Fluorescence sensor for detecting prostate specific antigen based on graphene oxide / nucleic acid aptamer as well as preparation method and application of fluorescence sensor

Abstract

The invention relates to the field of disease molecular diagnosis, and provides a graphene oxide / nucleic acid aptamer-based fluorescence sensor for detecting a prostate-specific antigen, a preparation method and application of the graphene oxide / nucleic acid aptamer-based fluorescence sensor, and a fluorescence sensor, the graphene oxide / nucleic acid aptamer-based fluorescence sensor is characterized in that the fluorescence sensor comprises an aptamer solution, a graphene oxide solution, fluorescence molecules and a buffer solution, the concentration of the aptamer in the fluorescence sensor is 8-11 [mu] M; the concentration of graphite oxide in the fluorescence sensor is 6-12 [mu] g / mL; the pH value of the buffer solution is 6.2 to 8.1; the nucleotide sequence of the aptamer is 5 '-TTTTAATTAAAGCTCGCCATAAATAGCTT-3', and the nucleotide sequence of the aptamer is 5 '-TTTTAATTAAAGCTCGCCATAAATAGCTT-3'; dependence of expensive reagents and complex operation is avoided, and high specificity and sensitivity are achieved.

Description

Technical Field

[0001] The present application belongs to the technical field of molecular diagnosis of diseases, and in particular, relates to a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamers, and a preparation method and application thereof. Background Art

[0002] Currently, prostate cancer is the second most common malignant tumor in the world and the sixth leading cause of cancer death in men. Its incidence has increased at an annual rate of 3% in recent years. To date, the diagnosis of prostate cancer mainly relies on technologies such as rectal ultrasound, puncture biopsy, computed tomography and magnetic resonance imaging. However, the sensitivity and specificity of these technologies are limited, often leading to underdiagnosis or overdiagnosis of cancer. In addition, some of these technologies are invasive and may cause a series of complications (such as infectious diseases). Tumor markers are a class of specific substances that exist in body fluids or tissues and are related to the development of tumors. Prostate-specific antigen (PSA) is a specific tumor marker approved by the US Food and Drug Administration (FDA). It shows specific elevation in prostate cancer patients and is helpful for the diagnosis of prostate cancer. The methods currently used for PSA detection include enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence, electrochemical detection and PCR technology. However, these technologies often rely on expensive reagents, complex and time-consuming operations, sophisticated equipment and professional operators, which indicates that it is necessary to develop an economical and simple PSA detection method.

[0003] Aptamers are oligonucleotide chains with high affinity for specific targets such as cells, nucleotides or proteins, which are identified and synthesized in vitro by the systematic evolution of ligands by exponential enrichment (SELEX) technique. Therefore, they are called chemical antibodies. Compared with antibodies commonly used in detection, aptamers have the characteristics of stability, non-immunogenicity, low cost, convenience of use, and easy modification. At present, a variety of sensors using aptamer detection have been developed, such as: electrochemical aptamer sensors, aptamer-based surface enhanced Raman spectroscopy, and aptamer-based fluorescence sensors. Fluorescence sensors are favored because of their high sensitivity, good reproducibility and easy operation. The establishment of fluorescence sensors is based on the energy transfer between donors and acceptors. Graphene oxide (GO) is a two-dimensional material with strong adsorption capacity for ssDNA. At the same time, GO is an excellent energy transfer receptor that can effectively quench the fluorescence of various fluorescent molecules labeled on the aptamer adsorbed on its surface through energy transfer. It is a commonly used fluorescence quencher, but GO has a weak adsorption capacity for dsDNA and aptamers after binding to the target. Therefore, PSA can be specifically detected by the fluorescence recovered from the GO surface after the aptamer binds to the target. At present, a large number of studies have shown that aptamers mainly achieve specific detection of targets through changes in their conformation after binding to the target. However, aptamers of different length sequences and conformations have different steric hindrance effects, which will cause corresponding changes in the binding force between the aptamer and the target and GO, resulting in certain limitations in the specificity and sensitivity of the constructed sensor. Summary of the invention

[0004] The purpose of the present application is to provide a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamers, and its preparation method and application, in order to solve the problem that aptamers with different length sequences and conformations have different steric hindrance effects, which will cause corresponding changes in the binding force between the aptamer and the target and GO, thereby resulting in certain limitations in the specificity and sensitivity of the constructed sensor.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In the first aspect, the present application provides a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer, the fluorescence sensor comprising an aptamer solution, a graphene oxide solution, a fluorescent molecule and a buffer solution, the concentration of the aptamer in the fluorescence sensor is 8 to 11 μM; the concentration of graphene oxide in the fluorescence sensor is 6 to 12 μg / mL; the pH of the buffer solution is 6.2 to 8.1;

[0007] The nucleotide sequence of the adapter is 5'-TTTTTAATTAAAGCTCGCCATCAAATAG CTTT-3'.

