Electrochemical aptamer sensor, preparation method and application thereof, and prostate specific antigen detection method

Abstract

The invention discloses an electrochemical aptamer sensor, a preparation method and application thereof and a prostate specific antigen detection method, and successfully develops a reliable electrochemical biological aptamer sensor which is used for ultra-sensitive detection of PSA (Prostate Specific Antigen). The aptamer is used as a biological recognition element to specifically recognize PSA. Nanoporous gold (NP-Gold) having a high specific surface area and excellent conductivity is used as a support material. According to the present invention, the double-strand specific nuclease (DSN) is adopted to reduce the noise signal, and the provided aptamer sensor shows excellent performance when the PSA in the clinical serum sample is determined, and has characteristics of high sensitivity, strong anti-interference ability and reliable stability. Due to the unique advantages, the aptamer sensor becomes a very potential choice for detecting the PSA.

Description

Technical Field

[0001] The present invention belongs to the technical field of the analysis of biological substances, and particularly relates to an electrochemical aptamer sensor and a preparation method and application thereof, as well as a prostate-specific antigen detection method. Background Art

[0002] Every year, millions of people around the world are diagnosed with prostate cancer (PCa), which is one of the most common solid malignant tumors in the world. The course of prostate cancer is long and the metastasis rate is high, and the treatment of the middle and late stages is extremely challenging. The prognosis of prostate cancer varies significantly depending on the patient's age, race, genetic background, and disease progression stage. Therefore, early diagnosis is crucial for reducing the mortality rate of prostate cancer and improving the treatment effect. Prostate-specific antigen (PSA) is a single-chain serine protease secreted by prostate epithelial cells with a molecular weight of 33-34 kDa, and it is considered to be one of the most reliable biomarkers for diagnosing prostate cancer. In addition, PSA is also regarded as the best biomarker for diagnosing prostatitis and benign prostatic hyperplasia. Therefore, the detection and identification of PSA are of great significance for the early diagnosis of prostate cancer.

[0003] Currently, there are a variety of traditional detection methods for the detection of PSA, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, surface-enhanced Raman spectroscopy (SERS), surface plasmon resonance, and mass spectrometry. However, these methods are time-consuming and require sophisticated and complex instrumentation. In addition, these traditional technologies not only have insufficient sensitivity but also have defects in the preparation, stability, and modification procedures of samples and antibodies. Therefore, establishing an innovative and reliable PSA detection method is crucial for the timely diagnosis of prostate diseases and effective treatment monitoring in clinical practice.

[0004] Compared with traditional detection methods, electrochemical biosensors have a variety of inherent advantages, such as high cost-effectiveness, fast response speed, simple operation, good reproducibility, and high sensitivity. When constructing a biosensor, it is crucial to select a suitable biorecognition element. Among different biorecognition elements (such as enzymes, antibodies, etc.), aptamers are widely used as specific biorecognition elements for screening and detecting tumor markers. An aptamer is a short single-stranded DNA or RNA sequence that can specifically bind to a specific target by folding into a three-dimensional structure. Compared with traditional immunorecognition molecules, aptamers have the advantages of higher stability, lower cost, simpler artificial synthesis and modification, smaller size, and higher specificity.

[0005] However, for the detection of PSA, no technical solutions for electrochemical biosensor with high sensitivity, selectivity, and reliability have been disclosed. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an electrochemical aptamer sensor, a preparation method and an application thereof, as well as a method for detecting prostate specific antigen. In the present invention, an aptamer sensor is developed based on aptamer-DNA double strand, nanoporous gold and double-strand specific nuclease (DSN) for the electrochemical determination of the prostate cancer biomarker PSA. Specifically, due to the high specific surface area and stability of nanoporous gold, it is selected as the substrate material, providing abundant binding sites for the effective immobilization of the aptamer-DNA double strand structure. At the same time, the characteristics of DSN are utilized to minimize the noise signal on the electrode surface.

[0007] As a first aspect of the present invention, there is provided a preparation method of an electrochemical aptamer sensor, comprising the following steps:

[0008] Step S1, preparation of NP-Gold / GCE electrode: Cover the glassy carbon electrode with nanoporous gold to form an NP-Gold / GCE electrode;

[0009] Step S2, preparation of dsDNA / NP-Gold / GCE electrode: Hybridize the aptamer and acDNA by heat treatment to form a double-stranded dsDNA structure; then, mix the formed dsDNA structure with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bond to a thiol group to obtain a mixed solution; subsequently, take the mixed solution and drop it onto the surface of the NP-Gold / GCE electrode prepared in Step S1, and incubate to construct a dsDNA / NP-Gold / GCE electrode.

[0010] Preferably, in Step S2, the aptamer and acDNA are mixed with Tris-MgSO 4 buffer (TM), activated at 85 °C to 100 °C, and then gradually cooled to room temperature. During this heat treatment process, the aptamer hybridizes with acDNA to form a double-stranded (dsDNA) structure. More preferably, the denaturation temperature is 90 °C.

[0011] Preferably, in Step S2, the incubation time is 3 to 18 hours, preferably 12 hours.

