Construction method and application of biosensor with double functions of autocatalytic enhancement and interface pollution resistance

Abstract

The invention discloses a construction method and application of a biosensor with double functions of autocatalysis enhancement and interface pollution resistance, and belongs to the technical fields of electrochemical luminescence, nano science and biological analysis. According to the preparation method disclosed by the invention, a Cu2O nano octahedron is loaded on an MXene nanosheet, and the MXene nanosheet is packaged in polyacrylamide hydrogel, so that the conductive hydrogel with autocatalytic signal enhancement and interface antifouling functions is prepared. Specifically, valence conversion between Cu < + > and Cu < 2 + > can catalyze S2O8 < 2-> to generate more SO4 <.->, so that signal self-enhancement is realized; meanwhile, the hydrogel is endowed with high conductivity by the MXene nanosheets, and the hydrogel has strong adsorption resistance due to the hydrophilicity of the hydrogel; in addition, an oligopeptide ligand KHRFNKDC is introduced to realize targeted immobilization of the antibody. On the basis, the linear range of the constructed ECL biosensor is 10 fg / mL to 100 ng / mL, the detection limit is 3.67 fg / mL, and a feasible method is provided for clinical detection of neuron-specific enolase.

Description

Technical Field

[0001] The invention discloses a construction method of a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling and its application in detecting neuron-specific enolase, belonging to the technical fields of electrochemiluminescence, nanoscience and bioanalysis. Background Art

[0002] Small cell lung cancer (SCLC) is a highly malignant tumor that develops rapidly and is prone to metastasis in its early stages, posing a serious threat to human health. Neuron-specific enolase (NSE), as a specific biomarker for SCLC, is of great value in the clinical diagnosis and subsequent treatment of SCLC patients. Currently, common methods for NSE detection include photo / electrochemical analysis and colorimetric analysis. Among them, electrochemiluminescence (ECL) has attracted widespread attention due to its low background signal, fast response time, and high controllability. Therefore, the development of an efficient ECL biosensor for the sensitive detection of NSE is of great significance.

[0003] The development of efficient luminophores is key to improving the detection performance of ECL sensors. Metal nanoclusters have become an emerging class of nanoluminescent materials due to their unique physical and chemical properties. Among them, silver nanoclusters, with their excellent biocompatibility and optical properties, large Stokes shift, and ease of labeling, are considered ideal candidates for luminophores. Furthermore, because the sequence and length of DNA are adjustable, precise control of the optical properties of silver nanoclusters with DNA as ligands can be achieved through chemical synthesis. Furthermore, silver nanoclusters with DNA as ligands possess excellent bioactivity and recognition capabilities, allowing for easy assembly with biomolecules to prepare efficient nanoluminescent probes.

[0004] In order to further improve the detection sensitivity of ECL biosensors, increasing the production of co-reactant free radicals and hindering the interfacial adsorption of interfering biomolecules are effective means. On the one hand, the valence state transition of multivalent metals can promote the redox of co-reactants to generate more co-reactant free radicals for ECL emission. In this study, Cu2O was introduced as a co-reactant promoter to catalyze the generation of more SO4 ·- At the same time, Cu2O can be connected to MXene nanosheets through amide bonds, effectively improving the electron transfer efficiency of the sensing interface and the loading capacity of the catalyst Cu2O. On the other hand, in order to solve the problem that a large number of biomolecules in serum affect the detection performance of biosensors through nonspecific adsorption and biooxidation, this study designed a hydrophilic polyacrylamide (PAM) hydrogel as an antifouling material, which effectively prevented the adsorption of interfering biomolecules on the electrode surface.

