An enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs and its application

Abstract

This invention relates to an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs and its applications. The sensor includes a working electrode, a reference electrode, and a counter electrode. The working electrode is prepared using the following process: S1: AuCuNPs and multi-walled carbon nanotubes are dispersed in tetrahydrofuran under ultrasonication to obtain a homogeneous solution of AuCuNPs-MWCNTs; S2: Pretreatment of the Au electrode; S3: The homogeneous solution is dropped onto the Au electrode and dried to obtain an AuCu@MWCNTs / Au electrode. This enzyme-free biosensor utilizes both the electrocatalytic properties of AuCuNPs and the rapid electron transfer properties of MWCNTs to achieve ultra-high sensitivity detection of glucose and hydrogen peroxide. According to this invention, an enzyme-free biosensor with ultra-high sensitivity, good selectivity, excellent stability, and reproducibility has been constructed, showing great application potential in clinical testing.

Description

Technical Field

[0001] This invention relates to the field of sensors, and more specifically to an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs and its applications. Background Technology

[0002] Glucose is a key metabolic product and primary energy source for maintaining the function of living cells and the body's systems, providing energy for physiological processes. Diabetes has long been mistakenly considered a disease with little impact on global health; however, it is now becoming one of the major threats to human health in the 21st century. Therefore, monitoring blood glucose levels and their changes is of great significance for the prevention and treatment of diabetes.

[0003] Hydrogen peroxide is one of the superoxides produced by abnormal oxygen metabolism in mitochondria. It readily penetrates cells, inducing apoptosis or necrosis. As a carcinogen listed by the World Health Organization, the detection of hydrogen peroxide plays an important role in tumor development and anti-cancer treatment.

[0004] Electrochemical analysis is a convenient method for quantitative and qualitative measurement of substances in solution. Compared with other detection methods such as fluorescence, spectroscopy, and chromatography, electrochemical sensing has the advantages of low cost, accuracy, convenience, and reproducibility. Currently, common biosensors can generally be divided into two types: enzyme-based sensors and enzyme-free sensors that do not require additional enzyme assistance. Enzyme-based sensors are generally based on enzyme-catalyzed reactions, such as glucose dehydrogenase and superoxide dismutase, and are used in many fields. However, dependence on enzyme activity limits their long-term stability and application in real-world environments. To address these issues, enzyme-free electrochemical biosensors have emerged, attracting widespread attention due to their excellent long-term stability, high electrocatalytic activity, and ease of operation. Early enzyme-free bioelectrochemical sensors were typically based on metal nanoparticles such as PtNPs, PdNPs, and AuNPs, or metal oxides such as NiO, CuO, and Co3O4. However, existing electrochemical sensors generally suffer from low sensitivity, low selectivity, and unreliable detection results. Therefore, it is crucial to develop novel glucose sensors and hydrogen peroxide sensors that are highly sensitive, selective, and provide reliable detection results. Summary of the Invention

[0005] The purpose of this invention is to provide an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs and its application, thereby solving the problem of the lack of highly sensitive, highly selective and reliable biosensors in the prior art. At the same time, it provides a universal method for high-performance electrochemical biosensors based on composite nanomaterials of multi-metal nanoparticles and carbon.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] According to a first aspect of the present invention, an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs is provided, comprising a working electrode, a reference electrode, and a counter electrode. The working electrode is prepared using the following process: S1: Preparation of the composite nanomaterial: AuCuNPs and multi-walled carbon nanotubes are dispersed in tetrahydrofuran under ultrasonic treatment to obtain a homogeneous solution of the composite nanomaterial AuCuNPs-MWCNTs; S2: Pretreatment of the Au electrode: The Au electrode is pretreated on suede with particles of 1.0 μm, 0.1 μm, and 0.05 μm in diameter. After polishing, the alumina powder is ultrasonically cleaned in ethanol and ultrapure water, and dried with nitrogen for later use; S3: Preparation of AuCu@MWCNTs / Au electrode: The uniform solution prepared in step S1 is dropped onto the Au electrode pretreated in step S2 to form the conductive layer of the electrode, and finally dried at room temperature to obtain AuCu@MWCNTs / Au electrode; wherein, the enzyme-free biosensor can achieve high-sensitivity detection of glucose and hydrogen peroxide by utilizing the electrocatalytic performance of AuCuNPs and the rapid electron transfer performance of MWCNTs.

[0008] According to a preferred embodiment of the present invention, the preparation of the composite nanomaterial AuCuNPs-MWCNTs in step S1 includes the following steps: A1: Synthesizing Au nanoparticles and dispersing them in hexane: HAuCl4·3H2O, o-xylene, and oleylamine are mixed to obtain an orange precursor solution, which is stirred in argon at 8-12°C for 10-15 minutes; then a clear solution A obtained by mixing tetrabutylammonium bromide, o-xylene, and oleylamine under ultrasonication is added, and the solution turns dark purple after a reduction reaction; then the mixed solution is stirred at 8-12°C for 1-2 hours; the solution obtained from the reaction is dissolved in ethanol, and Au nanoparticles are obtained by centrifugation, wherein the mass-to-volume ratio of HAuCl4·3H2O, o-xylene, and oleylamine is 100 mg:10 mL:10 mL; the molar volume ratio of tetrabutylammonium bromide, o-xylene, and oleylamine is 40 mg:1 mL:1 mL; the volume ratio of the precursor solution to the clear solution A is 2:1; A2: Synthesizing atomically ordered Au uCuNPs: A mixed solution B of copper acetate monohydrate, oleic acid, and trioctylamine was heated and then injected into a hexane solution of Au nanoparticles obtained in step A1. After the hexane was completely evaporated, the mixed solution was heated in argon at 110–130°C for 20–30 minutes. Then, before cooling to room temperature, the solution was rapidly heated to 190–210°C and reacted for more than 50 minutes. AuCuNPs were then collected by centrifugation. The mass-to-volume ratio of the mixed solution of copper acetate monohydrate, oleic acid, and trioctylamine was [not specified]. The ratio of the clarified solution B to the hexane solution of Au nanoparticles obtained in step A1 is 43 mg: 0.25 mL: 1.125 mL, and the volume ratio of the clarified solution B to the hexane solution of Au nanoparticles obtained in step A1 is 1.375 mL: 20 mL; A3: AuCuNPs and multi-walled carbon nanotubes are dispersed in tetrahydrofuran under ultrasonic treatment to obtain a homogeneous solution of AuCuNPs-multi-walled carbon nanotube composite material, wherein the mass-volume ratio of AuCuNPs, multi-walled carbon nanotubes and tetrahydrofuran is 1 mg: 0.95 mg: 1.2 mL.

