A method for detecting homocysteine ​​using a nanoporous sensor based on silver-sulfur polymerization.

Abstract

This invention discloses a method for detecting homocysteine ​​(Hcy) using a nanoporous sensor based on silver-sulfur polymerization. The method involves pretreating a homocysteine-containing solution with 4-dimethylaminocinnamaldehyde, then placing a sensor with a cylindrical nanoporous structure into the homocysteine-containing solution for polymerization. A linear equation is established between the ratio of conductivity before and after polymerization and the homocysteine ​​concentration. The concentration of homocysteine ​​in the test solution is then calculated using this linear equation. The test solution contains both homocysteine ​​and cysteine ​​and has undergone 4-dimethylaminocinnamaldehyde pretreatment before detection. The cylindrical nanopores of the sensor are modified with an Ag(Ⅰ) probe. This method utilizes a special pretreatment method to synergistically construct a functional probe and a special nanoporous structure, and amplifies the current signal change based on the silver-sulfur polymerization reaction, achieving rapid and accurate detection of Hcy.

Description

Technical Field

[0001] This invention belongs to the field of chemical / biological sensing technology, specifically relating to a method for selective detection of homocysteine ​​using a nanoporous sensor based on Ag(I)-thiol coordination polymer. Background Technology

[0002] Homocysteine ​​(Hcy) is a thiol-containing amino acid. In the human body, Hcy is typically generated by the demethylation of methionine, which then decomposes into cysteine ​​(Cys) through desulfurization. As an important biothiol, Hcy participates in many vital physiological processes and is an important indicator. In normal human blood, the Hcy concentration ranges from 5 μmol / L to 15 μmol / L. Maintaining a normal Hcy level helps maintain good biochemical balance in the body. Excessively high Hcy concentrations in the blood are termed hyperhomocysteinemia. However, due to its complex structure and susceptibility to interference from other amino acids, the development of rapid, simple, and accurate Hcy detection methods is of great significance for the field of life science research. Currently, analytical methods for the quantitative detection of Hcy mainly include high-performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis, and ion exchange chromatography. These methods have drawbacks such as long detection time, complex sample preparation procedures, high detection costs, and expensive detection instruments. Therefore, the development of a rapid, simple, accurate, and low-cost Hcy detection method has attracted widespread attention.

[0003] Compared to the sensors mentioned above, the biomimetic nanopore sensor inspired by ion channels has advantages such as less restriction of detectable substances, excellent specificity, and high sensitivity, and has greater application potential in the selective detection of Hcy.

[0004] Because Hcy and Cys have similar structures, Cys can interfere with the detection of Hcy. This problem has not been well solved in the current nanopore analysis sensors used to detect Hcy, especially whether it is possible to effectively avoid the interference of Cys and achieve high selectivity for Hcy detection.

[0005] Therefore, it is crucial to develop a highly selective detection method for homocysteine. To date, no biomimetic nanopore analytical sensing method based on the reaction of DMACA with Cys has been reported to effectively avoid the interference of Cys detection and to be applied to the selective detection of Hcy. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting homocysteine ​​using a nanoporous sensor based on silver-sulfur polymerization. This method utilizes the difference in reaction between 4-dimethylaminocinnamaldehyde and Cys and Hcy, eliminating the interference of Cys thiol groups on Hcy detection through a cyclization reaction. Simultaneously, by constructing a functional probe and a special nanoporous structure based on the silver-sulfur polymerization reaction, the change in current signal is amplified, achieving rapid, simple, accurate, and low-cost detection of Hcy.

[0007] To achieve the above-mentioned technical objectives, the present invention provides a method for detecting homocysteine ​​using a nanoporous sensor based on silver-sulfur polymerization, the method comprising the following steps:

[0008] 1) After pretreating a homocysteine-containing solution with 4-dimethylaminocinnamaldehyde, a sensor with a cylindrical nanopore structure was placed in the homocysteine-containing solution for polymerization reaction, and a linear equation was established between the ratio of conductivity before and after polymerization reaction and the homocysteine ​​concentration; the cylindrical nanopores of the sensor were modified with Ag(I) probes.

[0009] 2) Replace the homocysteine-containing solution with the test solution, measure the ratio of conductivity before and after the polymerization reaction, and then substitute the ratio into the linear equation to calculate the concentration of homocysteine ​​in the test solution; the test solution contains homocysteine ​​and cysteine.