[0005] In this work, an ultrasensitive ECL biosensor for the trace detection of NSE in serum was constructed using silver nanoclusters as luminescent probes, a Cu2O-MXene-PAM bifunctional hydrogel as an antifouling coating, and KHRFNKDC as an antibody-targeting immobilization component. Specifically, silver nanoclusters prepared with DNA as ligands exhibited stable cathodic ECL emission. A large amount of the co-reaction promoter, Cu2O, was immobilized on MXene nanosheets via an acylation reaction and subsequently encapsulated in a PAM hydrogel. The resulting Cu2O-MXene-PAM bifunctional hydrogel exhibited both antifouling properties and autocatalytic signal amplification. KHRFNKDC was designed for targeted antibody immobilization, improving antigen-antibody binding efficiency while preserving the target's bioactivity. This ECL biosensor achieved trace detection of NSE, which is of great value for the early clinical diagnosis of small cell lung cancer. Summary of the Invention

[0006] One of the technical tasks of the present invention is to make up for the shortcomings of the existing technology and prepare a conductive hydrogel with autocatalytic signal enhancement and interface antifouling properties for ECL analysis, thereby improving the detection sensitivity and accuracy of ECL biosensors;

[0007] The second technical task of the present invention is to construct an ECL biosensor with dual functions of autocatalytic enhancement and interfacial antifouling based on the above-mentioned hydrogel and antibody targeted immobilization strategy. The biosensor has excellent detection performance and long service life, uses low-cost raw materials, has a simple preparation process, and is safe to operate.

[0008] The third technical task of the present invention is to provide the use of a biosensor with the dual functions of autocatalytic enhancement and interfacial anti-fouling constructed by the construction method, namely, for the sensitive detection of NSE in a serum environment; the constructed biosensor has a wide linear range, a low detection limit and good specificity for NSE detection, and is expected to be used for effective clinical monitoring of small cell lung cancer, and has certain industrial prospects.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] 1. Construction of a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling

[0011] A glassy carbon electrode was polished to a mirror surface using alumina powder and washed sequentially with ultrapure water and ethanol. 6-10 μL of Cu2O-MXene-PAM hydrogel was modified on the polished electrode as an antifouling interface. 5 μL of a short peptide ligand, KHRFNKDC, at a concentration of 50 ng / mL was added dropwise for targeted antibody immobilization. 8 μL of a capture antibody solution at a concentration of 10 μg / mL, 8 μL of a NSE solution, and 8 μL of a silver nanocluster-KHRFNKDC-detection antibody solution at a concentration of 10 μg / mL were sequentially added to the electrode and incubated at 4°C for 2 h. This constructed a biosensor with the dual functions of autocatalytic enhancement and interfacial antifouling.

[0012] The Cu2O is prepared by dissolving 8.8 mL of 0.1 M CuCl2 in 300 mL of ultrapure water and stirring thoroughly; adding 7.7 g of sodium dodecyl sulfate and stirring until completely dissolved; sequentially dispersing 17.7 mL of 0.1 M NaOH and 54 mL of 0.2 M hydroxylamine hydrochloride in the resulting solution and stirring for 20 minutes; and allowing the mixed solution to stand for 5 hours, centrifuging it, and washing it three times with ethanol to obtain Cu2O;

[0013] The MXene is prepared by adding 1 g of Ti3AlC2 powder to 10 mL of an etching solution containing 6 mL of HCl and 1 mL of HF, and stirring at 41°C for 15 hours; adjusting the pH of the resulting solution to 6 with ultrapure water; dissolving 1.5 g of LiCl in 25 mL of ultrapure water, then adding the solution and stirring for 2 hours; centrifuging the resulting mixed solution and washing it three times with ultrapure water to obtain the MXene;

[0014] The Cu2O-MXene-PAM hydrogel is prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly dripping 1 mL of 3-aminopropyltriethoxysilane into the mixed solution and stirring at room temperature for 48 hours to obtain amino-modified Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen for 40 minutes; then adding 9.6 mL of NaOH and stirring at 60°C for 3 hours to obtain carboxylated MXene; The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM N-hydroxysuccinimide was added to activate its carboxyl groups. Aminated Cu2O was added to the mixed solution to carry out an acylation reaction. Acrylamide and bisacrylamide were added in sequence and stirred for 10 minutes. Under 365 nm ultraviolet light, 2-hydroxy-2-methylpropiophenone was added to form a gel to obtain a Cu2O-MXene-PAM hydrogel.