[0009] In step A2, to ensure the correct synthesis of AuCuNPs, the Cu / Au atomic ratio is 1:1.

[0010] The reference electrode is a calomel electrode, the counter electrode is a platinum wire electrode, and the electrolyte is a 0.1M alkaline solution with pH 12, selected from NaOH solution and KOH solution.

[0011] According to a second aspect of the present invention, an application of an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs in glucose detection is provided, comprising: providing an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs as described above, continuously adding glucose solutions of different concentrations to a detection system, using a chronoamperometry method to plot a current-time response curve based on the current response, and obtaining a standard curve of current change with glucose concentration; when a test solution containing glucose is added, by detecting the electrical signal and substituting it into the standard curve, the glucose concentration in the test solution can be obtained, thereby realizing the detection of glucose concentration in an enzyme-free system by the enzyme-free biosensor.

[0012] Preferably, the measuring voltage is selected as 0.40V.

[0013] In the aforementioned application, the enzyme-free biosensor has a sensitivity of 3.62 × 10⁻⁶ for glucose. 2 μA·mM -1 ·cm -2 The linear range is 8.0 × 10 -3 ~6.0×10 4 μM, detection limit is 1.3×10 -3 μM, S / N = 3.

[0014] According to a third aspect of the present invention, an application of an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs in the detection of hydrogen peroxide is provided, comprising: providing an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs as described above; continuously adding H2O2 solutions of different concentrations to a detection system; using a chronoamperometry method to plot a current-time response curve based on the current response; and obtaining a standard curve of current change with H2O2 concentration; when a test solution containing H2O2 is added, the concentration of H2O2 in the test solution can be obtained by detecting the electrical signal and substituting it into the standard curve, thereby realizing the detection of H2O2 concentration by the enzyme-free biosensor in an enzyme-free system. Finally, the sensor is co-incubated with HepG2 cells to verify its effectiveness in detecting trace amounts of H2O2 released by the cells.

[0015] Preferably, the measurement voltage is selected as -0.40V.

[0016] In the aforementioned application, the sensitivity of the enzyme-free H2O2 sensor is 1.335 × 10⁻⁶. 3 μA·mM -1 ·cm -2 The linear range is 1.0 × 10⁻⁶. -3 ~4.0×10 3 μM, detection limit is 1.0 × 10-4 μM, S / N = 3.

[0017] In the application described, in 50 ml, approximately 5 × 10 6 After adding 0.3 μM CHAPS to a solution of HepG2 cells, the H2O2 concentration measured by this biosensor was consistent with the results of a commercial hydrogen peroxide kit. When 5 nM H2O2 was added to the HepG2 cell sample, the recovery rate of the enzyme-free H2O2 sensor was 86.7%.

[0018] According to the inventors' previous research, the combination of Au and Cu can improve catalytic performance while reducing the high cost of Au and the instability of Cu. With increasingly stringent requirements for glucose and peroxide detection, more and more novel composite nanomaterials are being developed and applied to biosensors. For example, noble metal nanoparticles coupled to the surface of carbon materials are attracting increasing attention due to their synergistic effects.

[0019] Based on this, the inventors envisioned that coupling materials of AuCuNPs and MWCNTs might be better candidate materials for electrochemical sensors of glucose and H2O2. Therefore, the inventors successfully improved the catalytic activity of AuCuNPs by adjusting the atomic arrangement and specific surface area of ​​the particles, and for the first time developed a highly sensitive and selective biosensor based on a composite material of AuCuNPs and MWCNTs (AuCu@MWCNTs). It should be understood that the biosensor provided according to this invention can measure not only glucose and hydrogen peroxide, but also any other redox substances, such as ascorbic acid, dopamine, and reduced coenzyme I (NADH).

[0020] This invention utilizes X-ray diffraction (XRD) and transmission electron microscopy (TEM) to characterize the structure and morphology of the prepared materials. The electrochemical performance of AuCuNPs, MWCNTs, and AuCu@MWCNTs was compared using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Furthermore, the detection performance of the AuCu@MWCNTs-based electrode for glucose and hydrogen peroxide was evaluated using CV and chronoamperometry (CA). The synergistic effect resulting from the coupling of AuCuNPs and MWCNTs endows the sensor with excellent sensing performance. This simple, sustainable, and scalable synthetic strategy can be used to design other high-performance nanocomposites, thereby expanding the application of nanomaterials in electrochemistry.