[0008] As a possible preferred embodiment, the concentration of the aptamer is 0.02 pmol / μL; the concentration of the graphite oxide is 9×10 -6 μg / μL; the pH of the buffer solution is 7.4.

[0009] As a possible implementation manner, the graphite oxide is a single-layer lamellar structure and the thickness of the single layer is less than 1 μm, the graphite oxide has two characteristic absorption peaks, and the composition ratio of the ordered structure crystalline band and the disordered structure defect band on the surface of the graphite oxide is close; the graphite oxide surface has oxygen-containing functional groups.

[0010] As a possible implementation manner, the fluorescent molecule is labeled at the 5' end of the aptamer.

[0011] As a possible preferred implementation manner, the fluorescent molecule is 6-carboxyfluorescein.

[0012] In a second aspect, the present application provides a method for preparing a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer, comprising the following steps:

[0013] Aptamer screening and graphene oxide characterization and identification;

[0014] Prepare an aptamer solution and a graphene oxide solution, wherein the concentration of the aptamer solution is 10 pmol / μL, and the concentration of the graphene oxide solution is 0.5×10 -3 μg / μL;

[0015] The aptamer solution, graphene oxide solution and buffer solution were mixed evenly.

[0016] In a third aspect, the present application provides an application of a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer, wherein the application is to use the fluorescence sensor for detecting prostate-specific antigen in a sample to be tested; the detection comprises the following steps:

[0017] Mix the fluorescent sensor and the sample to be tested, and then place it at 27-47°C for reaction;

[0018] After the reaction is completed, the system is placed under a xenon lamp excitation light source with an excitation wavelength of 487 nm and an emission wavelength of 519 nm for fluorescence signal detection.

[0019] As a preferred embodiment, the reaction temperature is 37°C.

[0020] As a possible implementation, the detection recovery rate of the fluorescent sensor is 98.74% to 104.41%, and the relative standard deviation is 1.86% to 6.70%.

[0021] As a possible implementation, the linear range of the fluorescent sensor is: the concentration of prostate-specific antigen is 12.5-150 ng / mL, and the detection limit of the fluorescent sensor is 11.04 ng / mL.

[0022] The beneficial effects of the present invention are:

[0023] Based on the excellent properties of aptamers and GO, the present invention uses 6-FAM-labeled aptamers and GO to develop a convenient and cost-effective biosensor to quantitatively detect PSA by measuring the fluorescence changes in the system. At the same time, by evaluating the performance of the biosensor in detecting PSA in complex serum samples, it is determined that the sensor also has the potential to be used in clinical detection of PSA.

[0024] The detection range of the fluorescent sensor prepared by the invention for prostate specific antigen is 12.5-150 ng / mL, the detection lower limit is 11.04 ng / mL, the detection recovery rate is 98.74%-104.41%, and the relative standard deviation is 1.86%-6.70%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The characterization results of graphene oxide in the examples of the present invention are shown in Figure 1. (A) AFM image. (B) TEM image. (C) UV-visible absorption spectrum. (D) FT-IR spectrum. (E) Raman spectrum.

[0027] Figure 2 The feasibility analysis results of the sensor in the embodiment of the present invention. (A) Changes in the fluorescence value of the system with incubation time. (B) Fluorescence spectra of systems containing different components. (C) Fluorescence values ​​and statistical analysis of systems containing different components. (D) Fluorescence quenching efficiency of GO. (E) Fluorescence recovery effect of the sensor after adding PSA. Note: NS: no significant difference, ****p<0.0001, aptamer concentration is 20nM, GO concentration is 9μg / mL.

