SERS-FET dual-mode biosensor and its application in the detection of microcystin-LR

Abstract

The present invention relates to a SERS-FET dual-mode biosensor and its application in detecting microcystin-LR. The present invention develops a novel, simple, label-free SERS-FET dual-mode sensor by self-assembling AuNPs on the graphene surface to achieve high sensitivity and specific detection of MC-LR. Based on the SERS sensing mode, through the specific combination of MC-LR aptamer and MC-LR, the Raman fingerprint spectrum of MC-LR based on the SERS sensing mode is obtained, indicating that the SERS sensor can be used for the qualitative detection of MC-LR. Using the FET sensing mode, the detection limit of the G-FET biosensor in PBS and human serum is very low, the sensitivity is improved, and the quantitative detection of MC-LR is realized. In addition, the feasibility of the G-FET sensor is verified by analyzing MC-LR in real samples. Therefore, the combination of SERS-FET provides more options for MC-LR detection and promotes the safe monitoring of the water environment.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biosensing, and relates to a SERS-FET dual-mode biosensor and an application thereof in detecting microcystin-LR. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Microcystin-LR (MC-LR) is a common toxin that can strongly inhibit the activity of protein phosphatases and lead to liver tumor diseases. MC-LR pollution can also cause poisoning and death in wild animals, livestock, and poultry. Existing MC-LR detection methods have defects to varying degrees, such as time-consuming, cumbersome operation, low sensitivity, and most importantly, the inability to distinguish between MC-LR isomers. In order to achieve highly specific detection of biological molecules, people are working hard to develop a single biosensor. However, the specificity of a single biosensor is easily interfered by other molecules in complex system samples, thereby affecting the sensitivity of the target molecule. In order to improve the sensitivity and reliability of the sensor detection system, it is necessary to develop a dual-mode sensor with complementary advantages. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a SERS-FET dual-mode biosensor and its application in detecting microcystin-LR. The method of the present invention is simple to operate, low in cost, does not require labeling, and does not require the addition of fluorescent dyes. The combination of SERS and FET dual-mode sensors is suitable for qualitative and quantitative detection and has high sensitivity, selectivity and specificity, and good safety performance.

[0005] In a first aspect of the present invention, a SERS-FET dual-mode biosensor is provided. The SERS-FET dual-mode biosensor is composed of a glass substrate, an indium tin oxide (ITO) conductive film, an AuNPs / graphene sensing layer, and a sample pool, wherein the middle portion of the glass substrate is used as a channel, the upper surfaces of both sides of the glass substrate are covered with indium tin oxide (ITO) conductive films, the indium tin oxide (ITO) conductive films are covered with AuNPs / graphene sensing layers, the ITO conductive film on one side is used as a source level, and the ITO conductive film on the other side is used as a drain level.

[0006] Furthermore, the size of the glass substrate is 30x30x2mm; the size of the ITO conductive films on both sides is 30x12.5mm, and the thickness is 185nm.

[0007] Furthermore, the resistance of the ITO conductive film on both sides of the glass substrate is 1.0 KΩ.

[0008] Furthermore, the size of the channel is 30x5mm.

[0009] A second aspect of the present invention provides a method for preparing a SERS-FET dual-mode biosensor, comprising the following steps:

[0010] Graphene is prepared on a metal substrate by chemical vapor deposition, and then placed in an etching solution for etching. The graphene film after etching the metal substrate is cleaned and then transferred to a glass substrate covered with ITO conductive films on both sides. The graphene film is used as a bridge, and gold nanoparticles (AuNPs) are synthesized by reducing chloroauric acid with citrate in an aqueous phase. An array of AuNPs film is obtained by oil / water phase self-assembly and transferred to the surface of the graphene to form an AuNPs / graphene sensing layer.

[0011] Furthermore, the AuNPs film is uniform, array-like, and single-layer. The graphene is a single-layer graphene.

[0012] Furthermore, the chemical vapor deposition method uses metal copper as a substrate, methane as a carbon source, and hydrogen as an etching gas for growth. Preferably, the thickness of the metal copper is 200 um.