[0015] The NSE solutions of different concentrations are prepared by uniformly dispersing NSE at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffered saline solution having a pH of 7.4 to obtain NSE solutions;

[0016] The silver nanocluster-KHRFNKDC-detection antibody solution is prepared by mixing 8 μL of 1 mM AgNO3 with 15 μL of 0.1 mM DNA ligand, stirring at 0°C for 30 minutes; dropping 8 μL of NaBH4 into the mixture, reacting at 4°C for 3 hours to obtain silver nanoclusters; dispersing 200 μL of the short peptide ligand KHRFNKDC in 600 μL of the silver nanocluster solution, and shaking at 4°C for 1 hour; adding 100 μL of the detection antibody at a concentration of 10 μg / mL, and incubating at 4°C for 1 hour to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0017] 2. The use of a biosensor with dual functions of autocatalytic enhancement and interfacial anti-fouling constructed by the construction method is for the detection of NSE in a serum environment. A phosphate buffer solution containing 80-100 mM K2S2O8 and a pH of 7.4 is used as the detection solution. The constructed ECL biosensor is immersed in the solution, and an ECL signal test is performed using a three-electrode system comprising a working electrode, a reference electrode, and an auxiliary electrode. The scanning voltage range applied in the experiment is -1.6 to 0 V, and the photomultiplier tube high voltage is 600 V. A linear curve is drawn based on the measured ECL signal, and the detection range of the constructed biosensor is 10 fg / mL to 100 ng / mL, with a detection limit as low as 3.67 fg / mL. The results show that the constructed ECL biosensor has high stability, specificity, and reproducibility, as well as a long service life, and is suitable for trace detection of NSE in serum media.

[0018] Beneficial technical effects of the present invention:

[0019] 1. This invention prepares a conductive hydrogel with autocatalytic signal enhancement and interfacial antifouling properties for ECL analysis. The coreaction promoter, Cu2O, is immobilized on MXene nanosheets via an acylation reaction, effectively improving the electron transfer efficiency and Cu2O loading at the sensing interface. The complex is encapsulated in a polyacrylamide (PAM) hydrogel. Based on the efficient catalysis of Cu2O and the hydrophilic properties of the hydrogel, the prepared Cu2O-MXene-PAM conductive hydrogel exhibits excellent signal amplification and interfacial anti-adsorption capabilities, effectively enhancing the detection sensitivity and accuracy of the biosensor.

[0020] 2. This invention constructs a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling. Based on the prepared Cu2O-MXene-PAM conductive hydrogel and the targeted immobilization of KHRFNKDC on antibodies, the biosensor exhibits high detection sensitivity and accuracy, uses low-cost raw materials, has a simple preparation process, and is safe to operate.

[0021] 3. The biosensor constructed in the present invention has the dual functions of autocatalytic enhancement and interfacial antifouling, and has a wide linear range and low detection limit for the detection of NSE in serum media, a long service life, and high stability, specificity and reproducibility. It is expected to be used for the effective clinical monitoring of small cell lung cancer and has certain industrial prospects. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Any changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0023] Example 1 A method for constructing a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling: A glassy carbon electrode was polished to a mirror surface using alumina powder and washed sequentially with ultrapure water and ethanol; 6 μL of Cu2O-MXene-PAM hydrogel was modified on the polished electrode as an antifouling interface; 5 μL of a short peptide ligand KHRFNKDC with a concentration of 50 ng / mL was added dropwise for targeted immobilization of the antibody; 8 μL of a capture antibody solution with a concentration of 10 μg / mL, 8 μL of an NSE solution, and 8 μL of a silver nanocluster-KHRFNKDC-detection antibody solution with a concentration of 10 μg / mL were sequentially added dropwise to the electrode and incubated at 4°C for 2 h to construct a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling;

