Electrochemical sensor based on N-MLG coated Ni-Mn LDH / Cys composite material and preparation method and application thereof

Abstract

The invention discloses an electrochemical sensor based on an N-MLG coated Ni-Mn LDH / Cys composite material and a preparation method and application thereof, and belongs to the technical field of electrochemical sensors. The sensor adopts a three-electrode system, takes an N-MLG coated Ni-Mn LDH / Cys composite material modified screen-printed electrode (SPE) as a working electrode, takes Ag / AgCl as a reference electrode and takes a carbon electrode as a counter electrode, and detects copper ions by utilizing square wave anodic stripping voltammetry. By means of the high conductivity of N-MLG, the large specific surface area of Ni-Mn LDH and the specific chelating capacity of cysteine, a current signal is amplified, and the sensitivity is remarkably improved. The electrochemical sensor constructed by the invention has the outstanding advantages of low detection limit, wide linear range, high selectivity and the like in the aspect of sweat copper ion detection, is simple and convenient to operate, and has huge potential in personalized medical treatment and clinical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, and specifically relates to an electrochemical sensor based on nitrogen-doped graphene composite material, its preparation method, and its application. Background Technology

[0002] Currently, heavy metal ion (HMs) pollution is severe. Heavy metals are difficult to degrade and accumulate thousands of times through bioaccumulation and the amplification effect of the food chain, eventually reaching the human body and posing a serious threat to human health. As an essential trace element, both excessive and deficient copper ions can lead to corresponding diseases. High copper levels may cause Meniere's disease, Wilson's disease, Alzheimer's disease, etc.; copper deficiency may affect iron absorption, leading to anemia, osteoporosis, and other conditions. Therefore, monitoring copper levels in bodily fluids (such as blood, sweat, and urine) is crucial. 2+ Concentration has key clinical guiding significance for the early diagnosis and treatment of related diseases.

[0003] Currently, blood and urine tests are important standards for clinical assessment of copper metabolism status. However, these two methods cannot achieve continuous, real-time monitoring, and samples are easily contaminated. Furthermore, invasive blood sampling methods can pose risks to patients, especially newborns, the elderly, and those prone to fainting at the sight of blood. In contrast, sweat contains abundant biomarkers, and the human body produces a high level of sweat, making sweat samples easily obtainable. Therefore, the high level of sweat production in the human body allows for easy acquisition of sweat samples, enabling real-time, multiple, continuous, and dynamic monitoring.

[0004] Traditional detection techniques, such as atomic absorption spectrometry (AAS), high-performance liquid chromatography (HPLC), inductively coupled plasma mass spectrometry (ICP-MS), and graphite furnace atomic absorption spectrometry (GFAAS), offer high sensitivity and low detection limits. However, these methods are complex to operate, require expensive instruments, and necessitate specialized technical personnel. Electrochemical analysis methods, on the other hand, have gained widespread attention due to their short analysis time, low cost, high sensitivity, and ease of instrument miniaturization. For example, invention patent CN109298042A discloses a method for detecting copper ions using a flexible silver nanowire / gold nanoparticle composite electrode based on PDMS. This method utilizes the catalytic properties of noble metal nanomaterials (gold and silver) to achieve the detection of Cu ions in a buffer system. 2+ The detection range is 0.005-0.1 mg / L, or 5-100 ppb. It is only suitable for screening high concentrations of copper ions and cannot meet the needs for accurate, early monitoring of trace copper ions; furthermore, the detection environment of this patent is a simple tartaric acid buffer solution, requiring the addition of Bi... 3+As an auxiliary depositing agent to improve sensitivity, it has not been applied to testing in real, complex biological fluids (such as sweat, blood, and saliva). Furthermore, due to the complex composition of sweat and its extremely low copper ion concentration, traditional electrochemical methods exhibit poor selectivity, are easily affected by interference, and have high detection limits.

[0005] Therefore, there is an urgent need to develop an effective method to achieve simple, sensitive, rapid, and highly selective detection of ultra-low concentrations of copper ions in sweat. Summary of the Invention

[0006] This invention addresses the technical problems of poor selectivity, poor anti-interference ability, and high detection limit of existing electrochemical analysis methods by proposing an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for preparing an electrochemical sensor working electrode based on N-MLG@Ni-Mn LDH / Cys composite material, comprising the following steps:

[0009] (1) Pre-treat the SPE electrode;

[0010] (2) After uniformly dispersing multilayer graphene in deionized water, urea was added, followed by hydrothermal reaction. After centrifugation, washing, and drying, N-MLG was obtained.

