Method for detecting prostate cancer marker GSTP1 hypermethylation promoter DNA in urine

Abstract

The invention provides a method for rapidly and sensitively detecting GSTP1 (Glutathione S Transferase Protein 1) hypermethylated promoter DNA (Deoxyribose Nucleic Acid) of prostatic cancer in urine. Based on the liquid gate graphene transistor sensor, a bridge-shaped tetrahedral DNA probe is modified to the surface of a gold gate through a gold-sulfur bond, and a hypermethylated GSTP1 promoter converted by hydrosulphite can be selectively captured. Wherein DNA is treated by hydrosulfite, so that unmethylated cytosine (C) can be converted into uracil (U), and methylated cytosine (5-mC) is kept unchanged. According to the bridge-shaped tetrahedron DNA, due to the influence of electrostatic repulsion and space structure between two tetrahedrons, the transverse distance between curled ssDNA is easily controlled to improve the hybridization efficiency and reduce Debye shielding, the detection limit can be as low as 10-18 M, the wide linear detection range of 10 <-18 >-10 <-8 > M can be achieved, and the detection efficiency is greatly improved. The kit has a good detection effect in complex artificial urine and clinical practical sample detection, can effectively distinguish clinical urine samples of prostatic cancer patients and control groups, and has clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biosensors, and mainly to a method for detecting GSTP1 hypermethylated promoter DNA in urine for prostate cancer. Background Technology

[0002] Cancer screening, early diagnosis, and early treatment are key strategies for effectively mitigating the growing cancer burden. Although serum PSA screening for prostate cancer has been widely used since the early 1990s, only 30% to 40% of cases are diagnosed with cancer at initial biopsy, while the rest are diagnosed with benign prostatic hyperplasia or prostate intraepithelial neoplasia. DNA methylation is a stable, heritable covalent modification that occurs primarily against a CpG dinucleotide background. DNA hypermethylation has been shown to be one of the most common molecular alterations in prostate cancer, with over 90% of cancers exhibiting hypermethylated promoters in one or more genes, making it a promising biomarker for disease detection, diagnosis, and predicting treatment response.

[0003] Studies have shown that the sensitivity of GSTP1 hypermethylation in urinary sediment is 70%-83%, and the specificity is between 86%-100%, which is higher than that of PSA. Furthermore, unlike blood, urine is a good source for non-invasive in vitro diagnosis of urogenital diseases, effectively reducing the suffering caused by overdiagnosis. Although many technologies have been developed to detect DNA methylation over the past decade, these analytical methods face several challenges, including high cost and high technical complexity, which hinder the clinical translation of routine methods into standard practice. Therefore, it is necessary to develop a highly sensitive, rapid, selective, low-power, cost-effective, miniaturized, and portable biosensor to achieve rapid and accurate diagnosis of prostate cancer.

[0004] To meet the urgent need for non-invasive diagnosis of prostate cancer and address the aforementioned issues, various biosensors have been developed. These sensors immobilize biomolecular probes on the electrode surface of the sensor, allowing them to recognize targets in a buffer solution and generate specific physical or chemical signals. These signals are then converted into output signals for detection. Due to the continuous emergence of novel nanomaterials, graphene has attracted widespread attention because of its high carrier mobility, high specific surface area, good biocompatibility, and chemical stability, making it an excellent material for fabricating biosensors. Graphene-based liquid-gate graphene transistor sensors have already demonstrated excellent performance in the detection of various biomarkers, including DNA, RNA, proteins, and small molecules. The design principle of this invention involves modifying a bridge-shaped tetrahedral DNA probe onto the gate of a liquid-gate graphene transistor. This probe recognizes the hypermethylated GSTP1 promoter after bisulfite treatment. After bisulfite treatment, unmethylated cytosine (C) is converted to uracil (U), while methylated cytosine (5-mC) remains unchanged. In 0.1×PBS buffer solution, the probe binds to negatively charged methylated DNA, causing a change in the gate electrode potential, which in turn alters the channel current. Detection of the target analyte is achieved by measuring the shift in the Dirac point voltage of the transport curve or the change in the transistor channel current. Furthermore, due to the electrostatic repulsion and spatial structure between tetrahedral DNA molecules, the designed bridge-shaped tetrahedral DNA probe has the advantage of easily controlling the lateral distance between coiled ssDNA molecules to improve DNA hybridization efficiency. Summary of the Invention

[0005] This invention provides a small, easily integrated, portable detection device—a liquid-gate graphene transistor sensor—for detecting DNA methylation of the GSTP1 promoter, a urinary biomarker for prostate cancer. The device is simple and convenient to operate, requiring no professional personnel, and has a detection limit as low as 10. -18 M, can achieve 10 -18 ~10 -8 M's wide linear detection range provides excellent detection performance in both complex artificial urine environments and actual clinical sample testing.