[0012] Preferably, in Step S2, the constructed dsDNA / NP-Gold / GCE electrode is rinsed with ultrapure water to remove unbound dsDNA.

[0013] In the embodiment of the present invention, the aptamer is a PSA aptamer, and the sequence is as shown in SEQ ID NO.1.

[0014] The acDNA sequence is as shown in SEQ ID NO.2.

[0015] With the development of nanomaterials, various sensors based on different nanomaterials have been constructed to achieve the detection of low-concentration biomarkers. Among numerous nanomaterials, nanoporous gold (NP-Gold) has attracted extensive attention in the construction of biosensors due to its unique physical and chemical properties and high catalytic activity towards various electrochemical substances. Nanoporous gold has a unique three-dimensional bicontinuous nanoporous structure with a high specific surface area. This unique structure significantly increases the effective sensing area and provides abundant adsorption sites for the binding of biorecognition elements. In addition, nanoporous gold has excellent biocompatibility and conductivity. Moreover, the oxidized gold atoms on nanoporous gold can form covalent bonds with functional groups (such as amino (-NH2) and thiol (-SH)) on the surface of biomacromolecules. These properties make nanoporous gold very suitable for modifying various recognition components, such as aptamers.

[0016] As the second aspect of the present invention, it lies in providing a product obtained by the preparation method of an electrochemical aptasensor based on the synergistic effect of bispecific nuclease and nanoporous gold.

[0017] As the third aspect of the present invention, it lies in providing the application of the preparation method of an electrochemical aptasensor based on the synergistic effect of bispecific nuclease and nanoporous gold in the detection of prostate-specific antigen.

[0018] As the fourth aspect of the present invention, it lies in providing a method for detecting prostate-specific antigen, comprising the following steps:

[0019] Step 1, establishment of calibration curve: Drop PSA solutions with different concentrations onto the dsDNA / NP-Gold / GCE electrode, incubate at a temperature of 1 °C to 8 °C for 30 to 120 minutes to prepare the PSA / dsDNA / NP-Gold / GCE electrode; then, incubate the PSA / dsDNA / NP-Gold / GCE electrode with DSN at a temperature of 30 °C to 40 °C for 40 to 100 minutes; measure the current in phosphate buffer using the DPV method to establish a calibration curve reflecting the correlation between current response and PSA concentration;

[0020] In the embodiments of the present invention, the PSA solutions with different concentrations include solutions with several concentration values between 10 fg / mL and 10 ng / mL.

[0021] Step 2, detection of PSA: Drop the sample onto the dsDNA / NP-Gold / GCE electrode, incubate at a temperature of 1 °C to 8 °C for 30 to 120 minutes, preferably incubate at 4 °C for 60 minutes to prepare

[0022] PSA / dsDNA / NP-Gold / GCE electrode; Subsequently, add DSN, and incubate the electrode at a temperature of 30 °C to 40 °C for 40 to 100 minutes, preferably incubate for 60 minutes; Detect the current in phosphate buffer by DPV method, and substitute the recorded current response value into the calibration curve to determine the concentration of PSA.

[0023] In Steps 1 and 2, the first incubation condition is preferably to incubate at 4 °C for 60 minutes; the second incubation condition for adding DSN is preferably to incubate at 37 °C for 60 minutes.

[0024] The pH range of the phosphate buffer is 6 to 9, preferably PBS (50 mM, pH 7.0).

[0025] In Steps 1 and 2, the PSA solution is dropped onto the dsDNA / NP-Gold / GCE electrode and incubated. This incubation step is to promote the formation of the PSA-aptamer complex and to induce the folding of acDNA into a hairpin structure.

[0026] Preferably, in Steps 1 and 2, the prepared PSA / dsDNA / NP-Gold / GCE electrode is rinsed with ultrapure water to remove free PSA and PSA-aptamer complex; Add DSN to

[0027] the surface of the PSA / dsDNA / NP-Gold / GCE electrode and incubate. The dsDNA not bound to PSA is cleaved by DSN.

[0028] Preferably, in Steps 1 and 2, the amount of DSN used is 1 to 2.5 U, preferably 1 U.

[0029] In the embodiments of the present invention, the current value is detected by differential pulse voltammetry (DPV) in PBS (50 mM, pH 7.0) solution.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] (1) The present invention has successfully developed a reliable electrochemical aptasensor for ultrasensitive detection of PSA based on the synergistic effect of bispecific nuclease and nanoporous gold. An aptamer is used as a biorecognition element to specifically recognize PSA. Nanoporous gold (NP-Gold) with a high specific surface area and excellent conductivity is used as a support material. With the help of double-strand specific nuclease (DSN) to reduce the noise signal, the proposed aptasensor shows excellent performance in the determination of PSA in clinical serum samples, with high sensitivity and strong anti-interference ability. The minimum detection value of this aptasensor is 10 fg / mL, which can detect very low concentrations of PSA. The aptamer has a high affinity for PSA, and the proposed aptasensor has high selectivity for PSA.