[0011] (3) Disperse nickel nitrate hexahydrate and manganese nitrate tetrahydrate in deionized water to obtain solution I. After adjusting the pH of solution I, stir it evenly and then add N-MLG suspension to obtain solution II. Perform hydrothermal reaction, and after centrifugation, washing and drying, obtain N-MLG@Ni-Mn LDH composite material.

[0012] (4) The N-MLG@Ni-Mn LDH suspension was drop-coated onto the pretreated SPE electrode and dried to obtain the N-MLG@Ni-Mn LDH / SPE modified electrode;

[0013] (5) The N-MLG@Ni-Mn LDH / SPE modified electrode obtained in step (4) is immersed in cysteine ​​solution and incubated in the dark to obtain the N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode, which is the working electrode.

[0014] The pretreatment in step (1) includes immersing the SPE electrode in a (3-aminopropyl)triethoxysilane (APTES)-ethanol solution, followed by rinsing and drying to obtain the pretreated SPE electrode. The volume concentration of APTES in the APTES-ethanol solution is 2-5%, and the immersion time is 30-120 min.

[0015] In step (2), the mass ratio of multilayer graphene to urea is 1:3, the initial concentration of multilayer graphene is 2-5 mg / mL, the temperature of the hydrothermal reaction is 120-160℃, and the time is 3-5 h, and the drying is vacuum drying at a temperature of 60-80℃ for 8-12 h.

[0016] Ni in solution I described in step (3) 2+ and Mn 2+ The molar ratio is 1-3:1, and the pH of solution I is 9-10; the concentration of the N-MLG suspension is 2-5 mg / mL, and the amount added is based on 10 mL of N-MLG suspension, corresponding to a total mass of 0.83 g of metal salt; the hydrothermal reaction temperature is 120-180℃, and the time is 12-18 h; the drying is vacuum drying, at a temperature of 60-80℃, and for 8-12 h.

[0017] The concentration of the cysteine ​​solution in step (5) is 2-10 mM, and the incubation time is 8-12 h.

[0018] The present invention provides an electrochemical sensor working electrode based on N-MLG@Ni-Mn LDH / Cys composite material prepared by the preparation method described above.

[0019] Secondly, the present invention provides an electrochemical sensor, comprising a working electrode, a reference electrode, and a counter electrode, wherein the working electrode is the electrochemical sensor working electrode based on the N-MLG@Ni-Mn LDH / Cys composite material; the reference electrode is Ag / AgCl; and the counter electrode is a carbon electrode.

[0020] The present invention also provides the application of the electrochemical sensor in the detection of copper ions for non-disease diagnosis purposes, comprising the following steps: placing the electrochemical sensor in a container containing different concentrations of Cu 2+ In a buffer solution, different concentrations of Cu were collected. 2+ The electrochemical signal was obtained by applying a constant negative potential of -0.8V for 180 seconds. Then, Cu... 2+ Dissolution occurred within a potential range of -0.4V to 0.6V, and the values ​​of different Cu were measured. 2+ The peak current corresponding to the concentration was determined, and a linear fitting curve was plotted to establish Cu 2+Quantitative relationship between concentration and peak current for detecting Cu in the sample. 2+ The electrochemical signal is input into the fitting curve to obtain the Cu content in the sample. 2+ The concentration.

[0021] The Cu 2+ The concentration range is 0.1-500 ppb, the buffer solution is an HAc-NaHc buffer solution with a concentration of 0.1 M and pH=5; the electrochemical signal is the dissolution current value obtained by square wave anodic stripping voltammetry; the fitting curve is Cu 2+ When the concentration is between 0.1-100 ppb, the fitted curve is Y = 0.48689X + 93.1936. When Cu... 2+ When the concentration is between 100-500 ppb, the fitted curve is Y=0.24184X+114.71996.

