Method for noninvasive detection of prostate cancer

Abstract

The invention provides a liquid gate graphene transistor sensor capable of rapidly detecting a urine marker EN2 of prostatic cancer. The liquid gate graphene transistor comprises a substrate, and a gate electrode, a source electrode and a drain electrode which are arranged on the substrate; a graphene channel is arranged between the source electrode and the drain electrode, a long-chain aptamer with the strongest affinity with a target EN2 protein is determined according to the target EN2 protein, and the aptamer is fixed on the surface of a gold grid electrode of the transistor through an Au-S bond. When the EN2 protein is detected, the long-chain aptamer can be folded under the action of an object to be detected, the EN2 protein is positively charged in 0.1 * PBS, so that the liquid gate graphene transistor generates an output signal, high-sensitivity detection of the EN2 protein is realized, the low detection limit of 0.1 fg / m is realized, the detection time is 19 minutes, and the clinical urine sample detection accuracy reaches 100%. The method can be used for detecting the marker in the urine of the prostatic cancer, the marker can be obtained without an invasive mode, and noninvasive detection on the prostatic cancer is expected to be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to a method for non-invasive detection of prostate cancer. Background Art

[0002] Cancer is a major disease and the second leading cause of death worldwide. It seriously threatens human life and health. Early diagnosis and early treatment play a key role in prolonging the patient's life. The incidence and mortality of prostate cancer are rapidly increasing, making it one of the most common cancers in elderly men. To date, a variety of detection technologies have been developed for prostate cancer-specific antigen (PSA) screening, but elevated PSA levels may be associated with prostate hyperplasia (BPH) or prostatitis, and the use of PSA testing can easily lead to misdiagnosis of prostate cancer. Urine is a good source for in vitro diagnosis of various urogenital diseases, reducing overdiagnosis and pain for patients. However, unlike serum, urine contains higher amounts of various inorganic ions (K + , Na + , Cl - ,Mg 2+ and Ca 2+ ) and organic molecules such as urea, uric acid, creatinine, hippuric acid, ascorbic acid, etc., and the concentration of most of these components varies by more than 20 times, which makes it difficult to diagnose prostate cancer in urine. Therefore, the development of non-invasive and highly sensitive detection equipment has become a hot topic in the field of prostate cancer biosensor technology.

[0003] Enrailed-2 protein (EN2) is a transcription factor involved in the early embryonic development of prostate cancer. It is expressed in prostate cancer cells and secreted into urine. It has been reported that the expression level of EN2 is increased in prostate cancer tissues, and EN2 in urine has a higher specificity for prostate cancer than PSA. In addition, urine samples containing a large amount of EN2 protein can be obtained without invasive methods such as rectal examination, making non-invasive diagnosis of prostate cancer possible. At present, there are few studies on the detection of EN2 protein in urine, and these detection methods have problems such as low sensitivity, complex operation and dependence on large equipment. Therefore, it is necessary to develop a new type of simple, rapid and highly sensitive detection device for the detection of EN2 protein in urine to achieve non-invasive diagnosis of prostate cancer.

[0004] In order to solve the above problems, liquid-gate graphene transistor sensors are developed here. Monolayer graphene has become a preferred material for preparing biosensors due to its excellent properties such as high carrier mobility, high conductivity, good biocompatibility and chemical stability. Graphene-based transistor sensors have shown excellent performance in the detection of DNA, RNA, proteins and small molecules. However, the detection of EN2 protein, a urine marker for prostate cancer, has not yet been realized. The detection principle of the present invention is to modify the long-chain aptamer probe to the gate surface of the liquid-gate graphene transistor. In the solution, the EN2 protein binds to the aptamer probe to cause the probe to fold. In addition, since the EN2 protein is electrically neutral in 0.1×PBS buffer solution, the double electric layer interface properties between the transistor gate surface and the sample solution are changed under the dual effects of capacitance and gate potential changes, so that the channel current changes, and the detection of EN2 protein is achieved by measuring the transmission curve. The present invention detects clinical urine samples and finds that the diagnostic accuracy of prostate cancer can reach 100%, and it can distinguish prostate cancer from benign prostatic hyperplasia. Summary of the invention

[0005] The present invention mainly provides a method for non-invasive detection of prostate cancer. The sensor provided by the present invention is small in size, simple to operate, and is expected to achieve portable detection. The detection limit can be as low as 0.1 fg / mL, the detection time can be as short as 19 minutes, and the detection accuracy of clinical urine samples can reach 100%.