[0032] (2) The PSA biosensor prepared by the present invention has no decrease in the detected current value after being stored for 21 days and has excellent stability. The measured PSA concentration ranges from 91 to 112 fg / mL and has excellent reproducibility. These unique advantages make the aptasensor a very promising option for detecting PSA.

[0033] (3) The present invention provides a method for detecting prostate-specific antigen. The method is based on an electrochemical aptasensor that synergistically uses a bispecific nuclease and nanoporous gold, and has the characteristics of high sensitivity, good reproducibility, high selectivity, high stability, etc., providing a new way for the detection of PSA. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0035] Figure 1 It is a graph showing the test results of the detection in Example 2. Among them, A is a scanning electron microscope (SEM) image of nanoporous gold (NP-Gold). B is a transmission electron microscope (TEM) image of nanoporous gold. C is an X-ray diffraction (XRD) image of nanoporous gold. D is the X-ray photoelectron spectroscopy (XPS) of nanoporous gold, double-stranded DNA (dsDNA) / nanoporous gold. E is nanoporous gold and. F is the deconvoluted XPS spectrum of gold 4f of dsDNA / nanoporous gold. G is the deconvoluted XPS spectrum of S2p of double-stranded specific nuclease (DSN) / nanoporous gold. H is the cyclic voltammogram (CV) of a glassy carbon electrode (GCE), an NP-Gold / GCE electrode, a dsDNA / NP-Gold / GCE electrode, a prostate-specific antigen (PSA) / dsDNA / NP-Gold / GCE electrode, and a DSN / PSA / dsDNA / NP-Gold / GCE electrode in a 5 mM 6 4- / 3- solution (scanning rate: 50 mV s -1 ). I is the differential pulse voltammogram (DPV) of a GCE, an NP-Gold / GCE electrode, a dsDNA / NP-Gold / GCE electrode, a PSA / dsDNA / NP-Gold / GCE electrode, and a DSN / PSA / dsDNA / NP-Gold / GCE electrode in a phosphate buffer solution (PBS) (50 mM, pH 7.0).

[0036] Figure 2 ​For Example 2, polyacrylamide gel electrophoresis (PAGE) was used to demonstrate the affinity of the aptamer for PSA and the cleavage ability of double-stranded specific nuclease (DSN). Among them, in A, lane 1: aptamer; lane 2: mixture of aptamer and PSA. In B, lane 1: aptamer; lane 2: aptamer complementary DNA (acDNA); lane 3: mixture of aptamer and acDNA; lane 4: mixture of aptamer, acDNA and DSN; lane 5: mixture of aptamer and DSN (10 μM aptamer and acDNA, 100 μg / mL PSA, 1 U DSN).

[0037] Figure 3 For the optimization experiment of different experimental conditions in Example 3. Among them, A is the effect of pH value on the dsDNA / NP-Gold / GCE electrode. B is the effect of temperature on the dsDNA / NP-Gold / GCE electrode. C is the effect of the adsorption time of dsDNA on the dsDNA / NP-Gold / GCE electrode. D is the effect of the binding time between the aptamer and PSA on the PSA / dsDNA / NP-Gold / GCE electrode. E is the digestion time of DSN and F is the amount of DSN on the DSN / dsDNA / NP-Gold / GCE electrode.

[0038] Figure 4 For the experimental results of the performance study in Example 4. Among them, A is the DPV curve of the aptasensor after incubation with different concentrations of PSA in PBS (50 mM, pH 7.0) solution. B is the linear relationship between the current value and the logarithm of the PSA concentration.

[0039] Figure 5 For the experimental results of the anti-interference, reproducibility and stability of the aptasensor in Example 4. Among them, A is the anti-interference of the aptasensor (NC: negative control, that is, the aptasensor not incubated with PSA) (compared with the negative control for the interferent: P>0.05). B is the reproducibility of the aptasensor. C is the storage stability of the aptasensor.

[0040] Figure 6 Schematic diagram of the detection principle of the aptasensor provided by the present invention.

[0041] Figure 7 For the characterization result diagram of the sensor construction. Among them, (A) EIS curves of GCE, NP-Gold / GCE electrode, dsDNA / NP-Gold / GCE electrode, PSA / dsDNA / NP-Gold / GCE electrode, DSN / PSA / dsDNA / NP-Gold / GCE electrode in 5 mM [Fe(CN) 6 4- / 3- solution (frequency from 0.01 to 10 6 ​Hz). (B) CV curves of GCE, NP-Gold / GCE electrode, dsDNA / NP-Gold / GCE electrode, PSA / dsDNA / NP-Gold / GCE electrode, and DSN / PSA / dsDNA / NP-Gold / GCE electrode in PBS (50 mM, pH 7.0).

