Transistor sensor for detecting pancreatic cancer marker and preparation method thereof

Abstract

The invention provides a transistor sensor for detecting pancreatic cancer markers and a preparation method thereof. A trace amount of miRNA-196a in the solution can be detected by detecting the current change in the channel; through a synergistic anti-pollution design, bovine serum albumin (BSA) is used as a blocking agent, polyadenine (polyAn) forms a hydrophilic layer, and the bovine serum albumin (BSA) and the polyadenine (polyAn) complement each other and have a synergistic effect, so that non-specific adsorption of polluting biomolecules is avoided. The graphene transistor biosensor provided by the invention can be used for detecting an extremely trace amount of miRNA-196a with the concentration as low as 10 <-18 > M, the response time is about 18 minutes, and the R2 value of a fitting straight line is 0.998; and obvious and correct Dirac point voltage change signals exist in a complex plasma environment. Compared with the prior art, the technical scheme provided by the invention not only maintains the advantages of high sensitivity, good specificity and the like of the graphene transistor sensor, but also endows the sensor with the performance of resisting nonspecific adsorption of biomolecules due to the introduction of the anti-pollution material.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to a method for preparing a novel anti-pollution liquid-gate graphene transistor biosensor for detecting pancreatic cancer plasma exosome miRNA-196a. Background Art

[0002] In recent years, the incidence of pancreatic cancer has shown a clear upward trend. According to statistics from the National Cancer Center of China in 2021, pancreatic cancer ranks sixth in malignant tumor-related mortality. Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. Most PDACs are diagnosed in the late stage and are not eligible for therapeutic surgery, resulting in a diagnosis rate that is almost consistent with the mortality rate. Therefore, early diagnosis and screening of PDAC are key to reducing patient mortality. Exosomes (EVs) are small extracellular vesicles (30-200nm) released in large quantities by healthy and tumor cells through the endocytic pathway and enter the blood circulation, making intercellular communication and cargo transfer possible. More and more studies have shown that plasma exosomal miRNA markers for pancreatic cancer are expected to be used for pancreatic cancer detection, but the detection of low-expression levels of exosomal miRNAs in cancer remains a challenge, so it is of great significance to carry out detection in this regard.

[0003] Currently, methods for detecting specific exosomal miRNAs, such as reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry, usually take a long time to complete from sampling to results and require complex processing steps. In recent years, many sensor technologies have been developed to improve the limit of detection (LOD) and specificity of exosome detection, including graphene transistor sensors, electrochemical capacitive sensors, in situ fluorescence, and surface-enhanced Raman scattering, among which only a few studies specifically detect exosomal biomarkers in pancreatic patient samples.

[0004] Due to the complex plasma sensing environment and low expression levels of pancreatic cancer exosomal miRNA markers, existing methods still lack the specificity and ability to detect cancer. n ), polysorbate (Tween-20) and other anti-pollution designs, but there are disadvantages such as high cost, complex operation, and poor anti-pollution performance. Therefore, there is an urgent need to develop a diagnostic platform with strong anti-pollution properties, high specificity, high sensitivity, and the ability to specifically detect pancreatic cancer exosome miRNA in clinical samples. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of an anti-pollution liquid-gate graphene transistor biosensor for detecting pancreatic cancer plasma exosome miRNA-196a. In the present invention, a liquid-gate graphene transistor biosensor with synergistic anti-pollution properties is designed.

[0006] The present invention provides an anti-pollution liquid-gate graphene transistor biosensor for detecting pancreatic cancer plasma exosome miRNA-196a, comprising electronic-grade glass and a gate, a source and a drain arranged on the electronic-grade glass, wherein a graphene channel is arranged between the source and the drain; a thiol-modified DNA single-stranded probe, BSA, polyA are fixed in sequence on the surface of the gate. n The present invention fixes a single-stranded DNA probe on the surface of the graphene transistor gate electrode, and the DNA can hybridize with miRNA-196a through base complementary pairing, thereby changing the double electric layer interface characteristics between the transistor and the sample solution, causing the current in the graphene channel to change. By detecting the current change in the channel, the trace miRNA-196a in the solution can be detected; BSA is modified on the gate by adsorption, thereby improving the stability and biocompatibility of the gold electrode, and at the same time, as a blocker, it occupies the nonspecific binding site of the gate to avoid nonspecific adsorption; polyA n Forming van der Waals force adsorption with the gold surface, polyA n (Polyadenylic acid tail) has strong hydrophilicity due to its characteristics such as hydrogen bond formation with water molecules and negative charge of phosphate group, so that it forms a hydrophilic layer on the surface of the gate to avoid hydrophobic nonspecific adsorption of contaminating biological molecules. Through ultracentrifugation, plasma exosomes are extracted and lysed by 0.5% TritonX-100 for clinical sample testing.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a graphene transistor biosensor, comprising a substrate and a gate, a source and a drain arranged on the substrate; a graphene channel is arranged between the source and the drain; a CVD single-layer graphene is transferred to the graphene channel by a wet method; a single-stranded DNA biomolecular probe that can be complementary to the miRNA-196a base is fixed on the surface of the gate; polyadenine and bovine serum albumin are used as synergistic anti-pollution biomolecules to modify the gate.