[0028] Figure 3The optimization, sensitivity and specificity results of the GO-based fluorescent sensor in the examples of the present invention. (A) The effect of different GO concentrations on the fluorescence value and (F-F0) / F0. (B) The effect of PBS buffer with different pH values ​​on the fluorescence value and (F-F0) / F0. (C) The effect of different incubation temperatures on the fluorescence value and (F-F0) / F0. (D) The fluorescence intensity recovered by the sensor in the presence of different concentrations of PSA. (E) (F-F0) / F0 is linearly correlated with the PSA concentration. (F) The ratio of the fluorescence intensity (F-F0) / F0 of the GO-based fluorescent sensor in the presence of various proteins alone. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0030] Graphene oxide suitable for use in the present invention is screened and obtained, and the graphene oxide must meet the following conditions: the graphene oxide is a single-layer lamellar structure and the thickness of the single layer is less than 1 μm, the graphene oxide has two characteristic absorption peaks, and the ordered structure crystalline band and the disordered structure defect band on the surface of the graphene oxide have a similar composition ratio; the graphene oxide surface has oxygen-containing functional groups.

[0031] The screening is as follows:

[0032] GO was purchased from Xianfeng Nanotechnology Co., Ltd. Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai G2 F20 S-TWIN transmission electron microscope ( Figure 1 Fourier transform infrared (FT-IR) spectra were measured using a Nicoleti S10 FT-IR spectrometer. Atomic force microscopy (AFM) images were obtained using a Bruker Dimension Icon atomic force microscope ( Figure 1 Figure B). Raman spectra were measured using a Renishaw inVia microscope. UV-visible absorption spectra were measured using a MAPADA UV6300 UV-visible spectrophotometer. The characterization results of GO are shown in Figure 1 .

[0033] Depend on Figure 1 It can be seen that GO from Xianfeng Nanotechnology Co., Ltd. meets the requirements for graphene oxide in the present invention, and can therefore be applied to the following embodiments.

[0034] Prepare the aptamer solution as follows:

[0035] According to the sequence of the aptamer 5'-6-FAM-TTTTTAATTAAAGCTCGCCATCAAATA GCTTT-3', the corresponding dry powder was synthesized, and then dimethylnitrosoacetamide water was added to dissolve it to obtain an aptamer solution with a concentration of 10 μM. The solution was heated at 95°C for 5 minutes and then slowly cooled to room temperature to facilitate the formation of a specific secondary structure of the aptamer.

[0036] Example 1

[0037] This embodiment provides a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer, comprising an aptamer solution, a graphene oxide solution, a fluorescent molecule and a buffer solution. The concentration of the aptamer in the fluorescence sensor is 0.02 pmol / μL; the concentration of the graphene oxide in the fluorescence sensor is 9×10 -6 μg / μL; the pH of the buffer solution is 7.4.

[0038] The nucleotide sequence of the adapter is 5'-TTTTTAATTAAAGCTCGCCATCAAATAG CTTT-3'.

[0039] The fluorescent molecule is labeled at the 5' end of the aptamer, and the sequence of the aptamer after labeling is: 5'-6-FAM-TTTTTAATTAAAGCTCGCCATCAAATAGCTTT-3'.

[0040] Example 2

[0041] This embodiment discloses a method for preparing a fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer in Example 1, comprising the following steps:

[0042] S1. Prepare an aptamer solution and a graphene oxide solution, wherein the concentration of the aptamer solution is 10 pmol / μL, and the concentration of the graphene oxide solution is 0.5×10 -3 μg / μL;

[0043] S3. Mix the aptamer solution, graphene oxide solution and buffer solution evenly.

[0044] Example 3

[0045] The detection system of the fluorescent aptamer sensor of GO prepared in this embodiment is set to 500 μL, which contains 1 μL of aptamer solution (10 μM = 10 pmol / μL), 9 μL of GO solution (0.5×10 -3μg / μL) and 5 μL PSA solution, and the rest is PBS buffer, that is, PBS buffer is added to make up 500 μL. The fluorescence spectra and fluorescence values ​​of all systems were recorded using a F97PRO fluorescence spectrometer under a xenon lamp excitation light source with an excitation wavelength of 487 nm and an emission wavelength of 519 nm.

