Method for detecting depression marker serotonin by liquid gate transistor sensor

Abstract

The invention provides a method for detecting a depression marker serotonin by using a liquid gate transistor sensor, which is characterized in that a DNA probe is selected as a DNA single-stranded sequence, the tail end of the probe is modified with sulfydryl, and the sulfydryl is fixed on the surface of a gold gate through an Au-S bond. The detection principle is that the serotonin is captured on the surface of the gold gate after being identified by the probe, and the probe generates conformational change after identifying the serotonin, so that the tail end with negative electricity extends towards the direction far away from the surface of the gold gate, and the potential change of the surface of the gate is caused; the Dirac point voltage of the transmission curve of the liquid gate graphene transistor moves towards the negative gate voltage direction, and the serotonin content can be detected by detecting the voltage change of the Dirac point of the transfer curve in the channel or the channel current change. A clinical sample is detected by the sensor, and the serotonin level of a depression patient is relatively high and is consistent with a clinical diagnosis result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biosensing technology, and particularly relates to a method for detecting a marker of depression, serotonin, using a single-stranded DNA as a probe, a principle thereof and preparation thereof. BACKGROUND

[0002] Depression, also known as depressive disorder, is a mental disorder with high incidence, high clinical cure rate, low treatment acceptance rate and high recurrence rate. Patients may have self-injurious behavior, and may even have psychotic symptoms such as delusion and hallucination.

[0003] The detection method is a biosensor based on a liquid gate graphene transistor. The biosensor is a sensor that uses a biological molecule probe to modify an electrode, and converts the change in the concentration of the detected biological molecule marker into an output signal. Biosensing has many important applications, such as disease diagnosis, environmental monitoring, food safety inspection and human health management. The interaction between the detected biological molecule marker and graphene is important for all types of graphene transistor biosensors, because they work in solution. Various graphene transistor-based biosensors have received widespread attention in recent years.

[0004] Currently, the diagnosis of depression only relies on the comprehensive assessment of the patient by the doctor, including the patient's self-rating scale, clinical rating scale and clinical manifestations, and lacks scientific and objective data indicators for support, while the correlation theory between serotonin and depression has been supported by a large number of literatures in recent years.

[0005] The single-stranded DNA probe used in the present application is a single-stranded DNA (ssDNA) with a sequence of 5'-SH-CGACTGGTAGGCAGATAGGGGAAGCTGATT CGATGCGTGGGT CG-3' and consists of 44 bases. The application of DNA probe in biosensors is favored due to its high specificity and sensitivity. The sensor based on liquid gate graphene transistor combines the advantages of DNA probe, and can provide a rapid, sensitive and specific means for biological detection, with a detection limit of 10 - 15 M, and has played a good role in clinical practical application. The measurement of real samples has obtained a detection result that the serotonin of the patient with depression is high. SUMMARY

[0006] The purpose of this invention is to rapidly and sensitively detect serotonin using a specific sequence of single-stranded DNA and a biosensor based on a liquid-gate graphene transistor, thereby providing a reliable basis for the diagnosis of depression. The graphene transistor provided by this invention is small in size, easily integrated into a portable device, simple to operate even by non-professionals, and has high sensitivity, with a detection limit as low as 10. -15 M.

[0007] This invention provides a method for detecting serotonin, which uses a sequence of bases with a specific arrangement, including terminally modified thiol groups, to achieve fixation and recognition of serotonin on the electrodes of a liquid-gate graphene transistor. The liquid-gate graphene transistor includes electronic-grade glass and a gate, source, and drain electrode deposited on the electronic-grade glass. A graphene channel is provided between the source and drain electrodes. The gate surface is made of gold, which can react with sulfur at the probe tip to form an Au-S bond, thereby fixing the probe in the sensor.

[0008] This invention immobilizes a single-stranded DNA probe on the surface of a gate electrode. The DNA probe can capture positively charged serotonin and undergo a conformational change: the negatively charged probe tip extends away from the gold gate surface of the liquid-gate graphene transistor. This alters the electrical double-layer interface characteristics between the transistor and the sample solution, causing a voltage change in the channel. By detecting this voltage change in the channel, trace amounts of serotonin in the solution can be detected.

[0009] Preferably, the width of the graphene channel is 0.2~0.3mm and the length of the graphene channel is 4~8mm.

[0010] Preferably, the graphene channel material is CVD monolayer graphene.

[0011] Preferably, the gate, source, and drain are composed of a chromium layer and a gold layer. The chromium layer is located between the electronic-grade glass substrate and the gold layer.