[0022] Detection of Cu 2+ The working principle is as follows: Cu is detected using square wave anodic stripping voltammetry. 2+ The process mainly consists of two parts: pre-enrichment and stripping. In the pre-enrichment stage, Cu in the solution... 2+ Due to the strong chelating effect of the thiol (-SH) and amino (-NH2) groups in the cysteine ​​on the surface of the composite electrode, a stable Cu is formed. 2+ -Cys complexes, achieving Cu 2+ Directional capture on the electrode surface. Subsequently, Cu 2+ Driven by a constant negative potential, a reduction reaction occurs, depositing elemental copper (Cu). 0 It adheres firmly to the electrode surface. The high conductivity of nitrogen-doped multilayer graphene (N-MLG) provides a fast channel for electron transport, while the layered structure of Ni-Mn LDH significantly increases the electrode's specific surface area. The synergistic effect of these two elements effectively amplifies the Cu... 2+ The electrochemical signal was observed. After a brief settling period, the copper deposition layer on the electrode surface was uniformly distributed. During the dissolution phase, a positively increasing square wave potential scan was applied, and the enriched elemental copper underwent an oxidation reaction and was re-dissolved into the solution.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention provides a nitrogen-doped multilayer graphene N-MLG@Ni-Mn LDH nanocomposite material to modify the surface of screen-printed electrodes (SPEs), and modifies it with L-cysteine ​​to provide -SH-specific binding of Cu. 2+Nitrogen-doped multilayer graphene significantly enhances conductivity. Combined with the large specific surface area and abundant active sites of Ni-Mn LDH, it amplifies electrochemical signals. Finally, L-cysteine ​​is modified to specifically bind Cu through its surface amino (-NH2) and thiol (-SH) groups. 2+ This electrochemical sensor exhibits excellent performance in copper ion detection, unaffected by interference from other ions. It boasts advantages such as low detection limit, wide linear range, excellent stability, and high selectivity. The sensor constructed in this invention can achieve the detection of Cu ions without interference from other ions. 2+ It provides rapid, sensitive, and selective testing, and can be successfully applied to the detection of actual sweat.

[0025] 2. The N-MLG@Ni-Mn LDH composite material synthesized in this invention exhibits high electrical conductivity, large specific surface area, and abundant active sites, making it suitable for Cu... 2+ It provides abundant adsorption sites and electrocatalytic active centers, and effectively chelates copper ions through synergistic interaction with L-cysteine. It exhibits excellent response to copper ion detection, displaying a wide detection range of 0.1-500 ppb and a low detection limit of 0.24 ppb, while also demonstrating excellent selectivity, repeatability, and stability. The electrochemical sensor constructed in this invention can be practically applied to the detection of real sweat, achieving a recovery rate of 93.34% to 105.4%. The electrochemical sensor boasts fast response, low cost, good biocompatibility, and is non-toxic and odorless, making it suitable for use in the fabrication of flexible electrochemical sensors on skin surfaces. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a flowchart illustrating the fabrication process of the electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material in Example 1.

[0028] Figure 2 The N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1 was characterized by SEM, TEM, XRD and XPS.

[0029] Figure 3 Electrochemical testing of the electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1.

[0030] Figure 4 Comparison of SWASV responses for detecting the same concentration of copper ions using different modified electrodes.

[0031] Figure 5 The electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1, for different concentrations of Cu 2+ The SWASV response plot and the corresponding linear fit plot.

[0032] Figure 6 The stability, repeatability, reproducibility, and anti-interference performance of the electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1 were evaluated. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for preparing an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material, the overall synthesis process is as follows: Figure 1 As shown, the specific steps are as follows:

[0036] (1) SPE electrode pretreatment: Prepare an APTES-ethanol solution with a volume concentration of 2% and immerse the bare SPE electrode in (3-aminopropyl)triethoxysilane APTES solution. React at room temperature for 30 min. After removal, rinse thoroughly with anhydrous ethanol.

[0037] (2) Preparation of N-MLG: 0.1g of multilayer graphene was added to 50mL of deionized water and sonicated until uniformly dispersed to obtain a multilayer graphene dispersion. Then, 0.3g of urea was added and magnetically stirred until completely dissolved. The mixture was hydrothermally reacted at 160℃ for 3 hours. After centrifugation and washing, the mixture was finally vacuum dried at 60℃ for 12 hours to obtain nitrogen-doped multilayer graphene (N-MLG).