[0021] The key inventive point of this invention lies in the fabrication of a novel, high-performance enzyme-free biosensor using a nanocomposite material of ordered AuCu nanoparticles and MWCNTs. Most importantly, because this invention synthesizes lattice-ordered AuCu nanoparticles, which have a large specific surface area and fast ion-electron transfer rate, it achieves the most sensitive glucose and hydrogen peroxide sensor to date. Compared to AuCuNPs and MWCNTs alone, the coupling of AuCuNPs and MWCNTs gives the material faster electron transport and higher capacitance. Cyclic voltammetry results show that the enzyme-free glucose sensor based on AuCu@MWCNTs exhibits excellent sensitivity and selectivity. Simultaneously, the electrode's ability to quantitatively analyze glucose was detected using amperometric methods; the response curve shows that the electrode has a short response time (<5 s) and high sensitivity (362.3 μA·mM). -1 ·cm -2 It possesses numerous advantages, including a wide linear range (8 nM to 60 mM) and a low detection limit (1.3 nM, S / N = 3). Furthermore, the enzyme-free glucose electrode constructed in this invention exhibits good selectivity and repeatability. In serum sample detection, the excellent recovery rate demonstrates the accuracy and feasibility of the prepared sensor in detecting glucose in real samples. Simultaneously, this invention also used amperometric methods to detect the electrode's ability to quantitatively analyze H2O2; the response curve shows that this hydrogen peroxide sensor has high sensitivity (1335 μA·mM). -1 ·cm -2 The sensor exhibits numerous advantages, including a wide linear range (1 nM to 4 mM) and a low detection limit (0.1 nM, S / N = 3). In cell sample detection, the excellent recovery rate demonstrates the accuracy and feasibility of the prepared sensor in detecting hydrogen peroxide in real samples. Satisfactorily, the detection limit for hydrogen peroxide is 0.1 nM, representing the highest sensitivity to date. The enzyme-free biosensor successfully detected glucose concentration in serum samples and can also monitor the instantaneous concentration changes of trace amounts of hydrogen peroxide released from cell samples under drug stimulation. The detection results from these applications are consistent with those of commercial kits.

[0022] In summary, this invention presents the first novel enzyme-free biosensor based on a composite nanomaterial of AuCuNPs and MWCNTs. The electrode itself possesses the ability to electrocatalyze glucose, enabling glucose detection without the need for glucose oxidizing or reductizing enzymes. The electrochemical performance of the AuCu@MWCNTs electrode was analyzed using electrochemical impedance spectroscopy (EIS), revealing a low charge transfer impedance on the electrode surface, indicating rapid electron transfer—a crucial factor for highly sensitive detection. The catalytic mechanism and detection performance of the electrode were investigated using cyclic voltammetry (CV) and chronoamperometry (CA). The enzyme-free sensor constructed in this invention exhibits excellent selectivity, stability, and reproducibility in both glucose and H2O2 detection. The development of this enzyme-free sensor provides a general approach for developing high-performance electrochemical sensors based on multi-metal nanoparticles and carbon nanomaterials. According to the present invention, a universal enzyme-free biosensor with ultra-high sensitivity, good selectivity, excellent stability and reproducibility has been constructed. This sensor can also be applied to the detection of any other redox substances, including ascorbic acid, dopamine and reduced coenzyme I (NADH), and has great application potential in clinical testing. Attached Figure Description

[0023] Figure 1 The following are shown: (A) transmission electron microscopy analysis (scale bar 50 nm), (B) particle size statistical analysis and (C) XRD diffraction analysis of gold-copper bimetallic nanoparticles;

[0024] Figure 2 The morphology of AuCu@MWCNTs under transmission electron microscopy is shown;

[0025] Figure 3 The following are CV plots of (A) AuCu@MWCNTs / Au electrode at different scan rates in a 0.1M KCl solution containing 5mM K3[Fe(CN)6] (scan rates from top to bottom are 10, 25, 50, 75, 100, 125, 150, 175, 200 and 300 mV·s). -1 (B) and the corresponding peak current versus sweep rate curves;

[0026] Figure 4 The CV plots of AuCu@MWCNTs / Au electrode, MWCNTs / Au electrode, and bare gold electrode in 0.1M NaOH solution in the presence of 1mM glucose are shown separately (scan rate: 75mV·s). -1 Voltage variation range: -1.0V to +0.6V;

[0027] Figure 5The Nyquist plots of AuCu@MWCNTs / Au and MWCNTs / Au electrodes are shown (frequency range: 0.01Hz-100kHz, initial voltage: 0.4V, voltage variation amplitude: 10mV);

[0028] Figure 6 The current response of the AuCu@MWCNTs / Au electrode to continuously added 0.2 mM glucose (0.1 M NaOH) at different operating potentials (0.35, 0.40, 0.45 and 0.50 V) is shown.

[0029] Figure 7 The following figures are shown: (A) Current intensity variation of AuCu@MWCNTs / Au electrode at different glucose concentrations (0.1M NaOH); (B) Linear relationship between current intensity and glucose concentration from 8 nM to 60 mM (voltage: 0.40 V);

[0030] Figure 8 The current response of AuCu@MWCNTs / Au in 0.1M NaOH (voltage: 0.40V) to different substances (glucose, urea, sucrose, fructose, uric acid and glucose) is shown.

[0031] Figure 9 The current response of AuCu@MWCNTs / Au electrode to continuously added 0.2mM H2O2 (0.1M PB) at different operating potentials (-0.35, -0.40, -0.45 and -0.50V) is shown.

