An electrochemical aptamer sensor, a preparation method and application thereof, and a prostate specific antigen detection method

Abstract

The application discloses an electrochemical aptamer sensor and a preparation method and application thereof, and a prostate specific antigen detection method, and successfully develops a reliable electrochemical biological aptamer sensor for super-sensitivity detection of PSA. An aptamer is used as a biological recognition element to specifically recognize PSA. Nano-porous gold (NP-Gold) with high specific surface area and excellent conductivity is used as a support material. With the help of double-stranded specific nuclease (DSN), noise signals are reduced, the proposed aptamer sensor shows excellent performance in determination of PSA in clinical serum samples, has high sensitivity, strong anti-interference ability and reliable stability. These unique advantages make the aptamer sensor a potential choice for detection of PSA.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, and specifically relates to an electrochemical aptamer sensor, its preparation method and application, as well as a method for detecting prostate-specific antigens. Background Technology

[0002] Millions of people worldwide are diagnosed with prostate cancer (PCa) each year, one of the most common solid malignancies globally. Prostate cancer has a long course and a high metastasis rate, making treatment particularly challenging in its middle and late stages. The prognosis of prostate cancer varies significantly depending on the patient's age, race, genetic background, and disease stage. Therefore, early diagnosis is crucial for reducing prostate cancer mortality and improving treatment outcomes. Prostate-specific antigen (PSA), a single-chain serine protease secreted by prostate epithelial cells with a molecular weight of 33-34 kDa, is considered one of the most reliable biomarkers for diagnosing prostate cancer. Furthermore, PSA is also considered the best biomarker for diagnosing prostatitis and benign prostatic hyperplasia (BPH). Therefore, PSA testing and identification are of great significance for the early diagnosis of prostate cancer.

[0003] Currently, several traditional detection methods are used for PSA detection, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunosorbent assay (CISA), surface-enhanced Raman spectroscopy (SERS), surface plasmon resonance (SPR), and mass spectrometry. However, these methods are time-consuming and require sophisticated and complex instruments. Furthermore, these traditional techniques not only lack sensitivity but also have deficiencies in sample and antibody preparation, stability, and modification procedures. 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 to traditional detection methods, electrochemical biosensors offer several inherent advantages, such as high cost-effectiveness, fast response speed, ease of operation, good reproducibility, and high sensitivity. When constructing biosensors, selecting appropriate biorecognition elements is crucial. Among various biorecognition elements (such as enzymes and antibodies), 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 particular target by folding into a three-dimensional structure. Compared to traditional immunorecognition molecules, aptamers offer advantages such as higher stability, lower cost, simpler artificial synthesis and modification, smaller size, and higher specificity.

[0005] However, no electrochemical biosensor technology solution for PSA detection has been publicly disclosed that offers high sensitivity, selectivity, and reliability. Summary of the Invention

[0006] In view of the shortcomings of existing technologies, this invention provides an electrochemical aptamer sensor, its preparation method and application, as well as a method for detecting prostate-specific antigen (PSA). In this invention, an aptamer sensor based on aptamer-DNA double strands, nanoporous gold, and double-stranded specific nuclease (DSN) is developed for the electrochemical determination of the prostate cancer biomarker PSA. Specifically, nanoporous gold is selected as the substrate material due to its high specific surface area and stability, providing abundant binding sites for the effective immobilization of the aptamer-DNA double strand structure. Simultaneously, the characteristics of DSN are utilized to minimize noise signals on the electrode surface.

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

[0008] Step S1, Preparation of NP-Gold / GCE electrode: Nanoporous gold is coated on a glassy carbon electrode to form an NP-Gold / GCE electrode;

[0009] Step S2, preparation of the dsDNA / NP-Gold / GCE electrode: The aptamer and acDNA are hybridized by heat treatment to form a double-stranded dsDNA structure; then, the formed dsDNA structure is mixed with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bonds to thiol groups, and a mixture is obtained; subsequently, the mixture is dropped onto the surface of the NP-Gold / GCE electrode prepared in step S1, and after incubation, the dsDNA / NP-Gold / GCE electrode is constructed.