[0010] The superior selectivity and specificity of the detection method for Hcy in this invention is based on a special pretreatment method and the combined effect of a polymerization reaction with the sensor to amplify the change in current signal. Specifically, the analytes Hcy and Cys are structurally similar and difficult to distinguish. However, this invention uses 4-dimethylaminocinnamaldehyde (DMACA) to pretreat the analytes. DMACA reacts with Cys to form a stable five-membered heterocycle. After cyclization, the -SH group of Cys changes to -S-. In contrast, DMACA does not readily form a six-membered heterocycle when reacting with Hcy. Thus, the cyclization reaction eliminates the influence of the thiol group in Cys. The -SH group of Hcy-DMACA is retained after the reaction of Hcy with DMACA. This group undergoes a coordination polymerization reaction with the Ag(I) probe in the sensor, thereby accurately identifying Hcy within a nanoporous analytical sensing platform. The sensor of the present invention, modified with Ag(I) probe and featuring a cylindrical nanopore structure, utilizes supramolecular interactions such as metal-ligand and metal-metal interactions between Ag(I) and thiol compounds to form an Ag(I)-thiol coordination polymer with Ag(I) as nodes and thiol as ligands. This polymer occupies the effective diameter within the nanopore and has a relatively long molecular chain. Compared to bare pores without a fixed probe, this significantly induces a change in the current signal, thereby enabling rapid quantitative detection of Hcy.

[0011] As a preferred embodiment, the cylindrical nanopore structure of the sensor can be single-pore or multi-pore. The inventors have discovered that, when applied to the detection of Hcy, sensors employing a single-pore cylindrical nanopore structure exhibit higher selectivity and response values. The formation of single or multi-pore structures is achieved by controlling the number of ions in the ion bombardment device. When multiple ions bombard the film, multiple ion tracks are formed, thus obtaining a porous film. By controlling the device to reduce the number of incident ions and immediately stopping bombardment after one ion bombardment, a single ion track is formed, thereby fabricating a single nanopore.

[0012] As a preferred embodiment, the sensor with a cylindrical nanopore structure is first modified with cysteine ​​groups inside the pores, and then modified with Ag(I) probes via Ag-S bonds.

[0013] As a preferred embodiment, the fabrication of the sensor with the cylindrical nanopore structure includes the following steps:

[0014] (1) After bombarding the PET film with heavy ions and forming ion tracks inside it, the film is activated by irradiation with ultraviolet lamp, and then the tracks of the PET film are symmetrically etched with sodium hydroxide to form a PET film with cylindrical nanopores with through holes.

[0015] (2) The PET film with through cylindrical nanopores is immersed in an activation solution containing EDC, NHS and MES, and then cysteine ​​is added for graft modification to obtain a Cys / PET nanopore sensor.

[0016] (3) The Cys / PET nanopore sensor is immersed in a buffer solution containing AgNO3 and the Ag(I) probe is modified by Ag(I)-S bond to obtain the Ag(I) / Cys / PET nanopore sensor.

[0017] The principle behind this invention for preparing Ag(Ⅰ) / Cys / PET nanopore sensors is as follows: Due to the high energy of heavy ions, when heavy ions bombard a PET film, they create damage tracks within the film. Ultraviolet light can excite the chemical bonds in the PET molecules, increasing their reactivity. Because the tracked regions, after heavy ion bombardment and UV activation, have a different chemical structure than the unbombarded regions, their reactivity with sodium hydroxide is enhanced. Sodium hydroxide reacts chemically with the PET molecular chains, preferentially etching the tracked regions symmetrically from both sides of the PET film, gradually forming through-hole cylindrical nanopores. Furthermore, the PET channels after ion bombardment and alkaline symmetrical etching have different physical and chemical properties than the rest of the PET film. Under alkaline conditions, the ester groups in the PET channels react with hydroxide ions, breaking the ester bonds to generate carboxylates and ethylene glycol. The carboxylates further hydrolyze to obtain carboxyl groups, thus covering the PET channels with a large number of carboxyl groups. Then, a carbodiimide chemical linking method was used to immobilize Cys on the inner surface of cylindrical nanopores. The successfully modified Cys could modify the inner surface of the pores, covering it with a large number of thiol groups. Then, the thiol groups introduced by Cys formed Ag(I)-S bonds with Ag(I) to fix the Ag(I) probe on the inner surface of the Cys-modified nanopores. The inner surface of the pores after successful modification was modified again and covered with a large amount of Ag(I).

[0018] As a preferred embodiment, the heavy ion is Xe.

[0019] As a preferred embodiment, the ultraviolet lamp irradiation time is 9 to 12 hours.

