An aptamer electrochemical sensor for detecting homocysteine and a preparation method thereof

Abstract

The application belongs to the technical field of electrochemical analysis methods, and particularly relates to an aptamer electrochemical sensor for detecting homocysteine and a preparation method thereof. The preparation method of the aptamer electrochemical sensor for detecting homocysteine provided by the application comprises the following steps: preparing a single-walled carbon nanohorn modified carbon ionic liquid paste electrode, electrodepositing nano silver particles, incubating aptamers, and sealing non-specific sites that are not occupied, so as to obtain MCH / Apt / AgNPs / SWCNHs / CILPE. In the detection of Hcy, a wide linear range (0.5 nM-100 nM) and a low detection limit (0.1 nM) are exhibited, and meanwhile, good reproducibility, stability and anti-interference ability are also possessed, and the aptamer electrochemical sensor has been successfully applied to the detection of Hcy in human serum samples.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis methods, specifically relating to an aptamer electrochemical sensor for detecting homocysteine ​​and its preparation method. Background Technology

[0002] Homocysteine ​​(Hcy) is a sulfur-containing amino acid in the methionine metabolic cycle, with a normal physiological concentration of 5-15 μmol / L. Acquired deficiencies in folic acid, vitamin B12, and vitamin B6 can lead to insufficient or impaired activity of enzymes that metabolize homocysteine, resulting in elevated blood homocysteine ​​levels. Hyperhomocysteinemia is widely recognized as increasing the risk of cardiovascular disease, primarily through oxidative stress, endothelial dysfunction, lipid metabolism disorders, and the promotion of thrombosis, thus contributing to disease progression.

[0003] Traditional methods for Hcy detection include high-performance liquid chromatography (HPLC), fluorescence polarization immunoassay (FPIA), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry (MS). Electrochemical sensors, however, have become a research hotspot for Hcy detection due to their advantages such as rapid response, portability, low cost, and high sensitivity. The core of these sensors lies in electrode interface modification and signal response. By optimizing electron transfer rates and enhancing recognition specificity through modifiers, the biochemical reaction of Hcy is converted into a measurable electrical signal.

[0004] Electrochemical sensors have attracted much attention in the field of bioanalysis due to their high sensitivity and real-time detection capabilities, and the emergence of aptamer sensors has further improved the sensitivity and specificity of detection. Compared with traditional antibody-based recognition elements, aptamers are more specific, sensitive, and environmentally stable than proteins, and can resist interference factors such as pH and temperature, providing a more reliable solution for the identification of target analytes in complex samples. In the existing technology, Beitollahi et al. created an electrochemical sensor using oligonucleotides as aptamers. By modifying the surface of gold nanoparticles / glassy carbon electrodes (Au / GCE) with Hcy aptamers, they detected homocysteine ​​with a detection limit of 0.01 μM (S / N = 3) and achieved good analytical performance in a linear range of 0.05–20.0 μM. Chinese invention patent application number 202310812235.X discloses an electrochemical aptamer sensor for homocysteine ​​detection and its construction. The sensor is prepared by first incubating an aptamer on the surface of a screen-printed gold electrode (SPGE), and then adding 6-mercapto-1-hexanol (MCH) to block unoccupied blank sites on the electrode. The detection limit is 0.112 μM (S / N = 3) in the concentration range of 0.4 μM to 20 μM.

[0005] Although there have been some reports in the literature on the use of aptamer electrochemical sensors for homocysteine ​​detection, further optimization of the sensor's detection limit and linear range remains a hot topic for researchers. Summary of the Invention

[0006] Based on this, this application provides an aptamer electrochemical sensor for detecting homocysteine ​​and its preparation method, in order to solve the technical problems of low detection limit and low linear range of aptamer electrochemical sensors in the prior art.

[0007] A method for preparing an aptamer electrochemical sensor for detecting homocysteine ​​includes the following steps: S10. Prepare a carbon ion liquid paste electrode modified with single-walled carbon nanocorners, labeled as SWCNHs / CILPE; S20. Electrodeposition of silver nanoparticles: Using the SWCNHs / CILPE as the working electrode, reduction deposition was performed in AgNO3 solution using chronoamperometry. The electrode was washed with ultrapure water to obtain the substrate electrode, labeled as AgNPs / SWCNHs / CILPE.