[0010] Preferably, in step S2, the aptamer and acDNA are mixed with Tris-MgSO4 buffer (TM), activated at 85°C–100°C, and then gradually cooled to room temperature. During this heat treatment, the aptamer hybridizes with the 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 washed away with ultrapure water to remove unbound dsDNA.

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

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

[0015] With the development of nanomaterials, various sensors based on different nanomaterials have been constructed to detect low concentrations of biomarkers. Among numerous nanomaterials, nanoporous gold (NP-Gold) has attracted widespread attention in the construction of biosensors due to its unique physical and chemical properties, as well as its highly efficient catalytic activity towards various electrochemical substances. NP-Gold possesses 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. Furthermore, NP-Gold exhibits excellent biocompatibility and conductivity. Moreover, the oxidized gold atoms on NP-Gold can form covalent bonds with functional groups (such as amino (–NH2) and thiol (–SH)) on the surface of biomolecules. These properties make NP-Gold highly suitable for modifying various recognition components, such as aptamers.

[0016] As a second aspect of the present invention, a method for preparing an electrochemical aptamer sensor based on the synergistic effect of a dual-specific nuclease and nanoporous gold is provided, yielding a product.

[0017] As a third aspect of the present invention, it provides a method for preparing an electrochemical aptamer sensor based on the synergistic effect of a dual-specific nuclease and nanoporous gold, and its application in the detection of prostate-specific antigens.

[0018] As a fourth aspect of the present invention, a method for detecting prostate-specific antigen is provided, comprising the following steps:

[0019] Step 1, Establishment of calibration curve: PSA solutions of different concentrations were added to the dsDNA / NP-Gold / GCE electrode and incubated at 1℃~8℃ for 30~120 minutes to prepare the PSA / dsDNA / NP-Gold / GCE electrode; then, the PSA / dsDNA / NP-Gold / GCE electrode and DSN were incubated at 30℃~40℃ for 40~100 minutes; the current was measured in phosphate buffer using the DPV method to establish a calibration curve reflecting the correlation between the current response and the PSA concentration.

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

[0021] Step 2, PSA detection: Add the sample to the dsDNA / NP-Gold / GCE electrode and incubate at 1℃~8℃ for 30~120 minutes, preferably at 4℃ for 60 minutes, to prepare...

[0022] PSA / dsDNA / NP-Gold / GCE electrode; then, DSN is added, and the electrode is incubated at 30℃~40℃ for 40~100 minutes, preferably 60 minutes; the current is detected in phosphate buffer by DPV method, and the recorded current response value is substituted into the calibration curve to determine the concentration of PSA.

[0023] In steps 1 and 2, the first incubation is preferably carried out at 4°C for 60 minutes; the second incubation with added DSN is preferably carried out at 37°C for 60 minutes.

[0024] The phosphate buffer solution has a pH range of 6–9, 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 acDNA to fold 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 complexes; DSN is added to...

[0027] Incubate on the PSA / dsDNA / NP-Gold / GCE electrode surface. 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 this embodiment of the invention, the current value was detected in PBS (50mM, pH 7.0) solution by differential pulse voltammetry (DPV).

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

[0031] (1) This invention successfully developed a reliable electrochemical bioaptamer sensor based on the synergistic effect of a dual-specific nuclease and nanoporous gold for ultrasensitive detection of PSA. The aptamer is used as a biorecognition element to specifically recognize PSA. Nanoporous gold (NP-Gold), with its high specific surface area and excellent conductivity, is used as the support material. By utilizing a dual-stranded specific nuclease (DSN) to reduce noise signals, the proposed aptamer sensor exhibits excellent performance in measuring PSA in clinical serum samples, demonstrating high sensitivity and strong anti-interference capability. The minimum detection value of this aptamer sensor is 10 fg / mL, enabling the detection of extremely low concentrations of PSA. The aptamer has a high affinity for PSA, and the proposed aptamer sensor exhibits high selectivity for PSA.

[0032] (2) The PSA biosensor prepared in this invention exhibits excellent stability, with no decrease in detection current value after 21 days of storage. The measured PSA concentrations within the range of 91 to 112 fg / mL show excellent reproducibility. These unique advantages make this aptamer sensor a highly promising option for PSA detection.