[0020] As a preferred embodiment, the symmetrical etching time is 4–10 min, and the temperature is 50–60 °C. In this invention, if the symmetrical etching time is too short, not only will the channels not be fully penetrated, but the resulting pore size will also be too small, preventing the required Cys and Ag(Ⅰ) probes from entering and thus failing to detect them, or preventing the required Hcy probes from entering and thus hindering the polymerization reaction. As the etching time increases, the diameter of the cylindrical nanopores increases. If the pore size is too large, the change in the effective diameter and conductivity of the pores will be insignificant, interfering with the detection results. More preferably, the symmetrical etching time is 8–10 min, and even more preferably 10 min.

[0021] As a preferred embodiment, in step 2, the molar ratio of EDC, NHS, MES, and cysteine ​​is 50:250:100:(8-10), the pH of the activation solution is 4.7-5.5, and the grafting modification time is 1-2 hours. Within the molar ratio range selected in this invention, Cys can be successfully immobilized and modified within the nanopores using the carbodiimide chemical linking method, ensuring the grafting rate of Cys.

[0022] As a preferred embodiment, in step 3, the molar ratio of AgNO3 to cysteine ​​is (1–1.5):1, the impregnation time is 1–2 hours, and the impregnation process is carried out in a light-protected environment. The buffer solution is a sodium acetate-acetic acid buffer solution with a pH of 5.0–5.5. Performing the process in a light-protected environment effectively prevents Ag(I) from being reduced to metallic silver, thus avoiding its impact on the construction of the Ag(I) / Cys / PET nanoporous sensor.

[0023] As a preferred embodiment, the concentration of homocysteine ​​in the homocysteine-containing solution is 1 μmol / L to 30 μmol / L, and the linear equation is y = 0.91 - 0.025x; where y is the ratio of conductivity before and after the polymerization reaction, and x is the concentration of homocysteine ​​in μmol / L. In this invention, the conductivity of the Ag(Ⅰ) / Cys / PET nanopores before polymerization is defined as G0, and the conductivity of the Ag(Ⅰ) / Cys / PET nanopores after polymerization is G, and y equals G / G0. In this invention, as the Hcy concentration increases, G / G0 gradually decreases, and when the Hcy concentration is greater than 35 μmol / L, G / G0 tends to stabilize. Within the concentration range of 1 μmol / L to 30 μmol / L, G / G0 shows a good linear relationship with the corresponding Hcy concentration, and the correlation coefficient R0 is [value missing]. 2 The value is 0.99, and the detection limit can reach 1 μmol / L.

[0024] As a preferred embodiment, the polymerization reaction time is 30–80 min. With the increase of the polymerization reaction time, the polymer gradually occupies the pore space in the nanopores, the ion concentration within the pores decreases, and the conductivity of the nanopores begins to decrease. When the time increases to a certain extent, the polymer volume within the pores no longer increases, and the conductivity change rate tends to stabilize. A further preferred polymerization reaction time is 60–80 min.

[0025] As a preferred embodiment, the molar ratio of the homocysteine-containing solution to 4-dimethylaminocinnamaldehyde is 1:(1-1.2), wherein the homocysteine-containing solution is calculated based on homocysteine.

[0026] As a preferred approach, the selectivity and anti-interference ability of the Ag(Ⅰ) / Cys / PET nanopore analysis sensing platform were tested using control samples of other amino acids and thiols at 10-fold concentrations, respectively.

[0027] As a preferred approach, the standard addition method was used for detection in actual porcine serum samples. Different concentrations of Hcy were added to the treated porcine serum for quantitative detection, and the sensor's anti-interference capability in porcine serum was also examined.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The method of the present invention utilizes the reaction differences between 4-dimethylaminocinnamaldehyde and Cys and Hcy, and eliminates the interference of Cys thiol group on Hcy detection through cyclization reaction. At the same time, the current signal change is amplified based on silver-sulfur polymerization reaction through synergistically constructed functional probes and special nanoporous structures, so as to realize rapid, simple, accurate and low-cost detection of Hcy.

[0030] 2) During operation, the sensor of the present invention can form Ag(Ⅰ)-Hcy-DMACA coordination polymer chains through coordination bonding. These chains can hinder ion transport within the pores by means of their volume effect, and quantitative detection of Hcy can be achieved by detecting changes in conductivity within the pores.