[0024] The Cu2O is prepared by dissolving 8.8 mL of 0.1 M CuCl2 in 300 mL of ultrapure water and stirring thoroughly; adding 7.7 g of sodium dodecyl sulfate and stirring until completely dissolved; sequentially dispersing 17.7 mL of 0.1 M NaOH and 54 mL of 0.2 M hydroxylamine hydrochloride in the resulting solution and stirring for 20 minutes; and allowing the mixed solution to stand for 5 hours, centrifuging it, and washing it three times with ethanol to obtain Cu2O;

[0025] The MXene is prepared by adding 1 g of Ti3AlC2 powder to 10 mL of an etching solution containing 6 mL of HCl and 1 mL of HF, and stirring at 41°C for 15 hours; adjusting the pH of the resulting solution to 6 with ultrapure water; dissolving 1.5 g of LiCl in 25 mL of ultrapure water, then adding the solution and stirring for 2 hours; centrifuging the resulting mixed solution and washing it three times with ultrapure water to obtain the MXene;

[0026] The Cu2O-MXene-PAM hydrogel is prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly dripping 1 mL of 3-aminopropyltriethoxysilane into the mixed solution and stirring at room temperature for 48 hours to obtain amino-modified Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen for 40 minutes; then adding 9.6 mL of NaOH and stirring at 60°C for 3 hours to obtain carboxylated MXene; The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM N-hydroxysuccinimide was added to activate its carboxyl groups. Aminated Cu2O was added to the mixed solution to carry out an acylation reaction. Acrylamide and bisacrylamide were added in sequence and stirred for 10 minutes. Under 365 nm ultraviolet light, 2-hydroxy-2-methylpropiophenone was added to form a gel to obtain a Cu2O-MXene-PAM hydrogel.

[0027] The NSE solutions of different concentrations are prepared by uniformly dispersing NSE at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffered saline solution having a pH of 7.4 to obtain NSE solutions;

[0028] The silver nanocluster-KHRFNKDC-detection antibody solution is prepared by mixing 8 μL of 1 mM AgNO3 with 15 μL of 0.1 mM DNA ligand, stirring at 0°C for 30 minutes; dropping 8 μL of NaBH4 into the mixture, reacting at 4°C for 3 hours to obtain silver nanoclusters; dispersing 200 μL of the short peptide ligand KHRFNKDC in 600 μL of the silver nanocluster solution, and shaking at 4°C for 1 hour; adding 100 μL of the detection antibody at a concentration of 10 μg / mL, and incubating at 4°C for 1 hour to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0029] Example 2 A method for constructing a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling: A glassy carbon electrode was polished to a mirror surface using alumina powder and washed with ultrapure water and ethanol in sequence; 8 μL of Cu2O-MXene-PAM hydrogel was modified on the polished electrode as an antifouling interface; 5 μL of a short peptide ligand KHRFNKDC with a concentration of 50 ng / mL was added dropwise for targeted immobilization of the antibody; 8 μL of a capture antibody solution with a concentration of 10 μg / mL, 8 μL of an NSE solution, and 8 μL of a silver nanocluster-KHRFNKDC-detection antibody solution with a concentration of 10 μg / mL were sequentially added dropwise to the electrode and incubated at 4°C for 2 h to construct a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling;

[0030] The Cu2O is prepared by dissolving 8.8 mL of 0.1 M CuCl2 in 300 mL of ultrapure water and stirring thoroughly; adding 7.7 g of sodium dodecyl sulfate and stirring until completely dissolved; sequentially dispersing 17.7 mL of 0.1 M NaOH and 54 mL of 0.2 M hydroxylamine hydrochloride in the resulting solution and stirring for 20 minutes; and allowing the mixed solution to stand for 5 hours, centrifuging it, and washing it three times with ethanol to obtain Cu2O;