[0006] The liquid gate graphene transistor biosensor provided by this invention consists of a substrate and a gate, source, and drain on the substrate; graphene is placed between the source and drain as a channel; and a probe of bridge-shaped tetrahedral DNA is fixed on the surface of the gold gate.

[0007] The substrate is a glass substrate with a length and width of 1.2 cm.

[0008] The gate, source, and drain include a chromium layer with a thickness of 2-5 nm on the substrate surface and a gold layer with a thickness of 25-35 nm on the upper surface of the chromium layer.

[0009] The channel material is CVD-grown monolayer graphene, preferably with a length of 4-8 mm and a width of 2-3 mm.

[0010] The tetrahedral DNA probe has thiol groups modified at its tetrahedral base and fixed onto the gold grid surface.

[0011] This invention provides a method for preparing the above-mentioned graphene biosensor, comprising the following steps: (1) Chromium and gold layers were deposited on the surface of the glass substrate under vacuum conditions using a JSD-300 evaporation coating machine; (2) The cut graphene monolayer with a width of 2 mm and a length of 6 mm was transferred to the channel between the source and drain electrodes by a wet chemical method to obtain a graphene transistor. (3) By using a PCR instrument to react 8 mutually matched single strands of DNA at 95℃ for 10 min, and then cooling to 4℃ and holding for 30 s, tetrahedral DNA was successfully synthesized. ssDNA was added to the tetrahedral DNA and reacted at 37℃ for 2 h to obtain bridge-shaped tetrahedral DNA. The preferred molar ratio of tetrahedral DNA to ssDNA is 2:1. (4) The bridge-shaped tetrahedral DNA probe synthesized in step (3) is fixed on the gate surface of the graphene transistor obtained in step (2) by Au-S bond interaction, thus obtaining a graphene transistor biosensor that can be used for the detection of GSTP1 methylated DNA after bisulfite treatment.

[0012] This invention also provides the application of the liquid-gate graphene transistor prepared above in the detection of methylated DNA after bisulfite treatment, which has advantages such as operating voltage less than 1V, low detection limit, high sensitivity, fast detection time, wide linear detection range, and effective avoidance of Debye shielding effect. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the fabrication of the liquid gate graphene transistor in an embodiment of the present invention; Figure 2 This is a schematic diagram of the bridge-shaped tetrahedral DNA synthesis and modification process in an embodiment of the present invention; Figure 3 This is a schematic diagram of a liquid-gate graphene transistor biosensor in an embodiment of the present invention; Figure 4 This is a curve showing the transfer characteristics of GSTP1 methylated DNA biomolecules at different concentrations after bisulfite treatment, as detected by the liquid gate graphene transistor biosensor in this embodiment of the invention. Figure 5 This is a standard curve of the concentration of the marker for the Dirac point voltage change value of the graphene transistor biosensor in this embodiment of the invention; Figure 6 This is a current output characteristic curve of the liquid gate graphene transistor biosensor in this embodiment of the invention for detecting different concentrations of GSTP1 methylated DNA biomolecules treated with bisulfite. Figure 7 This is a schematic diagram of the detection principle of the liquid gate graphene transistor biosensor in an embodiment of the present invention; Figure 8 The above are the transfer characteristic curves of the liquid-gate graphene transistor biosensor in an artificial urine environment in this embodiment of the invention for detecting different concentrations of markers. Figure 9 This is a radial bar graph showing the detection of a liquid-gate graphene transistor biosensor in a clinical sample according to an embodiment of the present invention. Detailed Implementation

[0014] This invention provides a liquid-gate graphene transistor sensor for detecting GSTP1 methylated DNA, a urinary biomarker for prostate cancer; the transistor includes a substrate and three electrodes—gate, source, and drain—on the substrate surface; a CVD-grown monolayer graphene channel is attached between the source and drain electrodes; and a bridge-shaped tetrahedral DNA probe is fixed on the gate surface.

