Liquid gate graphene transistor glucose sensor with concanavalin modified gate

Abstract

The invention provides a liquid gate graphene transistor sensor with a grid modified by concanavalin. The liquid gate graphene transistor sensor is used for detecting glucose in real time. The liquid gate graphene transistor sensor comprises a substrate, the grid, a source and a drain, wherein the grid, the source and the drain are arranged on the substrate; a graphene channel is arranged between the source electrode and the drain electrode; and the surface of the grid is modified with concanavalin A biomolecules. The probe concanavalin is fixed on the surface of the grid electrode of the graphene transistor, the concanavalin can be combined with glucose in a solution with extremely high affinity, the conformation change of the concanavalin in the combination process causes the change of capacitance, the double-electric-layer interface characteristic between the transistor and the sample solution is changed, and the sensitivity of the transistor is improved. The current in the graphene channel is converted, and the glucose concentration is detected by detecting the change of the current in the channel. The channel current of the liquid gate graphene transistor sensor provided by the invention changes along with the change of glucose concentration, real-time detection is realized, the lowest detection limit of glucose can reach 10 <-19 > M, and the liquid gate graphene transistor sensor has very high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and particularly relates to a liquid-gated graphene transistor biosensor for detecting glucose and a preparation method thereof. Background Art

[0002] With the rapid development of sensor technology, field-effect transistors with two-dimensional material channels have received increasing attention due to their advantages such as label-free detection, fast response speed, simple operation, and easy integration. Graphene, due to its excellent physical and chemical properties such as large specific surface area, thermal conductivity, carrier mobility, and mechanical strength, is a functional nanomaterial widely used in the design of glucose sensors.

[0003] A biosensor uses biomolecules to modify an electrode, converts the concentration of the detected biomolecule recognized into an electrical signal, and the signal intensity is proportional to the concentration or content of the substance to be detected, so as to achieve the detection purpose. Biosensors have many important applications, such as medical analysis, environmental monitoring, food safety detection, etc. The interaction between biomolecules and graphene is important for all types of graphene transistor-based biosensors because they work in solution. Various graphene transistor-based biosensors have received extensive attention in recent years.

[0004] Glucose is one of the most widely distributed monosaccharides in nature. Both hypoglycemia and hyperglycemia can have an adverse impact on human health. Hypoglycemia can make people feel hungry and even unconscious, while hyperglycemia will increase the risk of diabetes to a certain extent. In addition, the determination of glucose content in fruits and vegetables is also one of the indicators to determine their quality and can be used as a reference for fruit and vegetable cultivation. Therefore, accurate, rapid, simple, and real-time blood glucose measurement is of great significance to industries such as clinical diagnosis and the food industry. Summary of the Invention

[0005] The purpose of the present invention is to provide a liquid-gated graphene transistor biosensor for detecting glucose in human body fluids, a preparation method thereof, and an application. The graphene transistor glucose biosensor provided by the present invention is small in volume, easy to be integrated into a portable device, simple to operate, and can be operated by non-professionals. It has high sensitivity, and the lowest detection limit of glucose can reach 10 -19 M.

[0006] The present invention provides a graphene transistor biosensor, including a substrate and a gate, a source, and a drain disposed on the substrate; a graphene channel is disposed between the source and the drain; concanavalin A capable of specifically binding to glucose is fixed on the surface of the gate.

[0007] Preferably, the width of the graphene channel is 2 - 3 mm, and the length of the graphene channel is 4 - 8 mm.

[0008] Preferably, the graphene channel material is monolayer graphene prepared by the CVD method.

[0009] Preferably, the gate, source, and drain independently include a chromium layer and a gold layer, and the chromium layer is located between the substrate and the gold layer.

[0010] Preferably, the thickness of the chromium layer is 3 - 6 nm, and the thickness of the gold layer is 30 - 60 nm.