[0013] Further, the graphene is transferred to a glass substrate with an ITO conductive film, so that it is placed on the channel, one side is in contact with the source electrode, and the other side is in contact with the drain electrode. After being naturally dried, the graphene / ITO / glass substrate is placed on a heating plate for baking to ensure that the moisture is completely removed and the graphene is better solidified on the graphene surface. Preferably, the baking temperature is 110-150°C for 15-30 minutes.

[0014] Furthermore, the AuNPs solution was added to a glass dish, and then n-hexane was transferred to the upper surface of the AuNPs solution to form an oil / water interface. Then, the ethanol solution was slowly introduced into the AuNPs / n-hexane solution to form an AuNPs layer at the oil / water interface. Next, the graphene / ITO / glass substrate was immersed in the mixed solution, and the AuNPs film formed was adsorbed at the oil / water interface. Due to electrostatic adsorption, the graphene and the AuNPs film are tightly combined.

[0015] Furthermore, the etching solution is 1M FeCl 3 Solution.

[0016] Furthermore, the volume of the AuNPs solution is 20 mL, and the concentration is 0.16 mM.

[0017] Furthermore, the volume of the n-hexane solution is 20 mL.

[0018] Furthermore, the volume of the ethanol solution is 40 mL.

[0019] A third aspect of the present invention provides a method for detecting microcystin-LR based on a SERS-FET dual-mode biosensor, the method comprising the following steps:

[0020] AuNPs / graphene / ITO / glass substrate is used as SERS substrate, MC-LR aptamer is modified on its surface to capture MC-LR molecules, MC-LR solution is dropped on AuNPs / graphene / ITO / glass substrate, laser excitation is used to obtain Raman spectrum of MC-LR, and qualitative detection of MC-LR is realized; AuNPs / graphene / ITO / glass substrate is used as FET substrate, sample pool is fixed on the channel surface in the middle of glass substrate, MC-LR solution is added into the sample pool, gate is inserted into the sample pool to form FET sensor, MC-LR aptamer is modified on the surface of AuNPs / graphene to capture MC-LR molecules, the FET sensor is inserted into electrode access circuit, source-drain electrode is connected to detection circuit through probe station to detect MC-LR, and quantitative detection of MC-LR is realized.

[0021] Furthermore, the sequence of the MC-LR aptamer is 5'-SHC 6 -GGCGCCAAACAGGACCACCATGACAATTACCCATACC ACCTCATTATGCCCCATCTCCGC-3', as shown in SEQ NO.1.

[0022] Preferably, the concentration of the MC-LR aptamer is 200 nM to 1 μM.

[0023] Furthermore, in order to prevent the occurrence of nonspecific binding, ethanolamine is introduced into the channel to encapsulate the unbound sites of the MC-LR aptamer on the surface of AuNPs / graphene; preferably, the concentration of ethanolamine is 10 mM to 100 mM.

[0024] Furthermore, the concentration of the MC-LR is 100 nM to 1 μM.

[0025] Furthermore, the laser wavelength is 633nm-785nm, and the objective lens is 10x-100x.

[0026] Furthermore, the sample pool is made of a PMMA plate with a size of 20×10×10 mm. The diameter of the circular hole in the middle of the PMMA plate is 0.5 cm for containing the solution.

[0027] Furthermore, during FET detection, the gate voltage range is set to -1V-1V, the step size is 0.05V~1V, and the constant voltage of the source-drain electrode is 0.1V~0.5V.

[0028] The combination of surface enhanced Raman scattering (SERS) and field effect transistors (FET) is a good choice for improving sensitivity and reliability. SERS and FET are simple to operate, highly integrated, and can provide label-free, highly stable, highly specific and highly sensitive detection of some small biological molecules. SERS is a highly specific fingerprint spectroscopy technology that can distinguish different molecules by unique molecular vibration modes. SERS mainly uses the local electromagnetic field enhancement caused by the surface plasmon resonance (SPR) of rough noble metals (such as Au, Ag, Cu) to increase the Raman signal. However, there are still some challenges for the quantitative detection of certain molecules by SERS. FET uses the electric field effect to control the current and has certain advantages in the quantitative detection of molecules. In addition, the outstanding performance of FET is to analyze the interaction state of biomolecules by establishing a molecular interaction kinetic model to achieve trace detection of biomolecules, which opens up a new way to improve the reliability of sensitivity in the field of sensors. The combination of SERS-FET provides more options for MC-LR detection and promotes the safe monitoring of water environment.