[0015] This invention does not limit the type of substrate used; electronic-grade glass, silicon wafers, or polyester resin can be selected. Electronic-grade glass (GL-10173-1.1) with a size of 10 × 10 mm is preferred. 2 ~14×14 mm 2 More preferably 12×12 mm 2 .

[0016] In this invention, the gate, source, and drain are placed on the same plane, and their specific positions are not particularly required; the three electrodes are preferably a chromium layer and a gold layer, with the gold layer located on top of the chromium layer. The thickness of the chromium layer is preferably 2-5 nm, more preferably 3 nm, and the thickness of the gold layer is 25-35 nm, more preferably 30 nm; the distance between the source and drain is preferably 0.2-0.3 mm, the width of the graphene channel is 0.2-0.3 mm, preferably 0.2 mm, and the length is 4-8 mm, preferably 6 mm. The graphene is preferably CVD-grown monolayer graphene.

[0017] This invention does not impose any special limitations on the wet transfer operation; any wet transfer technique for monolayer graphene well-known to those skilled in the art can be used. In this invention, the preferred technique for wet transfer of monolayer graphene is the one 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.

[0018] This invention also provides the synthesis of a bridge-shaped tetrahedral DNA probe. Eight paired single-stranded DNA molecules are reacted using a PCR thermal cycler at a temperature preferably 95°C for 10 min, then cooled to 4°C and held for 30 s to successfully synthesize tetrahedral DNA. ssDNA is added to the tetrahedral DNA and reacted at 37°C for 1.5–3 h, preferably 2 h, to obtain the bridge-shaped tetrahedral DNA. The preferred molar ratio of tetrahedral DNA to ssDNA is 2:1.

[0019] This invention also provides the application of the aforementioned liquid-gate graphene transistor in detection. The modified aptamer gate is immersed in a test solution, and the gate and graphene channel of the biosensor based on the liquid-gate graphene transistor are immersed in an electrolyte containing the analyte. In this invention, the electrolyte is preferably a 0.1×PBS solution with a pH of 7.4. In this invention, the gate and graphene channel are connected by the electrolyte, and the current in the graphene channel is controlled by the voltage input to the gate.

[0020] When using the graphene transistor biosensor provided by this invention to quantitatively determine the methylation of the GSTP1 promoter that has undergone bisulfite conversion, it is preferable to prepare a standard solution of the target analyte, measure the standard curve of current change value versus target analyte concentration, and determine the detection limit and detection range of the analyte based on the standard curve and the measured current change value.

[0021] The liquid-gate graphene transistor biosensor provided by this invention can be directly immersed in a solution containing the analyte for highly sensitive detection, exhibiting excellent stability and specificity. This sensor has extremely high sensitivity due to its very strong responsiveness to voltage changes; even a small voltage change will cause a corresponding current change. Furthermore, the detection limit of the liquid-gate graphene crystal provided by this invention is as low as 10⁻⁶. -18 M.

[0022] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It is to be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1

[0023] (1) Thermal evaporation coating: The substrate, electronic-grade glass, silicon wafer or polyester resin, preferably electronic-grade glass (GL-10173-1.1), is cut into pieces of 10×10 mm. 2 ~14×14 mm 2 More preferably 12×12 mm 2The glass slide is then ultrasonically cleaned sequentially with appropriate amounts of acetone, anhydrous ethanol, deionized water, and anhydrous ethanol. The ultrasonic cleaning time is preferably 10–30 minutes, more preferably 20 minutes. This invention does not impose a specific limitation on the power of the ultrasonic cleaning; any power known to those skilled in the art can be used. After drying or nitrogen blowing, the cleaned glass slide is adhered to a mask of a specific shape using high-temperature resistant adhesive. Preferably, 0.15–0.16 g of gold and 3–4 chromium particles are weighed and placed in a tungsten boat.