[0033] (3) This invention provides a method for detecting prostate-specific antigen. This method is based on an electrochemical aptamer sensor with the synergistic effect of a dual-specific nuclease and nanoporous gold. It has the characteristics of high sensitivity, good reproducibility, high selectivity and high stability, providing a new approach for the detection of PSA. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 The figures show the results of the detection experiments in Example 2. 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 an X-ray photoelectron spectroscopy (XPS) image of nanoporous gold and double-stranded DNA (dsDNA) / nanoporous gold. E is an image of nanoporous gold. F is the gold 4f deconvolution XPS spectrum of dsDNA / nanoporous gold. G is the S2p deconvolution XPS spectrum of double-stranded specific nuclease (DSN) / nanoporous gold. H shows the glassy carbon electrode (GCE), NP-Gold / GCE electrode, dsDNA / NP-Gold / GCE electrode, prostate-specific antigen (PSA) / dsDNA / NP-Gold / GCE electrode, and DSN / PSA / dsDNA / NP-Gold / GCE electrode at 5 mM [Fe(CN)6]. 4- / 3- Cyclic voltammetry (CV) curves in solution (scan rate: 50 mV s) -1 I represents the differential pulse voltammetry (DPV) curves of the GCE, NP-Gold / GCE, dsDNA / NP-Gold / GCE, PSA / dsDNA / NP-Gold / GCE, and DSN / PSA / dsDNA / NP-Gold / GCE electrodes in phosphate-buffered saline (PBS) solution (50 mM, pH 7.0).

[0036] Figure 2In Example 2, polyacrylamide gel electrophoresis (PAGE) was used to demonstrate the affinity of the aptamer for PSA and its cleavage ability by double-stranded specific nuclease (DSN). In lane A, lane 1 contained the aptamer; lane 2 contained a mixture of the aptamer and PSA. In lane B, lane 1 contained the aptamer; lane 2 contained the aptamer's complementary DNA (acDNA); lane 3 contained a mixture of the aptamer and acDNA; lane 4 contained a mixture of the aptamer, acDNA, and DSN; and lane 5 contained a mixture of the aptamer and DSN (10 μM aptamer and acDNA, 100 μg / mL PSA, 1 UDSN).

[0037] Figure 3 This section describes the optimization experiments under different experimental conditions in Example 3. A represents the effect of pH on the dsDNA / NP-Gold / GCE electrode. B represents the effect of temperature on the dsDNA / NP-Gold / GCE electrode. C represents the effect of dsDNA adsorption time on the dsDNA / NP-Gold / GCE electrode. D represents the effect of the binding time between the aptamer and PSA on the PSA / dsDNA / NP-Gold / GCE electrode. E represents the DSN digestion time, and F represents the effect of the DSN dosage on the DSN / dsDNA / NP-Gold / GCE electrode.

[0038] Figure 4 The results are from the performance study in Example 4. In this figure, A shows the DPV curves of the aptamer sensor after incubation with different concentrations of PSA in PBS (50 mM, pH 7.0) solution. B shows the linear relationship between the current value and the logarithm of the PSA concentration.

[0039] Figure 5 The results of the interference immunity, reproducibility, and stability experiments of the aptamer sensor in Example 4 are shown. A represents the interference immunity of the aptamer sensor (NC: negative control, i.e., the aptamer sensor not incubated with PSA) (compared to the negative control, P>0.05). B represents the reproducibility of the aptamer sensor. C represents the storage stability of the aptamer sensor.

[0040] Figure 6 This is a schematic diagram illustrating the detection principle of the aptamer sensor provided by the present invention.

[0041] Figure 7 Characterization results were constructed for the sensors. Among them, (A) GCE, NP-Gold / GCE electrode, dsDNA / NP-Gold / GCE electrode, PSA / dsDNA / NP-Gold / GCE electrode, and DSN / PSA / dsDNA / NP-Gold / GCE electrode were characterized at 5 mM [Fe(CN)6]. 4- / 3- EIS curves in solution (frequency range 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 (50mM, pH 7.0).

