A method for constructing and applying a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling.

Abstract

This invention discloses a method for constructing and applying a biosensor with dual functions of self-catalytic enhancement and interfacial antifouling, belonging to the fields of electrochemiluminescence, nanoscience, and bioanalytical technology. In this invention, Cu₂O octahedral nanosheets are loaded onto MXene nanosheets and encapsulated in a polyacrylamide hydrogel to prepare a conductive hydrogel with both self-catalytic signal enhancement and interfacial antifouling functions. Specifically, Cu₂O… + and Cu 2+ The valence state transition between them can catalyze S2O8 2‑ More SO4 is produced ·‑ This process achieved signal self-enhancement; simultaneously, MXene nanosheets endowed the hydrogel with high conductivity, and the hydrophilicity of the hydrogel gave it strong anti-adsorption capacity; furthermore, the short peptide ligand KHRFNKDC was introduced to achieve targeted antibody immobilization. Based on this, the constructed ECL biosensor has a linear range of 10 fg / mL to 100 ng / mL and a detection limit of 3.67 fg / mL, providing a feasible method for the clinical detection of neuron-specific enolases.

Description

Technical Field

[0001] This invention discloses a method for constructing a biosensor with dual functions of self-catalytic enhancement and interface antifouling, as well as its application in the detection of neuron-specific enolases, belonging to the fields of electrochemiluminescence, nanoscience, and bioanalysis. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly malignant tumor that progresses rapidly and is prone to early metastasis, 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 photochemical / electrochemical analysis and colorimetric analysis. Among these, electrochemiluminescence (ECL) has attracted widespread attention due to its low background signal, fast response time, and high controllability. Therefore, developing a highly efficient ECL biosensor for sensitive NSE detection is of great significance.

[0003] The development of highly efficient luminescent agents is crucial for improving the detection performance of ECL sensors. Metal nanoclusters have emerged as a new class of luminescent nanomaterials due to their unique physical and chemical properties. Among them, silver nanoclusters, with their good biocompatibility and optical properties, large Stokes shift, and ease of labeling, are considered ideal choices for luminescent agents. Furthermore, since the sequence and length of DNA are tunable, the optical properties of silver nanoclusters using DNA as a ligand can be precisely controlled through chemical synthesis. Simultaneously, silver nanoclusters using DNA as a ligand possess excellent bioactivity and recognition functions, and can be easily assembled with biomolecules to prepare highly efficient luminescent nanoprobes.

[0004] To further enhance the detection sensitivity of ECL biosensors, increasing the yield of co-reactant radicals and inhibiting interfacial adsorption of interfering biomolecules are effective approaches. On one hand, valence transitions of multivalent metals can promote the redox reaction of co-reactants, generating more co-reactant radicals for ECL emission. This study introduces Cu₂O as a co-reaction promoter to catalyze the generation of more SO₄²⁻. ·- Meanwhile, Cu2O can be linked to MXene nanosheets via amide bonds, effectively improving the electron transfer efficiency at the sensing interface and the loading of the Cu2O catalyst. On the other hand, to address the issue of a large number of biomolecules in serum affecting the detection performance of the biosensor through non-specific adsorption and bio-oxidation, this study designed a hydrophilic polyacrylamide (PAM) hydrogel as an antifouling material, effectively preventing the adsorption of interfering biomolecules on the electrode surface.

[0005] In this work, an ultrasensitive ECL biosensor for trace detection of NSE in serum was constructed using silver nanoclusters as luminescent probes, Cu2O-MXene-PAM bifunctional hydrogel as an antifouling coating, and KHRFNKDC as an antibody targeting and immobilization component. Specifically, the silver nanoclusters prepared with DNA as a ligand exhibited stable cathodic ECL emission; a large amount of co-reaction promoter Cu2O was immobilized on MXene nanosheets via acylation, and then encapsulated in PAM hydrogel. The prepared Cu2O-MXene-PAM bifunctional hydrogel not only has antifouling properties but also possesses autocatalytic signal amplification capabilities; KHRFNKDC was designed for antibody targeting and immobilization, which not only improved the antigen-antibody binding efficiency but also protected the bioactivity of the target. Based on this, the constructed ECL biosensor achieved trace detection of NSE, which has significant value for the early clinical diagnosis of small cell lung cancer. Summary of the Invention

[0006] One of the technical objectives of this invention is to overcome the shortcomings of the prior art by preparing a conductive hydrogel with self-catalytic signal enhancement and interfacial antifouling properties for ECL analysis, thereby improving the detection sensitivity and accuracy of the ECL biosensor.

