Simple and rapid manufacturing method of large-area transfer-free graphene field effect transistor biosensor

Abstract

The invention belongs to the technical field of field effect transistor biosensors, and discloses a simple and rapid manufacturing method of a large-area transfer-free graphene field effect transistor biosensor. The method comprises the following steps: preparing a graphene oxide film, performing hydrazine chemical reduction at 100 DEG C and one-step thermal annealing process at 200 DEG C to obtain a reduced graphene oxide film, depositing source and drain electrodes on the reduced graphene oxide film, and fixing a test cell to obtain a device for testing. The size of the RGO film can reach 4 * 4 cm < 2 > or even larger, the transfer process is omitted, and the RGO film can be directly prepared on a substrate. The device is simple and rapid in preparation process, high in economic benefit, mild in reduction process and beneficial to subsequent application on a flexible substrate. The result shows that the graphene with high carrier mobility is combined with the field effect transistor with the self-amplification effect, and high-sensitivity and rapid response can be generated to the concentration change of potassium ions.

Description

Technical Field

[0001] The invention belongs to the technical field of field effect transistor biosensors and relates to a simple and rapid method for manufacturing a large-area transfer-free graphene field effect transistor biosensor. Background Art

[0002] Potassium ion (K + ) is one of the main cations in biological systems and is essential for regulating body functions. Abnormal fluctuations in potassium concentration may affect the normal functions of the heart, kidneys, nervous system, cardiovascular system, etc. For cells, disturbed ion homeostasis, such as decreased extracellular sodium ion concentration or increased potassium ion concentration, will lead to cell apoptosis. Apoptosis is a process of programmed cell death that leads to many pathological conditions, such as cancer, neurodegenerative diseases, atherosclerosis, and autoimmune diseases. Therefore, effective detection of cellular environmental potassium levels is very important in medical health monitoring, and there is an urgent need to develop real-time sensing monitoring devices for ion detection. Compared with traditional electrochemical ion detection electrodes, highly sensitive and highly selective graphene field-effect transistor ion-selective sensors show great potential in real-time ion monitoring.

[0003] Hongmei Li et al. published an article titled Graphene field effect transistors for highly sensitive and selective detection of K + ions proposed that the ISFET sensor based on graphene has the advantages of high carrier mobility, high specific surface area, low noise, etc. Thanks to the single-layer two-dimensional structure of graphene and the self-amplification effect of FET, it is extremely sensitive to the charge changes generated by low-concentration targets on the surface, and is currently the main type of ISFET sensor application. However, although graphene-based ISFET has made great achievements in ion detection, some issues still need to be considered in practical applications.

[0004] Common methods for making graphene include mechanical exfoliation, chemical vapor deposition (CVD), and graphene oxide reduction. Among these methods, CVD is the most widely used and the graphene obtained is of high quality. For example, Hongmei Li et al. reported in their article Direct measurement of K +In the study of ion efflux from neuronal cells using a graphene-based ion sensitive field effect transistor, high-quality graphene is obtained by CVD, which can detect potassium ions discharged from cells with high sensitivity. However, the production process is complicated and expensive. In addition, for graphene, it has not yet been truly prepared at the large-area wafer level. Non-arraying cannot guarantee the consistency of performance between devices, nor can it achieve repeated measurements of the same ion or simultaneous measurements of multiple ions. For example, the graphene area prepared by Ibrahim Fakih et al. in the article High resolution potassium sensing with large-area graphene field-effect transistors is only 1×1cm 2 . Even if a large area of ​​CVD graphene is made, the complex transfer process is inevitable, and there will be problems with PMMA residues during the transfer process, such as the article Selective ion sensing with high resolution large area graphene field effect transistor arrays by Ibrahim Fakih et al. The graphene oxide reduction method can overcome these problems after the spin coating process. The graphene film is simple, economical, large-area and transfer-free to make, such as Walid-Madhat Munief et al. in the article Reduced graphene oxide biosensor platform for the detection of NT-proBNP biomarker inits clinical range and Dana Kadadou et al. in the article Detection of SARS-CoV-2 inclinical and environmental samples using highly sensitive reduced grapheneoxide (rGO)-based biosensor. They mentioned that arrayed devices that are easy to integrate were made, but high-temperature reduction conditions of more than 400°C were used, and flexible substrates such as PET and PI will deform at 200°C or even 150°C. High temperature limits its application on flexible substrates.