[0006] The present invention provides a liquid-gate graphene transistor biosensor, comprising a substrate and a gate, a source and a drain arranged on the substrate; a graphene channel is arranged between the source and the drain; a long-chain aptamer probe capable of specifically binding to an EN2 target is fixed on the surface of the gate.

[0007] The width of the graphene channel is 2 to 3 mm, and the length of the graphene channel is 4 to 8 mm; the graphene channel material is CVD single-layer graphene; the gate, source and drain include a chromium layer on the surface of the substrate and a gold layer on the upper surface of the chromium layer; the thickness of the chromium layer is 3 to 6 nm, and the thickness of the gold layer is 30 to 60 nm; the long-chain aptamer probe is fixed on the gate surface through thiol modification.

[0008] The present invention also provides a method for preparing the graphene transistor biosensor described in the above technical solution, comprising the following steps: (1) The gate, source and drain are deposited on the substrate surface by JSD-300 thermal evaporation coating, preferably under vacuum conditions, and the vacuum degree is preferably 8×10 -4 Below Pa; (2) transferring the cut single-layer graphene to the channel between the source and the drain to obtain a graphene transistor, wherein the graphene has a width of 2 mm and a length of 6 mm; (3) Fixing the long-chain aptamer probe on the gold gate surface of the graphene transistor obtained in step (2) through gold-sulfur bonding to obtain a graphene transistor biosensor.

[0009] The present invention also provides the use of the graphene transistor biosensor described in the above technical solution or the graphene transistor biosensor prepared according to the above technical solution in detecting the prostate cancer marker urine marker EN2.

[0010] The present invention provides a graphene transistor sensor for detecting EN2, a urine marker for prostate cancer. The transistor sensor comprises a glass substrate and a gate, a source, and a drain disposed on the substrate; a single-layer graphene conductive channel is disposed between the source and the drain; and the sensor surface is modified with a long-chain aptamer probe that can detect EN2, a urine marker for prostate cancer. The probe of the present invention is fixed to the surface of the gate electrode of the graphene transistor through an Au-S bond. The greatest advantage of the detection of the present invention is that it can realize non-invasive detection of prostate cancer, and EN2 protein can be secreted into the urine without an invasive screening procedure. During EN2 detection, the long-chain aptamer probe modified on the gate will fold under the action of EN2 protein, resulting in a decrease in the double electric layer between the gate and the solution and an increase in capacitance. Secondly, since EN2 protein is positively charged in 0.1×PBS buffer solution, the capacitance will also increase, causing the Dirac point voltage and the current in the graphene channel to change. By detecting the displacement of the Dirac point voltage and the change of current in the channel, the EN2 protein in the solution can be detected with a detection limit of 0.1 fg / mL and a shortest detection time of 19 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a schematic diagram of a graphene transistor biosensor in an embodiment of the present invention; Figure 2 A schematic diagram of preparing a graphene transistor in an embodiment of the present invention; Figure 3 Schematic diagram of the gold gate modification and hybridization process of the graphene transistor biosensor in an embodiment of the present invention; Figure 4 is a schematic diagram of a graphene transistor biosensor in an embodiment of the present invention; Figure 5 The graphene transistor biosensor in the embodiment of the present invention is a transfer characteristic curve when detecting different EN2 protein concentrations; Figure 6 is a standard curve of the change in Dirac point voltage and EN2 protein concentration of the graphene transistor biosensor in the embodiment of the present invention; Figure 7 This is a current output characteristic curve when detecting changes in EN2 protein concentration in an embodiment of the present invention; Figure 8 : is the selectivity transfer characteristic curve of the graphene transistor biosensor for EN2 protein detection in an embodiment of the present invention; Fig. 9 1 is a graph showing the Dirac point voltage displacement of the transfer characteristic curve of the graphene transistor biosensor for clinical urine samples and a statistical graph showing the PSA levels of different clinical serum samples in an embodiment of the present invention. DETAILED DESCRIPTION