[0011] Preferably, the concanavalin A biomolecule is immobilized on the surface of the gate through thiol modification.

[0012] The present invention also provides a method for preparing the graphene transistor biosensor described in the above technical solution, including the following steps:

[0013] (1) Prepare the gate, source, and drain on the surface of the substrate, such that there is a channel between the source and the drain;

[0014] (2) Lay the graphene flat on the channel between the source and the drain to obtain a graphene transistor;

[0015] (3) Immobilize the concanavalin A biomolecule probe on the surface of the gold gate of the graphene transistor obtained in step (2) to obtain a graphene transistor biosensor.

[0016] Preferably, the preparation of the gate, source, and drain in step (1) includes: successively evaporating a chromium layer and a gold layer on the surface of the substrate by thermal evaporation coating.

[0017] The present invention also provides the application of the graphene transistor biosensor described in the above technical solution or the graphene transistor biosensor prepared according to the above technical solution in glucose detection.

[0018] The present invention provides a graphene transistor biosensor, including a substrate and a gate, a source, and a drain disposed on the substrate; a graphene channel is provided between the source and the drain; the surface of the gate is modified with concanavalin A. In the present invention, the probe concanavalin A is fixed on the surface of the graphene transistor gate electrode. Concanavalin A can specifically bind to glucose in the solution, thereby changing the characteristics of the double - layer interface between the transistor and the sample solution, causing a change in the current in the graphene channel. By detecting the change in the current in the channel, trace glucose small molecules in the solution can be detected. The graphene transistor ion sensor provided by the present invention fixes the drain voltage and changes the gate voltage, and the lowest detection limit of glucose can reach 10 -19 M, and for different glucose concentrations, the current of the sensor changes in real - time, having a very high sensitivity. Brief Description of the Drawings

[0019] Figure 1 This is the schematic diagram of the graphene transistor biosensor in the embodiment of the present invention; Figure 2 This is the schematic diagram of the preparation process of the graphene transistor biosensor in the embodiment of the present invention; Figure 3 This is the schematic diagram of the three - electrode structure in the graphene transistor biosensor of Embodiment 1 of the present invention; Figure 4 This is the transfer characteristic curve when the graphene transistor biosensor detects the glucose concentration in Embodiment 3 of the present invention; Figure 5 This is the standard curve of the Dirac point voltage change value and the glucose biomolecule concentration of the graphene transistor biosensor in the embodiment of the present invention; Figure 6 This is the current output characteristic curve when detecting the change of glucose biomolecule concentration in the embodiment of the present invention; Figure 7 This is the standard curve of the change value of the channel current and the change of the glucose biomolecule concentration of the graphene transistor biosensor in the embodiment of the present invention. Detailed implementation manners

[0020] The present invention provides a graphene transistor glucose sensor, including a gate, a source and a drain disposed on the substrate; a graphene channel is disposed between the source and the drain; and a concanavalin A biomolecule is fixed on the surface of the gate.

[0021] The glucose biosensor based on graphene transistor provided by the present invention includes a substrate. The present invention has no special limitation on the type and source of the substrate, and a substrate for sensors well - known to those skilled in the art can be used. In a specific embodiment of the present invention, the substrate is preferably electronic - grade glass or PET, more preferably electronic - grade glass; in the present invention, the electronic - grade glass is preferably GL - 10173 - 1.1.

[0022] The present invention has no special limitation on the size of the substrate, which can be adjusted according to the size of the device. In the present invention, the length and width of the substrate are preferably independently 10 - 15 mm, more preferably 12 mm; the thickness of the substrate is preferably 2 mm.

[0023] The graphene transistor biosensor provided by the present invention includes a gate, a source, and a drain disposed on the substrate. In the present invention, the gate, the source, and the drain are preferably sequentially and spaced apart on the same surface of the substrate; the present invention has no special requirements for the specific positions of the gate, the source, and the drain, and they can be set according to the positions well-known in the art. In the present invention, a channel with a width of 2 - 3 mm is preferably formed between the source and the drain. The present invention has no special limitation on the shapes of the gate, the source, and the drain, and the shapes of the electrodes well-known to those skilled in the art can be adopted.