[0005] In summary, the main problems of graphene-based ISFET sensors can be summarized as follows: 1) The CVD method for preparing graphene is cumbersome, expensive, and has a complex transfer process, which will leave material residues that affect graphene performance. 2) It is impossible to achieve true large-area graphene levels and cannot be applied in arrays, which affects the performance consistency between devices. 3) The spin-coated graphene oxide reduction method that can achieve large-area transfer-free also requires high reduction conditions, and non-mild reduction conditions cannot be applied to flexible substrates. Summary of the invention

[0006] In order to improve the problems of the above-mentioned graphene-based ISFET sensor, the present invention designs a field effect transistor biosensor based on large-area, transfer-free graphene film materials. First, the graphene oxide film is made by spin coating, and then a uniform and large-area RGO film is obtained by hydrazine chemical reduction and one-step air thermal annealing process. The production process is simple, economical, transfer-free, and the reduction conditions are mild, and the temperature is controlled below 200°C; in addition, the simple and mild production method of large-area RGO film can also enable the sensor to be applied to flexible substrates, expanding the application range of the sensor.

[0007] The technical solution of the present invention:

[0008] A simple and rapid method for fabricating large-area transfer-free graphene field-effect transistor biosensors.

[0009] The large-area transfer-free graphene field effect transistor biosensor is composed of a large-area RGO film deposited on a glass substrate, a potassium ion selective membrane, a source-drain electrode made of gold or silver, and a test cell; the large-area RGO film is made by a graphene oxide film spin-coated on a glass substrate through a 100°C hydrazine chemical reduction and a one-step 200°C air thermal annealing process;

[0010] Step 1: Preparation of large-area graphene membrane

[0011] (1) hydrophilizing the glass slide for 300-400 seconds; mixing 3-aminopropyltriethoxysilane solution and ethanol at a volume ratio of 1:100, and immersing the hydrophilized glass slide therein for 2-3 hours;

[0012] (2) The soaked glass sheet is rinsed with ethanol and dried with nitrogen; a graphene oxide solution with a concentration of 1 mg / mL is coated on the surface of the glass sheet, first at a low speed of 450-500 rpm for 10-15 s, and then at a high speed of 1000-1300 rpm for 30-40 s, to obtain a graphene oxide film;

[0013] (3) In a sealed device, the graphene oxide film is first chemically reduced with hydrazine vapor at 100°C for 45 min-1 h, and then thermally annealed in air at 200°C for 20-30 min to obtain a reduced graphene oxide film, namely a large-area RGO film.

[0014] Step 2: Preparation of large-scale graphene field-effect transistors with high aspect ratio

[0015] A graphene channel with an aspect ratio of 10:1-5:1 is designed on the reduced graphene oxide film, gold paste or silver paste is used to coat both sides of the graphene channel as source and drain electrodes, copper wires are connected to lead out the source and drain electrodes, and the electrodes are dried at a temperature of 100-120°C; the rest of the RGO film is removed, and the rest of the parts except the graphene channel are encapsulated with polydimethylsiloxane to fix the test cell;

[0016] Step 3: Modification of ion-selective membrane

[0017] The formula of the potassium ion selective membrane mixture is taken and dissolved in tetrahydrofuran (THF) to obtain a potassium ion selective membrane mixture solution, and the concentration of the potassium ion selective membrane mixture is controlled to be 125-250 mg / ml; the potassium ion selective membrane mixture solution is evenly treated by ultrasonic treatment and then sealed and stored at 4°C; the potassium ion selective membrane mixture solution is cast onto a graphene channel by drop coating, and a layer of stable ISM is obtained after vacuum drying for 30-60 minutes, and the device is manufactured; finally, a graphene field effect transistor biosensor capable of detecting potassium ions is obtained.