[0042] Figure 8 This is a schematic diagram of the PSA aptamer secondary structure prediction. Detailed Implementation

[0043] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] Materials and reagents

[0045] All DNA used in this study was purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and purified by high-performance liquid chromatography (HPLC) before being lyophilized. This study used 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') modified with a thiol group (-SH) at the 3' end and methylene blue (MB) at the 5' end (SEQ ID NO.2). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Maclean Biotech Co., Ltd. (Shanghai, China). Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (TE) buffer (10 mM Tris-HCl, 1 mM EDTA), Tris-MgSO4 buffer™ (500 mM Tris-HCl, 80 mM MgSO4), 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-buffered saline (PBS, 50 mM) was prepared from Na2HPO4·12H2O and NaH2PO4·2H2O.

[0046] PSA, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) antigen, immunoglobulin G (IgG), bovine serum albumin (BSA), human epididymal protein 4 (HE4), cathepsin B (CB), and carbohydrate antigen 125 (CA125) were all purchased from Shanghai Lianke Biotechnology Co., Ltd. (Shanghai, China). Angiotensin-2 (Ang-2) was purchased from Shanghai Nuobao Biotechnology Co., Ltd. (Shanghai, China). Double-stranded specific nuclease (DSN) was purchased from Beijing Bio-Rad Biotechnology 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-methylenebisacrylamide, TBE buffer, 10% ammonium persulfate, and tetramethylethylenediamine) was performed, with TBE buffer as the electrophoresis buffer. The total sample volume 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 performed at 120 V for 45 minutes, and the gel was then imaged using a gel imaging system (ChemiScope 6200, Shanghai Qinxiang Scientific Instruments Co., Ltd.).

[0049] The detection principle of the electrochemical biosensor developed in this invention is as follows: Figure 6 As shown. In this study, acDNA capable of forming hairpin structures was used as a signal probe. Aptamers hybridized with acDNA to form dsDNA, which was then immobilized on the NP-Gold / GCE electrode surface via Au-S bonds. The electroactive marker MB located at the 5' end of the acDNA was far from NP-Gold, resulting in restricted electron transfer. When PSA was detected, the aptamer on the electrode surface specifically captured PSA and dissociated from the dsDNA into the solution. Subsequently, the single-stranded acDNA formed a stable hairpin structure, bringing the electroactive marker MB closer to the NP-Gold surface. Combined 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 interference from noise signals on the electrode surface, DSN was used to cleave dsDNA that was not bound to PSA, allowing the MB on the dsDNA to detach from the electrode. Furthermore, the formation of the hairpin structure prevented DSN from approaching the stem due to steric hindrance. The proposed electrochemical biosensor, utilizing DSN and NP-Gold, enables highly sensitive detection of PSA.

[0050] Example 1,

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

[0052] The bare glassy carbon electrode (GCE) was polished and cleaned according to the method described in previous studies. Specifically, the glassy carbon electrode was polished with alumina powder with a diameter of 30 nm, followed by ultrasonic cleaning (40 kHz frequency) in ultrapure water for 30 seconds. Then, nanoporous gold (NP-Gold) was prepared by dealloying gold / silver sheets (Au50Ag50, mass fraction, Sepp Leaf Products, USA) in concentrated nitric acid at 30°C for 30 minutes. The obtained nanoporous gold was then rinsed multiple times with ultrapure water, each rinse lasting 30 minutes. Next, the prepared nanoporous gold was coated onto the glassy carbon electrode to form the NP-Gold / GCE electrode. The electroactive area of ​​the NP-Gold / GCE electrode was calculated using methods described in the literature. Step 2: Preparation of the dsDNA / NP-Gold / GCE electrode.

[0053] PSA aptamer (2.0 μL, 10 μM) and acDNA (2.0 μL, 10 μM) were mixed with 4 μL of TM buffer and activated at 90 °C for 10 min, then gradually cooled to room temperature. During this heat treatment, the aptamer hybridized with acDNA to form a double-stranded (dsDNA) structure. The formed dsDNA structure (8 μL) was then mixed with TCEP (2 μL, 100 mM) solution at room temperature for 30 min to reduce disulfide bonds to thiol groups. Subsequently, 10 μL of this mixture was added dropwise to the surface of an NP-Gold / GCE electrode and incubated at 4 °C for 12 h. The dsDNA / NP-Gold / GCE electrode was successfully constructed by establishing Au-S bonds. Finally, unbound dsDNA was washed away with ultrapure water.