[0008] S30. After incubating the Hcy aptamer solution onto the surface of AgNPs / SWCNHs / CILPE, 6-mercapto-1-hexanol is added to the surface to block unoccupied nonspecific sites. After the incubation, the unreacted 6-mercapto-1-hexanol on the surface is washed off with ultrapure water to obtain an aptamer electrochemical sensor for detecting homocysteine, labeled as MCH / Apt / AgNPs / SWCNHs / CILPE.

[0009] Preferably, in the above preparation method, step S10, "preparing a carbon ion liquid paste electrode modified with single-walled carbon nano-angles" specifically refers to: Nano-graphite powder, single-walled carbon nano-angles, ionic liquid and paraffin oil are mixed and ground into a paste. The paste is then placed in an electrode tube, heated and polished to a smooth finish to obtain a carbon ionic liquid paste electrode modified with single-walled carbon nano-angles. The mass ratio of the single-walled carbon nano-angles to the nano-graphite powder is 5:(40~50).

[0010] Preferably, in the above preparation method, the mass ratio of the single-walled carbon nanoparticles to the nano-graphite powder is 5:45.

[0011] Preferably, in the above preparation method, the ionic liquid is 1-octylpyridine hexafluorophosphate, and the mass ratio of the ionic liquid to the nano-graphite powder is (20~30): (40~50).

[0012] Preferably, in the above preparation method, the amount of nano-graphite powder added per microliter of paraffin oil is 1.5 to 2 mg.

[0013] Preferably, in the above preparation method, in step S20, the concentration of the AgNO3 solution is 1 mM, the deposition potential of the "reduction deposition" is -0.2 V, the deposition time is 20 s, and the sampling interval is 0.1 s.

[0014] Preferably, in the above preparation method, in step S30, the concentration of the Hcy aptamer solution is 7.5 μM, and the sequence of the Hcy aptamer in the aptamer solution is: 5'-SH-(CH2)6 ACCA GCAC ATTC GATT ATAC CAGC TTATTCAA TTCA CAGC TATG TCCT ATAC CAGC TTAT TCAATT-3'.

[0015] Preferably, in the above preparation method, in step S30, the incubation is carried out at 4°C for 10–14 h; the sealing is carried out at room temperature for 20–40 min.

[0016] An aptamer electrochemical sensor for detecting homocysteine ​​is prepared using the method described above for preparing an aptamer electrochemical sensor.

[0017] A method for detecting homocysteine ​​includes the following steps: Construct aptamer electrochemical sensors as described above; The target compound Hcy was dropped onto the aptamer electrochemical sensor and incubated at room temperature for 30 min. After being gently rinsed with ultrapure water and allowed to stand and dry, it was used as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire as the counter electrode to form a three-electrode system. In a solution containing 5 mM [Fe(CN)6] 3- / 4- The differential pulse voltammetry method was used to record signal changes in the probe solution in 0.01 M pH 7.4 phosphate buffer to achieve quantitative analysis of Hcy. The scanning range was -0.2 to 0.5 V, the amplitude was 0.05 V, and the pulse period was 0.5 s.

[0018] Compared with the prior art, this application has at least the following advantages: The present invention provides a method for preparing an aptamer electrochemical sensor (hereinafter referred to as the aptamer electrochemical sensor) for detecting homocysteine, comprising preparing a carbon ion liquid paste electrode modified with single-walled carbon nanoparticles, electrodepositing silver nanoparticles, incubating the aptamer, and blocking unoccupied nonspecific sites to obtain MCH / Apt / AgNPs / SWCNHs / CILPE. In six parallel measurements using the same aptamer electrochemical sensor, the relative standard deviation (RSD) was 3.1%, indicating good reproducibility. After storage at 4 °C for one month, the ΔI value for Hcy detection was 97.71% of the initial current difference, indicating good stability. In the detection of interfering substances that may affect Hcy detection, it only showed a specific response to Hcy, indicating good anti-interference ability. Furthermore, the aptamer electrochemical sensor of this application has nano-silver particles electrodeposited on its surface. On the one hand, this can inhibit microbial adhesion and contamination, and significantly improve the long-term stability and service life of the aptamer sensor. On the other hand, compared with traditional nano-gold, nano-silver is inexpensive, which helps to save on the production cost of the aptamer electrochemical sensor.