[0018] The potassium ion selective membrane (K) in the literature H.Li et al. / Sensors and Actuators B 253 (2017) 759–765 was used. + -ISM) mixture, specifically the potassium ion selective membrane mixture, is mainly composed of 2wt.% valinomycin, 0.6wt.% sodium tetraphenylborate, 32.7wt.% polyvinyl chloride PVC and 64.7wt.% dioctyl sebacate DOS according to mass percentage.

[0019] The area ratio of the potassium ion selective membrane mixture solution to the graphene channel is 7.5-8 μL / cm 2 .

[0020] Beneficial effects of the present invention:

[0021] 1. The reduction method of graphene oxide (GO) used is mild. Under the condition of not giving up the performance of the device, the hydrazine chemical reduction at 200℃ and below and within 2h and the air thermal annealing process reduction conditions greatly shorten the device preparation time and reduce the high reduction conditions. It can also achieve the graphene oxide reduction effect of hydrazine chemical reduction of 15 hours or more or high temperature conditions of 400℃ and above mentioned in the literature, which can be proved by the relative content of carbon and oxygen in XPS characterization and the sensitivity of the detection material. The mild reduction conditions make it also applicable to the fields of flexible electronics and wearables.

[0022] 2. Transfer-free preparation conditions, the glass sheet is directly used as the substrate to prepare the membrane sensing area and the entire biosensor device, the process is simple and economical.

[0023] 3. Micro-nano processing technology can be used to prepare multiple standard devices that are easy to integrate in an array, ensuring the performance consistency of devices produced in batches;

[0024] 4. The ion selective sensor prepared by the present invention has the characteristics of being simple and fast (transfer-free), economical (spin coating of large-area RGO film), mild conditions (200°C and below), label-free, and having a rapid response to potassium ions. It is combined with the IT2805 semiconductor tester to realize real-time monitoring of potassium ions, which is conducive to the development of real-time continuous ion detection devices under physiological environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The large-area RGO film prepared by spin coating, where a is the area of ​​the prepared film, 4×4 cm 2 , b extends to the entire wafer area.

[0026] Figure 2 Figure 3 Characterization of RGO membrane. Among them, a is the SEM image of the membrane; b is the SEM image of the edge after ISM modification and RGO comparison; c is the Raman characterization image, through the increase of the double peak D / G value, it can be known that GO is successfully reduced to RGO; d is the XPS characterization image, which can clearly show the relative content of the main elements C, N, and O.

[0027] Figure 3 This is the AFM characterization image of the film, where a is the morphology of the film surface and b is a schematic diagram of the film thickness.

[0028] Figure 4 This is a physical picture of the large device produced, which mainly includes source and drain electrodes and reduced graphene oxide sensing channels. Figure 5 Schematic diagram of the microdevice, where a is a real picture of the microelectrode on half a glass sheet (the center circle is the graphene channel array); b is a microscope picture of the channel array of the microdevice.

[0029] Figure 6 Figure 3 is the electrical performance and ion measurement diagram of the large device, where a is a set of bipolar characteristic transfer curves of the device, and it can be seen that the electrical performance is very stable; b is the output curve of the device; c is the IT response curve of potassium ion detection at different concentrations; d is a linear fitting diagram of the changes in source-drain current caused by testing different potassium ion concentrations. For non-standardized large devices, a linear correlation of 0.995 is already very high.

[0030] Figure 7 This is the response of the device to potassium ions after drug stimulation of cell lysis and apoptosis, including a control group and a drug-treated group, where a is the IT response curve of the two groups of experiments, and b is a comparison chart of the source-drain current change values. DETAILED DESCRIPTION

[0031] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0032] Example 1

[0033] (1) Preparation of large-area RGO membrane

[0034] 1) Pretreatment of glass slide: Place the glass slide in an oxygen plasma cleaning machine for 300-400 seconds for hydrophilic treatment.