[0042] Figure 8 It is a schematic diagram for predicting the secondary structure of the PSA aptamer. Detailed implementation manners

[0043] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0044] Materials and reagents

[0045] All DNA used in this study was purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and was freeze-dried powder purified by high-performance liquid chromatography (HPLC). In this study, the PSA aptamer (5’-TTT TTA ATT AAA GCTCGC CAT CAA ATA GCT TT-3’) (SEQ ID NO.1) and the aptamer complementary DNA (acDNA) oligonucleotide (5’-MB-GAG CTT TAA TTA AAA GCT CTT T-SH-3’) (SEQ ID NO.2) modified with a thiol group (-SH) at the 3’ end and methylene blue (MB) at the 5’ end were used. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (TE) buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid) (EDTA), Tris-MgSO 4 buffer (TM) (500 mM Tris-HCl, 80 mM MgSO 4 ) and TBE buffer (89 mM Tris-HCl, 2 mM EDTA, pH 8.0) were all purchased from Sangon Biotech Co., Ltd. TE buffer was used to dilute DNA oligonucleotides. Phosphate buffer (PBS, 50 mM) was prepared from Na 2 HPO 4 ·12H 2 O and NaH 2 PO 4 ·2H 2 O.

[0046] PSA, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) antigen, immunoglobulin G (IgG), bovine serum albumin (BSA), human epididymis protein 4 (HE4), cathepsin B (CB), and carbohydrate antigen 125 (CA125) were all purchased from Shanghai Linko Biotechnology Co., Ltd. (Shanghai, China). Angiopoietin-2 peptide (Ang-2) was purchased from Shanghai Noble Biotechnology Co., Ltd. (Shanghai, China). Double-strand specific nuclease (DSN) was purchased from Beijing Biolegend Technology Co., Ltd. Human serum samples were provided by the Affiliated Hospital of Shandong First Medical University (Jinan, China).

[0047] Polyacrylamide gel electrophoresis (PAGE)

[0048] 12% polyacrylamide gel electrophoresis (containing water, 30% acrylamide - methylene bisacrylamide, TBE buffer, 10% ammonium persulfate, tetramethylethylenediamine) was used, with TBE buffer as the electrophoresis buffer. The total volume of the loaded sample was 13 μL, consisting of 10 μL nucleic acid sample, 1 μL GelRed dye (Beyotime, Shanghai), and 2 μL loading buffer (Beyotime, Shanghai). Electrophoresis was carried out at 120 V for 45 minutes, and then the gel was imaged using a gel imaging system (ChemiScope 6200, Shanghai Qinxiang Scientific Instrument Co., Ltd.).

[0049] The detection principle of the electrochemical biosensor developed in the present invention is as Figure 6 shown. In this study, acDNA that can form a hairpin structure was used as the signal probe. The aptamer hybridized with acDNA to form dsDNA, and then was immobilized on the surface of the NP-Gold / GCE electrode through Au-S bonds. The electroactive label MB located at the 5' end of acDNA was far from NP-Gold, resulting in restricted electron transfer. When detecting PSA, the aptamer on the electrode surface specifically captured PSA and dissociated from dsDNA into the solution. Subsequently, single-stranded acDNA formed a stable hairpin structure, making the electroactive label MB closer to the surface of NP-Gold. Combining with the high conductivity of NP-Gold, the electron transfer efficiency was significantly improved. DSN can specifically recognize and cleave dsDNA, but is inactive against single-stranded DNA or single-stranded and double-stranded RNA. To reduce the interference of noise signals on the electrode surface, DSN was used to cleave the dsDNA that did not bind PSA, enabling MB on the dsDNA to detach from the electrode. In addition, the formation of the hairpin structure can prevent DSN from approaching the stem due to steric hindrance effects. With the help of DSN and NP-Gold, the proposed electrochemical biosensor can achieve highly sensitive detection of PSA.

[0050] Example 1,

[0051] Step 1, Preparation of NP-Gold / GCE electrode

[0052] The bare glassy carbon electrode (GCE) was polished and cleaned according to the method described in detail in previous studies. The specific method was as follows: The glassy carbon electrode was polished with alumina powder with a diameter of 30 nm, and then it was placed in ultrapure water and sonicated (at a frequency of 40 kHz) for 30 seconds for standby. Subsequently, nanoporous gold (NP-Gold) was prepared by dealloying a gold / silver sheet (Au50Ag50, mass fraction, Sepp Leaf Products, USA) in concentrated nitric acid at 30 °C for 30 minutes. Then, the obtained nanoporous gold was rinsed with ultrapure water multiple times, with each rinse lasting 30 minutes. Next, the prepared nanoporous gold was covered on the glassy carbon electrode to form an NP-Gold / GCE electrode. The electroactive area of the NP-Gold / GCE electrode was calculated by the method in the literature. Step 2, Preparation of the dsDNA / NP-Gold / GCE electrode

[0053] The PSA aptamer (2.0 μL, 10 μM) and acDNA (2.0 μL, 10 μM) were mixed with 4 μL of TM buffer, activated at 90 °C for 10 minutes, and then gradually cooled to room temperature. During this heat treatment process, the aptamer hybridized with acDNA to form a double-stranded (dsDNA) structure. Then, the formed dsDNA structure (8 μL) was mixed with a TCEP (2 μL, 100 mM) solution at room temperature for 30 minutes to reduce the disulfide bond to a thiol group. Subsequently, 10 μL of this mixture was taken and dropped onto the surface of the NP-Gold / GCE electrode, and incubated at 4 °C for 12 hours. By establishing Au-S bonds, the dsDNA / NP-Gold / GCE electrode was successfully constructed. Finally, the unbound dsDNA was rinsed off with ultrapure water.