[0038] (3) Preparation of N-MLG@Ni-Mn LDH composite material: Weigh 0.58g of nickel nitrate hexahydrate and 0.25g of manganese nitrate tetrahydrate (Ni 2+ and Mn 2+The molar ratio of the reagents was 2:1. 20 mL of deionized water was added and the mixture was magnetically stirred until the reagents were fully dissolved. Then, NaOH was added dropwise until the pH of the solution was adjusted to 9.5 (the dropping rate should be controlled at one drop per second). The mixture was then magnetically stirred for 30 min to prepare a 5 mg / mL N-MLG suspension. 10 mL of the N-MLG suspension was poured into the homogenized solution above and hydrothermally reacted at 180 °C for 16 h. After centrifugation, the supernatant was discarded and the mixture was repeatedly washed to remove residual reagents and impurities. The mixture was then vacuum dried at 60 °C for 8 h to obtain the N-MLG@Ni-Mn LDH composite material.

[0039] The N-MLG@Ni-Mn LDH composite material prepared in this embodiment was characterized by TEM and SEM, and the results are as follows: Figure 2 As shown, Figure 2 a and 2b are TEM characterization images of the N-MLG@Ni-Mn LDH composite material. The translucent wrinkled sheet-like structure in the image corresponds to the nitrogen-doped multilayer graphene (N-MLG) substrate, and the dispersed particle aggregates anchored on its surface are Ni-Mn LDH. This morphology indicates that Ni-Mn LDH has been successfully loaded onto the surface of the N-MLG substrate, effectively suppressing its own aggregation behavior. Figure 2 The SEM image of c shows the macroscopic morphology of the N-MLG@Ni-Mn LDH composite material. The material exhibits a loose aggregated state formed by the stacking of lamellar structures. This structure provides abundant pores and a large specific surface area, providing ample space for the adsorption and diffusion of target substances, thereby enhancing its electrochemical sensing performance.

[0040] The crystal structure of the N-MLG@Ni-Mn LDH composite material was tested by XRD, and the results are as follows: Figure 2 As shown in d, it can be seen that its characteristic diffraction peaks (such as (003), (006), (012) crystal planes) match the standard card of Ni-Mn LDH (PDF#38-0715) to a high degree, indicating that the layered crystal structure of Ni-Mn LDH has been successfully formed in the N-MLG@Ni-Mn LDH composite material.

[0041] The elemental composition and surface functional groups of the N-MLG@Ni-Mn LDH composite material were characterized by XPS, and the results are as follows: Figure 2 e and Figure 2 As shown in f. Figure 2 XPS full spectrum analysis of the composite material revealed the presence of C, O, N, Ni, and Mn elements: C and N correspond to the N-MLG substrate, Ni and Mn correspond to the Ni-Mn LDH component, and the O element originates from the hydroxyl groups of LDH and the oxygen-containing functional groups of graphene, confirming the successful preparation of the target material. Figure 2f is the high-resolution peak spectrum of C1s, whose peaks can be decomposed into three characteristic components: 284.8 eV corresponds to C-C bonds (the framework carbon of graphene), 286.05 eV corresponds to CO bonds (oxygen-containing functional groups such as hydroxyl and ether bonds), and 288.84 eV corresponds to OC=O bonds (carboxyl functional groups). The presence of these oxygen-containing functional groups not only enhances the hydrophilicity of the material but also provides adsorption sites for target substances, which is beneficial to improving its electrochemical sensing performance. At the same time, the introduction of nitrogen (corresponding to N-MLG) can further regulate the electronic structure of carbon materials and optimize electron transport efficiency.

[0042] (4) Preparation of modified electrode: 5 μL of 5 mg / mL N-MLG@Ni-Mn LDH suspension was drop-coated onto the surface of the pretreated SPE working electrode and dried at room temperature to obtain N-MLG@Ni-Mn LDH / SPE modified electrode.

[0043] (5) Modification of cysteine: Dissolve 0.0121g of L-cysteine ​​in 10mL of 0.1M acetate-sodium acetate (HAc-NaHc) buffer solution with pH=5 and sonicate to dissolve. Immerse the modified electrode prepared in step (4) in the solution and incubate at 4℃ in the dark for 12h. After rinsing and drying, N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode is obtained.