[0012] The present invention provides a liquid-gate graphene transistor biosensor for detecting EN2 protein, a urine marker for prostate cancer, comprising a substrate and a gate, a source and a drain arranged on the surface of the substrate; a graphene channel is arranged between the source and the drain; a long-chain aptamer probe specifically binding to the EN2 target is fixed on the surface of the gate.

[0013] The liquid-gate graphene transistor-based prostate cancer urine marker EN2 protein biosensor provided by the present invention includes a substrate. The type and source of the substrate used in the present invention are not particularly limited, and electronic grade glass, silicon wafer or polyester resin can be used. In the specific embodiment of the present invention, electronic grade glass (GL-10173-1.1) or PET is preferred, and the size is 10×10 mm 2 ~14×14 mm 2 , preferably 12×12 mm 2 , the preferred thickness is 2 mm.

[0014] The liquid-gate graphene transistor prostate cancer urine marker EN2 protein biosensor provided by the present invention comprises a gate, a source and a drain arranged on the substrate. The gate, the source and the drain are placed on the same surface of the substrate; the present invention has no special requirements for the specific positions of the three electrodes, and the channel width between the source and the drain is preferably 0.2-0.3 mm.

[0015] The gate, source and drain are preferably independent chromium layers and gold layers, and the chromium layer is located between the substrate and the gold layer. The chromium layer and the gold layer are plated on the substrate surface by thermal evaporation using a JSD-300 evaporation coating system. The thermal evaporation coating is preferably carried out under vacuum conditions, and the vacuum degree of the vacuum is preferably 8 × 10 -4 Pa or less, more preferably 4 × 10 -4Pa; the evaporation current of the chromium layer is preferably 170-200 A, more preferably 175-190 A; the evaporation current of the gold layer is preferably 100-120 A, more preferably 105-110 A; the thickness of the evaporated gold is 20-30 nm, and the thickness of the evaporated chromium is about 2-3 nm. In the present invention, the chromium layer can make the gold layer firmly adhere to the surface of the substrate to prevent the gold layer from falling off due to subsequent operations.

[0016] The liquid gate graphene transistor prostate cancer urine marker EN2 protein biosensor provided by the present invention includes a graphene channel disposed between the source and drain electrodes. The width of the graphene channel is preferably 0.2 to 0.3 mm, and the length is preferably 4 to 8 mm. The graphene fills the gap between the source and drain electrodes and covers the surfaces of the two electrodes, preferably CVD single-layer graphene.

[0017] In the present invention, the modification amount of the gate surface aptamer probe molecules is preferably 1-2 mM, more preferably 1 mM.

[0018] In the present invention, the three-electrode structure of the gate, source and drain enables the voltage of the input gate to be used to control the channel current during the biomolecule detection process, thereby achieving an operating voltage lower than 1V.

[0019] The present invention also provides a method for preparing the liquid gate graphene transistor prostate cancer urine marker biosensor described in the above technical solution, comprising the following steps: (1) preparing a double gate, a source electrode and a drain electrode on the surface of a substrate, wherein a channel exists between the source electrode and the drain electrode; (2) Laying graphene on the source and drain to form a conductive channel to obtain a graphene transistor; (3) Immobilizing long-chain aptamer probes that specifically bind to the EN2 protein on the gate surface of the graphene transistor obtained in step (2) to obtain a graphene transistor biosensor.