[0029] The beneficial effects of the present invention are:

[0030] 1. Based on the existing methods for detecting MC-LR, such as bioassay, cytotoxicity assay, enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and protein phosphatase inhibition, these methods have defects to varying degrees, such as time-consuming, cumbersome operation, low sensitivity, and most importantly, the inability to distinguish between MC-LR isomers. In order to achieve highly specific detection of biological molecules, the technology of the present invention does not require labeling, is easy to operate, has a short detection time and high sensitivity, and solves the shortcomings of other detection technologies.

[0031] 2. The present invention attempts for the first time to detect MC-LR biomolecules by combining SERS-FET dual-mode biosensors, which solves the shortcomings of a single mode and transfers AuNPs into an array to the surface of graphene. The high specific surface area of ​​AuNPs and graphene, as well as the significant charge doping of AuNPs on graphene, increase the carrier concentration of graphene and significantly improve the sensitivity and reliability of the sensor detection system.

[0032] 3. The invention has a simple structure, is easy to operate, has strong practicability, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 Schematic diagram of the self-assembly process of AuNPs on graphene / ITO / glass substrate;

[0035] Figure 2 The immobilization process of MC-LR aptamer on AuNPs / graphene / ITO / glass substrate and the detection of MC-LR;

[0036] Figure 3 is the SERS spectrum of MC-LR on AuNPs / graphene / ITO / glass substrate;

[0037] Figure 4 Schematic diagram of the transmission characteristic curves and linear relationship of graphene sensors functionalized with and without MC-LR aptamers for detecting different concentrations of MC-LR in order to dilute MC-LR with PBS buffer;

[0038] Figure 5 The transmission characteristic curves of the graphene sensor functionalized with MC-LR aptamer in the presence of bovine serum albumin (BSA), MC-RR and MC-LR;

[0039] Figure 6 Schematic diagram of the transmission characteristic curves and linear relationship of graphene sensors functionalized with and without MC-LR aptamers for detecting different concentrations of MC-LR in order to dilute MC-LR with human serum albumin buffer;

[0040] Figure 7 Summary of graphs for the detection of MC-LR in real water samples using graphene sensors. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] As introduced in the background art, in order to overcome the deficiencies in the prior art, the present disclosure provides a method for qualitatively and quantitatively detecting microcystin-LR based on a SERS-FET dual-mode biosensor.

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present disclosure, the technical solution of the present disclosure will be described in detail below in combination with specific embodiments and comparative examples.

[0044] The reagents and consumables mentioned in the following examples are all commercially available products and can be purchased by those skilled in the art.

[0045] Example 1

[0046] In this embodiment, a method for qualitatively and quantitatively detecting microcystin-LR based on a SERS-FET dual-mode biosensor is provided. Figure 1 As shown, the specific steps include:

[0047] (1) Preparation of glass substrate:

[0048] The surface of the glass substrate is divided into two parts, one part is pure glass (the middle part) as the middle channel, and the other part of the glass surface is designed with indium tin oxide (ITO) conductive films on both sides, one side is the source and the other side is the drain.

[0049] (2) Preparation of graphene film

[0050] Graphene is prepared by chemical vapor deposition under low pressure and high temperature conditions, using metal copper as the substrate, methane as the carbon source, and hydrogen as the etching gas. To obtain a graphene film, the metal copper substrate needs to be etched. First, the graphene / copper is cut into 1×1cm size, and the graphene / copper is placed in the prepared etching solution to etch the metal copper, and the graphene film is cleaned with deionized water. After that, the graphene is transferred to a cleaned glass substrate with an ITO conductive film so that it is placed on the sensing channel. One side is in contact with the source and the other side is in contact with the drain. After drying naturally, the graphene / glass is placed on a heating plate for baking to ensure that the moisture is completely removed and that the graphene is better solidified on the graphene surface.