[0032] Figure 10 The following figures are shown: (A) Current intensity variation of AuCu@MWCNTs / Au electrode under different H2O2 concentrations (0.1 MPa); (B) Linear relationship between current intensity and H2O2 concentration from 1 nM to 4 mM (voltage: -0.40 V);

[0033] Figure 11 The amperometric response of AuCu@MWCNTs / Au in 0.1M PB (pH=7) (voltage: -0.40V) to HepG2 cells, 0.3μM CHAPS protein lysis buffer, HepG2 cells with added 0.3μM CHAPS protein lysis buffer, and HepG2 cells with continuously added 5nM H2O2 is shown. Detailed Implementation

[0034] Experimental materials

[0035] Copper acetate monohydrate (98%), oleylamine (70%), borane tert-butylamine complex (TBAB, 97%), urea (C3H8N2O2), potassium ferricyanide (K3[Fe(CN)6]), n-hexane, and o-xylene were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Sodium hydroxide (NaOH) and glucose (C6H2O) were also present. 12 O6), sucrose, fructose, and uric acid (C5H4N4O3) were purchased from Shanghai Aladdin Co., Ltd. Tetrachloroauric acid trihydrate (99%, Au: 50%), trioctylamine (98%), and oleic acid (90%) were purchased from Shanghai Adamas Reagent Co., Ltd. Sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4), and sodium dihydrogen phosphate (NaH2PO4) were purchased from Shanghai Maclean Co., Ltd. Glucose oxidase and goat serum were purchased from Guangzhou Solarbio Biotechnology Co., Ltd. Hydrogen peroxide (30%) was purchased from Shanghai General Reagent Co., Ltd. 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid hydrate (CHAPS, 99%) was purchased from Beijing Solarbio Technology Co., Ltd. Multi-walled carbon nanotubes (MWCNTs, purity >97%, diameter 10–20 nm, length 1–2 μm) were purchased from Shenzhen Nanotechnology Co., Ltd., China, requiring no further purification or modification.

[0036] Example 1: Synthesis and Characterization of AuCuNPs

[0037] Previous reports have indicated that increasing the degree of order in the internal structure can enhance the catalytic activity of AuCuNPs; therefore, the AuCuNPs used in this experiment are all ordered lattices.

[0038] 1.1 Synthesis of AuCuNPs

[0039] The synthesis of AuCuNPs involves two steps: pre-synthesis of AuNPs; and diffusion of newly generated Cu atoms into the AuNPs.

[0040] 1) Pre-synthesis of AuNPs: 200 mg HAuCl4·3H2O, 20 mL o-xylene, and 20 mL oleylamine were mixed in a 250 mL three-necked flask to obtain an orange precursor solution, which was stirred at 10 °C for 10 min under argon protection. 80 mg TBAB, 2 mL o-xylene, and 2 mL oleylamine were ultrasonically mixed, and the resulting milky white clear solution was injected into the orange precursor solution. The reduction reaction occurred immediately, and the color of the mixed solution rapidly turned deep purple. After stirring the solution at 10 °C for 1 hour, a black solution was obtained, which was dissolved in anhydrous ethanol. AuNPs were collected by centrifugation at 10,000 rpm for 10 min and washed three times with anhydrous ethanol. Finally, the precipitate was dispersed in 20 mL n-hexane.

[0041] 2) Diffusion of newly generated Cu atoms into AuNPs and their ordered arrangement via high temperature: 86 mg of copper acetate monohydrate, 0.5 mL of oleic acid, and 2.25 mL of trioctylamine were mixed in a 50 mL three-necked flask to obtain a clear solution. After reacting at 80 °C for 30 minutes, the solution was injected into the AuNPs prepared in step 1). The solution was heated and stirred at 80 °C until hexane completely evaporated, at which point the solution turned deep purple. Under argon protection, the solution was stirred at 120 °C for 20 minutes. Before cooling to room temperature, the solution was poured into a reaction vessel and rapidly heated to 250 °C for 1 hour. The resulting black solution was dissolved in anhydrous ethanol, and the AuCuNPs were collected by centrifugation (10,000 rpm, 10 minutes). The product was washed three times with anhydrous ethanol and then dried in a vacuum drying oven at 50 °C for later use.

[0042] According to this method, gold nanoparticles (AuNPs) were first synthesized as seeds. Then, newly generated Cu atoms were added to the AuNPs solution, and the Cu atoms dispersed into the AuNPs seeds to form AuCuNPs. To ensure the correct synthesis of AuCuNPs, the Cu / Au atom ratio was 1:1. During the subsequent heating process, the reduction reaction and the copper diffusion reaction occurred simultaneously on the gold surface, and the prolonged high-temperature environment provided sufficient thermal energy for the formation of an ordered crystal lattice. After the high-temperature reaction, a black viscous liquid was generated. The black powder obtained after dissolving the liquid in ethanol and centrifuging was the AuCuNPs.

[0043] 1.2 Characterization of AuCuNPs

[0044] In this embodiment, the morphological characteristics of the synthesized AuCuNPs were observed using transmission electron microscopy, such as... Figure 1 As shown in Figure A, AuCuNPs are spherical with an average particle size of approximately 7 nm. Furthermore, the particle size distribution is shown in the figure below. Figure 1 As shown in Figure B, the particle size of AuCuNPs is mainly concentrated in the range of 6–10 nm. The smaller particle size provides a larger specific surface area, thereby enhancing the catalytic activity of the nanoparticles.

[0045] To characterize AuCuNPs, XRD analysis was also performed in this embodiment. The results are as follows: Figure 1As shown in Figure C, the peak position (2θ value) of the 111 diffraction of the synthesized nanoparticles is at 41.34°, which is between 38.26° for pure Au and 43.29° for pure Cu. The peak position of the 200 diffraction is 48.46°, which is between 44.6° for pure Au and 50.43° for pure Cu, confirming the successful formation of the bimetallic system. Data for pure face-centered cubic Au (JCPDS 04-0784) and pure face-centered cubic Cu (JCPDS 04-0836) are from the Joint Committee on Powder Diffraction Standards (JCPDS). Furthermore, a superlattice peak appears at 201 (56.12°), which is absent in disordered face-centered cubic structures; in addition, there are obvious splitting peaks at 200 (48.46°) and 220 (79.26°), all indicating that the structure of AuCuNPs has transformed into an ordered face-centered cubic structure.