[0019] The aptamer electrochemical sensor of this application uses a nanocomposite of AgNPs, SWCNHs, and IL as a sensing platform to immobilize aptamer molecules, which not only enhances the sensor's response signal but also improves its stability. Under optimal experimental conditions, it exhibits a wide linear range (0.5 nM-100 nM) and a low detection limit (0.1 nM), and has been successfully applied to the detection of Hcy in human serum samples. Attached Figure Description

[0020] Figure 1 AgNPs / SWCNHs / CILPE (a), Apt / AgNPs / SWCNHs / CILPE (b), MCH / Apt / AgNPs / SWCNHs / CILPE (c), Hcy / MCH / Apt / AgNPs / SWCNHs / CILPE (d) in the presence of 5 mM [Fe(CN)6] 3- / 4- Differential pulse voltammogram of 0.01M phosphate buffer solution at pH 7.4.

[0021] Figure 2 AgNPs / SWCNHs / CILPE (a), Apt / AgNPs / SWCNHs / CILPE (b), MCH / Apt / AgNPs / SWCNHs / CILPE (c), Hcy / MCH / Apt / AgNPs / SWCNHs / CILPE (d) in the presence of 5 mM [Fe(CN)6] 3- / 4- Impedance plot of 0.01M phosphate buffer solution at pH 7.4.

[0022] Figure 3 The effect of SWCNHs content on the peak current of the substrate electrode (in the presence of 5 mM [Fe(CN)6)) 3- / 4- In a 0.01 M phosphate buffer solution with pH 7.4.

[0023] Figure 4 The effects of (A) deposition potential of silver nanoparticles on the peak current of the substrate electrode; and (B) deposition time of silver nanoparticles on the peak current of the substrate electrode (in the presence of 5 mM [Fe(CN)6]). 3- / 4- In a 0.01 M phosphate buffer solution at pH 7.4.

[0024] Figure 5 The effect of different pH values ​​on the change in peak current value.

[0025] Figure 6 The effect of different Hcy aptamer concentrations on the change in peak current value.

[0026] Figure 7 The effect of different sealing liquids on the change in peak current value.

[0027] Figure 8 The effect of different incubation times on the change in peak current value.

[0028] Figure 9 To detect the linear relationship between the peak current difference before and after homocysteine ​​and the concentration.

[0029] Figure 10 Research on anti-interference for aptamer sensors. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The technical solutions of this invention will be further described below with reference to the embodiments and accompanying drawings. This invention is not limited to the specific embodiments described below.

[0031] Unless otherwise defined, 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 application belongs. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] In one specific embodiment of this application, a method for preparing an aptamer electrochemical sensor for detecting homocysteine ​​includes the following steps: S10. Prepare a carbon ion liquid paste electrode modified with single-walled carbon nanocorners, labeled as SWCNHs / CILPE; Preferably, the "preparation of a carbon ion liquid paste electrode modified with single-walled carbon nano-angles" specifically refers to: Nano-graphite powder, single-walled carbon nano-angles, ionic liquid and paraffin oil are mixed and ground into a paste. The paste is then placed in an electrode tube, heated and polished to a smooth finish to obtain a carbon ionic liquid paste electrode modified with single-walled carbon nano-angles. The mass ratio of the single-walled carbon nano-angles to the nano-graphite powder is 5:(40~50).

[0033] This study uses homocysteine ​​(Hcy) as the target molecule and constructs a sensing platform with immobilized aptamers through innovative modification materials. In the selection of modification materials, silver nanoparticles (AgNPs) play a dual role: their high conductivity significantly accelerates electron transfer, providing an efficient electron transfer channel for the sensor; the Hcy aptamers screened using SELEX technology, with their terminal thiol (-SH) groups, can form Ag-S covalent bonds with AgNPs, to some extent avoiding the aptamer detachment problem caused by physical adsorption, thus ensuring the long-term stability of the recognition element. Single-walled carbon nanohorns (SWCNHs), as a novel carbon nanomaterial, possess a unique conical structure that can self-assemble into a three-dimensional porous network. This structure not only loads a large number of AgNPs, expanding the distribution of active sites, but also inhibits AgNP aggregation through steric hindrance, maintaining its highly dispersed state and conductivity; simultaneously, the three-dimensional porous network further increases the electrode specific surface area, providing more reaction interfaces for the binding of aptamers and targets. After the two are combined, the specific surface area of ​​the modified electrode is significantly increased, and the electron transfer rate is significantly accelerated due to the synergistic effect of the high conductivity of AgNPs and the porous structure of SWCNHs, providing an excellent interfacial basis for subsequent detection.