[0024] During evaporation, chromium is first evaporated, with a thickness preferably of 2-5 nm, more preferably 3 nm; then a gold layer is deposited, with a thickness preferably of 25-35 nm, more preferably 30 nm.

[0025] In this invention, the chromium layer ensures that the gold layer adheres firmly to the substrate surface, preventing the gold layer from peeling off during subsequent operations. In this invention, the thermal evaporation coating is preferably performed under vacuum conditions; the vacuum level is preferably 8 × 10⁻⁶. - 4 Pa or less, more preferably 4×10 Pa -4 Pa. The evaporation current of the chromium layer is preferably 170-200 A, more preferably 170-180 A; the evaporation current of the gold layer is preferably 100-130 A, more preferably 105-120 A.

[0026] The electrode shape, structure, and dimensions of the obtained device are as follows: Figure 1 As shown in the diagram, G stands for gate, S for source, and D for drain. A 6×2 mm graphene element is transferred between the source and drain to form a channel.

[0027] (2) Wet transfer of graphene.

[0028] 0.14 g of methyl methacrylate (PMMA) was dissolved in 4 mL of anisole and stirred on a magnetic stirrer to obtain a clear and transparent PMMA / anisole solution with a concentration of 35 mg / mL.

[0029] CVD monolayer copper-based graphene was cut into 12-12 mm pieces. 75 μL of the prepared PMMA / anisole solution was dropped onto the graphene surface and spin-coated. The spin coater was set to spin coat at 600 rpm for 3 s and 3000 rpm for 30 s. After spin coating, the surface was heated at 100 ℃ for 10 min to obtain PMMA / graphene.

[0030] Cut PMMA / graphene to a size of 6×2 mm, place the side coated with PMMA / anisole solution facing up in a pre-prepared 100 mg / mL ferric chloride solution until the copper substrate is completely etched.

[0031] Carefully transfer the PMMA / graphene sheet with the copper substrate etched off onto a glass slide and immerse it in deionized water for 5 minutes. Repeat the cleaning process 3 times to remove any residual ferric chloride solution from the PMMA / graphene. Take the device fabricated by thermal evaporation coating and clean the device surface with an oxygen plasma cleaner to improve the device's hydrophilicity.

[0032] 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 the moisture was no longer visible to the naked eye, it was placed on a hot plate and annealed at 75°C for 10 min and then at 105°C for 30 min to completely remove the moisture from the sample surface, thus obtaining PMMA / graphene / device.

[0033] After cooling, the device was immersed twice in acetone (PMMA / graphene / device), 5 minutes each time. Then, it was immersed in the acetone solution and heated at 75°C for 3 hours to completely remove the surface PMMA, yielding the desired graphene transistor. After heating, the graphene transistor was rinsed twice with deionized water, 5 minutes each time, to remove excess acetone, and then dried with nitrogen gas. Figure 1 The diagram shown is a schematic of thermal evaporation coating and graphene transfer.

[0034] (3) Preparation of bridge-shaped tetrahedral DNA probe: In this example, eight complementary single-stranded DNA molecules of the same concentration (preferably 10 μM) and volume (preferably 10 μL) were reacted in a PCR thermal cycler at a temperature preferably 95°C for 10 min, followed by rapid cooling to 4°C and holding for 30 s, yielding 80 μL of 1.25 μM tetrahedral DNA. Finally, one part ssDNA was added to twice the volume of the 1.25 μM tetrahedral DNA, and the reaction was carried out at a temperature preferably 37°C for 2 h to obtain the final bridge-shaped tetrahedral DNA probe. This probe was modified on a gold grid surface at room temperature for a reaction time preferably 10-13 h, more preferably 12 h. Figure 2 The diagram shown illustrates the synthesis and modification of a bridge-shaped tetrahedral DNA probe onto a gold grid. Example 2

[0035] The modified graphene transistor biosensor prepared in Example 1 was used to test target molecules, such as... Figure 3 The image shows a graphene transistor biosensor that has been fabricated and is ready for testing. The source, drain, and gate electrodes of the transistor are 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 in the computer.

[0036] Excess residue on the gate electrode surface was cleaned with 0.1×PBS solution. The cut PDMS groove, containing the gate and channel, was then attached to the device. 100 mL of 0.1×PBS solution was added to the groove. During testing, test solutions of specific concentrations were sequentially added to the PBS solution to obtain output signals at different concentrations.