[0051] (3) Self-assembly of AuNPs

[0052] Gold nanoparticles (AuNPs) were synthesized by reducing chloroauric acid with citrate in water. Figure 1 As shown, the AuNPs solution is added to a glass dish, and then n-hexane is transferred to the upper surface of the AuNPs solution to form an oil / water interface. Then, an ethanol solution is slowly introduced into the AuNPs / n-hexane solution with a syringe to form an AuNPs layer at the oil / water interface. Next, the graphene / ITO / glass substrate is immersed in the mixed solution, and the formed AuNPs film is adsorbed at the oil / water interface. Due to electrostatic adsorption, graphene is tightly combined with the AuNPs film.

[0053] In the step (1), the size of the glass substrate is 30x30x2mm, the size of the ITO on both sides is 30x12.5mm, and the thickness is 185nm.

[0054] In the step (1), the resistance of the ITO conductive film on both sides of the glass substrate is 1.0 KΩ.

[0055] The size of the sensing channel in step (1) is 30x5mm.

[0056] In step (2), the thickness of copper is 200 μm and the etching solution is 1 M FeCl 3 Solution, baking temperature is 110-150℃, 15-30min.

[0057] In the step (2), the cleaning agents used for cleaning the glass substrate are acetone, ethanol and deionized water. Each cleaning time is 10 to 20 minutes to remove impurities and organic matter on the surface.

[0058] The volume of the AuNPs solution selected in step (3) is 20 mL and the concentration is 0.16 mM.

[0059] The volume of the n-hexane solution selected in step (3) is 20 mL.

[0060] The volume of the ethanol solution selected in step (3) is 40 mL.

[0061] The size of the plate selected in step (3) is 90 mm in diameter.

[0062] Example 2

[0063] In this embodiment, a method for qualitatively detecting microcystin-LR based on the SERS sensing mode in the SERS-FET dual-mode biosensor in embodiment 1 is provided, comprising the following steps:

[0064] (1) Functionalization of AuNPs / graphene sensing layer

[0065] like Figure 2 As shown, the present invention selects a SH-modified MC-LR aptamer chain to connect AuNPs on one hand and capture MC-LR on the other hand. In order to prevent the occurrence of non-specific binding, ethanolamine is introduced into the sensor channel (channel) to encapsulate the unbound sites of the MC-LR aptamer on the AuNPs / graphene surface.

[0066] (2) SERS test

[0067] The MC-LR solution was sucked up with a pipette and dropped onto the channel surface in the middle part of the AuNPs / graphene / ITO / glass substrate. The laser emitted by the Raman instrument was irradiated into the surface of the MC-LR solution, and the MC-LR Raman spectrum was obtained by CCD to achieve qualitative detection of MC-LR.

[0068] The concentration of the MC-LR aptamer selected in step (1) is 200 nM, and the concentration of ethanolamine is 100 mM.

[0069] The sequence of the MC-LR aptamer selected in step (1) is

[0070] 5'-SHC 6 -GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3'(60nt)

[0071] The role of ethanolamine in step (1) is to encapsulate the sites that are not bound to the aptamer to prevent nonspecific adsorption.

[0072] The concentration of MC-LR selected in step (2) is 100 nM.

[0073] The laser wavelength selected in step (2) is 633 nm, the laser power is 14 mW, the excitation light energy is 5%, the spot size is 1 μm, the objective lens is 50x, and the integration time is 4 s.

[0074] Example 3

[0075] In this embodiment, a method for qualitatively detecting microcystin-LR based on the FET sensing mode in the SERS-FET dual-mode biosensor in Example 1 is provided, comprising the following steps:

[0076] (1) Preparation of G-FET

[0077] A sample cell was pasted on the AuNPS / graphene / ITO / glass substrate, and then the sample solution was added into the sample cell and the Ag / AgCl gate was inserted into the sample cell to form a graphene field effect transistor (G-FET) sensor.