[0008] (1) Preparation of liquid-gate graphene transistors

[0009] The graphene transistor is composed of a substrate, a gate, a source, a drain and a graphene channel. The transistor is made by a thermal evaporation coating method using a JSD-300 evaporation coating system. There is a graphene channel between the source and drain of the transistor. A single layer of graphene is flatly attached to the source and drain channels of the transistor to obtain a graphene transistor sensor.

[0010] (2) Preparation of anti-fouling liquid-gate graphene transistor biosensor for pancreatic cancer plasma exosomal miRNA-196a

[0011] Under room temperature and dark conditions, a single-stranded DNA probe with a thiol group at the 5′ end was modified onto the gate surface to form a stable Au-S bond between the thiol group and the gold electrode; n The affinity for the gold electrode surface was modified with BSA and polyA in sequence. n On the gate. Construction of anti-fouling liquid-gate graphene transistor biosensor for pancreatic cancer plasma exosomal miRNA-196a.

[0012] In the above preparation method, in step (1), a mask method vacuum coating is used, and the vacuum degree of the vacuum is 3× 10 -4 ~6 × 10 -4 Pa or less, more preferably 4 × 10 -4 Pa.

[0013] In the above preparation method, in step (1), the gate, source and drain are composed of a chromium layer and a gold layer, and the chromium layer is located between the substrate and the gold layer.

[0014] In the above preparation method, in the step (1), the evaporation current of the chromium layer is preferably 170-180A; and the evaporation current of the gold layer is preferably 110-120A.

[0015] In the above preparation method, in step (1), the thickness of the evaporated gold is 20 to 30 nm, and the thickness of the evaporated chromium is about 3 to 4 nm.

[0016] In the above preparation method, in step (1), the width of the graphene channel of the transistor is 0.2-0.3 mm, and the length of the graphene channel is 4-8 mm.

[0017] In the above preparation method, in step (1), the width of the single-layer graphene is 0.5 to 2 mm, and the length of the graphene is 4 to 6 mm.

[0018] In the above preparation method, in the step (1), the graphene is transferred by wet transfer to transfer the single-layer graphene to the channel between the source and the drain.

[0019] In the above preparation method, in step (1), after the transfer of graphene is completed, the transferred product needs to be annealed at a temperature of 100 to 130° C., preferably 120° C.

[0020] In the above preparation method, in step (2), the DNA single-stranded probe powder is dissolved in enzyme-free water, and the preparation concentration is preferably 1 mM. The gate incubation conditions are: immersing 10 mL of the probe at 28°C and incubating for 10 to 14 hours, more preferably, incubating for 12 hours.

[0021] In the above preparation method, in step (2), the solvent of BSA is 0.1×PBS solution, and the concentration of BSA is 0.05%~0.2%, and the preferred concentration is 0.1% BSA.

[0022] In the above preparation method, in step (2), the gate of the modified probe is immersed in 10 mL BSA, and the incubation conditions are: at 28°C, and the modification is carried out for 0.5 to 2 hours, more preferably 1 hour.

[0023] In the above preparation method, in step (2), polyA n The number of adenines in the polyadenine chain is 4 to 20, preferably 8, i.e. polyA8, and the sequence is "5¢-AAAAAAAA-3¢". The polyA8 solvent is 0.1×PBS solution, and the concentration is 1 to 10 mM, preferably 5 mM.

[0024] In the above preparation method, in step (2), the modified probe and the BSA gate are immersed in 10 mL polyA n The incubation conditions are: at 4°C for 0.5 to 2 hours, more preferably 1 hour.