[0031] 3) The method of the present invention is based on the selective detection of homocysteine ​​by a nanoporous sensor of Ag(I)-thiol coordination polymer. The quantitative detection of Hcy is achieved by detecting the change in conductivity. The linear response range of the Ag(I) / Cys / PET nanoporous sensor to Hcy is 1μmol / L to 30μmol / L, and the detection limit can reach 1μmol / L, which is lower than the normal Hcy content in the human body.

[0032] 4) The sensor of the present invention has excellent selectivity and anti-interference ability, and can be used for the detection of Hcy in complex serum samples. Using this method to detect Hcy in pig serum, the recovery rate is 102.5% to 105.9%, and the relative standard deviation (RSD) is 1.0% to 6.4%, which expands its application field.

[0033] 5) The method of the present invention has the advantages of simple device, high sensitivity, short time consumption and no labeling.

[0034] 6) The nanopore sensor prepared by etching PET as a substrate is covered with a large number of carboxyl groups on its surface, which facilitates the fixation of various recognition elements. At the same time, due to the good thermal stability and flexibility of PET material, PET nanopore sensor has great potential in the field of wearable sensing devices. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the construction of the Ag(Ⅰ) / Cys / PET nanoporous sensor and its selective detection of Hcy. The cross-sectional view of PET is shown.

[0036] Figure 2The following are SEM images (scale bar 500 nm) of the two ends of the cylindrical nanopores prepared at different etching times in Example 2: (A) and (E) have an etching time of 4 min; (B) and (F) have an etching time of 6 min; (C) and (G) have an etching time of 8 min; and (D) and (H) have an etching time of 10 min.

[0037] Figure 3 XPS spectra of the nanopore modification process in Example 1 (black line represents unmodified nanopores; red line represents Cys-modified nanopores; blue line represents Ag(Ⅰ) / Cys-modified nanopores).

[0038] Figure 4 (I) IV curves of Ag(Ⅰ) / Cys / PET nanopores before and after detecting Hcy-DMACA(A), Hcy(B), Cys-DMACA(C), Cys(D), and GSH(E) in Example 1; (F) IV curves of unmodified nanopores before and after detecting Hcy-DMACA.

[0039] Figure 5 Example 3 shows the change in the conductivity of nanopores under different polymerization reaction times.

[0040] Figure 6 (A) Effect of different concentrations of Hcy on the conductivity of Ag(Ⅰ) / Cys / PET nanopores prepared in Example 1; (B) Linear relationship between conductivity change and Hcy concentration.

[0041] Figure 7 The response of the Ag(Ⅰ) / Cys / PET nanopores prepared in Example 1 to different amino acids.

[0042] Figure 8 The response of the Ag(Ⅰ) / Cys / PET nanopores prepared in Example 1 to Hcy and different thiol interfering substances, where Hcy refers to Hcy-DMACA treated with DMACA. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments. Obviously, the embodiments described below are only a part of the embodiments, and all other embodiments obtained by those skilled in the art without creative effort are still within the scope of protection of the present invention.

[0044] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0045] The polyethylene terephthalate (PET) film (13 μm thick, biaxially oriented) used in the embodiments and comparative examples of this invention was purchased from Goodfellow GmbH, Germany; the Ag / AgCl electrode was purchased from Shanghai Xianren Instrument Co., Ltd.; the experimental water was ultrapure water (resistivity ≥18.3 MΩ·cm); all reagents used were analytical grade (AR); and the descriptions of amino acids in the following descriptions use English abbreviations.

[0046] Example 1

[0047] 1. Preparation of Ag(Ⅰ) / Cys / PET nanoporous sensors

[0048] Cylindrical nanopores were prepared using ion track etching, and a PET film (8.98 MeVu) was bombarded with a single heavy ion, Xe. -1 A PET film with a single track was obtained, and then activated by irradiation with a 312 nm UV lamp for 12 h on the front side and 9 h on the back side. Immediately after irradiation, the PET film was symmetrically etched with a 3 mol / L NaOH solution at 50 °C for 10 min to obtain a single-hole cylindrical pore. After etching, the PET film surface was rinsed with distilled water and left to stand overnight in ultrapure water.

[0049] Ag(I) / Cys / PET nanopores were chemically modified. Cys possesses both amino and thiol functional groups, and was immobilized on the inner surface of cylindrical nanopores via a carbodiimide chemical linkage method. Ag(I) was immobilized on the inner surface of the Cys-modified nanopores via Ag(I)-S bonds. First, a MES solution (0.1 mol / L, pH 4.7) containing 50 mmol / L EDC and 250 mmol / L NHS was placed in dispersion tanks on both sides of a PET film. After soaking for 1 hour, the mixture was removed, and the dispersion tanks were rinsed with ultrapure water. An 8 mmol / L Cys modification solution was prepared using 10 mmol / L PBS buffer (pH 7.4) and placed in the dispersion tanks on both sides of the PET film. After soaking for 1 hour, the Cys modification solution was removed, and the dispersion tanks were rinsed with ultrapure water. Subsequently, 1 mol / L NaF electrolyte solution was added to the dispersion tanks on both sides of the Cys-modified PET film, and current-voltage (IV) curves were measured to determine the success of the Cys modification.

