasing the loading capacity of the dual-terminal aptamer and ensuring the performance of the bioprobe layer. The bifunctional crosslinking agent is beneficial for improving the binding performance of the bioprobe layer and ensuring structural stability.

[0041] In some embodiments, the semiconductor channel layer comprises IGZO (indium gallium zinc oxide), which has high electron mobility, good stability, and a relatively simple manufacturing process.

[0042] In some embodiments, the field-effect transistor chip further includes: a substrate layer, a bottom gate layer, a top gate layer, an Al2O3 layer, a SiO2 layer, a source electrode, a drain electrode, a passivation layer, an electrode layer, and a via; the bottom gate layer is located on a portion of the surface of the substrate layer; the Al2O3 layer is located on the surface of the substrate layer and the surface of the bottom gate layer, and the SiO2 layer is located on the surface of the Al2O3 layer; the via penetrates through the Al2O3 layer and the SiO2 layer; the top gate layer is located in the via and contacts the substrate layer; the electrode layer is disposed on the surface of the top gate layer; the semiconductor channel layer is located on a portion of the surface of the SiO2 layer; the source electrode and the drain electrode are each independently located on a portion of the surface of the semiconductor channel layer and the SiO2 layer; the passivation layer is located on the surface of the source electrode, the drain electrode, the semiconductor channel layer, and the SiO2 layer. In some embodiments, the substrate layer includes a Si substrate passivation layer to protect the electrodes from corrosion.

[0043] According to another aspect of the present invention, the present invention also relates to a method for fabricating a field-effect transistor biosensor as described above, comprising the following steps: Obtain a field-effect transistor chip.

[0044] A surface modification layer is prepared on the surface of the field-effect transistor chip.

[0045] Under the action of a bifunctional crosslinking agent, the bi-terminal aptamer is anchored to the surface of the surface-modified layer to form a biological probe layer.

[0046] The fabrication method of the field-effect transistor biosensor of the present invention is simple and easy to implement. By coordinating the various steps, the distance between the target molecule binding site and the semiconductor surface can be reduced, thereby effectively overcoming the Debye shielding effect. This method can improve the sensitivity of the biosensor and reduce the detection limit of the device.

[0047] In some embodiments, the method for obtaining the field-effect transistor chip specifically includes: A bottom gate material, including Mo, is deposited on a substrate, and then coated with a first photoresist. Etching is performed using a first mask to form a bottom gate layer. An Al₂O₃ layer is deposited on the surface of the substrate and the bottom gate layer. A SiO₂ layer is then deposited on the surface of the Al₂O₃ layer. A semiconductor channel material, such as IGZO, is grown on the surface of the SiO₂ layer, coated with a second photoresist, and etched using a second mask to form a semiconductor channel layer. A via is fabricated penetrating the SiO₂ layer and the Al₂O₃ layer. A composite layer of Ti and Au is deposited on a portion of the surface of the semiconductor channel layer, a portion of the surface of the SiO₂ layer, and in the via to form a source, drain, and top gate layer. A passivation material, such as SU₈, is coated on a portion of the surface of the semiconductor channel layer, the source, the drain, and a portion of the SiO₂ layer to form a passivation layer. An electrode layer, such as an Ag / AgCl electrode, is fabricated on the surface of the top gate layer.

[0048] In some embodiments, the method for obtaining the field-effect transistor chip includes: (1) A layer of Mo is grown on a Si substrate, and then NR 1500 photoresist is spin-coated on its surface. The first mask is used for photolithography, exposure, development, dry etching, and photoresist removal to obtain the bottom gate layer.

[0049] (2) An Al2O3 layer is deposited on the surface of the substrate and the bottom gate layer by atomic layer deposition.

[0050] (3) A SiO2 layer is deposited on the surface of the Al2O3 layer. A relatively uniform IGZO thin film is then obtained by magnetron sputtering. After spin coating with positive photoresist, photolithography is performed using a second mask. After exposure, development, wet etching, and photoresist removal, the sensing area (semiconductor channel layer) is obtained.

[0051] (4) Create through holes that penetrate the SiO2 layer and the Al2O3 layer.

[0052] (5) Spin-coat a layer of NR 1500 photoresist, perform photolithography with the third mask, develop the pattern of the electrode area, grow a Ti / Au composite layer by electron beam evaporation, and then perform metal stripping to obtain the source, drain and top gate layers.

[0053] (6) Spin-coating SU8 adhesive, using the fourth mask for photolithography, exposure and development, and then fixing the SU8 adhesive on the surface of the source and drain electrodes to protect the electrodes and form a passivation layer.

[0054] (7) An Ag / AgCl electrode layer is fabricated on the surface of the top gate layer.

[0055] In some implementations, 2010 photoresist is used as a substrate to increase the adhesion of the thick adhesive used to fabricate the reservoir, requiring only longer pre-baking and post-baking times without development. The reservoir is then fabricated using photoresist and a mask, and hardened for 5-7 hours.

[0056] In some implementations, before using the device, the blue film on the silicon wafer is first peeled off, soaked in acetone for 20-40 minutes, and then soaked in anhydrous ethanol for 8-15 minutes. After removal, nitrogen gas is used to quickly dry the device and wash away the photoresist on the electrode surface.