[0054] Example 2: PSA detection, preparation of DSN / PSA / dsDNA / NP-Gold / GCE electrode

[0055] First, 10 μL of PSA solutions of varying concentrations were added dropwise to the dsDNA / NP-Gold / GCE electrode. The electrode was then incubated at 4°C for 60 minutes. This incubation step was performed to promote the formation of the PSA-aptamer complex and to induce acDNA folding 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 the electrode was incubated at 37°C for 60 minutes. Unbound dsDNA was cleaved by the DSN, and the current value was detected by differential pulse voltammetry (DPV) in PBS (50 mM, pH 7.0).

[0056] Example 2, Detection Test

[0057] 1. Detection method

[0058] 1.1 Structural Characterization and Electrochemical Analysis

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

[0060] All electrochemical experiments were conducted using a CHI 760E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). A traditional three-electrode configuration was employed, with a 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, PSA Detection

[0062] 10 μL of PSA solutions of different concentrations were added dropwise to the dsDNA / NP-Gold / GCE electrode and incubated at 4 °C for 60 min to obtain the PSA / dsDNA / NP-Gold / GCE electrode. In this example, the different concentrations of PSA solutions 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. 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 min. 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 serum sample evaluation, 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 added to a 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 using the DPV method. The recorded current response values ​​were substituted into the calibration curve to determine the PSA concentration.

[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 differences between groups. Data are 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 Adaptor Sensors

[0068] The morphology of nanoporous gold was characterized using SEM and TEM. For example... Figure 1 As shown in Figures A and B, the nanoporous gold exhibits an open three-dimensional nanoporous structure with a pore size of approximately 40 nm. This structure provides a larger surface area and more active sites for dsDNA loading. Figure 1 As shown in Figure C, XRD analysis of nanoporous gold revealed characteristic diffraction peaks at 2θ values ​​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 analysis was used to analyze the changes in elemental composition and the formation of Au-S bonds before and after the binding of nanoporous gold with dsDNA. Full-scan spectrum of nanoporous gold (… Figure 1 The middle D) shows distinct peaks corresponding to Au 4d (335 and 354 eV), C 1s (284 eV), and Au 4f (87.9 and 84.3 eV). Figure 1 As shown in Figure E, the deconvolution Au 4f spectrum of nanoporous gold exhibits a prominent peak at 84.3 eV, which is mainly attributed to elemental gold (Au0). After dsDNA assembly, the Au0 peak in the deconvolution Au 4f spectrum of dsDNA / NP-Gold shifts to 84.0 eV. Figure 1 The presence of F (in the middle) is mainly due to its interaction with the electronegative sulfur element on the nanoporous gold. An additional peak appears at 84.4 eV (…). Figure 1 The presence of F indicates that an Au-S bond is formed between dsDNA and the nanoporous gold. Furthermore, the S2p spectrum of dsDNA / NP-Gold shows a peak at 162.5 eV, corresponding to the Au-S bond. Figure 1 The presence of G further proves the formation of Au-S bonds.

[0069] During the preparation process, electrochemical impedance spectroscopy (EIS) was used. Figure 7 (A) and cyclic voltammetry (CV) Figure 1 H and Figure 7Part B) characterizes the GCE, NP-Gold / GCE, dsDNA / NP-Gold / GCE, PSA / dsDNA / NP-Gold / GCE, and DSN / PSA / dsDNA / NP-Gold / GCE electrodes. Figure 1 As shown in Figure H, compared with the GCE electrode, the redox peak current significantly increased after the electrode was modified with nanoporous gold, indicating that nanoporous gold possesses excellent conductivity. The assembly of dsDNA on the NP-Gold / GCE electrode led to a significant decrease in the redox peak current intensity. This phenomenon can be attributed to the interaction between the negatively charged dsDNA and [Fe(CN)6]. 3- / 4- Electrostatic repulsion between solutions was observed. Furthermore, this finding confirmed the successful immobilization of dsDNA on the NP-Gold / GCE electrode. Subsequently, the aptamer recognized and captured PSA, causing it to dissociate from the dsDNA. This resulted in a reduction in the amount of negatively charged DNA on the electrode surface, leading to a significant increase in the redox peak current. Conversely, after DSN attached to and cleaved dsDNA, the redox peak current of the DSN / PSA / dsDNA / NP-Gold / GCE electrode decreased. This may be because the insulating protein of DSN attached to dsDNA hindered interfacial electron transfer.