[0044] (6) Preparation of electrochemical sensor: Using N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode as modified working electrode, Ag / AgCl as reference electrode, and carbon electrode as counter electrode, an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material was obtained.

[0045] Example 2

[0046] A method for fabricating an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material, the specific steps of which are as follows:

[0047] (1) SPE electrode pretreatment: Prepare an APTES-ethanol solution with a volume concentration of 5% and immerse the bare SPE electrode in (3-aminopropyl)triethoxysilane APTES solution. React at room temperature for 90 min. After removal, rinse thoroughly with anhydrous ethanol.

[0048] (2) Preparation of N-MLG: 0.1g of multilayer graphene was added to 20mL of deionized water and sonicated until uniformly dispersed to obtain a multilayer graphene dispersion. Then, 0.3g of urea was added and magnetically stirred until completely dissolved. The mixture was then hydrothermally reacted at 150℃ for 3h. After centrifugation and washing, the mixture was finally vacuum dried at 70℃ for 10h to obtain nitrogen-doped multilayer graphene (N-MLG).

[0049] (3) Preparation of N-MLG@Ni-Mn LDH composite material: Weigh 0.45g of nickel nitrate hexahydrate and 0.38g of manganese nitrate tetrahydrate (Ni 2+ and Mn 2+ The molar ratio of the reagents was 1:1. 20 mL of deionized water was added and the mixture was magnetically stirred until the reagents were fully dissolved. Then, NaOH was added dropwise until the pH of the solution was adjusted to 10 (the dropping rate should be controlled at one drop per second). The mixture was then magnetically stirred for 30 min to prepare a 2 mg / mL N-MLG suspension. 10 mL of the N-MLG suspension was poured into the homogenized solution above and hydrothermally reacted at 160 °C for 12 h. After centrifugation, the supernatant was discarded and the mixture was repeatedly washed to remove residual reagents and impurities. The mixture was then vacuum dried at 70 °C for 12 h to obtain the N-MLG@Ni-Mn LDH composite material.

[0050] (4) Preparation of modified electrode: 5 μL of 5 mg / mL N-MLG@Ni-Mn LDH suspension was drop-coated onto the surface of the pretreated SPE working electrode and dried at room temperature to obtain N-MLG@Ni-Mn LDH / SPE modified electrode.

[0051] (5) Modification of cysteine: Dissolve 0.0121g of L-cysteine ​​solution in 5mL of 0.1M acetate-sodium acetate (HAc-NaHc) buffer solution with pH=5 and sonicate to dissolve. Immerse the modified electrode prepared in step (4) in the solution and incubate at 4℃ in the dark for 12h. After rinsing and drying, N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode is obtained.

[0052] (6) Preparation of electrochemical sensor: Using N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode as modified working electrode, Ag / AgCl as reference electrode, and carbon electrode as counter electrode, an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material was assembled.

[0053] Example 3

[0054] A method for fabricating an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material, the specific steps of which are as follows:

[0055] (1) SPE electrode pretreatment: Prepare an APTES-ethanol solution with a volume concentration of 3% and immerse the bare SPE electrode in (3-aminopropyl)triethoxysilane APTES solution. React at room temperature for 120 min. After removal, rinse thoroughly with anhydrous ethanol.

[0056] (2) Preparation of N-MLG: 0.1g of multilayer graphene was added to 25mL of deionized water and sonicated until uniformly dispersed to obtain a multilayer graphene dispersion. Then, 0.3g of urea was added and magnetically stirred until completely dissolved. The mixture was hydrothermally reacted at 120℃ for 5h. After centrifugation and washing, the mixture was finally vacuum dried at 80℃ for 8h to obtain nitrogen-doped multilayer graphene (N-MLG).