[0012] Preferably, the thickness of the chromium layer is 3~6nm and the thickness of the gold layer is 30~60nm.

[0013] Preferably, the single-stranded DNA biomolecular probe is immobilized on the surface of the gate by thiol modification.

[0014] Preferably, the thiol group is modified at the tip of the probe.

[0015] This invention also provides a method for fabricating the graphene transistor biosensor described in the above technical solution, comprising the following steps: A gate, source, and drain are fabricated on the substrate surface, with a channel existing between the source and drain. Graphene is laid flat on the channel between the source and drain to obtain a graphene transistor; A single-stranded DNA biomolecular probe is immobilized on the gold gate surface of the graphene transistor obtained in step (2) to obtain a graphene transistor biosensor.

[0016] In the above preparation method, the transistor substrate used in step (1) is preferably electronic-grade glass, silicon wafer, or PET, with electronic-grade glass being more preferred. The size of the electronic-grade glass is preferably 12mm × 12mm. In this invention, the electronic-grade glass is preferably GL-10173-1.1.

[0017] The glass substrate is pretreated by sequentially cleaning it with acetone, anhydrous ethanol, water, and anhydrous ethanol under ultrasonic conditions for 20-30 minutes, preferably 25 minutes. Drying is preferably performed by baking.

[0018] In the above preparation method, the preparation of the gate, source and drain in step (1) includes: depositing a chromium layer and a gold layer on the substrate surface in sequence using a thermal evaporation coating instrument. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the fabrication process of the graphene transistor in an embodiment of the present invention; Figure 2 This is a schematic diagram of the graphene transistor biosensor in an embodiment of the present invention; Figure 3 This is a schematic diagram of a graphene transistor in an embodiment of the present invention; Figure 4 This is the transfer characteristic curve of the graphene transistor biosensor detecting the concentration of serotonin biomolecules in an embodiment of the present invention; Figure 5 This is a standard curve showing the Dirac point voltage change value of the graphene transistor biosensor and the concentration of serotonin biomolecules in an embodiment of the present invention; Figure 6 This is the current output characteristic curve of the graphene transistor biosensor detecting changes in serotonin biomolecule concentration in an embodiment of the present invention; Figure 7 The water contact angle variation of the graphene transistor biosensor in this embodiment of the invention; Figure 8 This is the specificity curve of the graphene biosensor in serotonin and similar substances in an embodiment of the present invention; Figure 9 This is a comparison of the Dirac point of the graphene biosensor against other interfering substances and serotonin in an embodiment of the present invention; Figure 10This is a transfer characteristic curve of a simulated sample detected by a graphene biosensor in an embodiment of the present invention; Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and specific examples. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0021] The following are embodiments of certain implementations of the present invention, which are not intended to limit the scope of the present invention.

[0022] This invention provides a method for detecting serotonin, a biomarker for depression, using single-stranded DNA as a probe. The method uses a single-stranded DNA sequence to recognize serotonin; the DNA single-stranded probe is immobilized on the gate surface of a liquid-gate graphene transistor by end-modified thiol groups; the liquid-gate graphene transistor biosensor includes a substrate. This invention does not specifically limit the type and source of the substrate, but preferably uses electronic-grade glass, silicon wafers, or PET. In a specific embodiment of this invention, the substrate is preferably electronic-grade glass; in this invention, the electronic-grade glass is preferably GL-10173-1.1.

[0023] The present invention does not impose any particular 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 10-15 mm, more preferably 12 mm; the thickness of the substrate is preferably 2 mm.

[0024] The graphene transistor biosensor provided by this invention includes a gate, a source, and a drain on the substrate. In this invention, there are no special requirements for the specific positions of the gate, source, and drain; their positions can be designed according to the technical needs of the art. Preferably, the gate, source, and drain are sequentially spaced on the same surface of the substrate. The channel width of the source and drain is preferably 0.2~0.3 mm. This invention does not impose any special limitations on the shapes of the gate, source, and drain; electrode shapes well-known to those skilled in the art can be used.

[0025] In this invention, the gate, source, and drain preferably each independently comprises a chromium layer and a gold layer, with the chromium layer located between the electronic-grade glass substrate and the gold layer. In this invention, the thickness of the chromium layer in the gate, source, and drain is preferably 3-6 nm, more preferably 10 nm. The three-electrode structure of the gate, source, and drain enables the channel current to be controlled using the voltage of the input gate during serotonin detection, achieving an operating voltage below 1 V.