[0078] (2) Functionalization of AuNPs / graphene sensing layer

[0079] like Figure 2 As shown, the present invention selects a SH-modified MC-LR aptamer chain to connect AuNPs on one hand and capture MC-LR on the other hand. In order to prevent the occurrence of nonspecific binding, ethanolamine is introduced into the sensor channel to encapsulate the unbound sites of the MC-LR aptamer on the AuNPs / graphene surface.

[0080] (3) G-FET sensor detection MC-LR

[0081] The sensor is inserted into the electrode access circuit, and the source-drain electrode is connected to the detection circuit through a probe station to perform MC-LR detection, thereby realizing quantitative detection of MC-LR.

[0082] The material of the sample pool selected in step (1) is a solid PMMA plate with a size of 20×10×10 mm. The diameter of the circular hole in the middle of the PMMA plate is 0.5 cm, which is used to hold the solution.

[0083] The concentration of the MC-LR aptamer selected in step (2) is 200 nM, and the concentration of ethanolamine is 100 mM.

[0084] The sequence of the MC-LR aptamer selected in step (2) is

[0085] 5'-SHC 6 -GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3'(60nt)

[0086] The role of ethanolamine in step (2) is to encapsulate the sites that are not bound to the aptamer to prevent nonspecific adsorption.

[0087] In the step (3), the gate voltage range is set to -1V-1V, the step length is 0.05V, and the constant voltage of the source-drain electrode is 0.1V.

[0088] Figure 3 Schematic diagram of qualitative detection of microcystin-LR based on the SERS sensing mode in the SERS-FET dual-mode biosensor in Example 2. The SERS measurement results of MC-LR are shown in FIG. Figure 3 As shown, the SERS spectrum provides the unique “fingerprint” information of MC-LR on AuNPs / graphene / ITO / glass substrate, such as 640, 748, 757, 845, 889, 975, 1019, 1188, 1211, 1267, 1294, 1310, 1382, 1458, 1493, 1530, 1581 cm -1 The change in Raman shift can be attributed to the vibration mode of MC-LR molecules, which indicates that MC-LR molecules are captured by the specific binding of MC-LR aptamers on the surface of AuNPs.

[0089] Figure 4 Schematic diagram of quantitative detection of microcystin-LR by FET sensing mode in SERS-FET dual-mode biosensor in Example 3. In order to evaluate the performance of G-FET sensor, we measured the concentration of MC-LR in PBS buffer. -18 ~ 1x10 -8M of MC-LR solution was introduced into the sensor sequentially. As the concentration of MC-LR increased, the V CNP Gradually shifting to the right ( Figure 4 A). In general, the minimum detection limit of the sensor is estimated to be 3 times the standard deviation (3σ) of the sensor response to the blank solution. Here, the G-FET sensor showed a very low detection limit of about 0.62aM. The extremely low detection limit of this sensor can be attributed to the high specific surface area of ​​AuNPs and graphene. AuNPs produce significant charge doping on graphene, increasing the carrier concentration of graphene. Studying the interaction affinity between the aptamer and MC-LR is particularly important for the sensitivity of the sensor. Therefore, the equilibrium association constant (K a ) and the comprehensive coordination number (n) are calculated by the following equations (1-5). The interaction between the aptamer and MC-LR can be expressed by equation (1)

[0090]

[0091] [MC-LR] and [apt] indicate the concentrations of MC-LR and aptamer, respectively.

[0092] From equation (2), we can calculate K a

[0093]

[0094] Taking the logarithm of equation (2) yields equation (3)

[0095]

[0096] According to Lambert-Beer's law, Figure 4 ΔV in B CNP Proportional to the concentration of MC-LR.

[0097]

[0098] MC 0 is the V of G-FET before adding MC-LR CNP MCx is the V of MC-LR after adding x M CNP , M.C. 1 is the maximum concentration of MC-LR.

[0099] Finally, equation (4) can be simplified to equation (5)

[0100] y=nlg[MC-LR]+lgK a (5)

[0101] Therefore, K is calculated by y = 2.64x + 5.85 aThe value is 7.07×10 5 , n is 2.64( Figure 4 B) The high affinity between MC-LR and the aptamer indicates that the selected aptamer is suitable for the detection of MC-LR, and it is expected to obtain a sensor with high specificity. In addition, a control experiment was performed to test the inertness of AuNPs / graphene to MC-LR. Figure 4 CD shows graphene sensors without aptamer functionalization exposed to different concentrations of MC-LR. CNP The displacement of α was negligible (<3 mV), indicating that the sensor is suitable for MC-LR detection with high stability.