[0025] Compared with the prior art, the above technical solution conceived by the present invention can achieve the following beneficial effects:

[0026] (1) The present invention fixes the designed single-stranded DNA probe on the surface of the graphene transistor gate electrode, and can specifically detect trace miRNA-196a biomolecules in the solution by detecting the displacement of the Dirac point voltage and the current change in the channel, with a minimum detection limit of 10 -18 M, and has a good linear relationship.

[0027] (2) The present invention introduces the advantages of graphene transistors into the detection of miRNA-196a, making it very sensitive to voltage changes. A small voltage change will cause a corresponding current change, greatly shortening the detection time. The detection time can be controlled within 15 minutes. When different miRNA-196a biomolecule concentrations are added, the current of the sensor changes in real time, and it has a very high sensitivity.

[0028] (3) Compared with traditional detection methods, the present invention provides polyA n The synergistic anti-pollution design with BSA has a better anti-pollution effect than a single anti-pollution biological molecule, can be used in complex detection environments, and has greater application potential.

[0029] Compared with the existing technology, the above technical solution conceived by the present invention not only maintains the advantages of graphene transistor sensors such as high sensitivity, good specificity, simple operation and low cost, but also has the ability to resist nonspecific adsorption of biological molecules due to the introduction of anti-pollution materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the graphene transistor biosensor in Example 1 of the present invention; Figure 2 This is a schematic diagram of a device preparation process of a graphene transistor in Example 1 of the present invention; Figure 3 Schematic diagram of the gold gate modification process of the graphene transistor biosensor in Example 1 of the present invention Figure 4 This is a schematic diagram of a graphene transistor device in Example 1 of the present invention; Figure 5 Graphs showing water contact angles of differently modified gate electrodes in Example 2 of the present invention; Figure 6 The influence of the devices with different modifications in Example 2 of the present invention on the Dirac point voltage in plasma; Figure 7 The transfer characteristic curve of the anti-pollution liquid-gate graphene transistor biosensor in Example 2 of the present invention when detecting the concentration of miRNA-196a biomolecules; Figure 8 This is a correlation diagram of the output characteristic curve when the anti-pollution liquid-gate graphene transistor biosensor in Example 2 of the present invention detects the concentration of miRNA-196a biomolecules; Fig. 9 This is a standard curve of the change in channel current of the anti-pollution liquid-gate graphene transistor biosensor and the change in miRNA-196a biomolecule concentration in Example 2 of the present invention. Implementation

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Examples of certain embodiments of the present invention are given below, which are not intended to limit the scope of the present invention. Example

[0033] (1) Preparation of graphene transistors

[0034] Through the JSD-300 evaporation coating system, chromium and gold are sequentially plated onto the substrate in the evaporation coating instrument by the mask method. The transistor substrate used is preferably electronic grade glass, silicon wafer or PET, more preferably electronic grade glass; there is no special limitation on the size of the substrate glass, and the size is preferably 12 mm×12 mm. In the present invention, the electronic grade glass is preferably GL-10173-1.1. Among them, there is no special limitation on the specific parameters of the thermal evaporation coating method, and the corresponding parameters are based on the thickness of the prepared electrode.

[0035] In the embodiment of the present invention, the thermal evaporation coating is preferably carried out under vacuum conditions; the vacuum degree of the vacuum is 3× 10 -4 ~6 × 10 -4 Pa or less, more preferably 4 × 10 -4 Pa. In an embodiment of the present invention, the electrode is composed of a chromium layer and a gold layer, wherein the chromium layer is located between the substrate and the gold layer, the thickness of the chromium layer is 3-4 nm, the thickness of the gold layer is 20-30 nm, the evaporation current of the chromium layer is preferably 170-180 A; the evaporation current of the gold layer is preferably 110-120 A. The graphene channel is located between the source and the drain, the width of the channel is 0.2-0.3 mm, and the length of the channel is 4-8 mm. The shape and structure of the obtained electrode are as follows Figure 1 shown. Figure 1 In the figure, G is gate, S is source, and D is drain. The 0.25 mm wide channel between the source and the drain becomes the graphene channel after graphene is transferred.

[0036] (2) Graphene transfer

[0037] The present invention has no special limitation on the operation of the wet transfer, and the technical solution of wet transfer of single-layer graphene known to those skilled in the art can be used. In the present invention, the technical solution of wet transfer of single-layer graphene is preferably referred to the technical solution disclosed in: Chen Mu, Yan Yue, Zhang Xiaofeng, et al. Research Progress of Large-area Graphene Film Transfer Technology [J]. Journal of Aeronautical Materials, 2015, 35(2): 1-11.

