Liquid gate graphene transistor sensor for detecting vitamin C and preparation method thereof

Abstract

The invention provides a liquid gate graphene transistor vitamin C sensor with a gate modified by beta-lactoglobulin and a preparation method of the liquid gate graphene transistor vitamin C sensor. The liquid gate graphene transistor vitamin C sensor comprises a sensor template (a gate, a source and a drain are regularly arranged); a graphene channel is arranged between the source electrode and the drain electrode; and modifying protein on the gate surface. The detection probe is beta-lactoglobulin, the beta-lactoglobulin is fixed on the surface of a grid electrode of a graphene transistor by utilizing MPA, amino acid residues of protein can generate hydrogen-bond interaction with vitamin C molecules in a solution, and by measuring deviation of a Dirac point or current change of a graphene channel, the content of vitamin C in the solution can be detected. Trace vitamin C molecules in the solution can be detected; through multiple tests, the lowest detection limit of the vitamin C can reach 10 <-16 > M, current response of the sensor occurs instantly for vitamin C molecules with different concentrations, and the sensor has very high sensitivity, has real-time response in a range of 10 <-16 >-10 <-8 > M, and has good linear response.

Description

Technical Field

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

[0002] Vitamins are a class of trace organic compounds necessary for participating in biological metabolism and growth and development. Since these substances cannot be synthesized by the body itself, they must rely on food supply. The role of vitamins in the body is different from that of carbohydrates, fats, and proteins, but they play a crucial role in the biological metabolism process. When the body lacks vitamins, the material metabolism is disrupted. Because the physiological functions of various vitamins are different, the lack of different vitamins will lead to different diseases. Vitamin C has the function of preventing scurvy, so it is also called ascorbic acid, and is widely used as an antioxidant in food, pharmaceutical preparations, and cosmetics. At the same time, vitamin C plays a key role in biological processes such as scavenging free radicals, cancer prevention, and immune improvement. Especially in the central nervous system, vitamin C has an important regulatory effect on neurotransmitters, enzymes, and neuropeptides. Since the concentration of vitamin C in the central nervous system is in millimoles, and the concentration in body fluids is even lower, therefore, developing a simple, rapid, highly selective, and highly sensitive method for detecting vitamin C has important significance in the biomedical field.

[0003] Currently, the conventional detection methods for vitamin C include fluorescence detection, amperometric detection, chromatography, and spectrophotometry. The analysis techniques based on fluorescence detection and chromatography need to rely on large-scale instruments, which are expensive and complex to operate. Although amperometric detection can achieve rapid and sensitive quantitative analysis of vitamin C, its detection limit is high, and most of them use ascorbic acid oxidase for detection. The characteristic that the enzyme is easily inactivated makes the stability of amperometric detection poor.

[0004] With the vigorous development of transistors in biosensing, field-effect transistors based on graphene as the channel material are considered to have great development potential. Due to the unique two-dimensional planar structure of graphene and its good chemical stability and biological inertness, it shows great advantages in biosensing. In recent years, liquid-gated graphene field-effect transistors have been widely used in the detection of various substances, such as proteins, glucose, bacteria, antibiotics, etc. However, their application in vitamin detection is very few. Therefore, the inventor has created a method for detecting vitamin C using a liquid-gated graphene field-effect transistor, which has the advantages of simple operation, high sensitivity, low detection limit, etc. At the same time, the use of molecular recognition technology gets rid of the conventional thinking of detecting vitamin C using chemical reactions. Summary of the Invention

[0005] The object of the present invention is to provide a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate and a preparation method thereof. In the field of vitamin C detection, a field-effect transistor with a single-layer graphene covering as the channel material is fabricated using protein molecules as probes. This sensor has multiple advantages compared with traditional vitamin C detection methods: (1) β-lactoglobulin molecules have multiple binding sites with different affinities, can specifically bind to vitamin C, and have significant advantages such as being easy to modify, having good stability, being non-toxic and harmless, not being easily hydrolyzed and inactivated.

[0006] (2) This sensor combines the advantages of high sensitivity and real-time response of graphene transistor sensors, and can realize real-time detection of vitamin C.

[0007] (3) The working voltage of the liquid-gated graphene transistor sensor is usually less than 1V, which is beneficial to the application of wearable electronics.

[0008] (4) The liquid-gated graphene transistor sensor can maximize the integration of the advantages of both, thereby improving the sensing specificity, and the detection limit is reduced to 10 -16 M, with a good linear detection range.