[0057] In some embodiments, a surface modification layer is prepared on the surface of the field-effect transistor chip, specifically including: placing the field-effect transistor chip in an alcohol solution of aminosilane and performing a first incubation treatment; treating the field-effect transistor chip after the first incubation treatment with an alcohol solvent, and then performing a first drying treatment to obtain an aminosilane-modified field-effect transistor chip; placing the aminosilane-modified field-effect transistor chip in a PBS solution (phosphate buffer) of a bifunctional crosslinking agent and performing a second incubation treatment, followed by washing with PBS solution and a second drying treatment to obtain a field-effect transistor chip with a surface modification layer. In this invention, the semiconductor channel layer (sensing region) surface of the field-effect transistor chip contains hydroxyl groups. After wetting with the alcohol solution of aminosilane, silanization occurs, thereby changing the hydroxyl groups on the surface of the conductor channel layer to amino groups, forming an aminosilane layer; the alcohol solvent treatment can wash away high-concentration solution residues, ensuring the sensitivity of the device and the detection results; the drying treatment makes the silane-amino layer on the surface of the conductor channel layer more robust; the bifunctional crosslinking agent can further react to form stable amide bonds, allowing it to establish a stable connection with the sensing region of the device, forming a stable surface modification layer.

[0058] In some embodiments, the volume fraction of the aminosilane in the alcohol solution is 3% to 7%, for example, 3%, 4%, 5%, 6%, or 7%. The alcohol solvent includes anhydrous ethanol.

[0059] In some embodiments, the temperature of the first incubation treatment is 20~30℃, such as 20℃, 22℃, 25℃, 30℃, etc., the rotation speed of the first incubation treatment is 150~250r / min, such as 150r / min, 160r / min, 180r / min, 200r / min, 250r / min, etc., and the time of the first incubation treatment is 0.5~2h, such as 0.5h, 0.8h, 1h, 1.5h or 2h, etc.

[0060] In some embodiments, the alcohol solvent treatment includes washing and immersion. The surface of the semiconductor channel layer of the field-effect transistor chip is rinsed with an alcohol solvent to remove high concentrations of solution residue; then it is quickly immersed in anhydrous ethanol and repeatedly rinsed. Because anhydrous ethanol is highly volatile, the device should be kept in a liquid environment as much as possible throughout the process.

[0061] In some embodiments, the drying process includes nitrogen blowing and baking, wherein the baking temperature is 100-130°C (e.g., 100°C, 110°C, 120°C, or 130°C), and the baking time is 10-60 min (e.g., 10 min, 20 min, 30 min, 40 min, or 60 min). After the alcohol solvent treatment is completed, nitrogen is used for rapid drying to prevent the evaporation of anhydrous ethanol from leaving APTES residue on the device surface, forming white crystals that could affect the device's sensitivity and detection results.

[0062] In some embodiments, the concentration of the bifunctional crosslinking agent in the PBS solution is 0.8~1.2 mg / mL (e.g., 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, etc.), the concentration of the PBS solution is 8~12 mmol / L (e.g., 8 mmol / L, 10 mmol / L, 12 mmol / L, etc.), and the pH is 7.2~7.6 (e.g., 7.2, 7.3, 7.5, 7.6, etc.).

[0063] In some embodiments, because sulfo-SMCC is difficult to dissolve, the PBS solution of the bifunctional cross-linking agent can be sonicated in an ultrasonic machine at a temperature of 40–60°C (e.g., 40°C, 50°C, or 60°C) for 10–30 min (e.g., 10 min, 20 min, or 30 min). In some embodiments, the sonication is paused and the solution is shaken 2–4 times to help it dissolve more quickly.

[0064] In some embodiments, the rotation speed of the second incubation treatment is 150-200 r / min (e.g., 150 r / min, 160 r / min, 180 r / min, or 200 r / min), and the incubation time is 1-3 h (e.g., 1 h, 1.5 h, 2 h, 3 h). Since a relatively stable amino group has already formed in the sensing region of the device, under the aforementioned suitable second incubation conditions, a stable amide bond can be formed when reacting with sulfo-SMCC, allowing for a more stable connection with the sensing region of the device.

[0065] In some embodiments, the PBS solution used for washing has a concentration of 8–12 mmol / L and a pH of 7.2–7.6. In some embodiments, after the device is removed, it is rinsed thoroughly with a 10 mmol / L PBS solution (pH 7.4).

[0066] In some implementations, the second drying process uses nitrogen blowing to ensure the drying effect without affecting the performance of the device.

[0067] In some embodiments, the preparation of the bioprobe layer specifically includes: mixing a PBS solution of reducing agent and a bipolar aptamer powder, followed by a third incubation treatment to obtain a mixed system; adding the mixed system dropwise to the surface modification layer region of the field-effect transistor chip, and then performing a fourth incubation treatment to obtain the bioprobe layer. Since thiol groups are prone to oxidation during transport, the bipolar aptamer powder cannot be directly modified with thiol groups during modification; instead, the thiol groups at both ends of the bipolar aptamer powder need to be reduced after receipt. The above method ensures that the bioprobe layer is firmly attached to the surface of the device.

[0068] In some embodiments, the reducing agent in the PBS solution comprises tri-(2-formylethyl)phosphonic acid hydrochloride, the concentration of the reducing agent is 8-12 mmol / L (e.g., 8 mmol / L, 10 mmol / L, 12 mmol / L, etc.), the concentration of the PBS solution is 8-12 mmol / L (e.g., 8 mmol / L, 10 mmol / L, 12 mmol / L, etc.), and the pH of the PBS solution is 7.2-7.6 (7.2, 7.5, or 7.6, etc.).