[0057] (3) Preparation of N-MLG@Ni-Mn LDH composite material: Weigh 0.64g of nickel nitrate hexahydrate and 0.19g of manganese nitrate tetrahydrate (Ni 2+ and Mn 2+ The molar ratio of the reagents was 3:1. 20 mL of deionized water was added and the mixture was magnetically stirred until the reagents were fully dissolved. Then, NaOH was added dropwise until the pH of the solution was adjusted to 9 (the dropping rate should be controlled at one drop per second). The mixture was then magnetically stirred for 30 min to prepare a 3 mg / mL N-MLG suspension. 10 mL of the N-MLG suspension was poured into the homogenized solution above and hydrothermally reacted at 120 °C for 18 h. After centrifugation, the supernatant was discarded and the mixture was repeatedly washed to remove residual reagents and impurities. The mixture was then vacuum dried at 80 °C for 10 h to obtain the N-MLG@Ni-Mn LDH composite material.

[0058] (4) Preparation of modified electrode: 5 μL of 5 mg / mL N-MLG@Ni-Mn LDH suspension was drop-coated onto the surface of the pretreated SPE working electrode and dried at room temperature to obtain N-MLG@Ni-Mn LDH / SPCE modified electrode.

[0059] (5) Modification of cysteine: Dissolve 0.012g of L-cysteine ​​solution in 2mL of 0.1M acetate-sodium acetate (HAc-NaHc) buffer solution with pH=5 and sonicate to dissolve. Immerse the modified electrode prepared in step (4) in the solution and incubate at 4℃ in the dark for 12h. After rinsing and drying, N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode is obtained.

[0060] (6) Preparation of electrochemical sensor: Using N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode as modified working electrode, Ag / AgCl as reference electrode, and carbon electrode as counter electrode, an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material was assembled.

[0061] Example of implementation effect 1

[0062] The electrochemical performance of the N-MLG@Ni-Mn LDH / SPCE modified electrode, the N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode, and the bare SPE electrode prepared in Example 1 were tested, as follows:

[0063] The electrochemical performance of different modified electrodes was tested and investigated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), such as... Figure 3 As shown.

[0064] Figure 3 The CV curves of A show that the redox peak current of the N-MLG@Ni-Mn LDH / SPCE modified electrode is significantly higher than that of the bare SPE electrode, with a sharper peak shape. This is attributed to the synergistic effect of the high conductivity of nitrogen-doped multilayer graphene and the large specific surface area of ​​Ni-Mn LDH. However, the CV response of the N-MLG@Ni-Mn LDH / L-Cys / SPCE modified electrode is significantly weakened, possibly due to the cysteine ​​macromolecule hindering the contact between the probe and the active site. EIS testing (…) Figure 3 B) shows that the charge transfer resistance (Rct) of the bare SPE is approximately 2466 Ω, while the Rct of the N-MLG@Ni-Mn LDH / SPCE modified electrode and the N-MLG@Ni-Mn LDH / L-Cys / SPCE modified electrode decreases to 31 Ω and 69.38 Ω, respectively. This indicates that the synergistic effect of N-MLG and Ni-Mn LDH can reduce the charge transfer resistance at the electrode-electrolyte interface, while cysteine ​​modification introduces additional interfacial resistance, increasing Rct. This result is consistent with the conclusions of CV testing.

[0065] Example 2 of implementation results

[0066] The SWASV response of the electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1 was tested, as follows:

[0067] Copper ions were detected using square wave anodic stripping voltammetry (SWASV) with HAc-NaAc buffer as the electrolyte solution. The sample was first enriched at a constant potential of -0.8 V for 180 s, then allowed to stand for 1 min before square wave stripping scanning was performed in the range of -0.4 V to 0.6 V. Figure 4 The results showed that at the same concentration, the bare electrode had almost no dissolution peak, while the N-MLG@Ni-Mn LDH / SPCE modified electrode exhibited a significant dissolution peak at 0.228 V. This is attributed to its excellent charge transfer capability and abundant hydroxyl active sites. Further modification with cysteine ​​(Cys) resulted in Cu... 2+The peak current increased significantly, and the dissolution peak became sharper, indicating that the -SH group of Cys enhanced the dissolution effect on Cu. 2+ The chelation effect was observed. Results showed that the N-MLG@Ni-Mn LDH / L-Cys / SPCE modified electrode effectively enhanced the chelation effect on Cu. 2+ Detection performance.