[0009] The present invention provides a liquid-gated graphene transistor sensor for realizing real-time detection of vitamin C, specifically including the preparation of a β-lactoglobulin solution suitable for the graphene transistor sensor and having specific selectivity for vitamin C; how to modify the prepared β-lactoglobulin solution on the surface of the graphene transistor gate; constructing a new detection platform for the liquid-gated graphene transistor sensor with a β-lactoglobulin modified gate; and a specific detection application method.

[0010] (1) Preparation of a β-lactoglobulin solution suitable for the graphene transistor sensor and having specific selectivity for vitamin C: Weigh β-lactoglobulin powder and dissolve it in deionized water, shake it well to make it completely dissolve, and store the obtained β-lactoglobulin solution in the refrigerator at a low temperature.

[0011] (2) Preparation of the graphene transistor: Through the JSD-300 evaporation coating system, chromium and gold are sequentially deposited on the substrate in the evaporation coater by thermal evaporation coating to obtain the required initial transistor, and then a single-layer graphene is attached to the source-drain channel of the transistor. After cleaning, the desired graphene transistor sensor is obtained.

[0012] (3)Preparation of a liquid-gated graphene transistor sensor with a novel β-lactoglobulin modified gate: Under normal temperature and dark environment, drop the MPA solution onto the gate of the graphene transistor to fix MPA on the surface of the gold electrode. After the reaction is completed, wash the gate surface with deionized water and dry the moisture with high-purity nitrogen. Then drop the EDC / NHS mixed solution onto the gate. After the reaction is completed, wash the gate surface with deionized water again and dry it with nitrogen. Finally, drop the prepared β-lactoglobulin solution onto the gate surface. After the reaction is completed, rinse it with deionized water to remove excess protein and other residues, and let it dry naturally at room temperature to obtain a highly sensitive liquid-gated graphene transistor sensor with a β-lactoglobulin modified gate.

[0013] (4)Application of a liquid-gated graphene transistor sensor with a novel β-lactoglobulin modified gate: Use the electrochemical station 2400 to test the specific detection and concentration-dependent detection of vitamin C by the liquid-gated graphene transistor sensor with a β-lactoglobulin modified gate. The liquid-gated graphene transistor sensor with a β-lactoglobulin modified gate has a sensitive current response to vitamin C, and at the same time reaches an extremely low detection limit, has a wide linear detection range, far superior to the detection of vitamin C by other detection methods.

[0014] In the above preparation method, in step (1), the β-lactoglobulin powder was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0015] In the above preparation method, in step (1), the concentration of the prepared β-lactoglobulin solution is 0.05 g / ml.

[0016] In the above preparation method, in step (1), the low-temperature storage temperature is 2 to 4 °C.

[0017] In the above preparation method, in step (2), the transistor substrate used 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.

[0018] In the above preparation method, in step (2), clean and dry the substrate before use. The cleaning is preferably ultrasonic cleaning, and the specific operation is to perform ultrasonic cleaning with acetone, ethanol, deionized water, and ethanol in sequence for 15 - 30 min, most preferably 20 min. Then dry it, and the drying is preferably drying in an oven.

[0019] In the above preparation method, in step (2), the thermal evaporation coating is preferably carried out under vacuum conditions; the vacuum degree of the vacuum is preferably below 8×10 -4 Pa, more preferably 4×10 -4Pa. In the present invention, the evaporation temperature of the chromium layer is preferably 170 - 200 °C, more preferably 175 - 190 °C; the evaporation temperature of the gold layer is preferably 100 - 120 °C, more preferably 105 - 110 °C.

[0020] In the above preparation method, in step (2), the fabricated transistor includes a gate, a source, and a drain, and there is a channel of 0.1 - 0.4 mm between the source and the drain.

[0021] In the above preparation method, in step (2), the thickness of the evaporated gold is 60 - 100 nm, and the thickness of the evaporated chromium is about 7 - 13 nm.

[0022] In the above preparation method, in step (2), the transfer of the graphene preferably includes: transferring monolayer graphene on a metal substrate to the channel between the source and the drain by wet transfer.

[0023] In the above preparation method, in step (2), before transferring the graphene to the transistor, it is necessary to increase the hydrophilicity of the transistor (oxygen plasma ultraviolet irradiation).

[0024] In the above preparation method, in step (2), after the transfer of the graphene is completed, the transferred product needs to be annealed to obtain a graphene transistor.

[0025] In the above preparation method, in step (2), the obtained graphene transistor needs to be heated and soaked in acetone for 3 - 5 h, preferably 3 h, and then washed twice with deionized water after completion to remove excess impurities and keep it clean.