[0020] The present invention preferably cleans and dries the substrate before use. In the present invention, the cleaning is preferably ultrasonic cleaning, and more preferably acetone, ethanol, deionized water and ethanol are used in sequence for ultrasonic cleaning, and each cleaning time is preferably 10 to 30 minutes. The present invention has no particular limitation on the power of the ultrasonic cleaning, and the drying is preferably nitrogen drying.

[0021] The present invention prepares a gate, a source and a drain on the surface of a substrate so that a channel exists between the source and the drain. In the present invention, the preparation of the gate, the source and the drain preferably includes: sequentially depositing a chromium layer and a gold layer on the surface of the substrate by thermal evaporation coating. The specific parameters of the thermal evaporation coating method are not particularly limited, and the parameters well known to those skilled in the art can be used to prepare electrodes of the desired thickness.

[0022] After the gate, source and drain are prepared, the present invention lays the cut 2 mm×6 mm graphene on the channel formed by the source and drain to obtain a graphene transistor, such as Figure 2 As shown. In the present invention, the transfer method used for the graphene is wet transfer, preferably single-layer graphene grown on a copper substrate CVD. The graphene source of the present invention adopts a commercial product well known to those skilled in the art, or is prepared according to a preparation method well known to those skilled in the art. In the present invention, the technical solution for wet transfer of single-layer graphene is preferably referred to the technical solution disclosed in Chen Mu, Yan Yue, Zhang Xiaofeng, et al. Research Progress of Large-area Graphene Film Transfer Technology [J]. Journal of Aeronautical Materials, 2015, 35(2): 1-11.

[0023] After the transfer of graphene is completed, the present invention preferably anneals the transferred device to obtain a graphene transistor, and the annealing temperature is preferably 100-130°C, more preferably 120°C; the annealing time is preferably 20-40 minutes, more preferably 30 minutes. In the present invention, the annealing can remove moisture from the sample surface and make the graphene and the substrate more closely bonded.

[0024] After obtaining the graphene transistor, the present invention preferably fixes a long-chain aptamer probe on the gate surface of the graphene transistor to obtain a prostate cancer urine marker EN2 biosensor based on a liquid-gate graphene transistor. The synthesized long-chain aptamer will fold under the action of EN2 protein, such as Figure 3 As shown. In the present invention, the preferred aptamer probe is fixed on the gate surface by thiol modification. The probe concentration used in the present invention is preferably 1 μM, and the volume is preferably 10 μL. The well-known method of fixing the aptamer on the gold surface can be used.

[0025] The present invention also provides the application of the above-mentioned liquid gate graphene transistor in detection. The gate of the modified aptamer is immersed in the test liquid, and the gate and graphene channel of the biosensor based on the liquid gate graphene transistor are immersed in the electrolyte containing the object to be tested. In the present invention, the electrolyte of the EN2 protein is preferably 0.1×PBS solution, and the pH value is preferably 7.4. In the present invention, the gate and the graphene channel are passed through the electrolyte, and the voltage input to the gate is used to control the current of the graphene channel, such as Figure 1 shown.

[0026] When the graphene transistor biosensor provided by the present invention is used for quantitative determination of EN2 protein, it is preferred to prepare a standard solution containing EN2 protein, determine the standard curve of current change value EN2 protein concentration, and determine the detection limit and detection range of the analyte based on the standard curve and the current change value obtained in the test.

[0027] The liquid-gate graphene transistor prostate cancer marker EN2 biosensor provided by the present invention can be directly immersed in a solution containing the object to be detected for high-sensitivity detection, and has good stability and specificity. Since the sensor is very sensitive to voltage changes, a small voltage change will cause a corresponding current change, and the sensitivity is extremely high; and the liquid-gate graphene transistor provided by the present invention can detect EN2 protein as low as 0.1 fg / mL.