[0046] Example 4

[0047] Feasibility analysis of sensors

[0048] To determine the feasibility of this sensor, this example first prepared a PBS buffer + 1 μL aptamer solution (10 μM) system and recorded the fluorescence value of the system within 60 min to evaluate whether the labeled fluorescent molecules have self-quenching ability and whether the fluorescence reduction of the system is caused by self-quenching of the fluorescent molecules. Secondly, 500 μL detection systems containing different components were set up respectively: a: PBS buffer + 1 μL of aptamer solution (10 μM = 10 pmol / μL) + 5 μL PSA solution (1 mg / ml), b: PBS buffer + 1 μL of aptamer solution (10 μM), c: PBS buffer + 1 μL of aptamer solution (10 μM) + 5 μL PSA solution (1 mg / ml) + 9 μL of GO solution (0.5 μg / ml), d: PBS buffer + 1 μL of aptamer solution (10 μM) + 9 μL of GO solution (0.5 μg / ml), e: PBS buffer + 5 μL PSA solution (1 mg / ml), f: PBS buffer + 5 μL PSA solution (1 mg / ml) + 9 μL of GO solution (0.5 μg / ml). Each system was incubated for the same time, and the fluorescence spectra and fluorescence values ​​of all systems were measured and recorded to determine whether the constructed fluorescent aptamer sensor was feasible. Then, a PBS buffer + 1 μL aptamer solution (10 μM) + 9 μL GO solution (0.5 μg / ml) system was prepared, and the fluorescence value of this system was recorded every minute within 10 minutes to evaluate the efficiency and efficacy of GO-induced fluorescence quenching. Finally, a PBS buffer + 1 μL aptamer solution (10 μM) + 9 μL GO solution (0.5 μg / ml) system was prepared. After incubation for 5 minutes, 5 μL PSA solution (1 mg / ml) was added and incubated for a period of time. The fluorescence spectrum and fluorescence value of the system were measured and recorded to evaluate the minimum time and efficacy of fluorescence recovery of the sensor after the addition of PSA. The results are shown in the figure. Figure 2 After the introduction of GO, the fluorescence value of the system dropped rapidly, and the fluorescence quenching rate reached 95%. After the addition of PSA, the fluorescence value of the system gradually recovered and stabilized after 60 minutes. In addition, the PSA solution did not interfere with the fluorescence recovery of the system, indicating that the constructed fluorescence sensor is feasible.

[0049] Example 5

[0050] Optimization of sensor conditions, specificity and sensitivity analysis

[0051] Prepare a PBS buffer + 1 μL aptamer solution (10 μM) system and add different concentrations of GO to incubate for a period of time, then add 5 μL PSA solution (1 mg / ml), compare the (F-F0) / F0 ratio of the sensor at different GO concentrations (0, 3, 6, 9, 12 and 15 μg / mL) to select the optimal GO concentration. Prepare a PBS buffer + 1 μL aptamer solution (10 μM) + 5 μL PSA solution (1 mg / ml) + 9 μL GO solution (0.5 μg / ml) system and place it at different reaction temperatures (17, 27, 37, 47 and 57 ° C) to incubate and determine the optimal reaction temperature of the system. Prepare a sensor system containing PBS buffer (4.0, 5.0, 6.2, 7.4, 8.1) with different pH values ​​+ 1 μL of aptamer solution (10 μM) + 5 μL PSA solution (1 mg / ml) + 9 μL GO solution (0.5 μg / ml) to measure and calculate the (F-F0) / F0 of the corresponding system to determine the optimal buffer pH value required for sensor preparation. Prepare a sensor with PBS buffer + 1 μL of aptamer solution (10 μM) + 9 μL GO solution (0.5 μg / ml) and incubate it for 5 minutes and add a series of PSA solutions of different concentrations (12.5-250 ng / mL) prepared by continuously diluting the PSA standard solution. The PSA solutions of the corresponding concentrations are tested to evaluate the sensitivity and linear range of the sensor in detecting prostate-specific antigen PSA. In addition, the specificity of the sensor was evaluated using six tumor markers (alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), bovine serum albumin (BSA), carbohydrate antigen CA125 (CA125), carbohydrate antigen CA123 (CA153), and squamous cell carcinoma antigen (SCCA)). The concentration of tumor markers used in each system was 100 ng / ml. The results are shown in the figure. Figure 3 . The optimal GO concentration is 9 μg / mL. The buffer pH value of 7.4 is the best buffer reaction system. The temperature optimization results show that 37°C is the best temperature for sensor reaction detection. The sensor detection shows a good linear relationship when the PSA concentration is in the range of 12.5-150 ng / mL: y=0.03058×x+0.1905, r 2 =0.9913, and the detection limit (LOD) of this sensor is 11.04 ng / mL. In addition, the results of the sensor detecting PSA are significantly different from those of other serum tumor markers (P<0.05), indicating that the sensor has good specificity in detecting PSA.