[0026] In the above preparation method, in step (2), the graphene transistor can be placed at room temperature for natural drying.

[0027] In the above preparation method, in step (3), MPA solution (200 mM).

[0028] In the above preparation method, in step (3), EDC solution (50 mM), NHS solution (50 mM).

[0029] In the above preparation method, in step (3), the ratio of the EDC / NHS solution is preferably 1:1.

[0030] In the above preparation method, in step (3), wash with deionized water three times or more, preferably three times.

[0031] By detecting the current change in the channel of the liquid-gated graphene transistor through an electrochemical station 2400, trace vitamin C in the solution can be detected. The graphene transistor sensor provided by the present invention fixes the drain voltage and changes the gate voltage, and the lowest detection limit of vitamin C can reach 10-16 At M and different vitamin C concentrations, the current of the sensor changes instantaneously, with very high sensitivity. Description of the Drawings

[0032] Figure 1 It is a schematic diagram of a liquid-gated graphene transistor sensor with a β-lactoglobulin modified gate in an embodiment of the present invention; Figure 2 It is a schematic diagram of a three-electrode structure in a liquid-gated graphene transistor sensor with a β-lactoglobulin modified gate in an embodiment of the present invention; Figure 3 It is a practical assembly schematic diagram of a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate in Embodiment 3 of the present invention; Figure 4 It is a schematic diagram of the preparation process of a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate in an embodiment of the present invention; Figure 5 It is a transfer characteristic curve when a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate in Embodiment 3 of the present invention tests vitamin C; Figure 6 It is the Dirac point voltage change value of a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate in Embodiment 3 of the present invention; Figure 7 It is an output characteristic curve when a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate in Embodiment 3 of the present invention detects vitamin C molecules with different concentrations; Figure 8 It is the channel current change value of a liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate in Embodiment 3 of the present invention. Detailed Embodiments

[0033] The technical solutions of the present invention will be further described below in conjunction with embodiments.

[0034] The present invention proposes a liquid-gated graphene transistor sensor for detecting vitamin C and a preparation method thereof. The method uses β-lactoglobulin as a probe and modifies it onto a prepared graphene transistor to achieve specific real-time detection of vitamin C, that is, a novel liquid-gated graphene transistor vitamin C sensor with a β-lactoglobulin modified gate is obtained. Specifically, it includes the following steps and contents: (1) Preparation of β-lactoglobulin solution The present invention uses 0.02 g - 0.08 g of β-lactoglobulin powder, preferably 0.05 g. The weighed β-lactoglobulin powder is dissolved in deionized water, and after being fully dissolved, it is stored in a refrigerator at 2 - 4 °C.

[0035] (2) Preparation of Graphene Transistors Preparation of transistors. Chromium and gold are sequentially deposited onto a substrate in an evaporation coater through a JSD-300 evaporation coating system. The substrate is preferably electronic-grade glass GL-10173-1.1. Among them, the specific parameters of the thermal evaporation coating method are not particularly limited, and the corresponding parameters are based on the thickness of the required electrode that can be fabricated. In the present invention, the thermal evaporation coating is preferably carried out under vacuum conditions; the vacuum degree of the vacuum is preferably below 8×10 -4 Pa, more preferably 4×10 -4 Pa. In the present invention, the evaporation temperature of the chromium layer is preferably 170-200°C, more preferably 175-190°C; the evaporation temperature of the gold layer is preferably 100-120°C, more preferably 105-110°C. Finally, a transistor with the gate, source, and drain arranged in sequence is obtained.

[0036] Transfer of graphene. A polymethyl methacrylate (PMMA) film (400 nm - 600 nm, preferably 500 nm) is spin-coated on the pre-prepared single-layer graphene. Before attaching the graphene, the graphene needs to be soaked in a ferric chloride solution to remove the surface copper, specifically based on the transparency of the graphene. After soaking, annealing is carried out. After pre-treating a thin strip of graphene (width 2 - 3 mm, length 5 - 7 mm, preferably 0.2×6 mm 2 ), it is transferred onto the source and drain channels, enabling them to be conducted through the graphene, forming a graphite transistor sensor, as Figure 2 shown. At 70°C, the graphene transistor is soaked in acetone for 3 hours to dissolve the remaining substances on the graphene. After being washed twice with deionized water and naturally dried at room temperature, a stable I-V curve can be measured using a 2400 chemical station, and the Dirac point is stable between 0.2 - 0.7 V.