[0028] To further illustrate the present invention, the liquid-gate graphene crystal prostate cancer urine marker EN2 biosensor and its preparation method and application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1.

[0030] Thermal evaporation coating: Cut the electronic grade glass into 12 × 12 mm size, clean it with acetone, ethanol, deionized water and ethanol ultrasonically for 20 minutes, dry it with nitrogen, and then use high temperature glue to stick the glass piece on the mask of specific shape. Weigh appropriate amount of chromium and gold and put them into tungsten boat to prepare for vacuum thermal evaporation coating. When evaporating, evaporate chromium first, with a thickness of 3nm, and the evaporation current is preferably 170~200A; then evaporate the gold layer, with a thickness of 30nm, and the evaporation current is preferably 100~120A. The shape, structure and size of the obtained electrode are as follows: Figure 2 shown. Figure 2 In the figure, G is gate, S is source, and D is drain. The 0.25 mm wide channel between the source and the drain becomes the graphene channel after the graphene is transferred.

[0031] Wet transfer of single-layer graphene: Cut the CVD single-layer copper-based graphene to a size of 12×12 mm, drop 70 μL of the prepared 35 mg / mL PMMA / anisole solution on the graphene surface and spin coat, set the speed of the glue machine to 600 rpm and 3000 rpm, the spin coating time to 3 s and 30 s, and heat at 100 ℃ for 10 min after spin coating to obtain PMMA / graphene. Prepare 100 mg / mL ferric chloride solution, place the PMMA / graphene spin-coated with the PMMA / anisole solution facing up, and soak it in the ferric chloride solution for 2~4 h, preferably 3 h, to completely etch the copper substrate. Use a glass slide to transfer the PMMA / graphene sheet with the copper substrate etched off to deionized water and soak it for 5 minutes. Use deionized water to wash it three times to clean the residual ferric chloride solution on the PMMA / graphene. Take the vapor-deposited device and use an oxygen plasma cleaner to clean the device surface to improve the hydrophilicity of the device. The cleaning time is 3 to 5 minutes, preferably 4 minutes. Transfer the cleaned PMMA / graphene sheet to the device and lay it flat on the channel between the source and drain on the surface of the device. After natural drying, place it on a hot plate for annealing at 75 °C for 10 minutes and 105 °C for 30 minutes to completely remove the moisture on the sample surface to obtain a PMMA / graphene / electrode sheet. After cooling to room temperature, the PMMA / graphene / electrode sheet was washed twice with acetone, each time for 5 min, and then the PMMA / graphene / electrode sheet was placed in an acetone solution and heated at 75 °C for 3 h to remove the surface PMMA and obtain the required graphene transistor. The acetone residue and other impurities on the graphene transistor were washed with deionized water and dried with nitrogen. Finally, the graphene transistor was annealed at 120 °C for 30 min.

[0032] Immobilization of aptamer molecular probes: drop-coat 10 μL of 1 mM long-chain aptamer probe molecular solution onto the surface of the Gate; place in a 28 °C incubator and wash three times with 0.1× PBS buffer after 12 h; wash away the unimmobilized molecular probes and other impurities on the gate to obtain a liquid-gate graphene transistor that can be used for EN2 protein detection.

[0033] Example 2.

[0034] The target molecule was tested using the liquid-gate graphene transistor biosensor prepared in Example 1. The source, drain and gate electrodes of the graphene transistor were connected to two digital source meters (Keithley 2450). V G and source-drain voltage V DS It is controlled by a Labview program in the computer.

[0035] The modified gate electrode was thoroughly cleaned with 0.1× PBS solution to remove the residue left on the electrode. The test was carried out in a PDMS container filled with 100 mL of 0.1× PBS solution. During the test, EN2 solution of specific concentrations was added to the PBS solution in sequence to obtain output signals at different concentrations.