[0050] Next, an 8 mmol / L AgNO3 modification solution was prepared using 10 mmol / L sodium acetate-acetic acid buffer (NaAc-HAc buffe) at pH 5.0 and placed in the dispersion tanks on both sides of the PET film. After soaking in the dark for 1 hour, the AgNO3 modification solution was removed, and the dispersion tanks were rinsed with ultrapure water. Subsequently, 1 mol / L NaF electrolyte solution was added to the dispersion tanks on both sides of the PET film, and the IV curve was measured to detect whether Ag(Ⅰ) modification was successful and whether Ag(Ⅰ) / Cys / PET nanopores were formed.

[0051] Pretreatment method for biothiols using 2,4-dimethylaminocinnamaldehyde

[0052] Before the experiment, biothiols need to be pretreated. A series of solutions of 4-dimethylaminocinnamaldehyde (DMACA), Cys, Hcy, and GSH (glutathione) at concentrations of 100 mmol / L, 50 mmol / L, 10 mmol / L, 5 mmol / L, and 1 mmol / L were prepared using 0.1 mol / L carbonate buffer at pH 9.5.

[0053] Equal volumes of DMACA solution were mixed with Hcy solution, Cys solution, and GSH solution of the same concentration, and stirred at room temperature for 20 min to prepare Hcy-DMACA solution, Cys-DMACA solution, and GSH-DMACA solution of a certain concentration. These solutions were then stored in a light-protected environment for subsequent experiments.

[0054] 3. Ag(Ⅰ)-thiol polymerization method

[0055] Take 10 μL of Hcy solution with a concentration of n mmol / L (n is determined according to experimental requirements) and 5 μL of AgNO3 solution with a concentration of 2n mmol / L, and add them sequentially to 2 mL of sodium acetate-acetic acid buffer (10 mmol / L, pH 5.0). After shaking and mixing, let it stand in the dark for 1 h to obtain Ag(Ⅰ)-Hcy coordination polymer with Hcy and AgNO3 concentrations of 5n μmol / L.

[0056] By repeating the above steps, Ag(Ⅰ)-Cys coordination polymers with Cys and AgNO3 concentrations of 5 nμmol / L and Ag(Ⅰ)-Hcy-DMACA coordination polymers with Hcy-DMACA and AgNO3 concentrations of 5 nμmol / L can be obtained.

[0057] 4. Detection of Hcy by Ag(Ⅰ) / Cys / PET nanoporous sensor

[0058] The Ag(Ⅰ) / Cys / PET nanoporous sensor achieves quantitative analysis of Hcy through changes in current signal. A two-electrode system detects the current signal at both ends of the nanopores. A 1 mol / L NaF electrolyte solution is added to the dispersion tanks on both sides of the PET film. Using an Ag / AgCl electrode and the It function of an electrochemical workstation, the current signal across the pores is measured under steady-state voltage. The current magnitudes at different voltages are recorded, and an IV curve is plotted. By calculating the slope of the IV curve in the voltage range of -60 mV to 60 mV, the conductivity G0 before the polymerization reaction of the nanopores can be obtained.

[0059] A series of Hcy (0.1 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 35 μmol / L, 40 μmol / L, and 60 μmol / L) sodium acetate-acetic acid solutions with different concentrations and pH were prepared using DMACA-treated sodium acetate-acetic acid buffer solutions. 0.5 mL of the 200 μmol / L AgNO3 sodium acetate-acetic acid solution was added to the left side of the dispersion tank of the nanopores. 1.0 mL of the prepared DMACA-treated Hcy sodium acetate-acetic acid solutions was added to the right side of the dispersion tank. The mixture was allowed to stand in the dark for 1 h. After the polymerization reaction was complete, the solutions on both sides of the dispersion tank were aspirated, and the electrolytic cell was rinsed with ultrapure water. The current signal was detected, and the conductivity G of the nanopores after the polymerization reaction was calculated. The conductivity of Ag(Ⅰ) / Cys / PET nanopores before polymerization is defined as G0. The change in conductivity of the nanopores is reflected by the ratio of conductivity before and after polymerization, G / G0. At the same time, the response performance of the Ag(Ⅰ) / Cys / PET nanopore sensor to Hcy is evaluated by exploring the relationship between G / G0 and Hcy concentration.