[0034] When the content of the single-walled carbon nanoparticles as modifier is 10% of the mass of the nano-graphite powder, the response current value reaches its maximum, proving that a certain amount of single-walled carbon nanoparticles can effectively improve the surface reactivity of the electrode and amplify the electrical signal. Therefore, preferably, the mass ratio of single-walled carbon nanoparticles to the nano-graphite powder is 5:45.

[0035] Furthermore, the ionic liquid is 1-octylpyridine hexafluorophosphate, and the mass ratio of the ionic liquid to the nano-graphite powder is (20~30): (40~50).

[0036] Furthermore, the amount of nano-graphite powder added to each microliter of the paraffin oil is 1.5 to 2 mg.

[0037] S20. Electrodeposition of silver nanoparticles: Using the SWCNHs / CILPE as the working electrode, reduction deposition was performed in AgNO3 solution using the chronoamperometry method. The electrode was washed with ultrapure water to obtain the substrate electrode, which was labeled as AgNPs / SWCNHs / CILPE.

[0038] Specifically, using SWCNHs / CILPE as the working electrode, platinum wire as the auxiliary electrode, and a dual salt bridge (the inner salt bridge being a saturated potassium chloride solution and the outer salt bridge being a saturated potassium nitrate solution) as the reference electrode, reduction deposition is performed in AgNO3 solution using a chronoamperometry method. Preferably, the concentration of the AgNO3 solution is 1 mM. After completion, the electrode is removed and washed with ultrapure water to obtain a uniform silver deposition layer, labeled as the substrate electrode AgNPs / SWCNHs / CILPE. Preferably, the deposition potential for the "reduction deposition" is -0.2 V, the deposition time is 20 s, and the sampling interval is 0.1 s. It is worth noting that this application electrodeposits nano-silver particles on the surface of SWCNHs / CILPE. Because nano-silver possesses inherent broad-spectrum antibacterial properties, it can inhibit microbial adhesion and contamination, significantly improving the long-term stability and lifespan of the aptamer sensor—a characteristic not possessed by gold nanoparticles. Furthermore, the cost of nano-silver is lower than that of gold nanoparticles. Depositing nano-silver on the electrode surface can reduce the production cost of the aptamer sensor, which has certain economic significance.

[0039] S30. After incubating the surface of the AgNPs / SWCNHs / CILPE with the Hcy aptamer solution, 6-mercapto-1-hexanol is added to the surface to block unoccupied non-specific sites. Afterwards, the unreacted blocking solution is washed off with ultrapure water to obtain an aptamer electrochemical sensor for detecting homocysteine, labeled MCH / Apt / AgNPs / SWCNHs / CILPE. In this application, the 6-mercapto-1-hexanol (MCH) is the blocking solution with a concentration of 1 mM.

[0040] Preferably, the concentration of the Hcy aptamer solution is 7.5 μM. The Hcy aptamer solution is prepared using TE buffer. The sequence of the Hcy aptamer in this application is: 5'-SH-(CH2)6 ACCA GCAC ATTC GATTATAC CAGC TTAT TCAA TTCA CAGC TATG TCCT ATAC CAGC TTAT TCAATT-3'.

[0041] Further, in step S30, the incubation is carried out at 4°C for 10–14 h; the sealing is carried out at room temperature for 20–40 min.

[0042] Step S30 above specifically involves: dropping a 7.5 μM Hcy aptamer solution onto the surface of the AgNPs / SWCNHs / CILPE electrode and incubating it at 4 °C for 10–14 hours. Then, adding 1 mM 6-mercapto-1-hexanol (MCH) to the electrode surface to block non-specific binding sites for 20–40 min. After this, the unreacted MCH on the surface is washed off with ultrapure water, thus obtaining the aptamer sensor MCH / Apt / AgNPs / SWCNHs / CILPE, which is then stored at 4 °C for later use.

[0043] In another specific embodiment of this application, an aptamer electrochemical sensor for detecting homocysteine ​​is prepared using the above-described method for preparing an aptamer electrochemical sensor.

[0044] The aptamer electrochemical sensor of this application uses a nanocomposite of AgNPs, SWCNHs, and IL as a sensing platform to immobilize aptamer molecules, which not only enhances the sensor's response signal but also improves its stability. Under optimal experimental conditions, it exhibits a wide linear range (0.5 nM–100 nM) and a low detection limit (0.1 nM). In six parallel measurements using the same sensor, the relative standard deviation (RSD) was 3.1%, indicating good reproducibility. After storage at 4 °C for one month, the ΔI value for Hcy detection was 97.71% of the initial current difference, demonstrating good stability. In the detection of interfering substances that might affect Hcy detection, it showed a specific response only to Hcy, indicating good anti-interference ability. Furthermore, it has been successfully applied to the detection of Hcy in human serum samples.