[0037] The single-layer graphene is preferably sourced from copper-substrate CVD method single-layer graphene, which can be a commercially available product well-known to those skilled in the art or prepared according to the preparation methods well-known to those skilled in the art. Before attaching the graphene, the graphene needs to be soaked in ferric chloride for 2 - 4 h to remove the surface copper, and the time is preferably 3 h, specifically based on the transparency of the graphene. After soaking, annealing is carried out. Annealing can remove the moisture on the surface of the sample and at the same time make the combination of graphene and the substrate closer. 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.

[0038] (3) Preparation of a Novel Liquid-Gated Graphene Transistor Aptasensor Drop MPA solution onto the gate electrode of the graphene transistor and react for 10 - 13 hours, preferably 12 hours, under low - temperature and dark conditions. After the reaction is completed, rinse the gate with deionized water and then dry it with nitrogen. Then, drop the EDC / NHS mixed solution onto the gate and let it react fully for 1 - 2 hours, preferably 2 hours. After the reaction is completed, wash the gate with deionized water and dry it with nitrogen. Finally, drop the prepared β - lactoglobulin solution onto the gate and react for 10 - 12 hours, preferably 10 hours, under low - temperature conditions. After the reaction is completed, wash the gate with deionized water to remove the unfixed β - lactoglobulin solution and other residues. Dry it naturally at room temperature to obtain a graphene transistor sensor with a β - lactoglobulin solution - modified gate for highly sensitive and specific detection of vitamin C.

[0039] (4)Application of the novel liquid - gated graphene transistor aptasensor Use the electrochemical station 2400 to test the specific detection and concentration - dependent detection of vitamin C by the liquid - gated graphene transistor sensor with a β - lactoglobulin solution - modified gate. The probe β - lactoglobulin of the present invention is fixed on the surface of the graphene transistor gate electrode through MPA. The probe binds to vitamin C through amino acid residues, thereby changing the characteristics of the double - electric - layer interface between the transistor and the sample solution, causing a change in the current in the graphene channel, achieving an extremely low detection limit, having a wide linear detection range, and far superior to the traditional vitamin detection results.

[0040] The graphene transistor aptasensor provided by the present invention can directly detect the concentration of vitamin C molecules by being immersed in the solution to be tested, which is a label - free detection method; and it can perform highly sensitive detection on the sample and has good stability. The three - electrode structure and graphene channel of the graphene transistor aptasensor 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 liquid - gated graphene transistor sensor with a β - lactoglobulin solution - modified gate provided by the present invention can detect extremely trace vitamin C molecules with a concentration as low as 10 -16 M.

[0041] To further illustrate the present invention, the following examples are used to describe in detail the graphene transistor sensor 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.

[0042] The schematic diagram of the preparation process of the graphene transistor aptasensor in the present invention is as Figure 4 shown.

[0043] Example 1: Thermal evaporation coating: Cut the electronic-grade glass into pieces of 12×12 mm. Ultrasonically clean them successively with acetone, ethanol, deionized water, and ethanol for 20 minutes. After drying in an oven, paste the glass pieces on a mask plate with a specific shape using 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.

[0044] During evaporation, first evaporate chromium with a thickness of 8 nm, and then evaporate a gold layer with a thickness of 80 nm.

[0045] Wet transfer of single-layer graphene: Dissolve 250 mg of polymethyl methacrylate (PMMA) with a molecular weight of 99600 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. Cut the single-layer copper-substrate graphene obtained by electrochemical deposition method into pieces of 12 mm×12 mm. Drop 70 μL of the PMMA / anisole solution prepared by spin coating on the surface of the graphene. Set the rotation speed of the spin coater to 3000 rpm, the spin coating time to 30 s, and dry at room temperature for 30 min after spin coating to obtain PMMA / graphene.

[0046] Prepare a 100 mg / mL ferric chloride solution. Turn the side of the dried PMMA / graphene that has been spin coated with the PMMA / anisole solution upwards and place it in the ferric chloride solution to completely etch the copper substrate.

[0047] Use a glass slide to transfer the PMMA / graphene with the copper substrate etched away to deionized water and soak for 5 min. Change the deionized water twice, and wash the residual ferric chloride solution on the PMMA / graphene with deionized water; Take the electrode piece made by thermal evaporation coating, ultrasonically clean it successively with acetone, ethanol, water, and ethanol, and after drying, treat the surface of the electrode piece with oxygen plasma to improve the hydrophilicity of the electrode piece.