[0060] Furthermore, this method was used to evaluate the selectivity of the Ag(Ⅰ) / Cys / PET nanopore sensor for Hcy.

[0061] Later, using porcine serum, after processing, the supernatant was collected and added using the standard addition method. 100 μL of 1 mmol / L DMAC solution and 100 μL of a mixed solution of Hcy (concentrations of 5, 10, 15, and 20 μmol / L) and Cys (concentration of 100 μmol / L) were added, and the mixture was shaken well. The pH of the solution was adjusted to 9.5 using NaOH, and then stirred at room temperature for 20 min. 500 μL of the DMACA-treated serum sample was then taken and mixed thoroughly with 500 μL of 10 mmol / L acetate buffer (pH 5.0), and the pH was adjusted to 5.0 using sodium acetate. Subsequent experimental procedures were the same as described above.

[0062] The sensor prepared in Example 1 was tested:

[0063] 1) XPS spectral characterization of the nanopore modification process

[0064] The modification process of the Ag(Ⅰ) / Cys / PET nanopore sensor was characterized using X-ray photoelectron spectroscopy (XPS), such as... Figure 3 In the unmodified cylindrical nanopores (black line), only the O1s and C1s peaks are clearly visible, which are the O and C elements naturally present in the PET substrate material. After modifying the cylindrical nanopores with Cys (red line), the N 1s peak (around 399 eV) is clearly visible, proving the successful modification by introducing N. Further modification of the cylindrical nanopores with AgNO3 (blue line) reveals the Ag 3d peak near 370 eV, indicating the introduction of Ag into the nanopores, thus demonstrating the successful modification of Ag(I) within the nanopores.

[0065] 2) Electrochemical performance testing

[0066] The effects of Ag(Ⅰ) / Cys / PET nanopores on the ionic current and conductivity of three bio-thiols (Hcy, Cys, and GSH) after DMACA treatment were investigated. Figure 4 As shown in (C) and (D), after Cys is added to Ag(Ⅰ) / Cys / PET nanopores, due to the presence of thiol groups, it will also undergo coordination polymerization with Ag(Ⅰ) within the pores to form a polymer. After DMACA treatment, Cys undergoes a cyclization reaction. Figure 1 Cys-DMACA is generated and loses its thiol group. After being added to Ag(Ⅰ) / Cys / PET nanopores, it does not undergo coordination polymerization with Ag(Ⅰ), no polymer is generated in the pores, and the pore conductivity does not change significantly.

[0067] GSH cannot coordinate polymerize with Ag(I) to form coordination polymers at pH 5.0. For example... Figure 4 As shown in (E), the conductivity of the nanopores did not change significantly after GSH was added to the Ag(Ⅰ) / Cys / PET nanopores. To verify the immobilization effect of Ag(Ⅰ) modified on the inner surface of the nanopores on the coordination polymer, as shown in Figure (E),... Figure 4 As shown in (F), after Hcy-DMACA and Ag(Ⅰ) were added to the unmodified nanopores, the conductivity of the nanopores did not change significantly. This indicates that if the polymer cannot be fixed in the nanopores after formation, it will be discharged from the nanopores with the solution and will not cause significant changes in the ionic current and conductivity of the nanopores.

[0068] 3) Linear range and detection limit

[0069] A series of Hcy solutions of varying concentrations were prepared, and Hcy was quantitatively analyzed using an Ag(Ⅰ) / Cys / PET nanopore sensor. The G / G0 ratio was used to reflect changes in the nanopore conductivity in the experiment. The experimental results are as follows: Figure 6 As shown in (A), the G / G0 ratio gradually decreases with increasing Hcy concentration, and tends to stabilize when the Hcy concentration is greater than 35 μmol / L. Figure 6 As shown in (B), within the concentration range of 1 μmol / L to 30 μmol / L, G / G0 exhibits a good linear relationship with the corresponding Hcy concentration, with the linear equation y = 0.91 - 0.025x (μmol / L) and a correlation coefficient R. 2 The value is 0.99, and the detection limit can reach 1 μmol / L.