[0035] 2) APTES solution preparation: 3-aminopropyltriethoxysilane (APTES) solution. The silicon-oxygen bonds and amino groups at both ends of this substance are combined with the hydroxyl groups on the pretreated glass sheet and the carboxyl groups of graphene oxide (GO), respectively, which can make GO better and tightly combined with the glass substrate, and improve the stability of the obtained reduced graphene oxide (RGO) film. Dissolve APTES in ethanol at a volume ratio of 1:100 in a dry beaker and mix by ultrasonic.

[0036] 3) Soaking: Soak the glass slide in APTES for 2-3 hours, take it out, rinse it with ethanol, and blow it dry with nitrogen.

[0037] 4) Spin coating graphene oxide: Take 300-500 μL of 1 mg / mL GO solution and drop it onto the treated glass slide. Use a dropper to evenly distribute a suitable area on the glass slide. Wait for about 3 minutes to allow GO and APTES to fully bond. Adjust the speed of the desktop gel coater to a low speed of 450-500 rpm for 10-15 s and a high speed of 1000-1300 rpm for 30-40 s. Place the glass slide on the spin coating to obtain a GO film.

[0038] 5) Reduction: A combination of chemical reduction and thermal annealing is used to reduce the oxygen-containing groups on GO as much as possible, and GO is reduced to obtain an RGO film. First, a glass sheet GO film and a hydrazine hydrate solution are placed in a sealed device, and the sealed device is placed on a hot plate, heated at 100°C for 45min-1h, and the GO film is reduced with hydrazine vapor; then the glass sheet is taken out and placed on a hot plate, and air-annealed at 200°C for 20-30min to obtain a uniform and uniform film of about 4×4cm in size. 2 Large area RGO film.

[0039] (2) Preparation of large graphene field-effect transistor devices with high aspect ratio

[0040] 1) Reserved channel area: A large area of ​​uniform RGO film is obtained, and a channel with an aspect ratio of 10:1-5:1 is taken. All parts of the film outside the sensing area are scraped off with a blade.

[0041] 2) Conductive silver paste is used as the source and drain electrodes for large devices. The silver paste is coated on both sides of the sensing RGO film channel, and copper wires are connected to lead out the electrodes for subsequent testing. The device is placed on a hot table at 100-120°C to dry the silver paste. After drying, the organic thermosetting adhesive polydimethylsiloxane (PDMS) is used to encapsulate the silver paste source and drain to isolate the electrodes from being exposed to the subsequent test liquid environment.

[0042] 3) After the encapsulation PDMS is cured, the test pool is also fixed on the device with PDMS. The pool contains all the sensing channel areas, and the device is initially manufactured ( Figure 4 (Figure 1) The device before fixing the test pool.

[0043] (3) Preparation of graphene field effect transistor microdevices

[0044] 1) First, prepare the alignment symbol. For the 80×20μm channel sensing area of ​​the microdevice, this step is crucial for precise alignment of the tiny areas in each subsequent step. Take the glass sheet and put it into the oxygen plasma cleaner for hydrophilic treatment for 200-300s, apply S1805 photoresist, adjust the low speed of the desktop glue spreader to 1000-1200rpm, 8-10s, high speed 2000-2200rpm, 25-30s, evenly coat, and then put the glass sheet on the hot stage at 110-120℃ for 2min. Then photolithography for 30s, ZX238 developer for 30-40s, water rinse, and nitrogen blow dry. After patterning, put it into the magnetron sputtering gold electrode, Cr target 180W, pre-sputtering 60s, sputtering 30s, Au target 30W, sputtering 240s. Take out the glass sheet and remove the glue with NMP glue remover to obtain the alignment symbol.

[0045] 2) After the alignment symbol is prepared, a large-area RGO film is prepared according to the method in Example 1, and S1805 photoresist is evenly coated under the above conditions for photolithography and development, and oxygen plasma treatment is performed for 300-400s to etch away all the graphene except the channel part, remove the glue, and only the graphene film in the channel sensing area remains. S1805 photoresist is evenly coated again, photolithography and development are performed, magnetron sputtering is performed, gold is deposited as the source and drain electrodes of the microdevice, and the glue is removed.