[0038] Spin-coat a polymethyl methacrylate film (400-600 nm, preferably 500 nm, PMMA) on a single-layer copper-based graphene surface prepared in advance (the single-layer graphene is preferably a copper-based CVD single-layer graphene, the source of which is a commercially available product known to those skilled in the art, or prepared according to a preparation method known to those skilled in the art). After spin-coating PMMA, heat at 100 ° C for 10 minutes to fix PMMA. Before attaching graphene, soak one side of the graphene copper substrate in a ferric chloride solution of appropriate concentration for 2-4 hours to remove the surface copper, preferably for 3 hours, depending on the transparency of the graphene. After washing the graphene with PMMA three times with deionized water, lay the single-layer graphene flat on the graphene channel, and after annealing, obtain a graphene transistor. In the embodiment of the present invention, the annealing is first annealed at 60-70 °C for 10 minutes; the annealing time is preferably 20-40 minutes, more preferably 30 minutes, preferably 100-120 °C, more preferably 110 °C. After the annealing is completed, the moisture on the surface of the sample can be removed, and the graphene can be more closely combined with the electrode substrate. The width of the single-layer graphene is 0.5-2 mm, the length is 4-8 mm, preferably 1.5×6 mm.

[0039] Then, the prepared graphene transistor needs to be soaked in acetone at 70°C for 3 hours to dissolve the residual substances on the graphene. After washing with anhydrous ethanol and deionized water and drying at room temperature, the transfer of graphene is completed. The present invention preferably anneals the transferred device in a nitrogen atmosphere in a glove box to obtain a graphene transistor. In the present invention, the annealing temperature is preferably 100-130°C, more preferably 110°C; the annealing time is preferably 20-40 minutes, more preferably 30 minutes. In the present invention, the annealing can remove moisture from the surface of the sample and make the graphene and the electrode more closely bonded. The graphene transistor can measure stable IV The curve is shown in Figure 2, and the Dirac point is stable between 0.4 and 0.7 V. The graphene transistor sensor device is prepared as follows: Figure 2 shown.

[0040] (3) Probe, BSA, polyA n Modification of the gate of the graphene transistor sensor

[0041] The single-stranded DNA probe freeze-dried powder is dissolved in enzyme-free water, and the preparation concentration is preferably 1 mM. The thiol group modified at the 5¢ end forms an Au-S bond with the gold surface for fixation. The gate incubation conditions are: immerse in 10 mL of the probe and incubate for 10 to 14 hours at 28°C, more preferably, incubate for 12 hours. The solvent of BSA is 0.1×PBS solution, with a concentration of 0.15% to 0.2%, preferably 0.1% BSA. The gate of the modified probe is immersed in 10 mL BSA, and the incubation conditions are: at 28°C, the modification is 0.5 to 2 hours, more preferably 1 hour; polyA n The number of adenines in the polyadenine chain is 4 to 20, preferably 8, i.e. polyA8, with a sequence of "5¢-AAAAAAAA-3¢". The polyA8 solvent is 0.1×PBS solution, with a concentration of 1 to 10 mM, preferably 5 mM; the modified probe and BSA gate are immersed in 10 mL polyA n In the n The incubation conditions are: 4 °C for 0.5 to 2 hours, preferably 1 hour. Figure 3 The schematic diagram of the device structure is shown in Figure 4 shown. Example

[0042] Application of anti-fouling liquid-gate graphene transistor biosensor for pancreatic cancer plasma exosome miRNA-196a

[0043] The graphene transistor biosensor prepared in Example 1 was used to test the miRNA-196a target molecule. The source, drain and gate electrodes of the graphene transistor were connected to two digital source meters (Keithley 2450 / Keithley 2400). The gate voltage V G and source-drain voltage V DS It is controlled by a Labview program in the computer.

[0044] like Figure 1As shown in the figure, the detection principle is that the gate electrode and the graphene channel are connected by the electrolyte to form a "double capacitor" structure, and the voltage applied between the gate and the graphene channel is constant. The voltage applied to the gate is fixed with the DNA single-stranded probe, which is complementary to the base pairing of miRNA-196a, and the double electric layer interface characteristics are changed, which causes the change of the current in the graphene channel. By detecting the change of the current in the channel, the trace miRNA-196a in the solution can be quantitatively detected. The modified gate electrode is thoroughly washed with PBS solution to remove the residue left on the electrode. The test is carried out in a container containing 100 μL PBS solution. During the test, miRNA solutions of specific concentrations are added to the PBS solution in sequence to obtain output signals at different concentrations.