[0007] The second technical objective of this invention is to construct an ECL biosensor with dual functions of self-catalytic enhancement and interfacial antifouling based on the above-mentioned hydrogel and antibody targeting and immobilization strategy. It 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 objective of this invention is to provide the application of a biosensor with dual functions of self-catalytic enhancement and interface antifouling constructed by the aforementioned construction method, namely, for sensitive detection of NSE in the serum environment; the constructed biosensor has a wide linear range, 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 industrialization prospects.

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

[0010] 1. A method for constructing a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling.

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

[0012] The Cu2O was obtained 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 min; allowing the mixed solution to stand for 5 h, centrifuging, and washing three times with ethanol to obtain Cu2O.

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

[0014] The Cu2O-MXene-PAM hydrogel was prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly adding 1 mL of 3-aminopropyltriethoxysilane to the mixture and stirring at room temperature for 48 h to obtain aminated Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen protection for 40 min; continuing to add 9.6 mL of NaOH and stirring at 60 °C for 3 h to obtain carboxylated MXene; and then... The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM of N-hydroxysuccinimide was added to activate its carboxyl groups; aminated Cu2O was added to the above mixed solution to carry out an acylation reaction; acrylamide and bisacrylamide were added sequentially and stirred for 10 min; under 365 nm ultraviolet light irradiation, 2-hydroxy-2-methylphenylacetone was added to form a gel, resulting in Cu2O-MXene-PAM hydrogel;

[0015] The NSE solutions of different concentrations are obtained 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 buffer solution with a pH of 7.4.

[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 and stirring at 0 °C for 30 min; adding 8 μL of NaBH4 to the mixture and reacting at 4 °C for 3 h 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 h; adding 100 μL of 10 μg / mL detection antibody and incubating at 4 °C with shaking for 1 h to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0017] 2. The application of the biosensor constructed by the method described above, which possesses dual functions of self-catalytic enhancement and interfacial antifouling, is for the detection of NSE in serum environments. A phosphate buffer solution containing 80–100 mM K₂S₂O₈ at pH 7.4 is used as the detection solution. The constructed ECL biosensor is immersed in the solution, and a three-electrode system including a working electrode, a reference electrode, and an auxiliary electrode is used for ECL signal testing. The applied scanning voltage range is -1.6–0 V, and the photomultiplier tube voltage is 600 V. A linear curve is plotted based on the measured ECL signal, revealing that 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 indicate that the constructed ECL biosensor has high stability, specificity, and reproducibility, as well as a long service life, making it suitable for trace detection of NSE in serum media.

[0018] The beneficial technical effects of the present invention are as follows:

[0019] 1. This invention prepares a conductive hydrogel with self-catalytic signal enhancement and interfacial antifouling properties for ECL analysis; by immobilizing the co-reaction promoter Cu2O on MXene nanosheets through an acylation reaction, the electron transfer efficiency and Cu2O loading at the sensing interface are effectively improved; the complex is encapsulated in a PAM hydrogel, and 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 improving the detection sensitivity and accuracy of the biosensor;

[0020] 2. This invention constructs a biosensor with dual functions of self-catalytic enhancement and interfacial antifouling. Based on the prepared Cu2O-MXene-PAM conductive hydrogel and KHRFNKDC for targeted immobilization of antibodies, this biosensor exhibits high detection sensitivity and accuracy. The raw materials used are low-cost, the preparation process is simple, and the operation is safe.

[0021] 3. The biosensor constructed in this invention, which has dual functions of self-catalytic enhancement and interface antifouling, exhibits a wide linear range, low detection limit, long lifespan, and high stability, specificity, and reproducibility for the detection of NSE in serum media. It is expected to be used for effective clinical monitoring of small cell lung cancer and has certain industrialization prospects. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any changes made to the technical solutions of the present invention by those skilled in the art should fall within the scope of protection of the present invention.

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

[0024] The Cu2O was obtained 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 min; allowing the mixed solution to stand for 5 h, centrifuging, and washing three times with ethanol to obtain Cu2O.