[0046] 3) Packaging: Use SU8 photoresist to package the electrodes, adjust the low speed of the desktop photoresist machine to 1000-1200rpm for 8-10s, high speed to 2000-2200rpm for 25-30s, evenly coat, pre-bake at 130-135℃ for 2min, photolithography for 150s, post-bake at 130-135℃ for 2min, remove the glue with PGMEA glue remover for 1min, rinse with ethanol and water, blow dry with nitrogen, and package to ensure that only the channel sensing area is exposed to the solution in subsequent tests. Use silver paste to connect the copper wire to lead out the electrode for subsequent testing, seal all places except the channel sensing area with PDMS, and stick the test pool with PDMS.

[0047] (4) Modification of ion-selective membranes

[0048] 1) Configuration of potassium ion selective membrane solution: The potassium ion selective membrane (K + -ISM) mixture, consisting of valinomycin (2%, w / w), sodium tetraphenylborate (0.6%, w / w), polyvinyl chloride (PVC) (32.7%, w / w) and dioctyl sebacate (DOS) (64.7%, w / w). Weigh 10 mg valinomycin, 3 mg sodium tetraphenylborate, 163.5 mg polyvinyl chloride, 323.5 mg dioctyl sebacate, and dissolve all the above mixture in 2 mL tetrahydrofuran THF. The obtained K + -ISM solution was homogenized by ultrasonic treatment and stored at 4°C in a sealed container. Then 5-10 μL K + -ISM mixture is cast onto the graphene sensing channel of the large device, and after vacuum drying for 30-60 minutes, a stable ISM layer is obtained, and the ion selective field effect transistor sensor (ISFET) is completed. 3-5μL ISM mixture solution is dripped onto the micro device, and vacuum dried for 30-60 minutes to obtain a stable ISM layer. Finally, a standardized graphene field effect transistor micro device that can detect potassium ions is obtained.

[0049] 2) Add PBS solution to the test pool and soak it overnight to allow the RGO membrane in the sensing area to fully undergo nonspecific adsorption and other reactions, so that the device test system tends to be stable, and therefore the electrical performance of the operating device is also very stable.

[0050] Example 2

[0051] Ion selective sensor tests different concentrations of potassium ions

[0052] 1) First, prepare potassium ion solutions of different concentrations. Five linear points are selected for the potassium ion concentration gradient, which are 4μM, 16μM, 64μM, 252μM, and 1000μM. The initial PBS solution volume contained in the device test pool is 5000μL, and 200μL of different potassium ion concentration solutions are added each time. Therefore, by calculation, 104μM, 432μM, 1.792mM, 7.308mM, and 30mM potassium ion solutions are prepared and dripped into the test pool in turn to obtain the above five linear potassium ion concentrations.

[0053] 2) Device electrical performance test and determination of different concentrations of potassium ions: All electrical properties of the device are tested using Ag / AgCl electrodes as gate electrodes. The test instrument is IT2805 semiconductor tester. The three electrodes (source, drain, gate) of the device are connected. The transfer characteristics of the device are tested first. The instrument test parameters are set to gate voltage V g The scanning range is -0.3V-0.5V, and the source-drain voltage V ds The fixed setting is 0.2V, and 4000 points are set to test for 120s. The test is first scanned three times to stabilize the electro-activated device, and then scanned three times continuously to obtain the required bipolar transfer characteristic curve ( Figure 6 a).

[0054] 3) Then set the instrument parameters according to the gate voltage at the maximum transconductance of the transfer curve to fix the gate voltage V g is -0.12V, fixed source-drain voltage V ds The IT curve was scanned at 0.2 V. After the IT curve current was basically stable, 200 μL of potassium ion solution with concentrations of 104 μM, 432 μM, 1.792 mM, 7.308 mM, and 30 mM was added at every 100 points to obtain potassium ion concentrations of 4 μM, 16 μM, 64 μM, 252 μM, and 1000 μM. Finally, an equal amount of 200 μL of PBS solution was added to ensure that the current change was caused by the charge carried by the potassium ions in the device capture solution, and the final IT curve was obtained ( Figure 6 c).