[0070] 4) Selective testing

[0071] The selectivity of the sensor was studied by investigating the polymerization of different amino acids and biothiols in Ag(Ⅰ) / Cys / PET nanopores. Cysteine ​​(Cys), cystine, methionine (Met), arginine, glutamic acid, alanine, proline, glycine, aspartic acid, tyrosine, leucine, tryptophan, serine, threonine, valine, lysine, histidine, and glutathione (GHS) were selected as controls, with concentrations 10 times higher than the Hcy solution concentration. The conductivity of the sensor with modified probes but without polymerization was used as the control group. Figure 7 As shown, the pore conductivity decreased significantly in the presence of Hcy alone, while the other 18 control substances failed to coordinate and polymerize with Ag(Ⅰ) within the pores after treatment, and the pore conductivity remained unchanged. Therefore, the Ag(Ⅰ) / Cys / PET nanoporous sensor exhibits excellent selectivity for Hcy.

[0072] 5) Anti-interference test

[0073] Cysteine, glutathione, methionine (Met), and cystine were selected as interfering agents in the experiment, with concentrations 10 times higher than those in the Hcy solution. The interfering agent solution was mixed with the Hcy solution. The results are as follows: Figure 8 As shown, the addition of DMACA-treated Hcy and different interfering substances significantly reduced the pore conductivity, and the conductivity ratio G / G0 before and after the polymerization reaction was basically consistent with the conductivity ratio after polymerization of Hcy solution treated with DMACA alone in the pores. Therefore, the Ag(Ⅰ) / Cys / PET nanopore sensor exhibits excellent anti-interference performance for Hcy detection.

[0074] 6) Actual sample testing and recovery rate determination

[0075] This study investigated the application of an Ag(Ⅰ) / Cys / PET nanoporous sensor in the detection of homocysteine ​​(Hcy) in porcine serum samples. Different concentrations of Hcy were added to the treated porcine serum for quantitative detection, while 100 μmol / L Cys was added to interfere with the detection. The results showed that the recovery rate of Hcy was 102.5%–105.9%, and the relative standard deviation (RSD) was 1.0%–6.4%, both within a reasonable range. Therefore, the Ag(Ⅰ) / Cys / PET nanoporous sensor can be used for the detection of Hcy in complex serum samples.

[0076] Example 2

[0077] The effect of different etching times on the sensor aperture was investigated. Cylindrical nanopores were prepared using ion track etching, and multiple heavy ion Xe bombardments were performed on a PET film (8.98 MeVu). -1 Multiple track PET films were obtained, and then activated by irradiation with a 312 nm UV lamp for 12 h on the front side and 9 h on the back side. Immediately after irradiation, the PET films were symmetrically etched with a 3 mol / L NaOH solution at 50 °C for 4 min, 6 min, 8 min, and 10 min to obtain porous cylindrical PET films with different pore sizes. After etching, the PET film surface was washed with distilled water and left to stand overnight in ultrapure water. The cylindrical PET films were characterized by SEM, as shown below. Figure 2 As shown.

[0078] The nanopores on the left and right ends of the cylindrical nanopore PET film were characterized using scanning electron microscopy (SEM) at 4 min, 6 min, 8 min, and 10 min. Figure 2 The pore size increases with etching time. Specifically, the cylindrical nanopores etched for 4 min have a pore size of approximately 63.1 nm at both ends, those etched for 6 min have approximately 109.5 nm, those etched for 8 min have approximately 158.7 nm, and those etched for 10 min have approximately 201.3 nm. Based on the internal space effect, a cylindrical nanopore with an etching time of 10 min is further preferred for constructing an Ag(Ⅰ) / Cys / PET nanopore sensor.

[0079] Example 3

[0080] The only difference between this embodiment and Example 1 is that the polymerization reaction time is replaced with 30 min, 40 min, 50 min, 70 min, and 80 min, respectively. All other steps and conditions are the same. The effect of the Hcy polymerization reaction time on the change in conductivity within the sensor channel was investigated, and the results are as follows: Figure 5 As shown.

[0081] The results showed that when the reaction time was 30 min, the polymer occupied the pore space in the nanopores, the ion concentration in the pores decreased, and the conductivity of the nanopores began to decrease. When the polymerization reaction time was 60 min, the rate of change in conductivity tended to stabilize, indicating that the polymer volume in the pores no longer increased. Therefore, the optimal reaction time for coordination polymerization in the pores was 60 min.