[0054] Example 2, Detection of PSA, Preparation of the DSN / PSA / dsDNA / NP-Gold / GCE electrode

[0055] First, 10 μL of PSA solutions with different concentrations were dropped onto the dsDNA / NP-Gold / GCE electrode. Then the electrode was incubated at 4 °C for 60 minutes. This incubation step was carried out to promote the formation of the PSA-aptamer complex and to induce acDNA to fold into a hairpin structure. Subsequently, the prepared PSA / dsDNA / NP-Gold / GCE electrode was rinsed with ultrapure water to remove free PSA and the PSA-aptamer complex. Next, 1 U of DSN was added to the surface of the PSA / dsDNA / NP-Gold / GCE electrode and incubated at 37 °C for 60 minutes. The dsDNA not bound to PSA was cleaved by DSN, and the current value was detected by differential pulse voltammetry (DPV) in a PBS (50 mM, pH 7.0) solution.

[0056] Example 2, Detection Test

[0057] 1. Detection Method

[0058] 1.1. Structural Characterization and Electrochemical Analysis

[0059] The structural characteristics of nanoporous gold were analyzed using a scanning electron microscope (SEM, JSM-IT700 HR) and a transmission electron microscope (TEM, HT7800). The crystallographic properties were detected by X-ray diffraction (XRD, Rigaku SmartLab SE), while the surface chemical composition of the nanomaterials was studied by X-ray photoelectron spectroscopy (XPS, Kratos Axis Supra+).

[0060] All electrochemical experiments were carried out using a CHI 760E electrochemical workstation (Chenhua Instrument Co., Ltd., Shanghai, China). A traditional three-electrode configuration was adopted, with the modified glassy carbon electrode as the working electrode, a platinum sheet as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode.

[0061] 1.2. Detection of PSA

[0062] 10 μL of PSA solutions with different concentrations were dropped onto the dsDNA / NP-Gold / GCE electrode and incubated at 4 °C for 60 minutes to obtain the PSA / dsDNA / NP-Gold / GCE electrode. In this example, the PSA solutions with different concentrations included solutions with concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, and 10 ng / mL respectively. Then, 1 U of DSN was added to the surface of the PSA / dsDNA / NP-Gold / GCE electrode and incubated at 37 °C for 60 minutes. The current was measured in PBS (50 mM, pH 7.0) solution using the DPV method. A calibration curve was constructed to illustrate the correlation between the current response and the PSA concentration.

[0063] For the evaluation of serum samples, PSA was added to human serum samples at concentrations of 100 fg / mL, 10 pg / mL, and 1 ng / mL. Specifically, these serum samples containing different PSA concentrations were dropped onto the dsDNA / NP-Gold / GCE electrode and incubated at 4 °C for 60 minutes. Subsequently, 1 U of DSN was added, and the electrode was incubated at 37 °C for 60 minutes. The current was detected in PBS (50 mM, pH 7.0) solution by the DPV method. The recorded current response values were substituted into the calibration curve to determine the concentration of PSA.

[0064] 1.3. Statistical Analysis

[0065] All data were processed using GraphPad software. Student’s t-test or one-way ANOVA was used to analyze the differences between groups. Data were expressed as mean ± standard deviation (SD), and all experiments were repeated at least three times. P < 0.05 was considered statistically significant.

[0066] 2, Results and Discussion

[0067] 2.1 Characterization of the aptasensor

[0068] The morphology of nanoporous gold was characterized using SEM and TEM. As shown in A of Figure 1 and B of 1, nanoporous gold presented an open three-dimensional nanoporous structure with a pore size of approximately 40 nm. This structure provided a larger surface area and more active sites for the loading of dsDNA. As shown in C of Figure 1 , the XRD analysis of nanoporous gold showed characteristic diffraction peaks at 2θ of 38.30°, 44.40°, 64.78°, 77.72°, and 81.88°, corresponding to the (111), (200), (220), (311), and (222) crystal planes of metallic gold, respectively. XPS was used to analyze the changes in elemental species and the formation of Au–S bonds before and after the binding of nanoporous gold with dsDNA. The full-scan spectrum of nanoporous gold ( Figure 1 in D) showed distinct peaks corresponding to Au 4d (335 and 354 eV), C 1s (284 eV), and Au 4f (87.9 and 84.3 eV). As shown in E of Figure 1 , the deconvoluted Au 4f spectrum of nanoporous gold showed a prominent peak at 84.3 eV, which was mainly attributed to elemental gold (Au0). After the assembly of dsDNA, the Au0 peak in the deconvoluted Au 4f spectrum of dsDNA / NP-Gold shifted to 84.0 eV ( Figure 1 in F), mainly due to the interaction with the electronegative sulfur element on nanoporous gold. The additional peak that appeared at 84.4 eV ( Figure 1 in F) indicated the formation of Au–S bonds between dsDNA and nanoporous gold. In addition, the S2p spectrum of dsDNA / NP-Gold showed a peak at 162.5 eV, corresponding to Au–S ( Figure 1 in G), further proving the formation of Au–S bonds.