[0026] The gate, source, and drain electrodes described in this invention will be deposited using a JSD-300 evaporation coating system. The chromium and gold layers on the substrate are sequentially deposited onto the substrate using a mask method in the evaporation coating system. The specific parameters of the thermal evaporation coating system are not specifically limited, and the corresponding parameters are based on the thickness of the resulting electrodes.

[0027] In this embodiment of the invention, the operating environment of the thermal evaporation coating instrument is preferably a vacuum environment; the vacuum degree is preferably 3×10⁻⁶. -4 ~6×10 -4 Pa or less, more preferably 4×10 Pa -4 Pa. The electrode on the substrate is composed of a chromium layer and a gold layer, wherein the evaporation current of the chromium layer is preferably 170~180A; and the evaporation current of the gold layer is preferably 110~120A.

[0028] The graphene transistor biosensor provided by this invention includes a graphene channel disposed between the source and drain electrodes. In this invention, the channel formed between the source and drain electrodes as described in the above technical solution is the graphene channel. In this invention, the width of the graphene channel is preferably 0.2~0.3 mm, more preferably 0.25 mm; the length of the graphene channel is preferably 4~8 mm, more preferably 5~6 mm. In this invention, the graphene preferably fills the gap between the source and drain electrodes and covers the channel portion on the surface of the source and drain electrodes. In this invention, the graphene channel is preferably CVD monolayer graphene. In this invention, the graphene channel can improve the sensitivity of the sensor.

[0029] After the gate, source, and drain electrodes are fabricated, graphene is laid flat on the source and drain channels to obtain a graphene transistor. In this invention, the monolayer graphene on the metal substrate is preferably copper-based CVD monolayer graphene. This invention does not have a specific limitation on the source of the monolayer graphene on the metal substrate; commercially available products well known to those skilled in the art, or prepared according to methods well known to those skilled in the art, are acceptable.

[0030] This invention does not impose any special limitations on the operation of the transfer, and the wet transfer technique for monolayer graphene, which is well known to those skilled in the art, can be used. In this invention, the wet transfer technique for monolayer graphene is preferably based on the technique disclosed in Chen Mu, Yan Yue, Zhang Xiaofeng, et al. Research progress on large-area graphene film transfer technology [J]. Journal of Aeronautical Materials, 2015, 35(2): 1-11.

[0031] After the graphene transfer is completed, the transferred device is preferably annealed to obtain a graphene transistor. In this 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 this invention, annealing removes moisture from the sample surface and simultaneously allows the graphene to bond more tightly to the substrate.

[0032] After obtaining the graphene transistor, the present invention preferably immobilizes a single-stranded DNA biomolecular probe on the gate surface of the graphene transistor, thereby obtaining a liquid-gate graphene transistor biosensor that uses the DNA probe to detect serotonin. The DNA biomolecular probe can capture the target biomarker serotonin, such as... Figure 1 As shown. In this invention, the preferred DNA biomolecular probe is modified with thiol groups, allowing it to form Au-S bonds with the gold gate and be fixed to the gate surface.

[0033] In this invention, the amount of DNA probe molecule modification on the gate surface is preferably 1~2 μM, more preferably 1 μM.

[0034] The principle of the graphene transistor biosensor provided by this invention is as follows: Figure 2 As shown, the gate electrode and the graphene channel are connected by an electrolyte, forming a "double capacitor" structure. The voltage applied to the gate is constant, while the voltage applied to the channel will change the characteristics of the double layer interface due to the conformational and potential changes between the DNA biomolecule probe and serotonin. This will cause a shift in the Dirac point voltage and a change in the current in the graphene channel. By detecting the shift in the Dirac point voltage and the change in the current in the channel, trace amounts of serotonin biomolecules in the solution can be quantitatively detected.

[0035] This invention also provides the application of the graphene transistor biosensor described in the above technical solution in the detection of serotonin, a biomarker for depression. The graphene transistor biosensor's gate and graphene channel are immersed in an electrolyte containing the serotonin to be measured. In this invention, the electrolyte is preferably a 0.01×PBS and MgCl2 mixed solution; the pH value of the electrolyte is preferably 7.4. In this invention, the gate and graphene channel are connected through the electrolyte, and the current in the graphene channel is controlled by the voltage input to the gate, such as... Figure 3 As shown.

[0036] When using the graphene transistor biosensor provided by this invention to quantitatively determine serotonin, it is preferable to prepare a standard solution containing serotonin, measure the current change value and the standard curve of serotonin concentration, and determine the detection limit and detection range of the serotonin to be tested based on the standard curve and the current change obtained from the test.