[0052] Example 6

[0053] PSA testing of serum samples

[0054] The collected blood samples were centrifuged at 4°C (3000g / min, 5min), and the serum was collected and frozen at -80°C. The prepared serum was diluted 100 times, and then the PSA standard solution was added to obtain spiked serum samples containing different concentrations of PSA (0, 30, 45 and 60ng / mL). The sensor was used to detect and analyze spiked serum samples containing different concentrations of PSA to evaluate its performance in detecting PSA under complex conditions. The serum samples were obtained from the 416th Hospital of Nuclear Industry (Chengdu, China). The results are shown in Table 1. As can be seen from Table 1, the sensor detection recovery rate is 98.74% to 104.41%, and the relative standard deviation (RSD) is 1.86% to 6.70%. This sensor has good detection performance under complex samples.

[0055] Table 1 PSA detection in serum samples

[0056] sample Spiked amount (ng / mL) Measured value (ng / mL) Recovery rate (%) Relative standard deviation (%) 1 0 24.26±3.94 - - 2 30 55.72±2.11 104.87 6.70 3 45 68.70±0.83 98.74 1.86 4 60 86.91±2.63 104.41 4.19

[0057] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer, characterized in that: The fluorescent sensor comprises an aptamer solution, a graphene oxide solution, a fluorescent molecule and a buffer solution. The concentration of the aptamer in the fluorescent sensor is 8 to 11 μM; the concentration of graphene oxide in the fluorescent sensor is 6 to 12 μg / mL; and the pH of the buffer solution is 6.2 to 8.

1. The nucleotide sequence of the adapter is 5'-TTTTTAATTAAAGCTCGCCATCAAATAG CTTT-3'.

2. The fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer according to claim 1, characterized in that: The concentration of the aptamer is 0.02 pmol / μL; the concentration of the graphite oxide is 9×10 -6 μg / μL; the pH of the buffer solution is 7.

4.

3. The fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer according to claim 1, characterized in that: The graphite oxide is a single-layer lamellar structure with a thickness of less than 1 μm. The graphite oxide has two characteristic absorption peaks. The ordered structure crystalline band and the disordered structure defect band on the surface of the graphite oxide have a similar composition ratio. The graphite oxide surface has oxygen-containing functional groups.

4. The fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer according to claim 1, characterized in that: The fluorescent molecule is labeled at the 5' end of the aptamer.

5. The fluorescence sensor for detecting prostate-specific antigen based on graphene oxide / nucleic acid aptamer according to claim 1, characterized in that: The fluorescent molecule is 6-carboxyfluorescein.

6. A method for preparing the fluorescent sensor according to any one of claims 1 to 5, characterized in that: The preparation method comprises: Aptamer screening and graphene oxide characterization and identification; Prepare an aptamer solution and a graphene oxide solution, wherein the concentration of the aptamer solution is 10 pmol / μL, and the concentration of the graphene oxide solution is 0.5×10 -3 μg / μL; The aptamer solution, graphene oxide solution and buffer solution were mixed evenly.

7. An application of the fluorescence sensor according to any one of claims 1 to 5, characterized in that: The application is to use the fluorescent sensor for detecting prostate-specific antigen in a sample to be tested; the detection comprises the following steps: Mix the fluorescent sensor and the sample to be tested, and then place it at 27-47°C for reaction; After the reaction is completed, the system is placed under a xenon lamp excitation light source with an excitation wavelength of 487 nm and an emission wavelength of 519 nm for fluorescence signal detection.

8. The use of the fluorescence sensor according to claim 7, characterized in that: The reaction temperature was 37°C.

9. The use of the fluorescence sensor according to claim 7, characterized in that: The detection recovery rate of the fluorescent sensor is 98.74% to 104.41%, and the relative standard deviation is 1.86% to 6.70%.

10. The use of the fluorescence sensor according to claim 7, characterized in that: The linear range of the fluorescent sensor is: the concentration of prostate-specific antigen is 12.5-150 ng / mL, and the detection limit of the fluorescent sensor is 11.04 ng / mL.

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