[0069] During the preparation process, electrochemical impedance spectroscopy (EIS, Figure 7 in A) and cyclic voltammetry (CV, Figure 1 in H and Figure 7B) The GCE, NP-Gold / GCE, dsDNA / NP-Gold / GCE, PSA / dsDNA / NP-Gold / GCE, and DSN / PSA / dsDNA / NP-Gold / GCE electrodes were characterized. As Figure 1 shown in H, compared with the GCE electrode, after the electrode was modified with nanoporous gold, the redox peak current increased significantly, indicating that nanoporous gold has excellent conductivity. The assembly of dsDNA on the NP-Gold / GCE electrode led to a significant decrease in the intensity of the redox peak current. This phenomenon can be attributed to the electrostatic repulsion between the negatively charged dsDNA and the 6 3- / 4- solution. In addition, this finding also confirmed the successful immobilization of dsDNA on the NP-Gold / GCE electrode. Subsequently, the aptamer recognized and captured PSA, causing it to dissociate from dsDNA. This led to a decrease in the number of negatively charged DNAs on the electrode surface, so the redox peak current increased significantly. On the contrary, after DSN was attached and cleaved dsDNA, the redox peak current of the DSN / PSA / dsDNA / NP-Gold / GCE electrode decreased. The reason may be that the DSN insulating protein attached to dsDNA hindered the interfacial electron transfer.

[0070] The sensing performance of the aptasensor for PSA was studied using the DPV method in PBS (50 mM, pH 7.0) solution. As Figure 1 shown in I, the GCE and NP-Gold / GCE electrodes showed a straight line at -0.32 V without signal generation. When the dsDNA labeled with MB was immobilized on the surface of the NP-Gold / GCE electrode, an obvious MB electrical signal was generated. Subsequently, the binding of the aptamer to PSA caused a conformational change in acDNA, forming a stable hairpin structure, making MB closer to NP-Gold. These changes promoted electron transfer, thus increasing the electrochemical signal. After incubation with DSN, the dsDNA that did not capture PSA was cleaved. The electrical signal of the DSN / PSA / dsDNA / NP-Gold / GCE electrode decreased significantly, indicating that the interference of the noise signal (dsDNA not bound to PSA) was reduced, and the positive correlation between the PSA concentration and the electrical signal was enhanced.

[0071] The PAGE method was used to evaluate the affinity of the aptamer for PSA and the cleavage ability of DSN. In Figure 2 A, lane 1 represents the aptamer, and lane 2 represents the mixture of the aptamer and PSA. Compared with lane 1, the lighter band at the bottom of lane 2 indicates that the aptamer was consumed. In addition, a new band with a slower migration rate was observed (as Figure 2 shown by the red box in A), indicating the formation of the PSA-aptamer complex. As Figure 2 ​As shown in Lane 1 and Lane 2 of Panel B, the presence of the aptamer and acDNA is indicated respectively. The bright band in Lane 3 indicates that the aptamer has hybridized with acDNA to form a dsDNA structure. The discrete bands in the middle of Lane 4 indicate that the dsDNA has been cleaved by DSN. Lane 5 confirms that DSN is inactive against single-stranded DNA. The PAGE verification results are consistent with the CV and DPV analysis results.

[0072] Example 3, Optimization of Experimental Conditions

[0073] 3.1, Study on the pH Value of the Buffer Solution

[0074] To improve the electrochemical performance of the aptasensor, the experimental parameters were further optimized. In this study, the detection limit of prostate-specific antigen (PSA) is related to the intensity of the electrochemiluminescence signal of methylene blue (MB). To obtain the maximum electrochemiluminescence signal response value generated by the electroactive label MB molecules, the pH value of the buffer solution was explored. As Figure 3 shown in Panel A, the current response increases with the increase of pH value until the pH value reaches 7.0. Subsequently, when the pH value is higher than 7.0, the current response value gradually decreases. In addition, as the pH value increases, the peak potential shifts towards the negative direction. This shift is due to the Nernst slope of -59 mV / pH, which indicates that for every 1 unit increase in pH value, the peak potential will shift -59 mV towards the negative direction. Based on these results, the optimal pH value for detecting PSA was determined to be 7.0.

[0075] 3.2, Study on the Denaturation Temperature

[0076] The formation of double-stranded DNA (dsDNA) is closely related to temperature. To obtain the maximum amount of dsDNA, the denaturation temperature was investigated in this study. As Figure 3 shown in Panel B, when the temperature increases from 80 °C to 90 °C, the thermal motion of the molecules intensifies, resulting in the breakage of hydrogen bonds between bases. Therefore, the hairpin structures of the aptamer ( Figure 8 ) and the aptamer complementary DNA (acDNA) are disrupted. As the temperature gradually drops to room temperature, more dsDNA will be formed. When the temperature is higher than 90 °C, the electrochemiluminescence signal gradually weakens. Therefore, 90 °C was selected as the optimal denaturation temperature for DNA.