[0037] Transfer characteristic curve test: Applying a transfer characteristic curve to the sensor gate V GS Gate voltage, drain voltage applied to the drain (D) V DS (V DS =0.1V), with the source electrode (S) grounded as a reference voltage. As the gate voltage continuously changes from 0V to 1V, the channel current change is measured. The concentration of the analyte in the solution is changed sequentially for measurement, and the resulting transfer characteristic curve is shown below. Figure 4 As shown, the change in Dirac point voltage has a good linear relationship with the concentration of the analyte, such as... Figure 5 As shown, the R of the fitted line 2 Value: 0.987.

[0038] Output characteristic test: Source-drain voltage and gate voltage were both set to constant values ​​(V). DS =0.1 V and V GS =0.3 V), continuously measure the relationship between channel current and time. During the test, wait for the channel current to stabilize before adding the next concentration, starting from 10. -18 M to 10 -8 M changes sequentially; the resulting test results are as follows: Figure 6 As shown; according to Figure 6 It can be seen that changes in concentration cause significant changes in current. The magnitude of the current change reflects the magnitude of the concentration change, and the sensor's current changes instantly after the concentration is changed, demonstrating very high sensitivity.

[0039] Sensing principle analysis: such as Figure 7 As shown, the detection principle consists of a liquid-gate graphene transistor composed of a gate, source, drain, graphene channel, and electrolyte solution. A "double capacitor" structure is formed on both the gate and graphene channel surfaces. When the probe modified on the gate binds to the target material, the negative charge of the phosphate groups in the bases adds a negative voltage to the gate, causing a change in the potential of the gate capacitance layer. Since the gate surface capacitance and the graphene surface capacitance are connected in series, a change in the gate surface capacitance inevitably leads to a change in the graphene surface capacitance. This increases the positive charge in the graphene channel, resulting in p-type doping of the graphene and shifting the Dirac point voltage of the graphene to the right.

[0040] Transfer characteristic curve test in artificial urine: Different concentrations of the target substance were added to artificial urine diluted 1000 times, and the transfer characteristic curve was tested, such as... Figure 8 As shown, the transfer characteristic curve shifts to the right, which is consistent with the results of detection in PBS, verifying the reliability and practicality of the present invention.

[0041] Clinical Urine Sample Testing: To evaluate the clinical application value of this invention, we collected urine samples from 12 prostate cancer patients, 10 benign prostatic hyperplasia (BPH) patients, 2 BPH patients with prostatitis, and 2 healthy individuals for testing. DNA was extracted from all collected urine samples, which were then pretreated with bisulfite and stored at -20°C. The pretreated samples were diluted 100-fold with 0.1×PBS solution. By measuring the shift of the Dirac point voltage of the SGGT biosensor, biomarkers from prostate cancer patients' urine could be detected. The shift of the Dirac point voltage in the control group's clinical urine samples was less than 20 mV, while the shift in the cancer samples was greater than 20 mV. The shift of the Dirac point voltage in prostate cancer samples was significantly greater than that in the control group. Figure 9 As shown.

[0042] 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 detecting prostate cancer marker GSTP1 hypermethylated promoter DNA in urine, characterized by the following steps: fabrication of liquid gate graphene transistor; design of bridge-shaped tetrahedral DNA probe with special structure, high hybridization efficiency and high selectivity, and modification on the gate electrode of the transistor; and finally selective detection of hypermethylated GSTP1 promoter after bisulfite treatment, wherein bisulfite treatment of unmethylated DNA can convert unmethylated cytosine (C) to uracil (U), but 5-methylcytosine (5-mC) does not undergo conversion.

2. The method for detecting prostate cancer urine marker according to claim 1, wherein the bridge-shaped tetrahedral DNA probe is composed of two tetrahedral DNAs and an ssDNA "bridge" between the two tetrahedral DNAs, and there are three thiol groups at the top vertices of the tetrahedral bottom, which can form gold-sulfur bonds with the gold gate. Due to the electrostatic repulsion and spatial structure between the two tetrahedrons, the lateral distance between the curled ssDNA can be easily controlled, which can effectively improve the efficiency of capturing target objects and reduce Debye shielding.

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