[0068] Example of implementation effect 3

[0069] The electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1 was used to evaluate the performance of Cu at different concentrations. 2+ Detection

[0070] The results are as follows Figure 5 As shown, Cu produced by SWASV 2+ The dissolution current increases with increasing copper ion concentration. Furthermore, it can be observed that the dissolution peak potential gradually shifts to the right with increasing copper ion concentration. This may be because when Cu... 2+ As the concentration increases, Cu deposits on the electrode surface during the enrichment stage 0 The amount increases significantly, resulting in a thicker metal layer. This leads to increased Cu content during the dissolution process. 0 Overcoming the resistance of a thicker metal / solution interface is required, while localized Cu on the electrode surface is also necessary. 2+ Increased concentration causes the equilibrium potential of the oxidation reaction to shift to the positive direction.

[0071] Example of implementation effect 4

[0072] The performance evaluation of the electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1 is as follows:

[0073] 1. Selective assessment

[0074] Other interfering ions were added to a 0.1M HAc-NaHc buffer (pH=5) to simulate a real sweat environment. Cu in the buffer... 2+ The concentration was 100 ppb, and Mg was added at a molar concentration of 300 times. 2+ Zn 2+ Fe 3+ Ni 2+ Mn 2+ 50ppb Co 2 + Pb 2+ 50mM Na + 15mM K + And 20 mM lactate, 10 mM uric acid, 0.8 mg / mL glucose, and 0.1 mg / mL urea. Results are as follows... Figure 6As shown in Figure A, the selectivity of the substance was determined by calculating the ratio of the peak current with and without interfering substances. It was observed that the electrochemical sensor based on the N-MLG@Ni-Mn LDH / Cys composite material showed no significant change in peak current after the addition of interfering substances, with a relative standard deviation (RSD) of 6.87%. This indicates that the electrochemical sensor fabricated in this invention exhibits high selectivity and sensitivity for copper ion detection, meeting the anti-interference requirements in complex sweat environments.

[0075] 2. Reproducibility and repeatability assessment

[0076] The reproducibility and repeatability of sensors are also important indicators for evaluating sensor performance. Five sets of electrodes were prepared using the same method, and 100 ppb of Cu was tested in 0.1 M HAc-NaHc (pH=5) buffer solution. 2+ The SWASV response was used to investigate the reproducibility of the sensor. The results are as follows: Figure 6 As shown in Figure B, the peak current response of the five differently modified electrodes did not change significantly, with an RSD of 4.48%, indicating that the electrochemical sensor prepared in this invention has excellent reproducibility. Furthermore, the same electrode was repeatedly tested eight times, as shown in Figure B. Figure 6 As shown in Figure C, the peak current variation is extremely small, with a relative standard deviation (RSD) of only 2.8%. This indicates that the electrochemical sensor has excellent repeatability.

[0077] 3. Stability Assessment

[0078] Practical applications also require the sensor to maintain stability over long periods. The electrochemical sensor prepared in this invention was tested every five days for a total of 30 days. The results are as follows: Figure 6 As shown in Figure D, the peak current of the SWASV response of this sensor does not change significantly with increasing time, the RSD is 4.26%, and the relative standard error is also small. These results demonstrate that the electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in this invention exhibits good long-term stability and can meet the requirements for long-term storage in practical applications.

[0079] Application examples

[0080] The electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material prepared in Example 1 was used to detect Cu in actual samples. 2+ The testing details are as follows:

[0081] To evaluate the feasibility and reliability of the electrochemical sensor, it was tested in simulated sweat, and a standard addition method was used for quantitative copper analysis. First, the copper ion content in the simulated sweat was directly measured. Then, copper ion standard solutions of 50 ppb, 100 ppb, and 200 ppb were added to the simulated sweat, respectively. The corresponding dissolution currents were obtained using square wave anodic stripping voltammetry (SWASV). The peak dissolution current values ​​were then substituted into the linear regression equation to calculate the actual measured Cu. 2+ To improve detection accuracy, each sample was tested three times independently, and the average of the three results was taken as the concentration of Cu for that sample. 2+ The concentrations and results are shown in Table 1.