[0077] 3.3, Study on the Incubation Time

[0078] The loading amount of dsDNA on the electrode surface directly affects the detection limit and linear range of the aptasensor. In this study, the nanoporous gold (NP-Gold) nanostructure served as an effective carrier, providing more binding sites for the binding of dsDNA. To optimize the immobilization efficiency of dsDNA, cyclic voltammetry (CV) curves of dsDNA / NP-Gold / glassy carbon electrode (GCE) were studied in phosphate buffer solution (PBS, 50 mM, pH 7.0) at different dsDNA incubation times (0, 3, 6, 9, 12, 15, 18 h). As Figure 3 shown in C, the dsDNA / NP-Gold / GCE electrode with an incubation time of 0 h showed the maximum redox peak current of NP-Gold. As the dsDNA incubation time increased, the redox peak current of NP-Gold gradually decreased. The results showed that the dsDNA / NP-Gold / GCE electrode reached a saturated state at an incubation time of 12 h. Therefore, 12 h was selected as the optimal incubation time for dsDNA.

[0079] 3.4, Study on the binding time

[0080] The binding time of PSA to the aptamer is an important factor affecting the performance of the aptasensor. Therefore, the binding time of PSA was optimized in this study. Differential pulse voltammetry (DPV) curves of PSA / dsDNA / NP-Gold / GCE electrodes were studied in PBS (50 mM, pH 7.0) solution at different binding times (30, 60, 90, 120, and 150 min). As Figure 3 shown in D, as the incubation time increased, the peak current value of the electrochemical response gradually increased and reached the maximum at 60 min. Therefore, an incubation time of 60 min was determined as the optimal condition for forming the PSA-aptamer complex.

[0081] 3.5, Study on the cleavage time and dosage of DSN

[0082] The cleavage time and dosage of double-strand specific nuclease (DSN) are important factors affecting the performance of the aptasensor. The cleavage efficiency was evaluated by the current difference of the NP-Gold / GCE electrode before and after incubation with DSN. As Figure 3 shown in E, in the range of 20 to 100 min, the current difference increased with the increase of the cleavage time and reached the maximum at 60 min. Therefore, the optimal incubation time of DSN was 60 min. In addition, as Figure 3 shown in F, the current difference increased with the increase of the DSN dosage and reached the maximum at a dosage of 1 unit (U). Therefore, 1 U was selected as the optimal dosage of DSN.

[0083] Example 4, Performance study experiment

[0084] 4.1, Detection of PSA

[0085] Under the optimal parameter conditions, the proposed aptasensor was used to detect different concentrations of PSA. Figure 4 Figure A shows the correlation between the current value and the PSA concentration. As the PSA concentration increases, more aptamers are consumed, and more acDNA forms hairpin structures, resulting in enhanced electrical signals. In the range of 10 fg / mL to 10 ng / mL, there is a good linear correlation between the current value and the logarithm of the PSA concentration. As Figure 4 shown in Figure B, the linear regression equation is j (μA) = 2.4326 + 0.1428 × log C PSA (pg / mL), and the correlation coefficient is 0.9714. The minimum detection value of this aptasensor is 10 fg / mL.

[0086] The good performance of this aptasensor can be attributed to several factors. First, NP-Gold has a high specific surface area, providing abundant sites for the binding of dsDNA. Second, the excellent biocompatibility of NP-Gold creates a favorable microenvironment, ensuring the stability of dsDNA. Third, DSN is used to cleave the dsDNA that does not bind PSA, thereby reducing the interference of noise signals on the electrode surface. These findings indicate that the developed aptasensor exhibits excellent performance characteristics, including high sensitivity, low detection limit, and wide linear detection range.

[0087] 4.2, Anti-interference ability of the aptasensor

[0088] Anti-interference ability is one of the indispensable characteristics of aptasensors. To evaluate the anti-interference ability of the developed aptasensor, eight proteins were used as interfering proteins, including alpha-fetoprotein (AFP), angiopoietin-2 (Ang-2), carbohydrate antigen 125 (CA-125), carcinoembryonic antigen (CEA), immunoglobulin G (IgG), bovine serum albumin (BSA), cathepsin B (CB), and human epididymal protein 4 (HE4). The concentrations of both the interfering proteins and PSA were 100 pg / mL. The interfering proteins were added to the prepared dsDNA / NP-Gold / GCE electrode and incubated at 4 °C for 1 hour. After cleavage with DSN, DPV detection was performed in a PBS (50 mM, pH 7.0) solution. As Figure 5 shown in Figure A, the peak current values of the eight interfering proteins are the same as those of the negative control (NC) (P > 0.05), and are much lower than the peak current value of PSA. This indicates that the interfering proteins cannot be captured by the aptamer and do not cause changes in the acDNA structure. These results are attributed to the high affinity of the aptamer for PSA. The results show that the proposed aptasensor has high selectivity for PSA.