[0046] All the above results prove that the synthesis of ordered AuCuNPs was successfully completed in this embodiment.

[0047] Example 2: Electrode Preparation

[0048] The gold electrode (inner diameter 1.6 mm, outer diameter 6.35 mm) was polished sequentially on chamois leather with alumina powders of 1.0 μm, 0.1 μm, and 0.05 μm particle sizes, and then ultrasonically cleaned for 10 minutes each in ethanol and ultrapure water. The surface of the Au electrode was then purged with nitrogen to remove residual moisture and dust. The cleaned gold electrode was then placed in a desiccator overnight for subsequent experiments.

[0049] AuCu@MWCNTs / Au and MWCNTs / Au electrodes were prepared using a solvent casting method. To obtain the composite nanomaterial AuCu@MWCNTs, 1 mg of AuCuNPs and 0.95 mg of MWCNTs were dispersed in 1.2 mL of tetrahydrofuran under ultrasonic treatment to obtain a homogeneous solution until no obvious particles were observed in the system. Then, 100 μL of the homogeneous solution was dropped onto a clean bare gold electrode as a conductive layer, and finally dried overnight at room temperature to obtain the AuCu@MWCNTs / Au electrode. For comparison, a MWCNTs / Au electrode was also prepared. 0.95 mg of MWCNTs was dispersed in 1.2 mL of tetrahydrofuran under ultrasonic treatment to obtain a conductive layer. 100 μL of the conductive layer was dropped onto a clean bare gold electrode and dried overnight at room temperature to obtain the MWCNTs / Au electrode.

[0050] It should be understood that this embodiment is similar to the electrode preparation method in the prior patent (CN201811589008.0), but there are still obvious differences. One difference is that the preparation of the AuCu@MWCNTs composite nanomaterial of this invention does not use Pluronic F127 compared to the prior patent. This is because research in this invention has shown that adding Pluronic F127 significantly degrades the sensor's response performance, while removing it yields excellent electrochemical sensing performance. This may be because hydrophilic substances are unsuitable as sensing coatings for electrodes. Therefore, the electrode prepared according to the prior patent does not have the same sensing effect as this invention. Secondly, the prior patent uses a small section of PVC tubing over the electrode and further covers the composite nanomaterial layer with an ion-selective electrode sensing membrane (ISM). Thirdly, the two differ in their reaction mechanisms. This invention utilizes the electrocatalytic performance of AuCuNPs in the composite nanomaterial to detect glucose and H2O2, involving redox reactions, with MWCNTs primarily functioning as rapid electron transfer. The prior patent, however, utilizes the combined functions of AuCuNPs-MWCNTs, such as high capacitance and fast ion-electron transfer rate, without involving any redox reactions.

[0051] Example 3 Characterization of AuCuNPs and AuCu@MWCNTs

[0052] The morphological characteristics of AuCuNPs and AuCu@MWCNTs were observed using a JEOL 2100F (Japan) 200kV transmission electron microscope (TEM), and the particle size of AuCuNPs was statistically analyzed using ImageJ software. The X-ray diffraction (XRD) patterns of AuCuNPs were obtained using a RINT2000 vertical goniometer (Rigaku, Japan) under CuKα irradiation (λ = 0.15418 nm).

[0053] AuCuNPs and MWCNTs were dispersed in 1.2 mL of tetrahydrofuran under ultrasonic treatment to obtain AuCu@MWCNTs, and their morphological characteristics were observed by transmission electron microscopy. Figure 2 As shown, AuCuNPs were successfully coupled with MWCNTs, and the dispersion and adhesion effects were good.

[0054] Example 4: Characterization of the Bioelectrochemical Sensor

[0055] All electrochemical measurements were performed on a CHI 760D electrochemical workstation (Shanghai, Huachen). All electrochemical measurements used a three-electrode system, with a platinum wire as the auxiliary electrode, a calomel electrode (with saturated potassium chloride solution as the internal reference) as the reference electrode, and a prepared electrode as the working electrode.

[0056] The AuCu@MWCNTs / Au electrode or MWCNTs / Au electrode prepared in Example 2 were used as working electrodes, and electrochemical impedance spectroscopy (EIS) analysis of the above electrodes was performed in 0.1M CaCl2 at a frequency of 0.01 to 105 Hz. The detection potential was +0.40 V and the potential change was 10 mV.

[0057] 4.1 Scan Rate Analysis

[0058] The surface electrochemical kinetics of AuCu@MWCNTs / Au electrodes were analyzed in a 0.1M KCl solution with 5mM K3[Fe(CN)6] added, where K3[Fe(CN)6] was used to maintain the ionic strength in the solution. Figure 3 In the figure, A represents the CV curves of the AuCu@MWCNTs / Au electrode obtained at a series of scan rates, starting from 10 mV·s. -1 Increased to 300 mV·s -1 At that time, the corresponding redox peak current intensity also increases. Figure 3 Figure B illustrates the linear relationship between scan rate and peak current intensity. As shown in the figure, both the oxidation and reduction peak current intensities increase with increasing scan rate, with linear correlation coefficients of 0.992 and 0.997, respectively. The linear relationship between scan rate and anodic peak current intensity indicates a quasi-reversible surface-controlled process. Furthermore, due to the important role of electrode kinetics, a slight shift in the positive values ​​of the anodic peak and the negative values ​​of the cathode peak can be observed with changes in scan rate. This suggests that the electrochemical kinetics of the fabricated sensor is a diffusion-controlled process.

[0059] 4.2 Cyclic Voltmeter-Ammeter Analysis

[0060] To determine the electrocatalytic activity of AuCu@MWCNTs composite nanomaterials for glucose in an enzyme-free environment, cyclic voltammetry (CV) analysis was performed. During the experiment, the potential scan is performed in one direction and immediately reversed to the other; this process can be single or multiple cycles. This method, known as cyclic voltammetry, is one of the most commonly used electroanalytical methods. By analyzing the presence, shape, and position of redox peaks in the cyclic voltammetric curves, possible redox reactions in the system can be investigated.