[0045] In another specific embodiment of this application, a method for detecting homocysteine ​​includes the following steps: Construct aptamer electrochemical sensors as described above; The target compound Hcy was dropped onto the aptamer electrochemical sensor and incubated at room temperature for 30 min. After washing and drying with ultrapure water, it was used as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire as the counter electrode to form a three-electrode system. In a solution containing 5 mM [Fe(CN)6] 3- / 4- The differential pulse voltammetry method was used to record signal changes in the probe solution in 0.01 M pH 7.4 phosphate buffer to achieve quantitative analysis of Hcy. The scanning range was -0.2 to 0.5 V, the amplitude was 0.05 V, and the pulse period was 0.5 s.

[0046] In this application, the preferred volume of Hcy added to the electrode is 6 μL, and the incubation time is 30 min. The method for detecting homocysteine ​​in this application uses potassium ferricyanide / potassium ferrocyanide as the detection system. Based on the mechanism that the conformational change of the aptamer hinders electron transport and causes a decrease in current signal when Hcy binds to the aptamer, the quantitative analysis of Hcy is achieved by recording signal changes using differential pulse voltammetry (DPV).

[0047] It is worth noting that the process temperature and process time involved in the above embodiments are all temperatures or times used in the experiment. Any reasonable adjustments made by those skilled in the art based on the process temperature and process time provided by the present invention, within the error range, should be included within the protection scope of the present invention.

[0048] The technical solution and effects of the present invention will be further illustrated below through specific embodiments.

[0049] 1.1 Materials and Reagents DL-homocysteine, 95% pure, from Shanghai Aladdin Biochemical Technology Co., Ltd. The homocysteine ​​aptamer, with 66 base pairs, has the following sequence: 5'-SH-(CH2)6 ACCA GCAC ATTC GATTATAC CAGC TTAT TCAA TTCA CAGC TATG TCCT ATAC CAGC TTAT TCAATT-3', and is from Shanghai Sangon Biotech Co., Ltd. TE buffer, low EDTA, pH 8.0, from Shanghai Sangon Biotech Co., Ltd. Paraffin oil, analytical grade, from Sigma-Aldrich (USA); Single-walled carbon nanotubes, particle size: 30~100 nm, from Nanjing Xianfeng Nanotechnology Co., Ltd. Nano-graphite powder, flake size: ~400 nm, from Nanjing Xianfeng Nanotechnology Co., Ltd. Disodium hydrogen phosphate, potassium ferrocyanide, potassium ferricyanide, potassium chloride, and sodium chloride were all of analytical grade and were sourced from Sinopharm Group Co., Ltd. Glucose, analytical grade, from Xi'an Chemical Reagent Factory; Ascorbic acid, potassium nitrate, and silver nitrate were all of analytical grade and were sourced from Tianjin Kemeo Chemical Reagent Co., Ltd. The reduced glutathione and L-methionine both had a purity of 99% and were sourced from Shanghai Yuanye Biotechnology Co., Ltd. The ultrapure water used in the experiment was prepared in the laboratory; all reagents in this experiment did not require purification before use. An aptamer solution was prepared using TE buffer and stored at -20 °C for later use.

[0050] 1.2 Instruments The main instruments and equipment used in the experiment are as follows: Electrochemical workstation, model: CHI660E, manufacturer: Shanghai Chenhua Instrument Co., Ltd. Electrochemical workstation, model: PARSTAT 4000, manufacturer: Princeton Applied Research Corporation, USA; Saturated calomel electrode, model: CHI150, manufacturer: Shanghai Chenhua Instrument Co., Ltd. Platinum wire electrode, model: CHI115, manufacturer: Shanghai Chenhua Instrument Co., Ltd. Dual salt bridge electrode, model: 217 series, manufacturer: Shanghai Instrument & Electronics Scientific Instruments Co., Ltd. Magnetic stirrer, model: KMO2, manufacturer: IKA Group, Germany.

[0051] 1.3 Fabrication of aptamer sensors Weigh 0.045 g of nano-graphite powder, 0.005 g of single-walled carbon nano-angles and 0.025 g of ionic liquid, place them in an agate mortar and mix with 25 μL of paraffin oil. Grind thoroughly, then place a certain amount of the paste in an electrode tube with an inner diameter of 3 mm, heat for 2-3 min, press it on weighing paper and polish it smooth to obtain a carbon ionic liquid paste electrode modified with single-walled carbon nano-angles, labeled as SWCNHs / CILPE.