[0070] The sensing performance of the aptamer sensor for PSA was studied using the DPV method in PBS (50 mM, pH 7.0) solution. Figure 1 As shown in Figure I, the GCE and NP-Gold / GCE electrodes appear as a straight line at -0.32V, with no signal generation. When MB-labeled dsDNA is immobilized on the NP-Gold / GCE electrode surface, a significant MB electrochemical signal is generated. Subsequently, the binding of the aptamer to PSA leads to a conformational change in the acDNA, forming a stable hairpin structure that brings the MB closer to NP-Gold. These changes promote electron transfer, thereby increasing the electrochemical signal. After incubation with DSN, dsDNA that has not captured PSA is cleaved. The electrochemical signal at the DSN / PSA / dsDNA / NP-Gold / GCE electrode is significantly reduced, indicating reduced interference from noise signals (dsDNA not bound to PSA) and an enhanced positive correlation between PSA concentration and the electrochemical signal.

[0071] The PAGE method was used to evaluate the affinity of the aptamers for PSA and their cleavage ability for DSN. Figure 2 In lane A, lane 1 represents the aptamer, and lane 2 represents a mixture of the aptamer and PSA. Compared to lane 1, the fainter band at the bottom of lane 2 indicates that the aptamer has been consumed. Additionally, a new band with a slower migration rate (e.g., Figure 2 (As shown in the red box in section A), this indicates the formation of a PSA-aptamer complex. Figure 2As shown in Figure B, lanes 1 and 2 represent the presence of aptamers and acDNA, respectively. The bright band in lane 3 indicates that the aptamer has hybridized with acDNA to form a dsDNA structure. The dispersed band in the middle of lane 4 indicates that the dsDNA has been cleaved by DSN. Lane 5 confirms that DSN is inactive against single-stranded DNA. The PAGE validation results are consistent with the CV and DPV analysis results.

[0072] Example 3: Optimization of Experimental Conditions

[0073] 3.1 Study on pH value of buffer solution

[0074] To improve the electrochemical performance of the aptamer sensor, experimental parameters were further optimized. In this study, the detection limit of prostate-specific antigen (PSA) was correlated with the intensity of the methylene blue (MB) electrical signal. To obtain the maximum electrical signal response value generated by the electroactive marker MB molecule, the pH value of the buffer solution was investigated. Figure 3 As shown in Figure A, the current response increases with increasing pH until it reaches 7.0. Subsequently, the current response gradually decreases above 7.0. Furthermore, the peak potential shifts negatively with increasing pH. This shift is due to the Nernst slope of -59 mV / pH, indicating that for every unit increase in pH, the peak potential shifts negatively by -59 mV. Based on these results, the optimal pH for PSA detection was determined to be 7.0.

[0075] 3.2 Study on Deformation Temperature

[0076] The formation of double-stranded DNA (dsDNA) is closely related to temperature. To obtain the maximum amount of dsDNA, this study investigated the denaturation temperature. Figure 3 As shown in Figure B, when the temperature rises from 80℃ to 90℃, the thermal motion of molecules intensifies, leading to the breakage of hydrogen bonds between bases. Therefore, aptamers ( Figure 8 The hairpin structure of dsDNA and aptamer complementary DNA (acDNA) is disrupted. As the temperature gradually decreases to room temperature, more dsDNA is formed. When the temperature exceeds 90°C, the electrical signal gradually weakens. Therefore, 90°C is chosen as the optimal denaturation temperature for DNA.

[0077] 3.3, Research on incubation time

[0078] The loading of dsDNA on the electrode surface directly affects the detection limit and linear range of the aptamer sensor. In this study, nanoporous gold (NP-Gold) nanostructures served as an effective carrier, providing more binding sites for dsDNA. To optimize the dsDNA immobilization efficiency, cyclic voltammetry (CV) curves of the dsDNA / NP-Gold / glassy carbon electrode (GCE) were investigated in phosphate-buffered saline (PBS, 50 mM, pH 7.0) for different dsDNA incubation times (0, 3, 6, 9, 12, 15, 18 hours). Figure 3 As shown in Figure C, the dsDNA / NP-Gold / GCE electrode with an incubation time of 0 hours exhibits the maximum redox peak current of NP-Gold. With increasing dsDNA incubation time, the redox peak current of NP-Gold gradually decreases. These results indicate that the dsDNA / NP-Gold / GCE electrode reaches saturation at 12 hours of incubation. Therefore, 12 hours was selected as the optimal incubation time for dsDNA.