[0045] Sensing interface hydrophilicity test: Figure 5 As shown in the contact angle test of the gate sensing interface, the static water contact angle of the bare electrode is 88°, and after the electrode surface is modified with DNA single-stranded probes, the static water contact angle decreases to 39°. n Later, the static water contact angle decreased to 19°, which was due to the polyA n (Polyadenine tail) has strong hydrophilicity due to its hydrogen bond formation with water molecules and the negative charge of the phosphate group, which causes a significant decrease in the static water contact angle.

[0046] Anti-pollution test: Figure 6 As shown in the figure, the effect of different modified devices on the Dirac point voltage in plasma. BSA occupies the nonspecific binding site on the gate interface, blocking the nonspecific adsorption of proteins in plasma. At the same time, the hydrophilicity of polyA8 forms a hydrophilic layer on the gate surface, avoiding the hydrophobic nonspecific adsorption of contaminating biological molecules. Figure 6 As shown, after adding plasma, the modified BSA and polyA n The influence of signal error caused by plasma on the device will be greatly reduced, and it has excellent anti-pollution performance; at the same time, BSA and polyA n Significant Dirac point voltage change signals can be detected in a complex plasma environment.

[0047] Transfer characteristic curve test: The source-drain voltage is set to a constant value ( V DS = 0.1V), the channel current between the source and drain is measured when the gate voltage changes continuously from 0 V to 1 V I DS Then the concentration of miRNA-196a biomolecules in the solution was changed and measured in sequence; the transfer characteristic curve obtained is shown in Figure 7As shown in Figure 2, the change in the interface will change the potential on the device surface, causing the characteristic curve to move. Figure 7 It can be seen that as the concentration of miRNA-196a changes, the Dirac point voltage moves accordingly.

[0048] Dirac point voltage fitting curve drawing: Figure 7 The transfer characteristic curve in the above example is measured multiple times to obtain the average value of the Dirac point voltage change. The Dirac point voltage change value and the miRNA concentration show a good linear relationship, such as Figure 8 As shown, the fitting straight line R 2 The value is 0.998.

[0049] Output characteristics test: Source-drain voltage and gate voltage are set to constant values ​​( V DS = 0.1 V and V GS = 0.8 V), and continuously measure the relationship between channel current and time. During the test, wait until the channel current stabilizes before adding the next concentration. The concentration increases from 10 -18 M, 10 -16 M, 10 -14 M, 10 -12 M, 10 -10 M, 10 -8 M changes successively; the test results are as follows Fig. 9 shown; according to Fig. 9 It can be seen that the concentration change of miRNA-196a will cause a significant change in the current. The magnitude of the current change can reflect the magnitude of the concentration change. After the concentration changes, the current of the sensor will change over time. The response time is about 18 minutes, and the sensitivity is very high. The graphene transistor biosensor provided by the present invention can detect the concentration of miRNA-196a in the solution as low as 10 -18 Detection of extremely trace amounts of miRNA-196a in M.

[0050] Clinical sample test: plasma exosomes were extracted by ultracentrifugation at 4°C, preferably at 120,000 g and 70 min. After lysis with 0.5% TritonX-100, the exosomes were heated at 60-80°C for 3-10 min, preferably at 70°C for 5 min, to fully lyse the exosomes, and the lysed solution was tested for clinical samples. The clinical sample operating characteristic curve (ROC) showed that the area under the ROC was 0.98. This shows that in the clinical sample test, the designed transistor sensor can accurately distinguish pancreatic cancer patients from pancreatitis patients and healthy people.

[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be noted that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention.

Claims

1. A transistor sensor for detecting pancreatic cancer markers, wherein the gold gate surface of the sensor is modified with a DNA single-stranded probe that can detect early pancreatic cancer plasma exosome miRNA-196a, as well as bovine serum albumin (BSA) and polyadenine (polyA n ) Collaborative anti-pollution design. The detection principle is that through the combination between the marker and the probe, the charge carried by the marker itself causes the change of Dirac point voltage or channel current, and sensing is achieved by detecting the change of Dirac point voltage or channel current.

2. The anti-pollution design and modification method of the transistor sensor for detecting plasma exosome miRNA-196a in prostate cancer and early pancreatic cancer according to claim 1 is characterized in that BSA and polyA n Synergistic anti-fouling design, BSA as a blocking agent, polyA n A hydrophilic layer is formed, and the two complement each other and work synergistically to avoid nonspecific adsorption of contaminating biological molecules.

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