[0052] Electrodeposition of silver nanoparticles: Using SWCNHs / CILPE as the base electrode, platinum wire as the auxiliary electrode, and a double salt bridge (the inner salt bridge being a saturated potassium chloride solution and the outer salt bridge being a saturated potassium nitrate solution) as the reference electrode, reduction deposition was carried out in a solution containing 10 mL of 1 mM AgNO3 using a chronoamperometry method (constant potential -0.2 V, for 20 s, with a sampling interval of 0.1 s). After the deposition was completed, the electrode was removed and washed with ultrapure water to obtain a uniform silver deposition layer, which was labeled as the base electrode AgNPs / SWCNHs / CILPE.

[0053] 6 μL of 7.5 μM Hcy aptamer was dropped onto the surface of the AgNPs / SWCNHs / CILPE electrode and incubated at 4 °C for 12 hours. Then, 6 μL of 1 mM 6-mercapto-1-hexanol (MCH) was added to the electrode surface to block non-specific binding sites for 30 min. After that, the unreacted MCH on the surface was washed off with ultrapure water. The aptamer sensor MCH / Apt / AgNPs / SWCNHs / CILPE was thus prepared and stored at 4 °C for later use.

[0054] 1.4 Analytical Methods A 6 μL volume of Hcy was added to the electrode and incubated at room temperature for 30 min. The electrode was then gently rinsed with ultrapure water and allowed to air dry. This was used as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode, forming a three-electrode system. The system was prepared in an atmosphere containing 5 mM [Fe(CN)6]. 3- / 4- DPV detection was performed in a 0.01 M pH 7.4 phosphate buffer solution containing the probe solution, with a scan range of -0.2 to 0.5 V, an amplitude of 0.05 V, and a pulse period of 0.5 s.

[0055] 2.1 Electrochemical Characterization This paper investigates the electrochemical performance of different modified electrodes using DPV and EIS. The results of DPV are as follows: Figure 1 As shown, the substrate electrode AgNPs / SWCNHs / CILPE (curve a) exhibits the highest peak current value, which is attributed to the high conductivity of the nanocomposite. The Hcy aptamer, as an oligonucleotide chain, is non-conductive; therefore, after self-assembly of the Hcy aptamer, the peak current value of Apt / AgNPs / SWCNHs / CILPE (curve b) decreases significantly. MCH, as an organic compound that blocks non-specific sites, further hinders electron transfer, resulting in a continued decrease in the peak current value of MCH / Apt / AgNPs / SWCNHs / CILPE (curve c). When Hcy is incubated on the electrode surface and binds to its corresponding aptamer sites, the electron transfer rate is further slowed, leading to a further decrease in the response current signal (curve d). This result confirms the successful construction of a homocysteine ​​aptamer sensor, enabling specific recognition of homocysteine.

[0056] This application used electrochemical impedance spectroscopy to analyze the changes in electrode resistance of each modified electrode. Figure 2The AgNPs / SWCNHs / CILPE (curve a) shows only a small semicircle, with an Rct value of 11.5 Ω, reflecting the excellent conductivity of the substrate electrode. When Apt (curve b) and MCH (curve c) are progressively modified onto the electrode surface, and combined with Hcy (curve d), the Rct values ​​increase sequentially to 32.84 Ω, 37.13 Ω, and 77.75 Ω, respectively. With layer-by-layer modification and assembly of the electrode surface, the electrode interface impedance increases, and the electron transfer rate decreases, consistent with the DPV results.

[0057] 2.2 Optimization of Experimental Conditions This application investigated the effect of varying single-walled carbon nanoparticle content of the modifier on the performance of the substrate electrode in the range of 2.5% to 15%. Figure 3 When the SWCNHs content increased from 2.5% to 10%, the response current value increased. The response current value reached its maximum at a content of 10%, demonstrating that a certain amount of single-walled carbon nanoparticles can effectively improve the surface reactivity of the electrode and amplify the electrical signal. Therefore, 10% was chosen as the optimal content of the modifier.

[0058] Figure 4 The effects of deposition potential and deposition time on peak current of electrodeposited silver nanoparticles on the substrate electrode surface were investigated. Appropriate deposition potential and deposition time can effectively increase the effective surface area and catalytic activity of the substrate electrode. Experimental results show that the substrate electrode performance reaches its optimal value when the deposition potential is -0.2 V and the deposition time is 20 s.