[0031] The MXene is prepared by adding 1 g of Ti3AlC2 powder to 10 mL of an etching solution containing 6 mL of HCl and 1 mL of HF, and stirring at 41°C for 15 hours; adjusting the pH of the resulting solution to 6 with ultrapure water; dissolving 1.5 g of LiCl in 25 mL of ultrapure water, then adding the solution and stirring for 2 hours; centrifuging the resulting mixed solution and washing it three times with ultrapure water to obtain the MXene;

[0032] The Cu2O-MXene-PAM hydrogel is prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly dripping 1 mL of 3-aminopropyltriethoxysilane into the mixed solution and stirring at room temperature for 48 hours to obtain amino-modified Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen for 40 minutes; then adding 9.6 mL of NaOH and stirring at 60°C for 3 hours to obtain carboxylated MXene; The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM N-hydroxysuccinimide was added to activate its carboxyl groups. Aminated Cu2O was added to the mixed solution to carry out an acylation reaction. Acrylamide and bisacrylamide were added in sequence and stirred for 10 minutes. Under 365 nm ultraviolet light, 2-hydroxy-2-methylpropiophenone was added to form a gel to obtain a Cu2O-MXene-PAM hydrogel.

[0033] The NSE solutions of different concentrations are prepared by uniformly dispersing NSE at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffered saline solution having a pH of 7.4 to obtain NSE solutions;

[0034] The silver nanocluster-KHRFNKDC-detection antibody solution is prepared by mixing 8 μL of 1 mM AgNO3 with 15 μL of 0.1 mM DNA ligand, stirring at 0°C for 30 minutes; dropping 8 μL of NaBH4 into the mixture, reacting at 4°C for 3 hours to obtain silver nanoclusters; dispersing 200 μL of the short peptide ligand KHRFNKDC in 600 μL of the silver nanocluster solution, and shaking at 4°C for 1 hour; adding 100 μL of the detection antibody at a concentration of 10 μg / mL, and incubating at 4°C for 1 hour to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0035] Example 3 A method for constructing a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling: A glassy carbon electrode was polished to a mirror surface using alumina powder and washed sequentially with ultrapure water and ethanol; 10 μL of Cu2O-MXene-PAM hydrogel was modified on the polished electrode as an antifouling interface; 5 μL of a short peptide ligand KHRFNKDC with a concentration of 50 ng / mL was added dropwise for targeted immobilization of the antibody; 8 μL of a capture antibody solution with a concentration of 10 μg / mL, 8 μL of an NSE solution, and 8 μL of a silver nanocluster-KHRFNKDC-detection antibody solution with a concentration of 10 μg / mL were sequentially added dropwise to the electrode and incubated at 4°C for 2 h to construct a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling;

[0036] The Cu2O is prepared by dissolving 8.8 mL of 0.1 M CuCl2 in 300 mL of ultrapure water and stirring thoroughly; adding 7.7 g of sodium dodecyl sulfate and stirring until completely dissolved; sequentially dispersing 17.7 mL of 0.1 M NaOH and 54 mL of 0.2 M hydroxylamine hydrochloride in the resulting solution and stirring for 20 minutes; and allowing the mixed solution to stand for 5 hours, centrifuging it, and washing it three times with ethanol to obtain Cu2O;

[0037] The MXene is prepared by adding 1 g of Ti3AlC2 powder to 10 mL of an etching solution containing 6 mL of HCl and 1 mL of HF, and stirring at 41°C for 15 hours; adjusting the pH of the resulting solution to 6 with ultrapure water; dissolving 1.5 g of LiCl in 25 mL of ultrapure water, then adding the solution and stirring for 2 hours; centrifuging the resulting mixed solution and washing it three times with ultrapure water to obtain the MXene;