[0055] Example 3

[0056] Detection of potassium ions in the cellular environment

[0057] From the above, it can be seen that the prepared device can currently achieve the lowest μM level potassium ion detection, while the potassium ion concentration in the cell is about 140mM, and the potassium ion concentration outside the cell is 5mM, which is very suitable for clinical applications and health monitoring. After we prepare the device coated with the ion-selective membrane, we put it in the cell-to-cell clean bench for UV sterilization, and then incubate the cell active protein on the membrane to allow the cells to grow better. The protein is incubated in the incubator for 1h. Then take 300μL of culture medium containing Hela cells and add it to the device in the drug administration group, culture overnight, and no cell culture medium is added to the control group. After overnight, the IT curve of the drug administration group device is tested. After the curve runs steadily, the cell lysis solution Triton X-100 and the cell apoptosis reagent Rhein are added. Wait for 10-15 minutes, the cell membrane gradually becomes permeable, and the potassium ions are gradually released and captured by the device, generating a signal response. The control group device is operated in the same way, and the reagent is added at the same time after running steadily.

[0058] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized.

Claims

1. A simple and rapid method for making a large-area transfer-free graphene field-effect transistor biosensor, characterized in that: The large-area transfer-free graphene field effect transistor biosensor is composed of a large-area RGO film deposited on a glass substrate, a potassium ion selective membrane, a source-drain electrode made of gold or silver, and a test cell; the large-area RGO film is prepared by a graphene oxide film spin-coated on a glass substrate through a 100°C hydrazine chemical reduction and a one-step 200°C air thermal annealing process.

2. The method according to claim 1, characterized in that: The preparation steps of the large-area RGO membrane are as follows: (1) hydrophilizing the glass slide for 300-400 seconds; mixing 3-aminopropyltriethoxysilane solution and ethanol at a volume ratio of 1:100, and immersing the hydrophilized glass slide therein for 2-3 hours; (2) The soaked glass sheet is rinsed with ethanol and dried with nitrogen; a graphene oxide solution with a concentration of 1 mg / mL is coated on the surface of the glass sheet, first at a low speed of 450-500 rpm for 10-15 s, and then at a high speed of 1000-1300 rpm for 30-40 s, to obtain a graphene oxide film; (3) In a sealed device, the graphene oxide film is first chemically reduced with hydrazine vapor at 100°C for 45 min-1 h, and then thermally annealed in air at 200°C for 20-30 min to obtain a reduced graphene oxide film, namely a large-area RGO film.

3. The method according to claim 1, characterized in that: The preparation steps of the large-area transfer-free graphene field effect transistor biosensor are as follows: A graphene channel with an aspect ratio of 10:1-5:1 is designed on the reduced graphene oxide film, gold paste or silver paste is used to coat both sides of the graphene channel as source and drain electrodes, copper wires are connected to lead out the source and drain electrodes, and the electrodes are dried at a temperature of 100-120°C; the rest of the RGO film is removed, and the rest of the parts except the graphene channel are encapsulated with polydimethylsiloxane to fix the test cell; The formula of the potassium ion selective membrane mixture is taken and dissolved in tetrahydrofuran (THF) to obtain a potassium ion selective membrane mixture solution, and the concentration of the potassium ion selective membrane mixture is controlled to be 125-250 mg / ml; the potassium ion selective membrane mixture solution is evenly treated by ultrasonic treatment and then sealed and stored at 4°C; the potassium ion selective membrane mixture solution is cast onto a graphene channel by drop coating, and a layer of stable ISM is obtained after vacuum drying for 30-60 minutes, and the device is manufactured; finally, a graphene field effect transistor biosensor capable of detecting potassium ions is obtained.

4. The method according to claim 3, characterized in that: The formula of the potassium ion selective membrane mixture is mainly composed of 2wt.% valinomycin, 0.6wt.% sodium tetraphenylborate, 32.7wt.% polyvinyl chloride PVC and 64.7wt.% dioctyl sebacate DOS according to mass percentage.

5. The manufacturing method according to claim 3, characterized in that: The area ratio of the potassium ion selective membrane mixture solution to the graphene channel is 7.5-8 μL / cm 2 .

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