[0048] Transfer the washed PMMA / graphene to the washed electrode, and place it flat on the channel between the source and drain on the surface of the electrode piece. Let it dry naturally until no surface moisture can be observed by the naked eye, and then anneal it on a hot stage at 110 °C to completely remove the surface moisture of the sample to obtain PMMA / graphene / electrode piece.

[0049] 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 piece with acetone twice, 5 min each time, and then put the PMMA / graphene / electrode piece into 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.

[0050] Fixing the β-lactoglobulin probe: Drop MPA solution onto the gate electrode of the graphene transistor, react for 12 hours under low-temperature and dark conditions. After the reaction is completed, rinse the gate with deionized water and then dry it with nitrogen. Then, drop the EDC / NHS mixed solution onto the gate, allow it to react fully for 2 hours. After the reaction is completed, wash the gate with deionized water and dry it with nitrogen. Finally, drop the prepared β-lactoglobulin solution with a concentration of 0.05 g / ml onto the gate, react for 10 hours under low-temperature conditions. After the reaction is completed, wash the gate with deionized water to remove the unfixed β-lactoglobulin solution and other residues. Let it dry naturally at room temperature to obtain a graphene transistor sensor with a β-lactoglobulin solution-modified gate for highly sensitive and specific detection of vitamin C.

[0051] Example 2: Use the graphene transistor sensor with a β-lactoglobulin solution-modified gate prepared in Example 1 to test the concentration of vitamin C.

[0052] The source, drain, and gate electrodes of the graphene transistor are connected to two combined Keithley data source meters (Keithley 2400), and the gate voltage V G and the source-drain voltage V DS are controlled by a Labview program in a computer.

[0053] Transfer characteristic curve test: The source-drain voltage is set to a constant value (V DS = 0.1 V), and when the gate voltage continuously changes from 0.1 V to 1 V, measure the change in the channel current I DS between the source and the drain, and then change the concentration of vitamin C in the solution and measure successively; the obtained transfer characteristic curves are as Figure 5 shown. The change in the interface will cause a change in the potential on the surface of the device, thus causing the characteristic curve to shift. It can be seen from Figure 5 that as the concentration of vitamin C increases, the current changes.

[0054] Output characteristic test: Both the source-drain voltage and the gate voltage are set to a constant value (V DS = 0.1 V and V G = 0.7 V), and continuously measure the relationship image between the channel current and time. During this period, after the channel current stabilizes, add vitamin C with different concentrations to make the concentration change successively from 10 -16 M, 10 -14 M, 10 -12 M, 10 -10 M, 10 -8 M; the obtained test results are as Figure 7 shown; according to Figure 7It can be seen that the change in the concentration of vitamin C will cause an obvious change in the current. The magnitude of the current change can reflect the magnitude of the concentration change. Moreover, after changing the vitamin C concentration, the current of the sensor changes immediately, with very high sensitivity. The graphene transistor sensor provided by the present invention can detect extremely trace vitamin C molecules with a concentration as low as 10 -16 M in solution.

[0055] Standard curve plotting: The source-drain voltage and the gate voltage are both set to a constant value (V DS = 0.1 V and V G = 0.7 V). Vitamin C solutions with different concentrations are prepared respectively. The change values of the channel current of the solution-gated graphene transistor sensor with the gate modified by β-lactoglobulin solution are measured respectively when the vitamin C molecule concentration is 10 -16 M 、10 -14 M、10 -12 M、10 -10 M、10 -8 M. The test is repeated 3 - 5 times and the average value is taken. A curve is plotted with the vitamin C 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 vitamin C concentration. When detecting the concentration of the vitamin C to be measured, the concentration of vitamin C can be obtained according to the change value of the current in the graphene channel and the standard curve.

[0056] The above is only the preferred embodiment of the present invention and does not impose any form of limitation on the present invention. It should be noted that for those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention.

Claims

1. A liquid-gated graphene vitamin C transistor sensor with a β-lactoglobulin modified gate, comprising the transistor sensor used (the separate regular arrangement of the gate, source, and drain); the setting of a graphene channel between the source and the drain; and a molecular probe capable of detecting vitamin C is modified on the surface of the gate.

2. The β-lactoglobulin modified gate liquid-gated graphene transistor vitamin C sensor according to claim 1, the detection principle of which is that vitamin C molecules have hydrogen bond interactions with the amino acid residues of the protein on the gate surface, causing the Dirac point of the transfer curve to shift, and real-time sensing is achieved by measuring the shift of the Dirac point or the change in the graphene channel current.

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