[0069] In some embodiments, the third incubation treatment time is 0.5 to 1.5 hours, for example, 0.5 hours, 1 hour, or 1.5 hours. The rotation speed of the third incubation treatment is 150 to 200 r / min. A suitable third incubation treatment time can ensure effective dissolution and mixing.

[0070] In some embodiments, the concentration of the biterminal aptamer powder is 8~12 μmol / mL, such as 8 μmol / mL, 8.5 μmol / mL, 9 μmol / mL, 10 μmol / mL, 11 μmol / mL or 12 μmol / mL.

[0071] In some embodiments, the temperature of the fourth incubation treatment is 3~8℃ (e.g., 2℃, 5℃, 8℃, 10℃, etc.), and the time of the fourth incubation treatment is 8~15h (e.g., 8h, 10h, 12h, 15h, etc.). Suitable fourth incubation conditions can ensure the stable connection of the biological probe layer.

[0072] In some implementations, the first incubation, the second incubation, and the third incubation are each carried out independently in a shaker.

[0073] In some embodiments, the (3-aminopropyl)triethoxysilane of the present invention is derived from Aladdin Shanghai Co., Ltd.

[0074] In some embodiments, the sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide of the present invention is derived from Shanghai Yuanye Biotechnology Co., Ltd.

[0075] In some embodiments, the phosphate-buffered saline (PBS) of the present invention is sourced from Shanghai Yuanye Biotechnology Co., Ltd.

[0076] In some embodiments, the tri-(2-formylethyl)phosphine hydrochloride (TCEP) of the present invention is derived from Shanghai Sangon Biotech Co., Ltd.

[0077] In some embodiments, the di-terminal aptamer powder (di-terminal thiol-modified probe nucleic acid sequence) and the single-terminal aptamer powder used in the comparative example of the present invention were synthesized and purified by Shanghai Sangon Biotech Co., Ltd. The synthetic sequence of the di-terminal aptamer powder is: 5' HS-TTTTTTTTTAAGACGGGAAGACCCCG-SH 3'. The synthetic sequence of the single-terminal aptamer powder is: 5' HS-TTTTTTTTTAAGACGGGAAGACCCCG 3'. In some embodiments, the di-terminal and single-terminal aptamer powders were synthesized using the phosphoramidite method.

[0078] In some embodiments, the preparation methods of bi-ended aptamer powder and single-ended aptamer powder include the following steps: I. Preparations before synthesis 1. Selection of solid support Single-end modified probe: Synthesis started at the 3' end using a standard CPG carrier (500 Å pore size).

[0079] Dual-end modified probe: 3'-thiol modified CPG carrier (Thiol-Modifier C6 SS CPG) was selected.

[0080] The carrier is pre-linked with thiol-modified groups via disulfide bonds.

[0081] 2. Phosphite monomer preparation Conventional dA, dC, dG, and dT monomers are prepared according to standard concentrations (0.1M acetonitrile solution).

[0082] 5' Thiol-terminal modification: Add 5'-Thiol-Modifier C6 phosphoramide monomer (for 5' end modification of single-end / double-end probes).

[0083] 3. Synthesizer parameter settings The coupling time of the mercapto monomer was extended to 300s (30s for conventional bases), and the temperature was increased to 50°C to improve the reaction efficiency.

[0084] Open the dithiol modification channel (the dual-end probe needs to call both the 3' carrier and 5' monomer ports simultaneously).

[0085] II. Solid-phase synthesis cycle steps (adding a cycle for each base) 1. Deprotection: The CPG support was treated with 3% trichloroacetic acid (TCA) solution for 30 seconds to remove the 5'-dimethoxytriphenylmethyl (DMT) protecting group and expose the free 5'-hydroxyl group.

[0086] 2. Activation and Coupling: 1) The phosphoramide monomer is mixed with the tetrazolium activator to form a highly active phosphoramide tetrazolium intermediate.

[0087] 2) The intermediate condenses with the exposed 5'-hydroxyl group of the support to form a phosphite triester bond.

[0088] The standard base coupling time is 30 s. 5' end thiol modification: A thiol monomer is replaced at the end of the sequence synthesis, extending the coupling time to 300 s.

[0089] 3. Capping: Unreacted 5'-hydroxyl 15s was treated with a mixture of acetic anhydride and N-methylimidazole (volume ratio 1:1) to acetylate and block short-chain byproducts, thereby improving the yield of full-length probes.

[0090] 4. Oxidation: Treatment with 0.02M tetrahydrofuran solution of iodine for 30 seconds converts the phosphite triester bond into a stable phosphodiester bond. Strict time control is necessary to prevent thiol group oxidation.

[0091] The loop endpoint is determined as follows: Single-end probe sequence: After the last base is added, 5' mercapto monomer is coupled.

[0092] Dual-ended probe sequence: The 3' end is provided with a thiol group by the carrier, and a 5' thiol monomer still needs to be added after coupling with the terminal base.

[0093] III. Post-processing and purification 1. Cutting and removing protection 1) Cutting: Treat with concentrated ammonia (28% by mass) at 55°C for 16 hours to break the ester bonds between DNA and the CPG vector.