[0079] 3.4, Research in conjunction with time

[0080] The binding time of PSA to the aptamer is a crucial factor affecting the performance of aptamer sensors. Therefore, this study optimized the PSA binding time. Differential pulse voltammetry (DPV) curves of the PSA / dsDNA / NP-Gold / GCE electrode were investigated in PBS (50 mM, pH 7.0) solution at different binding times (30, 60, 90, 120, and 150 min). Figure 3 As shown in Figure D, the peak current value of the electrochemical response gradually increases with increasing incubation time, reaching its maximum value at 60 minutes. Therefore, an incubation time of 60 minutes was determined to be the optimal condition for the formation of the PSA-aptamer complex.

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

[0082] The cleavage time and amount of double-stranded specific nuclease (DSN) are important factors affecting the performance of aptamer sensors. The cleavage efficiency was evaluated by the current difference before and after DSN incubation using an NP-Gold / GCE electrode. Figure 3 As shown in Figure E, within the range of 20 to 100 minutes, the current difference increases with increasing cutting time, reaching its maximum at 60 minutes. Therefore, the optimal incubation time for DSN is 60 minutes. Furthermore, as... Figure 3 As shown in Figure F, the current difference increases with the increase of DSN dosage, reaching its maximum value when the dosage is 1 unit (U). Therefore, 1U is selected as the optimal dosage of DSN.

[0083] Example 4, Performance Study Experiment

[0084] 4.1 PSA Detection

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

[0086] The superior performance of this aptamer sensor can be attributed to several factors. First, NP-Gold possesses a high specific surface area, providing abundant binding sites for dsDNA. Second, NP-Gold's excellent biocompatibility creates a favorable microenvironment, ensuring the stability of dsDNA. Third, DSN is used to cleave unbound PSA-binding dsDNA, thereby reducing interference from electrode surface noise signals. These findings indicate that the developed aptamer sensor exhibits excellent performance characteristics, including high sensitivity, low detection limit, and a wide linear detection range.

[0087] 4.2 Anti-interference capability of body sensors

[0088] Interference resistance is an indispensable characteristic of aptamer sensors. To evaluate the interference resistance of the developed aptamer sensor, eight proteins were used as interfering proteins, including alpha-fetoprotein (AFP), angiotensin-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 concentration of both interfering proteins and PSA was 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 DSN digestion, DPV was detected in PBS (50 mM, pH 7.0). Figure 5 As shown in Figure A, the peak current values ​​of the eight interfering proteins were the same as those of the negative control (NC) (P>0.05) and significantly lower than that of PSA. This indicates that the interfering proteins cannot be captured by the aptamers and do not cause changes in acDNA structure. These results are attributed to the high affinity of the aptamers for PSA. The results demonstrate that the proposed aptamer sensor exhibits high selectivity for PSA.

[0089] 4.3 Reproducibility and Stability of Adaptive Sensors

[0090] To further investigate the reproducibility of the aptamer sensor, five electrodes were prepared under identical conditions for detecting PSA at a concentration of 100 fg / mL. Figure 5 As shown in Figure B, the measured current values ​​of the five DSN / PSA / dsDNA / NP-Gold / GCE electrodes ranged from 2.284 to 2.291 μA. This indicates that the measured PSA concentrations ranged from 91 to 112 fg / mL, with an average of 105.2 fg / mL and a relative standard deviation of 0.8%. These findings confirm the accuracy and excellent reproducibility of the aptamer sensor developed in this study. The stability of the aptamer sensor 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 DSN digestion, the current was measured using the DPV method in PBS solution. Figure 5 As shown in Figure C, after 21 days of storage, the current value of the aptamer sensor to the PSA did not decrease, and the change in relative current value was within 5%. These results indicate that the proposed aptamer sensor exhibits excellent stability, which can be attributed to the good biocompatibility of the NP-Gold nanomaterials.