[0102] Figure 5 The specificity of G-FET for MC-LR was further tested by electrical detection of bovine serum albumin (BSA), MC-RR and MC-LR. Figure 5 As shown in A and B, at the same concentration of 100 nM, the electrical response of the graphene sensor induced by MC-LR was significantly higher than that of BSA and MC-RR. The excellent specificity of the sensor is mainly attributed to the high affinity between the aptamer and MC-LR.

[0103] Figure 6 In order to confirm the feasibility of MC-LR detection in complex components, the G-FET sensor was tested in human serum. Figure 6 As shown in A, V CNP The shift of V of the G-FET sensor is closely related to the concentration of MC-LR in human serum. CNP Gradually shifts to the left. V of MC-LR CNP It showed a good linear dependence on the logarithm of concentration and exhibited good sensing performance in serum. In addition, we also found that with the increase of MC-LR concentration, V CNP With PBS V CNP This can be explained by the fact that MC-LR shows an opposite charge. In contrast to PBS, MC-LR in serum is negatively charged, resulting in V CNP Shift to the left. The affinity and comprehensive coordination number n between MC-LR and aptamer can be calculated from formula (1-5): K a =1.4x10 6 , n = 1.12( Figure 6 B). The minimum detection limit of the sensor for MC-LR in serum is 0.91aM, indicating that the graphene sensor can be used to detect trace amounts of MC-LR in serum.

[0104] Table 1 shows the detection of MC-LR in real water samples by G-FET sensor. Compared with buffer and serum, real water samples are very complex systems containing many interfering molecules such as biopesticides, bioherbicides, insecticides, body secretions and bioproductivity agents. The applicability and practical application ability of the developed G-FET biosensor in detecting MC-LR using the standard addition method were evaluated. We selected deionized water (DI water), tap water (Tap water) and fresh lake water after centrifugal filtration as real water samples, and added low concentrations of MC-LR to these three water samples. The MC-LR concentrations of each water sample were 50pM, 100pM and 175pM, respectively. The developed G-FET sensor had a high detection rate for the three samples. As shown in the table, in DI water, Tap water and Fresh lake water, the recovery rate was 92.59% to 106.94%, and the standard deviation was 4.3% to 12.8%, indicating that the graphene sensor has high sensitivity and accuracy in the quantitative determination of MC-LR. In addition, the relative standard deviation (RSD) of the measured values ​​of MC-LR is very low in DI water, Tap water, and Fresh lake water, indicating that the graphene sensor has high stability and can be widely used.

[0105]

[0106] a 50 pM MC-LR was added to DI water, Tap water and Fresh lake water, respectively.

[0107] b 100 pM MC-LR was added to DI water, Tap water and Fresh lake water, respectively.

[0108] c 175 pM MC-LR was added to DI water, Tap water and Fresh lake water, respectively.

[0109] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Texas College <120> SERS-FET dual-mode biosensor and its application in the detection of microcystin-LR <130> 2022 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 60 <212> DNA <213> Artificial sequence <400> 1 ggcgccaaac aggaccacca tgacaattac ccataccacc tcattatgcc ccatctccgc 60