[0094] 2) Deprotection: Simultaneous removal of base protecting groups (benzoyl group of A / C, isobutyryl group of G) and cyanoethyl phosphate protecting group.

[0095] 3) Reduction treatment: Add 50mM TCEP solution (pH 8.0) and shake at room temperature for 2 hours to reduce disulfide bonds (changing -SS- to -SH). 2. HAP purification (4°C) 1) Pretreatment of the chromatography column: Hydroxyapatite (HAP) packing material was suspended in 0.01M sodium phosphate buffer (pH 6.8), degassed by sonication, and then packed into the chromatography column (column bed volume ≥ 5 times the sample volume). The column was washed with 5 column volumes of equilibration buffer (0.01M sodium phosphate and 0.1M NaCl, pH 6.8) until the baseline was stable.

[0096] 2) Sample loading and binding: Dissolve the crude DNA sample (containing the cut / deprotected probe) in equilibration buffer and load the sample at a flow rate of 0.5 mL / min. The DNA probe is adsorbed by the electrostatic interaction between calcium ions and HAP and the affinity of phosphate groups, while short-chain impurities, proteins, etc. are washed away by the buffer.

[0097] 3) Gradient elution Low-salt washing of impurities: Single-stranded DNA fragments, salt and organic solvent residues were washed away using 0.12M sodium phosphate and 0.3M NaCl buffer (flow rate 1 mL / min).

[0098] Target probe elution: Linear gradient elution was used (0.2→0.4M sodium phosphate, flow rate of 0.5mL / min), and the concentration was adjusted according to the probe modification type.

[0099] Due to the enhanced negative charge of the double-terminated thiol-modified probe, the elution salt concentration needs to be increased by 0.05~0.1M compared to the conventional method.

[0100] 4) Post-processing: The eluent was concentrated and desalted using an ultrafiltration centrifuge tube (10 kDa molecular weight cutoff); it was then replaced with TE buffer (pH 8.0) containing 1 mM EDTA to prevent thiol oxidation; the solution was aliquoted into cryovials, pre-frozen at -80°C, and then freeze-dried under vacuum to obtain DNA dry powder, namely, double-ended aptamer powder and single-ended aptamer powder.

[0101] The embodiments of the present invention use the above-described bi-ended aptamer powder, while the comparative examples use the above-described single-ended aptamer powder.

[0102] The following explanation, combined with specific embodiments and comparative examples, further illustrates the point.

[0103] The field-effect transistor biosensors according to various embodiments of the present invention include a field-effect transistor chip, a surface modification layer 12, and a biological probe layer 13; the field-effect transistor chip includes: a semiconductor channel layer 5, a substrate layer 1, a bottom gate layer 2, a top gate layer 6, an Al2O3 layer 3, a SiO2 layer 4, a source electrode 8, a drain electrode 7, a passivation layer 9, an electrode layer 10, and a via 11; the bottom gate layer 2 is located on a portion of the surface of the substrate layer 1; the Al2O3 layer 3 is located on the surface of the substrate layer 1 and the surface of the bottom gate layer 2. SiO2 layer 4 is located on the surface of Al2O3 layer 3; via 11 penetrates Al2O3 layer 3 and SiO2 layer 4; top gate layer 6 is located in via 11 and is in contact with substrate layer 1; electrode layer 10 is disposed on the surface of top gate layer 6; semiconductor channel layer 5 is located on a portion of the surface of SiO2 layer 4; source 8 and drain 7 are each independently located on a portion of the surface of semiconductor channel layer 5 and SiO2 layer 4; passivation layer 9 is located on the surface of source 8, drain 7, semiconductor channel layer 5 and SiO2 layer 4. Surface modification layer 12 is located on the surface of semiconductor channel layer; biological probe layer 13 includes a bipolar aptamer, the 5' end and 3' end of which both have a modification group -SH, and the bipolar aptamer is connected to surface modification layer 11 through the modification group -SH.

[0104] Example 1 Methods for fabricating field-effect transistor biosensors, such as Figure 1a , Figure 1b and Figure 2a As shown. Includes the following steps: 1. Obtaining Field-Effect Transistor Chips A Mo layer is grown on a Si substrate 1, then NR 1500 photoresist is spin-coated onto its surface, and photolithography is performed using a first mask. The process includes exposure, development, dry etching, and photoresist removal to obtain the bottom gate layer 2. Figure 1a As shown in (a1).

[0105] An Al2O3 layer 3 is deposited on the surfaces of substrate layer 1 and bottom gate layer 2 using atomic layer deposition. For example... Figure 1a As shown in (a2).

[0106] A SiO2 layer 4 is deposited on the surface of the Al2O3 layer 3. A relatively uniform IGZO thin film is then obtained by magnetron sputtering, followed by spin-coating of a positive photoresist. Photolithography is performed using a second mask, and after exposure, development, wet etching, and photoresist removal, the semiconductor channel layer 5 is obtained. Figure 1a As shown in (a3).

[0107] A through-hole 11 is fabricated, penetrating between the SiO2 layer 4 and the Al2O3 layer 3. For example... Figure 1a As shown in (a4).

[0108] A layer of NR 1500 photoresist is spin-coated, and photolithography is performed using a third mask. After developing the pattern in the electrode area, a Ti / Au composite layer is grown by electron beam evaporation, followed by metal lift-off to obtain the source 8, drain 7, and top gate layer 6. Figure 1a As shown in (a5).