[0089] 4.3 Reproducibility and Stability of the Aptasensor

[0090] To further investigate the reproducibility of the aptasensor, five electrodes were prepared under the same conditions for detecting PSA at a concentration of 100 fg / mL. As Figure 5 shown in B, the measured current values of the five DSN / PSA / dsDNA / NP-Gold / GCE electrodes were in the range of 2.284 to 2.291 μA. This indicates that the measured PSA concentration was in the range of 91 to 112 fg / mL, with an average value of 105.2 fg / mL and a relative standard deviation of 0.8%. These findings confirm that the aptasensor developed in this study is accurate and has excellent reproducibility. The stability of the aptasensor was evaluated by storing the prepared dsDNA / NP-Gold / GCE electrodes at 4 °C for 0, 7, 14, and 21 days respectively. The stored dsDNA / NP-Gold / GCE electrodes were incubated with PSA at a concentration of 1 ng / mL. After cleavage with DSN, the current was measured using the DPV method in PBS solution. As Figure 5 shown in C, after 21 days of storage, the current value of the aptasensor for PSA did not decrease, and the change in the relative current value was within 5%. These results indicate that the proposed aptasensor exhibits excellent stability, which can be attributed to the good biocompatibility of the NP-Gold nanomaterial.

[0091] 4.4 Detection of Prostate-Specific Antigen (PSA) in Serum

[0092] To evaluate the detection effect of the developed aptasensor on actual samples, PSA was added to undiluted serum at final concentrations of 100 fg / mL, 10 pg / mL, and 1 ng / mL respectively. Then, the proposed aptasensor was used to detect the PSA concentration in undiluted serum, and the corresponding current values were obtained. The data thus obtained were substituted into the calibration curve ( Figure 4 shown in B) to calculate the PSA concentration. As shown in Table 1, the concentrations detected by the aptasensor were very close to the concentrations added to the undiluted serum, with a deviation rate of less than 5%. In addition, the successful detection of PSA in human serum also indicates that the constructed aptasensor has good anti-interference performance against serum components. Based on these results, the proposed aptasensor is a reliable tool for detecting PSA in human serum without sample pretreatment.

[0093] Table 1 Detection of PSA in Serum

[0094]

[0095] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical aptamer sensor, characterized in that: The steps include: Step S1, preparation of NP-Gold / GCE electrode: covering nanoporous gold on a glassy carbon electrode to form a NP-Gold / GCE electrode; Step S2, preparation of dsDNA / NP-Gold / GCE electrode: hybridize the aptamer and acDNA through heat treatment to form a double-stranded dsDNA structure; then, mix the formed dsDNA structure with tri(2-carboxyethyl)phosphine hydrochloride to reduce the disulfide bond to a thiol group to obtain a mixed solution; then, drop the mixed solution onto the surface of the NP-Gold / GCE electrode prepared in step S1, and construct the dsDNA / NP-Gold / GCE electrode after incubation.

2. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: In step S2, the aptamer and acDNA are mixed with Tris-MgSO4 buffer, activated at 85°C to 100°C, and then gradually cooled to room temperature.

3. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: In step S2, the incubation is carried out at a temperature of 1°C to 8°C for 3 to 18 hours.

4. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: In step S2, the constructed dsDNA / NP-Gold / GCE electrode is rinsed with ultrapure water to remove unbound dsDNA.

5. The method for preparing an electrochemical aptamer sensor according to claim 1, characterized in that: The aptamer is a PSA aptamer, and the sequence is shown in SEQ ID NO.1; the acDNA sequence is shown in SEQ ID NO.

2.

6. The product obtained by the method for preparing the electrochemical aptamer sensor according to any one of claims 1 to 5.

7. Application of the preparation method of the electrochemical aptamer sensor according to any one of claims 1 to 5 in the detection of prostate specific antigen.

8. A method for detecting prostate-specific antigen, characterized in that: The steps include: Step 1, establishment of a calibration curve: PSA solutions of different concentrations are dripped onto the dsDNA / NP-Gold / GCE electrode and incubated at 1°C to 8°C for 30 to 120 minutes; then, the PSA / dsDNA / NP-Gold / GCE electrode and DSN are incubated at 30°C to 40°C for 40 to 100 minutes; the current is measured in a phosphate buffer using the DPV method to establish a calibration curve reflecting the correlation between the current response and the PSA concentration; The PSA solutions of different concentrations include solutions with several concentration values ​​between 10fg / mL and 10ng / mL; Step 2, detection of PSA: add the sample to the dsDNA / NP-Gold / GCE electrode and incubate at 1°C to 8°C for 30 to 120 minutes; then, add DSN and incubate the electrode at 30°C to 40°C for 40 to 100 minutes; detect the current in phosphate buffer by the DPV method, and substitute the recorded current response value into the calibration curve to determine the concentration of PSA.

9. The method for detecting prostate-specific antigen according to claim 8, characterized in that: In step 1 and step 2, the pH range of the phosphate buffer is 6-9.

10. The method for detecting prostate-specific antigen according to claim 8, characterized in that: In step 1 and step 2, the amount of DSN used is 1 to 2.5 U.

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