[0059] The performance of the aptamer sensor in this study was closely related to the pH of the buffer solution, and the influence of pH 5.0 to pH 9.0 on sensor performance was discussed. Observation Figure 5 The results showed that both excessive acidity and alkalinity affected the activity of the aptamer, and the difference in response current before and after Hcy binding to the aptamer was greatest at pH 7.4. Therefore, a buffer solution with pH 7.4 was selected for subsequent detection.

[0060] The concentration of the aptamer solution also affects sensor performance. For example... Figure 6 As shown, ΔI gradually increases as the aptamer solution concentration increases from 1 μM to 7.5 μM. However, when the concentration exceeds 7.5 μM, ΔI decreases instead. This may be because high concentrations of aptamer can cause spatially active sites to be buried due to intermolecular interactions, making it impossible to effectively recognize Hcy molecules, resulting in lower matching efficiency and a decrease in peak current difference. Therefore, a 7.5 μM aptamer solution is selected as the optimal reaction concentration.

[0061] This application also investigated the effects of three different blocking solutions—1-hexathiol (1-HT), bovine serum albumin (BSA), and 6-mercapto-1-hexanol (MCH)—on the detection of the aptamer sensor. Figure 7 The results showed that using MCH to block the non-specific active sites at the electrode interface significantly improved the binding effect of Hcy molecules and aptamers. Therefore, MCH was selected as the optimal blocking solution.

[0062] like Figure 8 As shown, this application investigated the effect of Hcy incubation time on the peak current difference in the constructed aptamer sensor. When the incubation time increased from 10 min to 30 min, ΔI gradually increased, and after 30 min, the peak current difference remained essentially unchanged, indicating that the direct reaction between the Hcy molecule and the aptamer essentially reached saturation after 30 min. Ultimately, 30 min was selected as the optimal incubation time.

[0063] 2.3 Standard Curve Under optimal conditions, Hcy standard samples of different concentrations were detected using the aptamer sensing platform MCH / Apt / AgNPs / SWCNHs / CILPE. Figure 9 The response current difference of the aptamer sensor increases with increasing Hcy concentration. Within the range of 0.5-100 nM, the response current difference exhibits a good linear relationship with Hcy concentration, and its linear regression equation is ΔI(μA) = 0.042c(nM) + 2.17(r). 2 =0.9975), with a detection limit of 0.1 nM.

[0064] 2.4 Reproducibility, stability, and interference This application used the same MCH / Apt / AgNPs / SWCNHs / CILPE electrode to perform six parallel measurements of 50 μM Hcy, with a relative standard deviation (RSD) of 3.1% for ΔI. The results indicate that the sensor exhibits good reproducibility. Furthermore, to investigate the stability of this aptamer sensor, after storing the modified electrode at 4 °C for one month, the ΔI value for Hcy detection remained at 97.71% of the initial current difference. This result demonstrates the good stability of the aptamer sensing platform MCH / Apt / AgNPs / SWCNHs / CILPE.

[0065] Interfering substances that may affect Hcy detection were detected, and the anti-interference capability of the constructed aptamer sensor was investigated. For example... Figure 10As shown, this aptamer sensor exhibits a specific response to 50 μM Hcy compared to 140 mM sodium chloride (NaCl), 5 mM glucose (Glu), 50 μM ascorbic acid (AA), 50 μM methionine (Met), and 10 μM glutathione (GSH).

[0066] 2.5 Actual Sample To evaluate the practical application effect of the aptamer sensor, human serum samples were diluted 250-fold with 0.01 M PBS and detected using DPV. Table 1 shows that the actual detected homocysteine ​​concentrations in blood samples 1, 2, and 3 were 11.37 μM, 6.79 μM, and 5.95 μM, respectively. Spike recovery experiments were also performed, with recoveries ranging from 97.62% to 101.04%. This indicates that the aptamer can be successfully applied to the detection of human serum samples.