[0038] The Cu2O-MXene-PAM hydrogel is prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly dripping 1 mL of 3-aminopropyltriethoxysilane into the mixed solution and stirring at room temperature for 48 hours to obtain amino-modified Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen for 40 minutes; then adding 9.6 mL of NaOH and stirring at 60°C for 3 hours to obtain carboxylated MXene; The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM N-hydroxysuccinimide was added to activate its carboxyl groups. Aminated Cu2O was added to the mixed solution to carry out an acylation reaction. Acrylamide and bisacrylamide were added in sequence and stirred for 10 minutes. Under 365 nm ultraviolet light, 2-hydroxy-2-methylpropiophenone was added to form a gel to obtain a Cu2O-MXene-PAM hydrogel.

[0039] The NSE solutions of different concentrations are prepared by uniformly dispersing NSE at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffered saline solution having a pH of 7.4 to obtain NSE solutions;

[0040] The silver nanocluster-KHRFNKDC-detection antibody solution is prepared by mixing 8 μL of 1 mM AgNO3 with 15 μL of 0.1 mM DNA ligand, stirring at 0°C for 30 minutes; dropping 8 μL of NaBH4 into the mixture, reacting at 4°C for 3 hours to obtain silver nanoclusters; dispersing 200 μL of the short peptide ligand KHRFNKDC in 600 μL of the silver nanocluster solution, and shaking at 4°C for 1 hour; adding 100 μL of the detection antibody at a concentration of 10 μg / mL, and incubating at 4°C for 1 hour to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0041] Example 4: Use of a biosensor with dual functions of autocatalytic enhancement and interfacial anti-fouling constructed by the construction methods described in Examples 1, 2, and 3 for the detection of NSE in a serum environment. A phosphate buffered saline solution containing 80 mM K2S2O8 and a pH of 7.4 is used as the detection solution. The constructed ECL biosensor is immersed in the solution, and an ECL signal is tested using a three-electrode system comprising a working electrode, a reference electrode, and an auxiliary electrode. The scanning voltage range applied in the experiment is -1.6 to 0 V, and the photomultiplier tube high voltage is 600 V. A linear curve is drawn based on the measured ECL signal, and the detection range of the constructed biosensor is 10 fg / mL to 100 ng / mL, with a detection limit as low as 3.67 fg / mL. The results show that the constructed ECL biosensor has high stability, specificity, and reproducibility, as well as a long service life, and is suitable for trace detection of NSE in serum media.

[0042] Example 5: Use of a biosensor with dual functions of autocatalytic enhancement and interfacial anti-fouling constructed by the construction methods described in Examples 1, 2 and 3 for the detection of NSE in a serum environment. A phosphate buffered saline solution containing 90 mM K2S2O8 and a pH of 7.4 is used as the detection solution. The constructed ECL biosensor is immersed in the solution, and an ECL signal test is performed using a three-electrode system comprising a working electrode, a reference electrode and an auxiliary electrode. The scanning voltage range applied in the experiment is -1.6 to 0 V, and the photomultiplier tube high voltage is 600 V. A linear curve is drawn based on the measured ECL signal, and the detection range of the constructed biosensor is 10 fg / mL to 100 ng / mL, with a detection limit as low as 3.67 fg / mL. The results show that the constructed ECL biosensor has high stability, specificity and reproducibility, as well as a long service life, and is suitable for trace detection of NSE in serum media.

[0043] Example 6 Application of a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling constructed by the construction methods described in Examples 1, 2, and 3 for NSE detection in serum environments

[0044] A phosphate buffer solution containing 100 mM K2S2O8 and a pH of 7.4 was used as the detection solution. The constructed ECL biosensor was immersed in the solution, and the ECL signal was tested using a three-electrode system consisting of a working electrode, a reference electrode, and an auxiliary electrode. The scanning voltage range applied in the experiment was -1.6 to 0 V, and the photomultiplier tube high voltage was 600 V. A linear curve was drawn based on the measured ECL signal, and the detection range of the constructed biosensor was 10 fg / mL to 100 ng / mL, with a detection limit as low as 3.67 fg / mL. The results showed that the constructed ECL biosensor has high stability, specificity, and reproducibility, as well as a long service life, and is suitable for trace detection of NSE in serum media. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the construction process of the ECL biosensor with dual functions of self-catalytic enhancement and interfacial anti-fouling (MXene nanosheets: MXene NSs; KHRFNKDC: KFC; capture antibody: Ab1; detection antibody: Ab2; glassy carbon electrode: GCE).