[0109] SU8 adhesive is spin-coated, and photolithography is performed using a fourth mask. After exposure and development, the SU8 adhesive is fixed on the surfaces of the source and drain electrodes to protect them and form a passivation layer 9. Figure 1a As shown in (a6).

[0110] An Ag / AgCl electrode layer 10 is fabricated on the surface of the top gate layer 6. For example... Figure 1a As shown in (a7).

[0111] A photoresist and mask were used to create a reservoir for hardening the film for 6 hours. Before using the device, the blue film on the silicon wafer was peeled off, soaked in acetone for 30 minutes, and then soaked in anhydrous ethanol for 10 minutes. After removal, nitrogen gas was used to quickly dry the device and wash away the photoresist on the electrode surface.

[0112] 2. Preparation of surface modification layer 12 A 5% (v / v) APTES solution was prepared by mixing anhydrous ethanol and (3-aminopropyl)triethoxysilane (APTES). The obtained field-effect transistor chip was immersed in the APTES solution and then incubated in a shaker at 25°C and 180 rpm for 1 hour. The device surface was then rinsed with anhydrous ethanol to remove any high-concentration solution residue, followed by rapid immersion and rinsing in anhydrous ethanol. After rinsing, the device was quickly dried with nitrogen gas to prevent the evaporation of anhydrous ethanol from leaving APTES residue on the device surface and forming white crystals. The dried device was then placed in an oven at 110°C for 30 minutes for surface silanization.

[0113] A 1 mg / mL solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfo-SMCC) was prepared using 10 mmol / L (pH 7.4) PBS. The solution was then sonicated at 50°C for 20 min. The device was immersed in the prepared sulfo-SMCC solution and incubated on a shaker at 180 rpm for 2 h. After removal, the device was rinsed thoroughly with 10 mmol / L (pH 7.4) PBS and dried with nitrogen gas to form surface modification layer 12. Figure 1a As shown in (a8).

[0114] 3. Preparation of biological probe layer 13 Tris(2-formylethyl)phosphine hydrochloride particles were dissolved in 10 mmol / L PBS (pH 7.4) to prepare a 10 mmol / L tris(2-formylethyl)phosphine hydrochloride solution. The bipolar aptamer powder (DNA probe powder) was dissolved in the tris(2-formylethyl)phosphine hydrochloride solution to a concentration of 10 μmol / mL, and incubated on a shaker for 1 h to obtain a mixed system. This mixed system was then dropped into the surface modification layer region of the aforementioned device and incubated at 4 °C for 12 h to form the biological probe layer 13. Figure 1a As shown in (a8).

[0115] Example 2 The fabrication method of a field-effect transistor biosensor includes the following steps: 1. Obtaining Field-Effect Transistor Chips The method is the same as in Example 1.

[0116] 2. Preparation of surface modification layer 12 A 6% (v / v) APTES solution was prepared by mixing anhydrous ethanol and (3-aminopropyl)triethoxysilane (APTES). The obtained field-effect transistor chip was immersed in the APTES solution and then incubated in a shaker at 24°C and 200 rpm for 1.5 h. The device surface was then rinsed with anhydrous ethanol to remove any high-concentration solution residue, followed by rapid immersion and rinsing in anhydrous ethanol. After rinsing, the device was quickly dried with nitrogen gas to prevent the evaporation of anhydrous ethanol from leaving APTES residue on the device surface and forming white crystals. The dried device was then placed in an oven at 120°C for 20 min for surface silanization.

[0117] A 1 mg / mL solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfo-SMCC) was prepared using 10 mmol / L (pH 7.4) PBS. The solution was placed in an ultrasonic machine at 45 °C and sonicated for 25 min. The device was then immersed in the prepared sulfo-SMCC solution and incubated in a shaker at 200 rpm for 1.5 h. After removal, the device was rinsed thoroughly with 10 mmol / L (pH 7.4) PBS and dried with nitrogen gas to form the surface modification layer 12.

[0118] 3. Preparation of biological probe layer 13 Tris(2-formylethyl)phosphine hydrochloride particles were dissolved in 10 mmol / L PBS (pH 7.4) to prepare a 10 mmol / L tris(2-formylethyl)phosphine hydrochloride solution. The bipolar aptamer powder (DNA probe powder) was then dissolved in the tris(2-formylethyl)phosphine hydrochloride solution to a concentration of 10 μmol / mL. The mixture was incubated in a shaker for 1.5 h to obtain a mixed system. This mixed system was then dropped into the surface modification layer region of the aforementioned device and incubated at 6 °C for 10 h to form the biological probe layer 13.

[0119] Example 3 The fabrication method of a field-effect transistor biosensor includes the following steps: 1. Obtaining Field-Effect Transistor Chips The method is the same as in Example 1.

[0120] 2. Preparation of surface modification layer 12 A 3% (v / v) APTES solution was prepared by mixing anhydrous ethanol and (3-aminopropyl)triethoxysilane (APTES). The obtained field-effect transistor chip was immersed in the APTES solution and then incubated in a shaker at 30°C and 250 rpm for 0.5 h. The device surface was then rinsed with anhydrous ethanol to remove any high-concentration solution residue, followed by rapid immersion and rinsing in anhydrous ethanol. After rinsing, the device was quickly dried with nitrogen gas to prevent the evaporation of anhydrous ethanol from leaving APTES residue on the device surface and forming white crystals. The dried device was then placed in an oven at 130°C for 10 min for surface silanization.