[0024] In the present invention, the gate, the source, and the drain preferably independently include a chromium layer and a gold layer, and the chromium layer is located between the substrate and the gold layer. In the present invention, the thickness of the chromium layer in the gate, the source, and the drain is preferably independently 3 - 6 nm, more preferably 10 nm; the thickness of the gold layer in the gate, the source, and the drain is preferably independently 30 - 60 nm, more preferably 50 nm. In the present invention, the chromium layer enables the gold layer to firmly adhere to the surface of the substrate, avoiding the shedding of the gold layer during later operations.

[0025] In the present invention, the three-electrode structure of the gate, the source, and the drain enables the use of the voltage input to the gate to control the channel current during the detection of copper ions, achieving an operating voltage below 1V.

[0026] The graphene transistor biosensor provided by the present invention includes a graphene channel disposed between the source and the drain. In the present invention, the channel formed between the source and the drain in the above technical solution is the graphene channel. In the present invention, the width of the graphene channel is preferably 2 - 3 mm, more preferably 2.5 mm; the length of the graphene channel is preferably 4 - 8 mm, more preferably 5 - 6 mm. In the present invention, the graphene preferably fills the gap between the source and the drain and covers the channel part on the surface of the source and drain electrodes. In the present invention, the graphene channel is preferably CVD single-layer graphene. In the present invention, the graphene channel can improve the sensitivity of the sensor.

[0027] The biosensor based on graphene transistor provided by the present invention includes concanavalin A biomolecules fixed on the surface of the gate. The present invention utilizes the specific binding ability of concanavalin A biomolecules to glucose, and the concanavalin A biomolecules on the gate have high selectivity for glucose.

[0028] In the present invention, the modification amount of concanavalin A biomolecules on the surface of the gate is preferably 2 - 3 mg / ml, more preferably 2 mg / ml.

[0029] The schematic diagram of the sensor based on graphene transistor provided by the present invention is as Figure 1As shown, the electrolyte conducts between the gate electrode and the graphene channel, forming a "double capacitor" structure. The voltage applied between the gate and the graphene channel is constant. The voltage applied to the gate changes the interfacial characteristics of the double electric layer when glutathione complexes with lead ions, and at the same time causes a change in the current in the graphene channel. By detecting the change in the current in the channel, trace lead ions in the solution can be quantitatively detected.

[0030] The schematic diagram of the sensor based on the graphene transistor provided by the present invention is as Figure 1 As shown, the electrolyte conducts between the gate electrode and the graphene channel, forming a "double capacitor" structure. The voltage applied between the gate and the graphene channel is constant. The voltage applied to the gate changes the conformation of concanavalin A when it specifically binds to glucose, resulting in a change in the interfacial characteristics of the double electric layer, and at the same time causing a displacement of the Dirac point voltage and a change in the current in the graphene channel. By detecting the displacement of the Dirac point voltage and the change in the current in the channel, trace glucose in the solution can be quantitatively detected.

[0031] The present invention also provides a preparation method for the graphene transistor glucose biosensor described in the above technical solution, including the following steps:

[0032] (1) Prepare a gate electrode, a source electrode, and a drain electrode on the surface of the substrate, such that there is a channel between the source electrode and the drain electrode;

[0033] (2) Lay graphene flat on the source electrode and the drain electrode to form a conductive channel, obtaining a graphene transistor;

[0034] (3) Fix a concanavalin A biomolecular probe on the surface of the gate of the graphene transistor obtained in step (2), obtaining a graphene transistor biosensor.