[0061] The reaction background of the enzyme-free system is an alkaline solution with pH=12, such as 0.1M NaOH or KOH solution. After the addition of glucose, electrochemical characterization can be performed directly. After adding glucose standard solutions of different concentrations to the measurement system in sequence, the electrical signal can be measured immediately.

[0062] To compare the electrocatalytic performance of AuCu@MWCNTs and MWCNTs, this example investigated the cyclic voltammetry of AuCu@MWCNTs / Au, MWCNTs / Au, and bare gold electrodes in a 0.1 M NaOH background solution in the presence of 1 mM glucose. Since the three electrodes were prepared using similar methods, the change in current peak value can be considered to be solely related to the electrochemically active surface, thus directly inferring the difference in electrocatalytic performance between AuCu@MWCNTs and MWCNTs. The CVs of the AuCu@MWCNTs / Au, MWCNTs / Au, and bare gold electrodes were approximately 75 mV·s⁻¹. -1 The scan rate was recorded, and the voltage variation range was determined by the position of the redox peak from -1.0V to +0.6V. The CV results for the AuCu@MWCNTs / Au electrode are as follows: Figure 4 As shown by the dotted line, the anodic peak (peak I) at -0.15V represents the oxidation of Cu(0) to Cu(I) and the oxidation of Cu(0) to Cu(II). Furthermore, at the anodic peak (peak II) at 0.23V, oxidation reactions of Au(0) to Au(I) and Cu(II) to Cu(III) occur. At a potential of 0.43V (peak III), the oxidation of Cu(II) to Cu(III) occurs, where Cu(III) is considered the electron transfer medium and can assist in the oxidation of glucose. In alkaline solutions, when the applied potential is greater than 0V, Au-OH is generated. Au-OH can help oxidize the electroadsorption-derived intermediates of glucose. In this process, it generates free active Au sites, directly oxidizing glucose and producing an oxidation current peak at 0.2–0.4V. In the negative scan of the electrode, two distinct reduction peaks were observed at -0.12 V (peak IV) and -0.39 V (peak V), which are attributed to the conversions of Au(I) / Au(0) and Cu(II) / Cu(I), respectively. The cathode peak VI at -0.77 V in the negative scan can be attributed to the reduction of Cu(I) to Cu(0). Furthermore, compared with the MWCNTs / Au electrode (dashed line), the AuCu@MWCNTs / Au electrode (short dotted line) exhibits a higher current intensity, which implies better electrocatalytic performance and a faster electron transfer rate.

[0063] 4.3 Electrochemical Impedance Analysis

[0064] In this embodiment, the impedance spectra of gold electrodes with AuCu@MWCNTs and MWCNTs as sensing layers were studied respectively. The Nyquist plots of the AuCu@MWCNTs / Au electrode and the MWCNTs / Au electrode were recorded with AC current at open-circuit voltage in the frequency range of 0.01Hz to 100kHz.

[0065] Figure 5Impedance spectra of two electrodes in 0.1 M NaCl with a voltage variation of 5 mV are shown. The corresponding fitted equivalent circuit is as follows. Figure 5 As shown. The circuit consists of a solution resistor (R). s ), charge transfer resistance (R) on the electrode surface ct ) and double-layer capacitors (C dl It consists of a parallel combination of R and a constant phase element (CPE). s R represents the internal resistance of the electrode. ct The difficulty of ion-electron transfer at the Au electrode interface reflects the magnitude of resistance to electrochemical reactions occurring at the electrode and electrolyte surfaces. (This is in contrast to MWCNTs(R...)) ct =57.0Ω) compared to AuCu@MWCNTs(R ct =45.3Ω) of R ct A lower value indicates faster electron transport. Double-layer capacitance C dl This is related to electrode stability. Another important parameter for determining the sensing capability of a sensor is capacitance, which is related to the ion-electron transduction rate of the functional nanomaterial coated electrode. From Figure 5 It can be seen that the imaginary part (-Z") of the impedance of the AuCu@MWCNTs / Au electrode is much lower than that of the MWCNTs / Au electrode, indicating that AuCu@MWCNTs has a higher capacitance than MWCNTs. The low-frequency capacitance can be calculated using the formula C = 1 / (2πfZ"), where f is the frequency and Z” is the imaginary part of the impedance. According to this equation, the calculated capacitance of AuCu@MWCNTs is 114.7 μF, which is 6.6 times that of MWCNTs (17.3 μF). Therefore, it can be concluded that, due to the synergistic effect, the coupling of AuCuNPs and MWCNTs greatly improves the electron transfer and ion-electron transduction capabilities of the materials.

[0066] 4.4 Evaluation of the response performance of the enzyme-free biosensor to glucose

[0067] Previous cyclic voltammetry (CV) analyses demonstrated the excellent electrocatalytic activity of AuCu@MWCNTs / Au in alkaline solutions. Chronoamperometry has been reported to offer higher sensitivity and selectivity under hydrodynamic conditions compared to cyclic voltammetry; therefore, this example utilizes chronoamperometry, employing the AuCu@MWCNTs / Au electrode to determine the glucose concentration in an enzyme-free system. First, the electrode's response to the same glucose concentration at different voltages was examined to select the optimal voltage for subsequent experiments. 0.2 mM glucose was continuously added under stirring conditions, and the corresponding current response was recorded.