[0037] The graphene transistor serotonin biosensor provided by this invention can detect serotonin concentration by direct immersion in the test solution, providing a label-free detection method. It also exhibits high sensitivity and good stability. The three-electrode structure and graphene channel of the graphene transistor serotonin biosensor make it highly sensitive to voltage changes; even a small voltage change will trigger a corresponding current change, resulting in high sensitivity. Furthermore, the graphene transistor biosensor provided by this invention can detect serotonin concentrations as low as 10... -15 Detection of trace amounts of serotonin biomolecules in M.

[0038] To further illustrate the present invention, the graphene transistor serotonin biosensor, its preparation method, and its application provided by the present invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example

[0039] Thermal evaporation coating: Cut electronic-grade glass into 12×12mm pieces, and ultrasonically clean it in sequence with acetone, ethanol, deionized water and ethanol for 20 minutes. After drying with nitrogen, use high-temperature adhesive to attach the glass sheet to a mask of a specific shape. Weigh an appropriate amount of chromium and gold and put them into a tungsten boat to prepare for vacuum thermal evaporation coating.

[0040] Chromium is evaporated first during evaporation: the thickness is 3nm.

[0041] Gold layer is deposited again: 30nm thick.

[0042] The obtained electrode shape, structure and size are as follows Figure 3 As shown. Figure 3 In the diagram, G stands for gate, S for source, and D for drain. The 0.25mm wide channel between the source and drain is formed by transferring graphene and thus creating the graphene channel.

[0043] Wet transfer of monolayer graphene: 250 mg of methyl methacrylate (PMMA) with a molecular weight of 99600 g / mol was dissolved in 5 mL of anisole and stirred on a magnetic stirrer to obtain a clear and transparent PMMA anisole solution with a concentration of 50 mg / mL.

[0044] CVD monolayer copper-based graphene was cut to a size of 12×12mm. 100μL of the prepared PMMA / anisole solution was dropped onto the graphene surface and spin-coated. The spin coater was set to a speed of 600 and 3000 rpm, and the spin coating time was 3s and 30s. After spin coating, the surface was heated at 100℃ for 10min to obtain PMMA / graphene.

[0045] Prepare a 100 mg / mL ferric chloride solution. Spin-coat the PMMA / graphene with the PMMA / anisole solution facing up and place it in the ferric chloride solution to completely etch away the copper substrate.

[0046] The PMMA / graphene with the copper substrate etched off was transferred to deionized water and soaked for 5 minutes using a glass slide. It was then rinsed twice with deionized water to remove the residual ferric chloride solution from the PMMA / graphene. The device fabricated by thermal evaporation coating was ultrasonically cleaned with acetone, ethanol and deionized water respectively. After being dried with nitrogen, the device surface was cleaned with an oxygen plasma cleaner to improve the hydrophilicity of the device.

[0047] The cleaned PMMA / graphene sheet was transferred to the cleaned device and laid flat on the channel between the source and drain electrodes on the device surface. After air drying until no surface moisture was visible to the naked eye, it was placed on a hot plate at 110°C for annealing to completely remove the surface moisture of the sample, thus obtaining the PMMA / graphene / electrode sheet.

[0048] After cooling to room temperature, excess PMMA / graphene was removed from both sides of the channel using tweezers. The PMMA / graphene / electrode sheet was then rinsed twice with acetone for 5 minutes each time. Next, the PMMA / graphene / electrode sheet was immersed in an acetone solution and heated at 70°C for 3 hours to remove surface PMMA, yielding the desired graphene transistor. After 3 hours, the graphene transistor was rinsed with deionized water and allowed to air dry.

[0049] Immobilization of single-stranded DNA biomolecular probes: 10 μL of 1 μM DNA single-stranded biomolecular probe solution was dropped onto the surface of the gate, and washed three times with PBS buffer after 18 hours; the unimmobilized DNA biomolecular probes and other impurities on the gate were washed away to obtain the graphene transistor serotonin biosensor. Example

[0050] The graphene transistor biosensor prepared in Example 1 was used to test the serotonin target molecule. The source, drain, and gate electrodes of the graphene transistor were connected to two digital source meters (Keithley 2450). The gate voltage... V G Source-drain voltage V DS It is controlled by a LabVIEW program on the computer.