[0036] Transfer characteristic curve test: The source-drain voltage is set to a constant value ( V DS = 0.1 V), and continuously measure the relationship between the channel current and the concentration of the substance to be tested. After the channel current stabilizes, the concentration of EN2 protein is increased from 0.1 fg / mL, 1 fg / mL, 0.01 pg / mL, 0.1 pg / mL, and 1 pg / mL in sequence; the test results are shown in the figure. Figure 5 shown; according to Figure 5 It can be seen that changes in cobalt ion concentration will cause the Dirac point voltage to change, and the magnitude of the change can reflect the magnitude of the concentration change.

[0037] Standard curve drawing: The source-drain voltage is set to a constant value ( V DS = 0.1 V), prepare test solutions of different concentrations, test the Dirac point voltage change value of the graphene transistor biosensor at concentrations of 0.1 fg / mL, 1 fg / mL, 0.01 pg / mL, 0.1 pg / mL, and 1 pg / mL, respectively, repeat the test 3 to 5 times, take the average value, and draw a curve with the concentration of the test substance as the horizontal axis and the Dirac point voltage change value as the vertical axis. It can be seen from the curve that the Dirac point voltage change value has a good linear relationship with the EN2 concentration, and the R of the fitting line is 2 The values ​​are 0.971 respectively. Figure 6 shown.

[0038] Output characteristics test: Source-drain voltage and gate voltage are set to constant values ​​( V DS = 0.1 V and V G =0.4V), and continuously measure the relationship between channel current and time. During the test, wait for the channel current to stabilize before adding 10 μL of the next concentration, and the concentration changes from 0.1 fg / mL to 0.1 ng / mL in sequence; the test results are as follows Figure 7 shown; according to Figure 7 It can be seen that changes in EN2 concentration will cause significant changes in the current, and after changing the concentration, the current of the sensor changes in real time, with very high sensitivity.

[0039] Selective transfer characteristic test curve drawing: The source-drain voltage is set to a constant value ( V DS = 0.1 V), and the transfer characteristic curves of different interfering substances with the same concentration were measured. After the channel current stabilized, 10 μL of the next interfering substance was added, and finally the same concentration of EN2 protein was added. After multiple measurements, it was found that the liquid-gate graphene transistor prepared in Example 1 had a very obvious response to EN2 protein, but had no obvious response to other interfering molecules, such as Figure 8 shown.

[0040] Clinical urine sample detection: In order to evaluate the clinical application value of the present invention. We collected urine samples from 20 prostate cancer patients, 3 prostate hyperplasia patients, 2 prostate hyperplasia with prostatitis patients and 3 healthy people for detection. All urine collection processes did not require rectal examination. Before the test, 1.5 mL of urine was centrifuged at 10,000 × g for 5 min, the supernatant was taken and diluted 1 million times. By measuring the displacement of the Dirac point voltage of the SGGT biosensor, EN2 protein in the urine of prostate cancer patients can be detected. The displacement of the Dirac point voltage of the control group urine clinical samples was less than 22 mV, while the displacement of the cancer samples was greater than 35 mV. The displacement of the Dirac point voltage of prostate cancer was significantly greater than that of the control group, such as Fig. 9 shown.

[0041] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be noted that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention.

Claims

1. A liquid-gate graphene transistor sensor for detecting the prostate cancer urine marker EN2, wherein the gate surface of the transistor is modified with a long-chain aptamer probe of the EN2 marker, which can be used for non-invasive detection of early prostate cancer.

2. The sensor according to claim 1 has the following working principle: When the EN2 protein binds to the EN2 aptamer probe modified on the gate, the long-chain aptamer probe will fold under the action of the EN2 protein, resulting in a decrease in the double electric layer between the gate and the solution and an increase in capacitance. Secondly, since the EN2 protein is positively charged in the 0.1×PBS buffer solution, the gate potential increases. Both effects can effectively cause the displacement of the Dirac point voltage of the transfer characteristic curve to move in the direction of the negative gate voltage, and sensing is achieved by measuring the transistor transfer curve.

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