[0068] Figure 6The current response of the AuCu@MWCNTs / Au electrode under continuous addition of 0.2 mM glucose to 0.1 M NaOH and at different operating potentials (0.35, 0.40, 0.45, and 0.50 V) was demonstrated. The current response decreased in the potential range of 0.40 to 0.50 V and increased in the potential range of 0.35 to 0.40 V, with the best current response at 0.40 V. Therefore, a constant potential of 0.40 V was used as the optimal potential for subsequent enzyme-free glucose quantification experiments.

[0069] Figure 7 Figure A shows the current-time curves of the AuCu@MWCNTs / Au electrode with different concentrations of glucose solution in 0.1M NaOH continuously added under continuous stirring, with a selected measurement voltage of 0.40V. Figure 7 As shown in Figure A, even in the presence of only 1.3 nM glucose, the prepared electrode achieved a stable current response within 5 seconds. Within a concentration range of 8 nM to 100 mM, the current intensity increased with increasing glucose concentration. Furthermore, over a wide range from 8 nM to 60 mM, the current intensity of the electrode exhibited a linear correlation with the glucose concentration in the system (R0). 2 =0.997)(e.g. Figure 7 (As shown in B in the figure). Meanwhile, the AuCu@MWCNTs / Au electrode exhibits higher sensitivity (362.3 μA·mM). -1 ·cm -2 The results demonstrate that, due to the synergistic effect of AuCuNPs and MWCNTs, the AuCu@MWCNTs / Au electrode exhibits excellent sensitivity, selectivity, and detection limit for enzyme-free glucose detection.

[0070] 4.5 Selectivity and Reproducibility Analysis

[0071] To investigate the selectivity of the AuCu@MWCNTs / Au electrode, this embodiment detected changes in the electrode's electrical signal after adding glucose (1 mM), urea (1 mM), sucrose (1 mM), fructose (1 mM), and uric acid (0.1 mM) to the background solution.

[0072] The ability to distinguish between target and interfering substances is a key factor in determining the quality of a sensor. Therefore, in this embodiment, the selectivity of the AuCu@MWCNTs / Au electrode was tested in 0.1M NaOH and 0.1M PB solutions, respectively. First, the electrode's anti-interference ability in an enzyme-free system was tested. The following substances—glucose, urea, sucrose, fructose, uric acid, and glucose—were added sequentially to a 0.1M NaOH background solution. The results are as follows: Figure 8As shown, a significant increase in current intensity was observed after the addition of glucose. However, no change in current intensity was observed after the addition of urea, sucrose, fructose, and uric acid. These results demonstrate the excellent selectivity of the AuCu@MWCNTs / Au electrode, a crucial prerequisite for its ability to perform practical sample analysis.

[0073] The reproducibility of a single electrode was calculated by recording five consecutive current responses of the same electrode to 1 mM glucose in a background solution. The relative standard deviations (RSDs) for the same electrode in the enzyme-free system were 2.9% and 2.5%, respectively. Five parallel electrodes were prepared to evaluate the inter-electrode reproducibility of the AuCu@MWCNTs / Au electrode for the determination of 1 mM glucose in 0.1 M NaOH or 0.1 M PB solution. Measurements were performed on five different AuCu@MWCNTs / Au electrodes under the same conditions, and the current responses were recorded; the calculated RSDs were 2.2% and 2.3%, respectively. These results demonstrate that the enzyme-free glucose sensor based on AuCu@MWCNTs / Au exhibits good single-electrode and inter-electrode reproducibility.

[0074] 4.6 Serum Sample Analysis

[0075] In the actual sample analysis, the standard addition method was used. First, 0.5 mL of goat serum was added to 50 mL of NaOH solution (0.1 M). Then, different concentrations of glucose standard solution were continuously added to the above system to obtain the current response of glucose concentration in the serum sample. The recovery rate was calculated based on the standard curve in the enzyme-free system. The glucose concentration of the original goat serum was detected using a glucose concentration detection kit (Applygen, China).

[0076] To verify the feasibility of the enzyme-free biosensor invented in this experiment in the analysis of actual samples, goat serum dilution was used as the reaction background. Glucose was continuously added using the standard addition method, and the electrode response was recorded. First, 0.5 mL of goat serum was injected into 50 mL of 0.1 M NaOH, and AuCu@MWCNTs / Au was used as the working electrode for electrochemical detection. After adding 0.5 mL of goat serum, the change in electrode current was not significant, indicating that the glucose content in the goat serum was extremely low, which is consistent with the results obtained using a glucose detection kit. Then, different volumes of 1 M glucose solution were added to the detection system to achieve final glucose concentrations of 1 mM, 1.5 mM, and 2.5 mM, and the current intensity was recorded. Substituting the current intensities into the previously prepared standard curve, the recoveries of the target analyte were obtained as 103.8%, 95.7%, and 97.2%, respectively. The results demonstrate that AuCu@MWCNTs / Au exhibits good glucose detection capability even in real samples with complex backgrounds.

[0077] Table 2. Glucose recovery rate in real samples determined by AuCu@MWCNTs / Au electrode (n=3)

[0078]

[0079] 4.7 Evaluation of the response performance of the enzyme-free biosensor to H2O2

[0080] Previously, chronoamperometry was used to characterize the response of the AuCu@MWCNTs / Au electrode to different concentrations of glucose in an enzyme-free system. To demonstrate the versatility of this enzyme-free system design, this example also uses chronoamperometry to determine the H2O2 concentration in the enzyme-free system using the AuCu@MWCNTs / Au electrode. First, the electrode's response to the same concentration of H2O2 at different voltages was examined to select the optimal voltage for subsequent experiments. 0.2 mM H2O2 was continuously added under stirring conditions, and the corresponding current response was recorded.

[0081] Figure 9 The current response of the AuCu@MWCNTs / Au electrode under continuous addition of 0.2mM H2O2 to 0.1M PB and at different operating potentials (-0.35, -0.40, -0.45, and -0.50V) was demonstrated. A constant potential of -0.40V was used as the optimal potential for subsequent enzyme-free H2O2 quantification experiments.