[0046] Figure 2 (A) Fluorescence excitation and emission spectra, and (B) UV absorption spectrum of silver nanoclusters (Ag NCs).

[0047] Figure 3 (A) High-resolution transmission electron microscopy image of AgNCs (inset: lattice fringe analysis) and (B) particle size distribution.

[0048] Figure 4 This is the X-ray diffraction spectrum of Cu2O.

[0049] Figure 5 X-ray photoelectron spectrum of Cu2O, including: (A) full-area X-ray photoelectron spectrum; (B) high-resolution X-ray photoelectron spectrum of Cu 2p region; (C) high-resolution X-ray photoelectron spectrum of O 1s region.

[0050] Figure 6 This is a scanning electron microscope image of Cu2O.

[0051] Figure 7 X-ray diffraction spectra of Ti3AlC2 and MXene NSs.

[0052] Figure 8 Transmission electron microscopy image of MXene NSs.

[0053] Figure 9 Infrared spectra of MXene NSs and carboxylated MXene NSs (MXene NSs-COOH).

[0054] Figure 10 Scanning electron microscope image of Cu2O-MXene-PAM hydrogel and corresponding elemental mapping map.

[0055] Figure 11 ECL intensity-voltage curves of (a) GCE, (b) Ag NCs / GCE, (c) Cu2O / GCE, (d) Ag NCs / Cu2O / GCE in phosphate buffer solution containing K2S2O8, and (e) ECL intensity-voltage curve of Ag NCs / Cu2O / GCE in phosphate buffer solution (inset: magnified effect of the relevant curves).

[0056] Figure 12 Cyclic voltammograms of (a) Ag NCs / GCE and (b) AgNCs / Cu2O / GCE in phosphate buffer solution containing K2S2O8.

[0057] Figure 13 This is the electron paramagnetic resonance spectrum of the ECL reaction system.

[0058] Figure 14 Static water contact angle diagrams of (a) GCE and (b) Cu2O-MXene-PAM hydrogel / GCE.

[0059] Figure 15 (A) ECL intensity graphs of biosensors with modified hydrogels (pink curve) and unmodified hydrogels (blue curve) incubated in different concentrations of fetal bovine serum, and (B) the corresponding signal decay rates.

[0060] Figure 16 Fluorescence imaging of ITO and Cu2O-MXene-PAM hydrogel / ITO after incubation in fluorescein isothiocyanate-coupled bovine serum albumin solution (indium tin oxide: ITO).

[0061] Figure 17 Figure 2 Characterization of the construction process of the biosensor with dual functions of autocatalytic enhancement and interfacial antifouling based on (A) cyclic voltammetry and (B) electrochemical impedance spectroscopy.

[0062] Figure 18 Condition optimization diagram of the biosensor based on the dual functions of autocatalytic enhancement and interfacial anti-fouling, including: (A) K2S2O8 concentration optimization result diagram; (B) test solution pH optimization result diagram; (C) MXene NSs concentration optimization result diagram; (D) Cu2O concentration optimization result diagram.

[0063] Figure 19(A) ECL intensity curve and (B) corresponding calibration curve of the biosensor based on the dual functions of autocatalytic enhancement and interfacial antifouling after incubation with different concentrations of NSE; where a to h refer to concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively.

[0064] Figure 20 (A) Stability, (B) specificity, and (C) reproducibility of the biosensor based on the dual functions of autocatalytic enhancement and interfacial antifouling.