[0067] Table 1. Detection results of Hcy in samples by MCH / Apt / AgNPs / SWCNHs / CILPE (n=3) Blood sample 1 Blood sample 2 Blood sample 3 Detection value / nM 45.48 27.14 23.81 scalar dosage / nM 50.00 50.00 50.00 Detected value after spiking / nM 96.00 76.43 72.62 Recovery rate / % 101.04 98.58 97.62 Relative standard deviation / % 0.57 0.64 2.61 In summary, this paper successfully constructed an aptamer sensor based on MCH / Apt / AgNPs / SWCNHs / CILPE. Using the nanocomposite of AgNPs, SWCNHs, and IL as a sensing platform to immobilize aptamer molecules significantly improved the specific surface area of ​​the modified electrode and increased the aptamer immobilization sites at the electrode interface, thereby enhancing both the electrode response signal and the sensor's stability. The sensor was successfully constructed using a SWCNHs content of 10%, a nanosilver particle deposition potential of -0.2 V, a deposition time of 20 s, and a 0.01 M pH 7.4 phosphate buffer solution (containing 5 mM [Fe(CN)6]). 3- / 4- The sensor performance was optimal when the aptamer concentration was 7.5 μM and 6-mercapto-1-hexanol (MCH) was used as the blocking solution, and the Hcy incubation time was 30 min, with a linear range of 0.5 nM-100 nM and a detection limit of 0.1 nM. The constructed electrochemical sensor can be used to detect homocysteine ​​in human serum samples.

[0068] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing an aptamer electrochemical sensor for detecting homocysteine, characterized in that, Includes the following steps: S10. Prepare a carbon ion liquid paste electrode modified with single-walled carbon nanocorners, labeled as SWCNHs / CILPE; S20. Electrodeposited silver nanoparticles: Using the SWCNHs / CILPE as the working electrode, reduction deposition was carried out in AgNO3 solution using the chronoamperometry method. The electrode was washed with ultrapure water to obtain the substrate electrode, which was labeled as AgNPs / SWCNHs / CILPE. S30. After incubating the Hcy aptamer solution onto the surface of AgNPs / SWCNHs / CILPE, 6-mercapto-1-hexanol is added to the surface to block unoccupied nonspecific sites. After the incubation, the unreacted 6-mercapto-1-hexanol on the surface is washed off with ultrapure water to obtain an aptamer electrochemical sensor for detecting homocysteine, labeled as MCH / Apt / AgNPs / SWCNHs / CILPE.

2. The preparation method according to claim 1, characterized in that, In step S10, the "preparation of a carbon ion liquid paste electrode modified with single-walled carbon nano-angles" specifically refers to: Nano-graphite powder, single-walled carbon nano-angles, ionic liquid and paraffin oil are mixed and ground into a paste. The paste is then placed in an electrode tube, heated and polished to a smooth finish to obtain a carbon ionic liquid paste electrode modified with single-walled carbon nano-angles. The mass ratio of the single-walled carbon nano-angles to the nano-graphite powder is 5: (40~50).

3. The preparation method according to claim 2, characterized in that, The mass ratio of the single-walled carbon nanoparticles to the nano-graphite powder is 5:

45.

4. The preparation method according to claim 2, characterized in that, The ionic liquid is 1-octylpyridine hexafluorophosphate, and the mass ratio of the ionic liquid to the nano-graphite powder is (20~30): (40~50).

5. The preparation method according to claim 2, characterized in that, The amount of nano-graphite powder added per microliter of the paraffin oil is 1.5~2 mg.

6. The preparation method according to claim 1, characterized in that, In step S20, the concentration of the AgNO3 solution is 1 mM, the deposition potential of the "reduction deposition" is -0.2 V, the deposition time is 20 s, and the sampling interval is 0.1 s.

7. The preparation method according to claim 1, characterized in that, In step S30, the concentration of the Hcy aptamer solution is 7.5 μM.

8. The preparation method according to claim 1, characterized in that, In step S30, the incubation is carried out at 4°C for 10–14 h; the sealing is carried out at room temperature for 20–40 min.

9. An aptamer electrochemical sensor for detecting homocysteine, characterized in that, The aptamer electrochemical sensor was prepared using the method described in any one of claims 1 to 8.

10. A method for detecting homocysteine, characterized in that, Includes the following steps: Construct the aptamer electrochemical sensor as described in claim 9; The target compound Hcy was dropped onto the aptamer electrochemical sensor and incubated at room temperature for 30 min. After being gently rinsed with ultrapure water and allowed to stand and dry, it was used as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire as the counter electrode to form a three-electrode system. In a solution containing 5 mM [Fe(CN)6] 3- / 4- The differential pulse voltammetry method was used to record signal changes in the probe solution in 0.01 M pH 7.4 phosphate buffer to achieve quantitative analysis of Hcy. The scanning range was -0.2 to 0.5 V, the amplitude was 0.05 V, and the pulse period was 0.5 s.

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