[0035] The present invention prepares a gate electrode, a source electrode, and a drain electrode on the surface of the substrate, such that there is a channel between the source electrode and the drain electrode. In the present invention, the preparation of the gate electrode, the source electrode, and the drain electrode preferably includes: successively evaporating a chromium layer and a gold layer on the surface of the substrate by thermal evaporation coating.

[0036] The present invention does not have special limitations on the specific parameters of the thermal evaporation coating method. Using parameters well-known to those skilled in the art, electrodes with the required thickness can be prepared. In the present invention, the thermal evaporation coating is preferably carried out under vacuum conditions; the degree of vacuum of the vacuum is preferably 8×10 -4 Pa or less, more preferably 4×10 -4 Pa. In the present invention, the evaporation current of the chromium layer is preferably 170 - 200 A, more preferably 170 - 180 A; the evaporation temperature of the gold layer is preferably 100 - 130 A, more preferably 105 - 120 A.

[0037] Before use, the substrate of the present invention is preferably cleaned and dried. In the present invention, the cleaning is preferably ultrasonic cleaning, and more preferably ultrasonic cleaning is sequentially performed with acetone, ethanol, deionized water, and ethanol. In the present invention, the ultrasonic cleaning time of the acetone, ethanol, and deionized water is independently preferably 10 to 30 minutes, and more preferably 20 minutes. The present invention has no special limitation on the power of the ultrasonic cleaning, and the power well-known to those skilled in the art can be used. In the present invention, the drying is preferably drying in an oven.

[0038] After the gate, source, and drain are fabricated, the present invention lays graphene flat on the source and drain channels to obtain a graphene transistor. In the present invention, the laying of the graphene preferably includes: transferring a single-layer graphene on a metal substrate to the channel between the source and drain by wet transfer. In the present invention, the single-layer graphene on the metal substrate is preferably single-layer graphene prepared by the CVD method on a copper substrate. The present invention has no special limitation on the source of the single-layer graphene on the metal substrate, and commercially available products well-known to those skilled in the art or products prepared according to the preparation methods well-known to those skilled in the art can be used.

[0039] The present invention has no special limitation on the operation of the wet transfer, and the technical scheme of wet-transferring single-layer graphene well-known to those skilled in the art can be used. In the present invention, the technical scheme of wet-transferring single-layer graphene preferably refers to the technical scheme disclosed in Chen Mu, Yan Yue, Zhang Xiaofeng, etc. Research progress of large-area graphene film transfer technology [J]. Journal of Aeronautical Materials, 2015, 35(2): 1-11.

[0040] After the transfer of graphene is completed, the present invention preferably anneals the transferred device to obtain a graphene transistor. In the present invention, the annealing temperature is preferably 100 to 130 °C, and more preferably 110 °C; the annealing time is preferably 20 to 40 minutes, and more preferably 30 minutes. In the present invention, the annealing can remove the moisture on the surface of the sample and at the same time make the combination between graphene and the substrate closer.

[0041] After obtaining the graphene transistor, the present invention preferably fixes concanavalin A biomolecules on the surface of the gate of the graphene transistor to obtain a biosensor based on the graphene transistor. In the present invention, the concanavalin A biomolecules are preferably fixed on the gate surface after being modified with mercapto groups. The present invention has no special limitation on the operation of fixing the concanavalin A biomolecules, and the method of fixing on the gold surface well-known to those skilled in the art can be used.

[0042] The present invention also provides the application of the graphene transistor-based biosensor described in the above technical solution or the graphene transistor-based biosensor prepared according to the above technical solution in glucose detection. The gate and the graphene channel part of the graphene transistor-based biosensor are immersed in the electrolyte containing the glucose to be detected. In the present invention, the electrolyte is preferably 0.1× PBS; the pH value of the electrolyte is preferably 7 to 7.4. In the present invention, the gate and the graphene channel are connected through the electrolyte, and the voltage input to the gate is used to control the current of the graphene channel.