Claims

1. A method for detecting microcystin-LR based on a SERS-FET dual-mode biosensor, characterized in that: The SERS-FET dual-mode biosensor is composed of a glass substrate, an indium tin oxide (ITO) conductive film, an AuNPs / graphene sensing layer, and a sample pool. The middle part of the glass substrate is used as a channel, the upper surfaces of both sides of the glass substrate are covered with indium tin oxide (ITO) conductive films, the indium tin oxide (ITO) conductive films are covered with AuNPs / graphene sensing layers, the ITO conductive film on one side is used as a source electrode, and the ITO conductive film on the other side is used as a drain electrode; Graphene is prepared on a metal substrate by chemical vapor deposition, and then placed in an etching solution for etching. The graphene film after etching the metal substrate is cleaned and then transferred to a glass substrate with ITO conductive films on both sides. The graphene film is used as a bridge, and gold nanoparticles (AuNPs) are synthesized by reducing chloroauric acid with citrate in an aqueous phase. An array of AuNPs film is obtained by oil / water phase self-assembly, and then transferred to the surface of graphene to form an AuNPs / graphene sensing layer. The method comprises the following steps: using AuNPs / graphene / ITO / glass substrate as a SERS substrate, modifying a microcystin-LR (MC-LR) aptamer on the surface thereof for capturing MC-LR molecules, dropping a MC-LR solution on the AuNPs / graphene / ITO / glass substrate, using laser de-excitation to obtain a Raman spectrum of MC-LR, and realizing qualitative detection of MC-LR; using AuNPs / graphene / ITO / glass substrate as a field effect transistor FET substrate, fixing a sample pool on the channel surface in the middle of the glass substrate, adding the MC-LR solution into the sample pool, inserting a gate into the sample pool to form a FET sensor, modifying a MC-LR aptamer on the surface of AuNPs / graphene for capturing MC-LR molecules, inserting the FET sensor into an electrode access circuit, and connecting a source-drain electrode to a detection circuit through a probe station to detect MC-LR, and realizing quantitative detection of MC-LR; The size of the glass substrate is 30x30x2mm; the size of the ITO conductive films on both sides is 30x12.5mm and the thickness is 185nm; the size of the channel is 30x5mm; the resistance of the ITO conductive films on both sides of the glass substrate is 1.0KΩ; The concentration of the MC-LR aptamer is 200nM to 1μM; The sequence of the MC-LR aptamer is 5'-SHC6-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3'.

2. The method according to claim 1, characterized in that Ethanolamine was introduced into the channel to encapsulate the unbound sites of the MC-LR aptamer on the AuNPs / graphene surface.

3. The method according to claim 2, characterized in that The concentration of ethanolamine is 10 mM to 100 mM.

4. The method according to claim 1, characterized in that: The concentration of the MC-LR is 100 nM to 1 μM; The laser wavelength is 633nm~785nm, 10x~100x objective lens; The sample pool is made of a PMMA plate with a size of 20×10×10 mm. The diameter of the circular hole in the middle of the PMMA plate is 0.5 cm, which is used to hold the solution. The gate voltage range was set to -1V-1V with a step size of 0.05V to 1V, and the constant voltage of the source-drain electrodes was 0.1V to 0.5V.

5. The method according to claim 1, characterized in that The chemical vapor deposition method uses metal copper as a substrate, methane as a carbon source, and hydrogen as an etching gas for growth.

6. The method according to claim 5, characterized in that The thickness of the metal copper is 200 μm.

7. The method according to claim 1, characterized in that The graphene is transferred to a glass substrate with an ITO conductive film so that it is placed on the channel with one side in contact with the source and the other side in contact with the drain. After drying naturally, the graphene / ITO / glass substrate is placed on a heating plate for baking to ensure that the moisture is completely removed and that the graphene is better solidified on the graphene surface.

8. The method according to claim 7, characterized in that The baking temperature is 110-150°C and the time is 15-30 minutes.

9. The method according to claim 1, characterized in that: The AuNPs solution was added to a glass dish, and then n-hexane was transferred to the upper surface of the AuNPs solution to form an oil / water interface. The ethanol solution was then slowly introduced into the AuNPs / n-hexane solution to form an AuNPs layer at the oil / water interface. Next, the graphene / ITO / glass substrate was immersed in the mixed solution, and the AuNPs film formed was adsorbed at the oil / water interface.

10. The method according to claim 1, characterized in that The etching solution is 1MFeCl3 solution.

11. The method according to claim 9, characterized in that The volume of AuNPs solution was 20 mL, and the concentration was 0.16 mM.

12. The method according to claim 9, characterized in that The volume of the n-hexane solution was 20 mL.

13. The method according to claim 9, characterized in that The volume of the ethanol solution was 40 mL.

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