[0121] A 1 mg / mL solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfo-SMCC) was prepared using 10 mmol / L (pH 7.4) PBS. The solution was placed in an ultrasonic machine at 55 °C and sonicated for 20 min. The device was then immersed in the prepared sulfo-SMCC solution and incubated in a shaker at 150 rpm for 3 h. After removal, the device was rinsed thoroughly with 10 mmol / L (pH 7.4) PBS and dried with nitrogen gas to form the surface modification layer 12.

[0122] 3. Preparation of biological probe layer 13 Tris(2-formylethyl)phosphine hydrochloride particles were dissolved in 10 mmol / L PBS (pH 7.4) to prepare a 10 mmol / L tris(2-formylethyl)phosphine hydrochloride solution. The bipolar aptamer powder (DNA probe powder) was then dissolved in the tris(2-formylethyl)phosphine hydrochloride solution to a concentration of 8 μmol / mL. The solution was incubated in a shaker for 0.5 h to obtain a mixed system. This mixed system was then dropped into the surface modification layer region of the aforementioned device and incubated at 3 °C for 15 h to form the biological probe layer 13.

[0123] Example 4 The fabrication method of a field-effect transistor biosensor includes the following steps: 1. Obtaining Field-Effect Transistor Chips The method is the same as in Example 1.

[0124] 2. Preparation of surface modification layer 12 A 7% (v / v) APTES solution was prepared by mixing anhydrous ethanol and (3-aminopropyl)triethoxysilane (APTES). The obtained field-effect transistor chip was immersed in the APTES solution for wetting, and then incubated in a shaker at 20°C and 150 rpm for 2 hours. The device surface was then rinsed with anhydrous ethanol to remove high-concentration solution residue, followed by rapid immersion and rinsing in anhydrous ethanol. After rinsing, the device was rapidly dried with nitrogen gas to prevent the evaporation of anhydrous ethanol from leaving APTES residue on the device surface and forming white crystals. The dried device was then placed in an oven at 100°C for 60 minutes for surface silanization.

[0125] A 1 mg / mL solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfo-SMCC) was prepared using 10 mmol / L (pH 7.4) PBS. The solution was placed in an ultrasonic machine at 45 °C and sonicated for 35 min. The device was then immersed in the prepared sulfo-SMCC solution and incubated in a shaker at 200 rpm for 1 h. After removing the device, it was rinsed thoroughly with 10 mmol / L (pH 7.4) PBS and dried with nitrogen gas to form the surface modification layer 12.

[0126] 3. Preparation of biological probe layer 13 Tris(2-formylethyl)phosphine hydrochloride particles were dissolved in 10 mmol / L PBS (pH 7.4) to prepare a 10 mmol / L tris(2-formylethyl)phosphine hydrochloride solution. The bipolar aptamer powder (DNA probe powder) was then dissolved in the tris(2-formylethyl)phosphine hydrochloride solution to a concentration of 12 μmol / mL. The mixture was incubated in a shaker for 1.5 h to obtain a mixed system. This mixed system was then dropped into the surface modification layer region of the aforementioned device and incubated at 8 °C for 8 h to form the biological probe layer 13.

[0127] Comparative Example 1 The fabrication method of the field-effect transistor biosensor differs from that in Example 1 in that: Using a single-ended aptamer (single 5' site modification -SH), such as Figure 2b As shown.

[0128] Experimental Example I. Biosensor Characteristic Transfer Curve The characteristic transfer curve test results of the biosensor in Example 1 of this invention are as follows: Figure 3a and Figure 3b As shown. Figure 3a The Id-Vg curve is shown when the leakage voltage is between 0.005V and 1V. Figure 3b This is the Id-Vd curve when the gate voltage is between 0.3V and 0.9V. (From...) Figure 3a and Figure 3b It can be seen that the leakage current increases with increasing drain voltage and gate voltage, and the curves intersect at the origin, exhibiting a good linear relationship. This indicates that a good ohmic contact is successfully formed between the source, drain, and IGZO film of this device. Furthermore, curves with consistent linear trends are obtained when different gate voltages are applied, demonstrating that the device can be effectively controlled by the gate voltage.

[0129] II. Validation of Bioprobe Modification The success of applying the double-ended modified probe to the device surface was verified, such as... Figure 4 As shown. Since the DNA solution itself is negatively charged at pH 7.4, the transfer characteristic curve of the IGZO-FET should shift to the right and the drain current should decrease after the introduction of additional DNA. The current-voltage characteristic curve shows that the Id-Vg curve hardly changes after the APTES-SMCC modification step. After modifying both double-ended and single-ended aptamer trapping probes, the Id decreases because the trapping probes introduce additional electrons, proving that the aptamer probes are successfully connected to the sensing region (semiconductor channel layer) of the device.

[0130] III. Comparison between dual-ended modified probes and traditional single-ended modified probes Figure 5a A functionalized atomic force microscopy (AFM) image of a dual-ended functionalized probe. Figure 5b This is an atomic force microscopy image of a single-ended probe functionalized. The results show that the surface roughness of the dual-ended functionalized probe is significantly reduced compared to that of conventional single-ended modified biological probes.