[0043] When using the graphene transistor biosensor provided by the present invention for the quantitative determination of glucose, it is preferable to prepare a standard solution containing glucose, measure the standard curve of the current change value and the glucose concentration, and determine the detection limit and detection range of the glucose to be detected according to the standard curve and the measured current change value.

[0044] The graphene transistor glucose biosensor provided by the present invention can directly detect the glucose concentration by being immersed in the solution to be detected, which is a label-free detection method; and it can perform high-sensitivity detection on samples and has good stability. The three-electrode structure and the graphene channel of the graphene transistor glucose biosensor provided by the present invention make it very sensitive to voltage changes, and a very small voltage change will cause a corresponding current change, with high sensitivity; and the graphene transistor biosensor provided by the present invention can detect extremely trace glucose molecules with a concentration as low as 10 -19 M in the solution.

[0045] In order to further illustrate the present invention, the following examples are used to describe in detail the graphene transistor glucose biosensor provided by the present invention, its preparation method and application, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1:

[0047] Thermal evaporation coating: Cut the electronic-grade glass into a size of 12×12 mm, ultrasonically clean it with acetone, ethanol, deionized water and ethanol for twenty minutes in sequence, dry it with nitrogen, and then paste the glass sheet on a mask plate with a specific shape with high-temperature glue. Weigh an appropriate amount of chromium and gold and put them into a tungsten boat to prepare for vacuum thermal evaporation coating.

[0048] First evaporate chromium during evaporation: The thickness is 3 nm.

[0049] Then evaporate the gold layer: The thickness is 30 nm.

[0050] The obtained electrode shape, structure and size are as Figure 3 shown. Figure 3Among them, G is the gate, S is the source, and D is the drain. After transferring the 0.25-mm-wide channel between the source and the drain to graphene, it becomes the graphene channel.

[0051] Wet transfer of single-layer graphene:

[0052] Dissolve 250 mg of methyl methacrylate (PMMA) with a molecular weight of 99,600 g / mol in 5 mL of anisole, and stir on a magnetic stirrer to obtain a clear and transparent PMMA / anisole solution with a concentration of 50 mg / mL.

[0053] Cut the CVD single-layer copper substrate graphene to a size of 12×12 mm, drop 100 μL of the prepared PMMA / anisole solution on the surface of the graphene and spin-coat it. Set the rotation speeds of the spin coater to 600 and 3000 rpm, and the spin-coating times to 3 s and 30 s. After spin-coating, heat it at 100 °C for 10 min to obtain PMMA / graphene.

[0054] Prepare a 100 mg / mL ferric chloride solution. Place the side of the PMMA / graphene that has been spin-coated with the PMMA / anisole solution facing up in the ferric chloride solution to completely etch away the copper substrate.

[0055] Use a glass slide to transfer the PMMA / graphene sheet with the etched copper substrate to deionized water and soak it for 5 min. Wash it with deionized water twice to wash away the residual ferric chloride solution on the PMMA / graphene. Take the device made by thermal evaporation coating, ultrasonically clean it with acetone, ethanol, and deionized water respectively, dry it with nitrogen, and then clean the surface of the device with an oxygen plasma cleaner to improve the hydrophilicity of the device.

[0056] Transfer the washed PMMA / graphene sheet to the washed device, place it flat on the channel between the source and the drain on the surface of the device, and let it dry naturally until no surface moisture can be observed with the naked eye. Then place it on a hot stage and anneal it at 110 °C to completely remove the surface moisture of the sample and obtain the PMMA / graphene / electrode sheet.

[0057] After cooling to room temperature, use tweezers to remove the excess PMMA / graphene on both sides of the channel. Then wash the PMMA / graphene / electrode sheet with acetone twice, 5 min each time. Then place the PMMA / graphene / electrode sheet in an acetone solution and heat it at 70 °C for 3 h to remove the surface PMMA and obtain the required graphene transistor. After 3 h, wash the graphene transistor with deionized water and dry it naturally.