[0082] Figure 10 Figure A shows the current-time curves of the AuCu@MWCNTs / Au electrode with different concentrations of H2O2 solution continuously added to 0.1M PB under continuous stirring, with a selected measurement voltage of -0.40V. The results indicate that AuCu@MWCNTs / Au can respond to H2O2 concentrations above 0.1 nM. Figure 10 As shown in Figure A, within the concentration range of 8 nM to 10 mM, the current intensity decreases with increasing H₂O₂ concentration. Furthermore, over a wide range from 1 nM to 4 mM, the electrode current intensity is linearly correlated with the H₂O₂ concentration in the system (R0). 2 =0.995)(e.g. Figure 10 (As shown in B in the figure). Meanwhile, the AuCu@MWCNTs / Au electrode exhibits higher sensitivity (1335 μA·mM). -1 ·cm -2 The results demonstrate that, due to the synergistic effect of AuCuNPs and MWCNTs, the AuCu@MWCNTs / Au electrode exhibits advantages such as high sensitivity, wide detection range, and low detection limit in the enzyme-free detection of H2O2.

[0083] 4.8 Cell Sample Analysis

[0084] In actual sample analysis, the standard addition method was used. First, approximately 5 × 10⁻⁶... 6 One HepG2 cell was added to 50 mL of 0.1 M PB solution. Then, 0.3 μM CHAPS solution was added to the above system, and the current response to the H2O2 concentration in the cell sample was obtained. Simultaneously, the H2O2 concentration in the cells was detected using a commercial hydrogen peroxide detection kit (Applygen, China). The results showed that after treatment with 0.3 μM CHAPS solution, the cells released approximately 20 nM of H2O2, consistent with the electrochemical method described above. The current response to the H2O2 concentration in the cell sample was obtained by continuously adding 5 nM H2O2 standard solution. Figure 11 As shown. Recovery rates were calculated based on the standard curve in the enzyme-free system. The results indicate that the recovery rate of the fabricated sensor was 86.7% when 5 nM H₂O₂ was added to the cell sample.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. An application of an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs in glucose detection, characterized in that, include: An enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs is provided. Different concentrations of glucose solutions are continuously added to the detection system. A chronoamperometric method is used to plot the current-time response curve based on the current response, and a standard curve of current change with glucose concentration is obtained. When a test solution containing glucose is added, the glucose concentration in the test solution can be obtained by detecting the electrical signal and substituting it into the standard curve, thus realizing the detection of glucose concentration in an enzyme-free system using the enzyme-free biosensor. The enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs includes a working electrode, a reference electrode, and a counter electrode; the working electrode is prepared using the following process: S1: Preparation of composite nanomaterials: AuCuNPs and multi-walled carbon nanotubes are dispersed in tetrahydrofuran under ultrasonic treatment to obtain a homogeneous solution of composite nanomaterials AuCuNPs-MWCNTs. S2: Pretreatment of the Au electrode: The Au electrode was polished on chamois leather with alumina powder of 1.0 μm, 0.1 μm and 0.05 μm particle sizes, then ultrasonically cleaned in ethanol and ultrapure water, and dried with nitrogen for later use; S3: Preparation of the AuCu@MWCNTs / Au electrode: The homogeneous solution prepared in step S1 was dropped onto the Au electrode after pretreatment in step S2 to form a conductive layer of the electrode. It was dried at room temperature to obtain the AuCu@MWCNTs / Au electrode, which serves as the working electrode; wherein, the enzyme-free biosensor utilizes both the electrocatalytic performance of AuCuNPs and the rapid electron transfer performance of MWCNTs to achieve highly sensitive detection of glucose and hydrogen peroxide; the reference electrode is a calomel electrode, the counter electrode is a platinum wire electrode, and the electrolyte for detecting glucose is a 0.1M alkaline solution with pH 12, selected from NaOH solution and KOH solution; The electrolyte used for detecting hydrogen peroxide was a pH 7, 0.1 M phosphate buffer solution.

2. The application according to claim 1, characterized in that, The measured voltage was set to 0.40 V.

3. The application according to claim 1, characterized in that, In the aforementioned application, the enzyme-free biosensor has a sensitivity of 3.62 × 10⁻⁶ for glucose. 2 μA·mM -1 ·cm -2 The linear range is 8.0 × 10 -3 ~6.0×10 4 μM, detection limit is 1.3×10 -3 μM, S / N=3.

4. An application of an enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs in the detection of hydrogen peroxide, characterized in that, include: An enzyme-free biosensor based on the composite nanomaterial AuCuNPs-MWCNTs as described in claim 1 is provided. Different concentrations of H2O2 solutions are continuously added to the detection system. A chronoamperometric method is used to plot the current-time response curve based on the current response, and a standard curve is obtained showing the change in current with H2O2 concentration. When a test solution containing H2O2 is added, the concentration of H2O2 in the test solution can be obtained by detecting the electrical signal and substituting it into the standard curve, thus realizing the detection of H2O2 concentration in an enzyme-free system using the enzyme-free biosensor.

5. The application according to claim 4, characterized in that, The measurement voltage was selected as -0.40 V; the sensitivity of the enzyme-free biosensor to H2O2 was 1.335 × 10⁻⁶. 3 μA·mM -1 ·cm -2 The linear range is 1.0 × 10⁻⁶. -3 ~4.0×10 3 μM, detection limit is 1.0 × 10 -4 μM, S / N=3.

6. The application according to claim 4, characterized in that, The enzyme-free biosensor can be used to monitor the instantaneous concentration changes of trace amounts of hydrogen peroxide released from cell samples under chemical reagent stimulation.

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