[0131] IV. Comparison of detection responses between dual-ended and single-ended modified probes To further explore the advantages of dual-ended probe modification, the biosensor of Example 1 of this invention was tested at a concentration of 10... -16 Up to 10 -6 The Id-Vg standard concentration gradient curve and real-time current response of NMP22 at g / mL were plotted, and a single-ended modified probe was used as a control. The test results are as follows: Figure 6a , 6b As shown in 6c, the biosensor of Example 1 is effective at a concentration of 10 -16 Up to 10 -6 The Id-Vg standard concentration gradient curve of NMP22 at g / mL is shown in Figure 1. Figure 6a As shown, the single-end modified probe pairs well with a concentration of 10... -16 Up to 10 -6 The Id-Vg standard concentration gradient curve of NMP22 at g / mL is shown in Figure 1. Figure 6b As shown, the real-time current response is compared to, for example Figure 6c As shown in the figure. The results indicate that the response of the double-ended modified probe device is significantly improved compared to the single-ended modified probe, demonstrating that the double-ended modified probe effectively overcomes the Debye length and improves the device sensitivity.

[0132] V. Detection Limits and Response Speed The biosensor described in Example 1 was used for real-time dynamic testing of DNA standards at different concentrations. The samples used in this experiment ranged from 10... -13 mol / L to 10 -21 DNA standards were serially diluted 10-fold with mol / L, using voltages of 0.5V for both Vd and Vg. During detection, different concentrations of the standard were injected into the sensing region (semiconductor channel layer), and the current was monitored for an extended period. Finally, the drain current for each concentration was analyzed over 100 seconds, yielding the following results: Figure 7b The Id-Time real-time response curve is shown below. Figure 7bAs we can see, the device can clearly distinguish samples of different concentrations, and the fluctuations in the curves did not cause overlap. These results are consistent with the sensitivity experiments described above, demonstrating the good stability of the biosensor and further proving its sensitivity in distinguishing DNA standards at 10-fold concentrations. Ultimately, this also reflects the extremely low detection limit of this method: 10... -21 mol / L.

[0133] like Figure 7a As shown, a bar chart characterizes the device drain current magnitude when detecting each concentration. This invention uses the average drain current over 100 seconds as a benchmark and calculates the standard deviation of the drain current over 100 seconds to evaluate fluctuations. Figure 7a As can be seen, the higher the concentration, the smaller the average drain current. The currents between different concentrations can be completely distinguished with small errors, proving that the biosensor of the present invention has good dynamic stability.

[0134] like Figure 7c The image shows the Id / I0-Time graph obtained by calculating the ratio of the corresponding drain current Id to the drain current I0 obtained when detecting PBS solution, based on the Id-Time detection results of different concentrations of standard samples. Figure 7c As can be seen, with the gradual increase of sample concentration, the number of target sequences complementary to the probe bases also gradually increases. When the concentration changes, Id / I0 changes rapidly, reflecting the current influence brought by the DNA standard. Therefore, the device has a fast response speed; that is, when the concentration changes, the drain current response of the device changes rapidly. The difference in Id / I0 between different concentrations also represents the degree of differentiation between the two, and each concentration of DNA standard can be clearly distinguished. This demonstrates that the biosensor of the present invention has good dynamic stability and response speed, exhibits high stability and reliability when detecting samples, and the change in sample concentration is rapidly reflected in the drain current response.

[0135] Figure 7d The display shows the current change response during the detection of four different DNA standards. Figure 7dAs can be seen, the target sequence standard elicits a response greater than 95%; single-base mismatch standards, due to the relatively large amount of non-specific binding, also introduce additional electrons into the sensing region, leading to a decrease in drain current, with a current change response of approximately 28%; double-base mismatch and completely non-complementary DNA standards elicit current change responses of around 20%. This demonstrates that the target sequence has a far greater impact on the drain current than other incompletely complementary sequences, proving that the significant current change is indeed due to specific base complementarity pairing, rather than the use of samples different from the PBS solution causing a large change in drain current; thus, it proves that the end-modified probe of this invention has good specificity.

[0136] As can be seen from the above, the IGZO-FET biosensor using the dual-end modification method of this invention has a significant improvement in both detection response and detection limit compared to the IGZO-FET biosensor using the traditional single-end modification method.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A field effect transistor biosensor, characterized by, The field effect transistor chip includes a semiconductor channel layer, a surface modification layer on the surface of the semiconductor channel layer, and a biological probe layer.

2. The field effect transistor biosensor of claim 1, wherein, At least one of the following features (1) to (2) is included: (1) The biological probe in the biological probe layer has a U-shaped structure; (2) The double-end aptamer in the biological probe layer is used to complementarily pair with a target molecule to form a binding body, and the vertical distance between the binding body and the semiconductor channel layer is less than the Debye length.

3. The field effect transistor biosensor of claim 1, wherein, At least one of the following features (1) to (3) is included: (1) The surface modification layer includes an aminosilane layer, and the -NH2 of the aminosilane layer is connected to the modified group -SH of the double-end aptamer through a bifunctional crosslinking agent; (2) The surface modification layer includes an aminosilane layer, and the aminosilane layer includes 3-aminopropyltriethoxysilane; (3) The bifunctional crosslinking agent in the surface modification layer includes 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid succinimidyl sodium salt.