Claims

1. A method for constructing a biosensor with dual functions of autocatalytic enhancement and interface antifouling, characterized in that: A glassy carbon electrode was polished to a mirror-like surface using alumina powder and washed sequentially with ultrapure water and ethanol; 6-10 μL of Cu2O-MXene-polyacrylamide hydrogel was modified on the polished glassy carbon electrode as an antifouling interface; 5 μL of a short peptide ligand KHRFNKDC with a concentration of 50 ng / mL was added for targeted immobilization of the antibody; 8 μL of a capture antibody solution with a concentration of 10 μg / mL, 8 μL of a neuron-specific enolase solution, and 8 μL of a silver nanocluster-KHRFNKDC-detection antibody solution with a concentration of 10 μg / mL were added sequentially to the electrode and incubated at 4°C for 2 h. This constructed a biosensor with the dual functions of autocatalytic enhancement and interfacial antifouling.

2. The method for constructing a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling according to claim 1, characterized in that: The Cu2O is prepared by dissolving 8.8 mL of 0.1 M CuCl2 in 300 mL of ultrapure water and stirring thoroughly; adding 7.7 g of sodium dodecyl sulfate and stirring until completely dissolved; sequentially dispersing 17.7 mL of 0.1 M NaOH and 54 mL of 0.2 M hydroxylamine hydrochloride in the resulting solution and stirring for 20 minutes; and allowing the mixed solution to stand for 5 hours, centrifuging it, and washing it three times with ethanol to obtain Cu2O.

3. The method for constructing a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling according to claim 1, characterized in that: The MXene is prepared by adding 1 g of Ti3AlC2 powder to 10 mL of an etching solution containing 6 mL of HCl and 1 mL of HF, and stirring at 41°C for 15 hours. The pH of the resulting solution is adjusted to 6 with ultrapure water. 1.5 g of LiCl is dissolved in 25 mL of ultrapure water, then poured into the above solution and stirred for 2 hours. The resulting mixed solution is centrifuged and washed three times with ultrapure water to obtain MXene.

4. The method for constructing a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling according to claim 1, wherein: The Cu2O-MXene-polyacrylamide hydrogel is prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly dripping 1 mL of 3-aminopropyltriethoxysilane into the mixed solution and stirring at room temperature for 48 hours to obtain amino-modified Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen for 40 minutes; then adding 9.6 mL of NaOH and stirring at 60°C for 3 hours to obtain carboxylated MXene; The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM N-hydroxysuccinimide was added to activate its carboxyl groups. Aminated Cu2O was added to the mixed solution to carry out acylation reaction. Acrylamide and bisacrylamide were added in sequence and stirred for 10 min. Under 365 nm ultraviolet light, 2-hydroxy-2-methylpropiophenone was added to form a gel to obtain Cu2O-MXene-polyacrylamide hydrogel.

5. The method for constructing a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling according to claim 1, characterized in that: The neuron-specific enolase solutions of different concentrations are obtained by uniformly dispersing neuron-specific enolase at concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL in a phosphate buffered saline solution with a pH of 7.

4.

6. The method for constructing a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling according to claim 1, characterized in that: The silver nanocluster-KHRFNKDC-detection antibody solution is prepared by mixing 8 μL of 1 mM AgNO3 with 15 μL of 0.1 mM DNA ligand, stirring at 0°C for 30 minutes; dropping 8 μL of NaBH4 into the mixture, reacting at 4°C for 3 hours to obtain silver nanoclusters; dispersing 200 μL of the short peptide ligand KHRFNKDC in 600 μL of the silver nanocluster solution, and shaking at 4°C for 1 hour; adding 100 μL of the detection antibody at a concentration of 10 μg / mL, and incubating at 4°C for 1 hour to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

7. Use of a biosensor with dual functions of autocatalytic enhancement and interface anti-fouling constructed by the construction method of claim 1, characterized in that: Application for the detection of neuron-specific enolase.

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