[0058] Fixed concanavalin A biomolecular probe: 10 μL of 2 mg / ml concanavalin A solution was drop-coated onto the surface of the gate, and after 3 hours, it was washed three times with PBS buffer; the un-fixed concanavalin A biomolecular probe and other impurities on the gate were washed away to obtain a graphene transistor glucose biosensor.

[0059] Example 2

[0060] The graphene transistor biosensor prepared in Example 1 was used to test glucose target molecules. The source, drain, and gate electrodes of the graphene transistor were connected to two digital source meters (Keithley 2450), and the gate voltage V G and the source-drain voltage V DS were controlled by a Labview program in a computer.

[0061] The modified gate electrode was thoroughly washed with PBS solution to remove the residues left on the electrode. The test was carried out in a container containing 100 μL of PBS solution. During the test, a solution of concanavalin A with a specific concentration was successively added to the PBS solution to obtain output signals at different concentrations.

[0062] Transfer characteristic curve test: The source-drain voltage was set to a constant value (V DS = 0.1 V), and when the gate voltage continuously changed from 0 V to 1 V, the change in the channel current I DS between the source and the drain was measured, and then the concentration of glucose in the solution was changed and measured successively; the obtained transfer characteristic curve is as Figure 4 shown. The change in the interface will change the potential on the surface of the device, thereby shifting the characteristic curve. According to Figure 4 it can be seen that as the concentration of glucose changes, the Dirac point voltage changes accordingly.

[0063] Output characteristic test: Both the source-drain voltage and the gate voltage were set to a constant value (V DS = 0.1 V and V G = 0.2 V), and the relationship image between the channel current and time was continuously measured. During this period, after the channel current stabilized, the glucose concentration was increased, and the concentration was changed from 10 -19 M, 10 -17 M, 10 -15 M, 10 -13 M, 10 -11 M, 10 -9 M, 10 -7 M in sequence; the obtained test results are as Figure 6 shown; according to Figure 6It can be seen that the change in glucose concentration will cause an obvious change in current. The magnitude of the current change can reflect the magnitude of the concentration change. Moreover, after changing the glucose concentration, the current of the sensor changes immediately, and the sensitivity is very high. The graphene transistor biosensor provided by the present invention can detect extremely trace glucose with a concentration as low as 10 -19 M in solution.

[0064] Standard curve plotting: The source-drain voltage and the gate voltage are both set to a constant value (V DS = 0.1V and V G = 0.2V). Glucose solutions with different concentrations are prepared respectively according to the method in Example 3. The change values of the channel current of the graphene transistor biosensor are measured respectively when the glucose concentrations are 10 -19 M, 10 -17 M, 10 -15 M, 10 -13 M, 10 -11 M, 10 -9 M, 10 -7 M. The test is repeated 3 to 5 times, and the average value is taken. A curve is plotted with the glucose concentration as the abscissa and the change value of the channel current as the ordinate. It can be seen from the curve that there is a good linear relationship between the change value of the channel current and the glucose concentration. The R 2 value of the fitted straight line is 0.989. When detecting the concentration of the glucose to be measured, the concentration of glucose can be obtained according to the change value of the current in the graphene channel and the standard curve.

[0065] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. It should be noted that for those of ordinary skill in the art in this technical field, several improvements can be made without departing from the principle of the present invention.

Claims

1. A liquid-gated graphene transistor glucose sensor with a concanavalin A modified gate, comprising a substrate, and a gate, a source and a drain disposed on the substrate; a single-layer graphene conductive channel is disposed between the source and the drain; a concanavalin A biomolecular probe capable of detecting glucose is modified on the surface of the gate.

2. The liquid-gated graphene transistor sensor for detecting glucose according to claim 1, wherein The detection principle is that the special affinity of concanavalin A for glucose enables glucose to be captured by the concanavalin A biomolecular probe, thereby causing a change in the channel current to achieve sensing.

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