4. The field effect transistor biosensor of claim 1, wherein, At least one of the following features (1) to (2) is included: (1) The semiconductor channel layer includes IGZO; (2) The field effect transistor chip further includes a substrate layer, a bottom gate layer, a top gate layer, an Al2O3 layer, an SiO2 layer, a source, a drain, a passivation layer, an electrode layer, and a via; the bottom gate layer is located on part of the surface of the substrate layer; the Al2O3 layer is located on the surface of the substrate layer and the surface of the bottom gate layer, and the SiO2 layer is located on the surface of the Al2O3 layer; the via penetrates through the Al2O3 layer and the SiO2 layer; the top gate layer is located in the via and contacts the substrate layer; the electrode layer is arranged on the surface of the top gate layer; the semiconductor channel layer is located on part of the surface of the SiO2 layer; the source and the drain are each independently located on part of the surface of the semiconductor channel layer and the SiO2 layer; the passivation layer is located on the surface of the source, the drain, the semiconductor channel layer, and the SiO2 layer.

5. The method for fabricating a field-effect transistor biosensor as described in any one of claims 1 to 4, characterized in that, The method includes the following steps: Obtaining a field effect transistor chip; Preparing a surface modification layer on the surface of the field effect transistor chip; Anchoring a double-end aptamer to the surface of the surface modification layer under the action of a bifunctional crosslinking agent to form a biological probe layer.

6. The method of claim 5, wherein the step of forming the field effect transistor biosensor is performed by a process comprising: The method for obtaining the field effect transistor chip specifically includes: Depositing a bottom gate material on a substrate to form a bottom gate layer; Depositing an Al2O3 layer on the surface of the substrate and the bottom gate layer, and depositing an SiO2 layer on the surface of the Al2O3 layer; Growth of a semiconductor channel material on the surface of the SiO2 layer to form a semiconductor channel layer; Making a via penetrating through the SiO2 layer and the Al2O3 layer; Depositing a composite layer of Ti and Au in the partial surface of the semiconductor channel layer, the partial surface of the SiO2 layer and the via hole, forming a source electrode, a drain electrode and a top gate layer; coating a passivation material on the partial surface of the semiconductor channel layer, the source electrode, the drain electrode and the partial surface of the SiO2 layer, forming a passivation layer; Manufacturing an electrode layer on the surface of the top gate layer.

7. The method of claim 5, wherein the step of forming the field effect transistor biosensor is performed by a process comprising: Preparation of the surface modification layer on the surface of the field effect transistor chip, specifically comprising: placing the field effect transistor chip in an alcohol solution of amino silane and performing a first incubation treatment; performing alcohol solvent treatment on the field effect transistor chip after the first incubation treatment, and then performing a first drying treatment to obtain an amino silane-modified field effect transistor chip; placing the amino silane-modified field effect transistor chip in a PBS solution of a bifunctional crosslinking agent and performing a second incubation treatment, and then performing PBS solution washing and a second drying treatment to obtain a field effect transistor chip with a surface modification layer. ​ 8. The method of claim 7, wherein the step of forming the field effect transistor biosensor is performed by a process comprising: At least one of the following features (1) to (8) is included: (1) In the alcohol solution of amino silane, the volume fraction of amino silane is 3% to 7%; (2) The temperature of the first incubation treatment is 20 to 30°C, the rotation speed of the first incubation treatment is 150 to 250 r / min, and the time of the first incubation treatment is 0.5 to 2 h; (3) The alcohol solvent treatment includes washing and soaking; (4) The drying treatment includes nitrogen blowing and oven drying, the temperature of the oven drying is 100 to 130°C, and the time of the oven drying is 10 to 60 min; (5) In the PBS solution of the bifunctional crosslinking agent, the concentration of the bifunctional crosslinking agent is 0.8 to 1.2 mg / mL, the concentration of the PBS solution is 8 to 12 mmol / L, and the pH is 7.2 to 7.6; (6) The rotation speed of the second incubation treatment is 150 to 200 r / min, and the time of the second incubation treatment is 1 to 3 h; (7) The concentration of the PBS solution used in the PBS solution washing is 8 to 12 mmol / L, and the pH is 7.2 to 7.6; (8) The second drying treatment uses nitrogen blowing.

9. The method of claim 5, wherein the field effect transistor biosensor is prepared by the steps of: Preparation of the biological probe layer specifically comprises: mixing a PBS solution of a reducing agent and a double-end aptamer powder to perform a third incubation treatment, obtaining a mixed system; dropping the mixed system to the surface modification layer region of the field effect transistor chip, and then performing a fourth incubation treatment to obtain a biological probe layer.

10. The method of claim 9, wherein the step of forming the field effect transistor biosensor is performed by a process comprising: At least one of the following features (1) to (4) is included: ​ (1) In the PBS solution of the reducing agent, the reducing agent includes tri- (2-formyl ethyl) phosphine hydrochloride, the concentration of the reducing agent is 8 to 12 mmol / L, the concentration of the PBS solution is 8 to 12 mmol / L, and the pH of the PBS solution is 7.2 to 7.6; (2) The time of the third incubation treatment is 0.5 to 1.5 h; (3) The concentration of the double-end aptamer powder is 8 to 12 umol / mL; (4) The temperature of the fourth incubation treatment is 3 to 8°C, and the time of the fourth incubation treatment is 8 to 15 h.

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