[0091] 4.4 Detection of prostate-specific antigen (PSA) in serum

[0092] To evaluate the detection performance of the developed aptamer sensor on real samples, PSA was added to undiluted serum at final concentrations of 100 fg / mL, 10 pg / mL, and 1 ng / mL, respectively. The proposed aptamer sensor was then used to detect the PSA concentration in the undiluted serum, and the corresponding current values ​​were obtained. These data were then substituted into the calibration curve (…). Figure 4 (B) The PSA concentration was calculated. As shown in Table 1, the concentration detected by the aptamer sensor was very close to the concentration added to undiluted serum, with a deviation rate of less than 5%. Furthermore, the successful detection of PSA in human serum also indicates that the constructed aptamer sensor has good anti-interference performance against serum components. Based on these results, the proposed aptamer sensor 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 description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemical aptamer sensor, characterized in that, Includes the following steps: Step S1, Preparation of NP-Gold / GCE electrode: Nanoporous gold is coated on a glassy carbon electrode to form an NP-Gold / GCE electrode; Step S2, preparation of the dsDNA / NP-Gold / GCE electrode: The aptamer and acDNA are hybridized by heat treatment to form a double-stranded dsDNA structure; then, the formed dsDNA structure is mixed with tris(2-carboxyethyl)phosphine hydrochloride to reduce the disulfide bonds to thiol groups, and a mixture is obtained; subsequently, the mixture is dropped onto the surface of the NP-Gold / GCE electrode prepared in step S1, and after incubation, the dsDNA / NP-Gold / GCE electrode is constructed. In step S2, the aptamer and acDNA are mixed with Tris-MgSO4 buffer and activated at 85 ℃~100 ℃, and then gradually cooled to room temperature; in step S2, the mixture is incubated at 1 ℃~8 ℃ for 3~18 hours. The aptamer is a PSA aptamer, and its sequence is shown in SEQ ID NO.1; The aptamer-specific capture of PSA on the dsDNA / NP-Gold / GCE electrode surface dissociates from the dsDNA into the solution, and the single-stranded acDNA forms a stable hairpin structure; in order to reduce the interference of noise signals on the electrode surface, the dsDNA that is not bound to PSA is cleaved using DSN. The formation of the hairpin structure can prevent the DSN from approaching the stem due to the steric hindrance effect; The proposed electrochemical aptamer sensor uses DSN to reduce noise signals, has a minimum detection value of 10 fg / mL, and can detect extremely low concentrations of PSA. The aptamer has a high affinity for PSA, and the proposed aptamer sensor has high selectivity for PSA.

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

3. The method for preparing the electrochemical aptamer sensor according to claim 1, characterized in that, The acDNA sequence is shown in SEQ ID NO.

2.

4. The product obtained by the preparation method of the electrochemical aptamer sensor according to any one of claims 1 to 3.

5. The application of the preparation method of the electrochemical aptamer sensor according to any one of claims 1 to 3 in the detection of prostate-specific antigens.

6. A method for detecting prostate-specific antigen using an electrochemical aptamer sensor prepared by any one of claims 1 to 3, characterized in that, Includes the following steps: Step 1, Establishment of calibration curve: PSA solutions of different concentrations were added to the dsDNA / NP-Gold / GCE electrode and incubated at 1℃~8℃ for 30~120 minutes; then, the PSA / dsDNA / NP-Gold / GCE electrode and DSN were incubated at 30℃~40℃ for 40~100 minutes; the current was measured in 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 several concentration values ​​between 10 fg / mL and 10 ng / mL; Step 2, PSA detection: Add the sample to the dsDNA / NP-Gold / GCE electrode and incubate at 1 ℃~8 ℃ for 30~120 minutes; Subsequently, DSN was added, and the electrode was incubated at 30 ℃~40 ℃ for 40~100 minutes; The current was detected in phosphate buffer using the DPV method, and the recorded current response value was substituted into the calibration curve to determine the concentration of PSA.

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

8. The method for detecting prostate-specific antigen according to claim 6, characterized in that, In steps 1 and 2, the amount of DSN used is 1~2.5 U.

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