[0082] The above results demonstrate that this invention constructs an Ag(I) / Cys / PET nanoporous sensor for the selective detection of Hcy based on Ag(I)-thiol coordination polymerization. The Ag(I)-thiol coordination polymerization reaction was investigated using characterization methods. Since Ag(I) can undergo coordination polymerization with thiols to form coordination polymers, the addition of 4-dimethylaminocinnamaldehyde-treated Hcy into the Ag(I) / Cys / PET nanoporous channels generates an Ag(I)-Hcy-DMACA coordination polymer, which hinders ion transport within the channels, thus reducing the channel conductivity. Quantitative detection of Hcy is achieved by detecting changes in conductivity. The linear response range of the Ag(I) / Cys / PET nanoporous sensor for Hcy is 1 μmol / L to 30 μmol / L, with a detection limit as low as 1 μmol / L, lower than the normal Hcy content in the human body. Furthermore, this sensor exhibits excellent selectivity and anti-interference properties, is not easily affected by other amino acids and biothiols during detection, and can be used for Hcy detection in complex serum samples. In addition, the sensor has advantages such as low manufacturing cost, mass production capability, simple operation, no need for large instruments, and fast detection speed of only 90 minutes, making it a highly promising biosensing platform.

Claims

1. A method for detecting homocysteine based on silver-sulfur polymerization in a nanopore sensor, characterized by: The method comprises the following steps: 1) After the solution containing homocysteine is pretreated by 4-dimethylaminocinnamaldehyde, the sensor with a cylindrical nanochannel structure is placed in the solution containing homocysteine to perform a polymerization reaction, and a linear equation of a ratio of conductivities before and after the polymerization reaction to a homocysteine concentration is established; the cylindrical nanochannel of the sensor is modified with an Ag(I) probe; 2) The solution containing homocysteine is replaced by a to-be-tested liquid, a ratio result of the conductivities before and after the polymerization reaction is determined, and then the ratio result is substituted into the linear equation to calculate the homocysteine concentration in the to-be-tested liquid; the to-be-tested liquid contains homocysteine and cysteine; the sensor with the cylindrical nanochannel structure is first modified with a cysteine group in the nanochannel, and then modified with the Ag(I) probe through an Ag-S bond; the preparation of the sensor with the cylindrical nanochannel structure comprises the following steps: (1) after a PET film is bombarded by heavy ions and ion tracks are formed in the PET film, the PET film is activated by irradiation of an ultraviolet lamp, and then the tracks of the PET film are symmetrically etched by sodium hydroxide to form a PET film with a cylindrical nanochannel having a through hole; (2) the PET film with the cylindrical nanochannel having the through hole is immersed in an activation solution containing EDC, NHS and MES, and then cysteine is added for graft modification to obtain a Cys / PET nanochannel sensor; (3) the Cys / PET nanochannel sensor is immersed in a buffer solution containing AgNO3 to modify the Ag(I) probe through an Ag(I)-S bond to obtain an Ag(I) / Cys / PET nanochannel sensor.

2. The method for detecting homocysteine based on a silver-sulfur polymerization nanochannel sensor according to claim 1, characterized in that: the irradiation time of the ultraviolet lamp is 9-12 h; the symmetric etching time is 4-10 min, and the temperature is 50-60°C.

3. The method for detecting homocysteine based on silver-sulfur polymerization nanochannel sensor according to claim 2, characterized in that: In step (2), the molar ratio of EDC, NHS, MES and cysteine is 50:250:100:(8-10), the pH of the MES activation buffer solution is 4.7-5.5, and the graft modification time is 1-2 h.

4. The method for detecting homocysteine based on silver-sulfur polymerization nanochannel sensor according to claim 3, characterized in that: In step (3), the molar ratio of AgNO3 and cysteine is (1-1.5):1, the immersion time is 1-2 h, and the immersion process is in a light-proof environment, and the buffer solution is a sodium acetate-acetic acid buffer solution with a pH of 5.0-5.

5.

5. The method for detecting homocysteine based on silver-sulfur polymerization nanochannel sensor according to claim 1, characterized in that: The concentration of homocysteine in the solution containing homocysteine is 1-30 μmol / L, and the linear equation is y=0.91-0.025x; wherein y is the ratio of the conductivities before and after the polymerization reaction, and x is the concentration of homocysteine, in units of μmol / L.

6. The method for detecting homocysteine based on silver-sulfur polymerization nanochannel sensor according to claim 5, characterized in that: The polymerization time is 30-80 min.

7. The method for detecting homocysteine based on silver-sulfur polymerization nanochannel sensor according to claim 5, characterized in that: The molar ratio of the solution containing homocysteine to 4-dimethylaminocinnamaldehyde is 1:(1-1.2), wherein the solution containing homocysteine is calculated based on homocysteine.

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