[0051] The modified gate electrode was thoroughly cleaned with PBS solution to remove any residues remaining on the electrode. The test was performed in a container containing 100 μL of PBS solution. During the test, serotonin solutions of specific concentrations were sequentially added to the PBS solution at concentrations of 10... -15 M, 10 -13 M, 10 -11 M, 10-9 M, 10 -7 M, thus obtaining the output signal at different concentrations.

[0052] Principle explanation: According to Figure 2 As can be seen, after the probe captures positively charged serotonin, the conformation changes, and the probe tip extends away from the gold gate surface, causing the potential of the gold gate surface of the liquid gate graphene transistor to increase. The Dirac point voltage of the transfer curve of the liquid gate graphene transistor shifts towards the negative gate voltage direction. The serotonin content can be detected by detecting the voltage change at the Dirac point of the transfer curve or the channel current change in the channel.

[0053] Transfer characteristic curve test: The source-drain voltage was set to a constant value ( V DS =0.1V), measure the communication current between the source and drain when the gate voltage changes continuously from 0V to 1V. I DS Changes in the voltage will alter the potential at the device surface, thus shifting the characteristic curve. Figure 4 It can be seen that the Dirac point voltage shifts with changes in serotonin concentration, according to... Figure 5 It can be seen that the change in Dirac point voltage and serotonin concentration have a good linear relationship.

[0054] Output characteristic test: After setting the source-drain voltage and gate voltage to constant values, the channel current versus time graph was continuously measured. During the test, the channel current was allowed to stabilize before adding the next concentration. The test results are shown below. Figure 6 As shown. According to Figure 6 It can be seen that changes in serotonin concentration cause significant changes in current, and the current of the sensor changes instantly after the concentration changes, demonstrating high sensitivity.

[0055] Water contact angle test: The water contact angle of the gate before and after modification was tested. The modified single-stranded DNA probe formed a strong hydrophilicity with the gold surface. Figure 7 It can be seen that the water contact angle after modification is smaller than that before modification, and the hydrophilicity is increased.

[0056] Specificity Curve Test: Single-stranded DNA bioprobes exhibit excellent specificity due to their unique sequence design. They are immersed in a solution of biomolecules similar to serotonin, namely uric acid (a small molecule like serotonin), dopamine (a neurotransmitter like serotonin), L-tryptophan (a precursor of serotonin), 5-hydroxyindole-3-acetic acid (a metabolite of serotonin), and 5-hydroxytryptophan (a precursor of serotonin). Non-serotonin substances do not completely pair with the single-stranded DNA bioprobe used to detect serotonin, therefore they do not cause a shift in the Dirac point. Based on... Figure 8 It can be seen that only immersion in an environment marked by serotonin markers results in a significant shift in the Dirac point. According to... Figure 9 It can be seen that the Dirac point offset value caused by serotonin is many times that of other interfering substances, which indicates that the prepared sensor has good selectivity for serotonin.

[0057] Simulated sample testing: A serotonin solution of a specific concentration is added to artificial serum, with concentrations of 10... -15 M, 10 -13 M, 10 -11 M, 10 -9 M, 10 -7 M, thus obtaining the output signal under different simulated sample concentrations. According to Figure 10 It can be seen that the Dirac point voltage shifts as the concentration of the simulated serotonin sample changes.

[0058] Real sample testing: Testing of real samples from 12 patients revealed elevated serotonin levels in the blood of patients with depression, consistent with clinical diagnoses.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. It should be noted that those skilled in the art can make several improvements without departing from the principles of the present invention.

Claims

1. A method for the preparation and detection of liquid-gated graphene transistor sensor for depression marker serotonin detection, characterized in that: The gold gate, the source and the drain are arranged on the substrate, and a single-layer graphene conductive channel is arranged between the source and the drain; The single-stranded DNA probe is fixed by using a terminal modification method, and the sequence of the gold gate is 5'-SH-CGACTGGTAGGCAGATAGGGGAAGCTGATTCGATGCGTGGGTG-3', the terminal modification is a mercapto group, and the probe fixing amount is 1-2 μM.

2. The method of claim 1, wherein the step of detecting is characterized by, When the probe captures the serotonin, the conformation of the probe changes, the terminal of the probe extends away from the surface of the gold gate due to the negative electricity of the probe itself, the surface potential of the gold gate of the liquid gate graphene transistor is increased, the Dirac point voltage of the transfer curve of the liquid gate graphene transistor is moved to the negative gate voltage direction, and the content of the serotonin can be detected by detecting the voltage change of the Dirac point of the transfer curve in the channel or the change of the channel current.

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