[0082] Table 1 Cu in artificial sweat 2+ Spiked recovery experiment

[0083] Table 1 shows that the RSD after spiked analysis ranged from 1.72% to 4.61%, and the recoveries ranged from 93.34% to 105.4%. These results demonstrate that the electrochemical sensor based on the N-MLG@Ni-Mn LDH / Cys composite material prepared in this invention exhibits reliability and accuracy in the analysis of actual sweat samples and can be effectively applied to the analysis of Cu in real sweat samples. 2+ Testing.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a working electrode for an electrochemical sensor based on N-MLG@Ni-Mn LDH / Cys composite material, characterized in that, Includes the following steps: (1) Pre-treat the SPE electrode; (2) After uniformly dispersing multilayer graphene in deionized water, urea was added, followed by hydrothermal reaction. After centrifugation, washing, and drying, N-MLG was obtained. (3) Disperse nickel nitrate hexahydrate and manganese nitrate tetrahydrate in deionized water to obtain solution I. After adjusting the pH of solution I, stir it evenly, then add N-MLG suspension and carry out hydrothermal reaction. After centrifugation, washing and drying, N-MLG@Ni-MnLDH composite material is obtained. (4) The N-MLG@Ni-Mn LDH suspension was drop-coated onto the pretreated SPE electrode and dried to obtain the N-MLG@Ni-MnLDH / SPE modified electrode; (5) The N-MLG@Ni-Mn LDH / SPE modified electrode obtained in step (4) is immersed in cysteine ​​solution and incubated in the dark to obtain the N-MLG / Ni-Mn LDH / L-Cys / SPCE modified electrode, which is the working electrode.

2. The preparation method according to claim 1, characterized in that: The pretreatment in step (1) includes immersing the SPE electrode in an APTES-ethanol solution, rinsing and drying it to obtain the pretreated SPE electrode. The volume concentration of APTES in the APTES-ethanol solution is 2-5%, and the immersion time is 30-120 min.

3. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of multilayer graphene to urea is 1:3, and the initial concentration of multilayer graphene is 2-5 mg / ml; the hydrothermal reaction temperature is 120-160℃ and the time is 3-5 h; the drying is vacuum drying at a temperature of 60-80℃ for 8-12 h.

4. The preparation method according to claim 3, characterized in that: In step (3), solution I contains Ni 2+ and Mn 2+ The molar ratio is 1-3:1, and the pH of solution I is 9-10; the concentration of the N-MLG suspension is 2-5 mg / mL, and the amount added is based on 10 mL of N-MLG suspension, corresponding to a total mass of 0.83 g of metal salt; the hydrothermal reaction temperature is 120-180℃, and the time is 12-18 h; the drying is vacuum drying, at a temperature of 60-80℃, and for 8-12 h.

5. The preparation method according to claim 4, characterized in that: In step (5), the concentration of the cysteine ​​solution is 2-10 mM, and the incubation time is 8-12 h.

6. An electrochemical sensor working electrode based on N-MLG@Ni-Mn LDH / Cys composite material prepared by the preparation method according to any one of claims 1-5.

7. An electrochemical sensor, comprising a working electrode, a reference electrode, and a counter electrode, characterized in that: The working electrode is the electrochemical sensor working electrode based on N-MLG@Ni-Mn LDH / Cys composite material as described in claim 6; the reference electrode is Ag / AgCl; and the counter electrode is a carbon electrode.

8. The application of the electrochemical sensor according to claim 7 in the detection of copper ions.

9. The application according to claim 8, characterized in that, The process includes the following steps: placing the electrochemical sensor according to claim 6 in a container containing different concentrations of Cu. 2+ In a buffer solution, different concentrations of Cu were collected. 2+ Electrochemical signals were analyzed, linear fitting curves were plotted, and Cu in the test sample was detected. 2+ The electrochemical signal is input into the fitting curve to obtain the Cu content in the sample. 2+ The concentration.

10. The application according to claim 9, characterized in that: The Cu 2+ The concentration range is 0.1-500 ppb, the buffer solution is an HAc-NaHc buffer solution with a concentration of 0.1 M and pH=5; the electrochemical signal is the dissolution current value obtained by square wave anodic stripping voltammetry; the fitting curve is Cu 2+ When the concentration is between 0.1-100 ppb, the fitted curve is Y = 0.48689X + 93.1936. When Cu... 2+ When the concentration is between 100-500 ppb, the fitted curve is Y=0.24184X+114.71996.

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