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

[0026] The Cu2O-MXene-PAM hydrogel was prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly adding 1 mL of 3-aminopropyltriethoxysilane to the mixture and stirring at room temperature for 48 h to obtain aminated Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen protection for 40 min; continuing to add 9.6 mL of NaOH and stirring at 60 °C for 3 h to obtain carboxylated MXene; and then... The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM of N-hydroxysuccinimide was added to activate its carboxyl groups; aminated Cu2O was added to the above mixed solution to carry out an acylation reaction; acrylamide and bisacrylamide were added sequentially and stirred for 10 min; under 365 nm ultraviolet light irradiation, 2-hydroxy-2-methylphenylacetone was added to form a gel, resulting in Cu2O-MXene-PAM hydrogel;

[0027] The NSE solutions of different concentrations are obtained 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 buffer solution with a pH of 7.4.

[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 and stirring at 0 °C for 30 min; adding 8 μL of NaBH4 to the mixture and reacting at 4 °C for 3 h 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 h; adding 100 μL of 10 μg / mL detection antibody and incubating at 4 °C with shaking for 1 h to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0029] Example 2: A method for constructing a biosensor with dual functions of self-catalytic enhancement and interfacial antifouling. A glassy carbon electrode was polished to a mirror finish using alumina powder and then washed sequentially with ultrapure water and ethanol. 8 μL of Cu2O-MXene-PAM hydrogel was applied to the polished electrode as an antifouling interface. 5 μL of a 50 ng / mL short peptide ligand KHRFNKDC was added for antibody targeting and immobilization. 8 μL of a 10 μg / mL capture antibody solution, 8 μL of NSE solution, and 8 μL of a 10 μg / mL silver nanocluster-KHRFNKDC-detection antibody solution were sequentially added to the electrode and incubated at 4°C for 2 h, thus constructing a biosensor with dual functions of self-catalytic enhancement and interfacial antifouling.

[0030] The Cu2O was obtained 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 min; allowing the mixed solution to stand for 5 h, centrifuging, and washing three times with ethanol to obtain Cu2O.

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

[0032] The Cu2O-MXene-PAM hydrogel was prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly adding 1 mL of 3-aminopropyltriethoxysilane to the mixture and stirring at room temperature for 48 h to obtain aminated Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen protection for 40 min; continuing to add 9.6 mL of NaOH and stirring at 60 °C for 3 h to obtain carboxylated MXene; and then... The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM of N-hydroxysuccinimide was added to activate its carboxyl groups; aminated Cu2O was added to the above mixed solution to carry out an acylation reaction; acrylamide and bisacrylamide were added sequentially and stirred for 10 min; under 365 nm ultraviolet light irradiation, 2-hydroxy-2-methylphenylacetone was added to form a gel, resulting in Cu2O-MXene-PAM hydrogel;

[0033] The NSE solutions of different concentrations are obtained 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 buffer solution with a pH of 7.4.

[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 and stirring at 0 °C for 30 min; adding 8 μL of NaBH4 to the mixture and reacting at 4 °C for 3 h 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 h; adding 100 μL of 10 μg / mL detection antibody and incubating at 4 °C with shaking for 1 h to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

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

[0036] The Cu2O was obtained 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 min; allowing the mixed solution to stand for 5 h, centrifuging, and washing three times with ethanol to obtain Cu2O.

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

[0038] The Cu2O-MXene-PAM hydrogel was prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly adding 1 mL of 3-aminopropyltriethoxysilane to the mixture and stirring at room temperature for 48 h to obtain aminated Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH and reacting under nitrogen protection for 40 min; continuing to add 9.6 mL of NaOH and stirring at 60 °C for 3 h to obtain carboxylated MXene; and then... The MXene was dispersed in 2 mL of ultrapure water, and 50 μL of a mixture containing 40 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM of N-hydroxysuccinimide was added to activate its carboxyl groups; aminated Cu2O was added to the above mixed solution to carry out an acylation reaction; acrylamide and bisacrylamide were added sequentially and stirred for 10 min; under 365 nm ultraviolet light irradiation, 2-hydroxy-2-methylphenylacetone was added to form a gel, resulting in Cu2O-MXene-PAM hydrogel;

[0039] The NSE solutions of different concentrations are obtained 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 buffer solution with a pH of 7.4.

[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 and stirring at 0 °C for 30 min; adding 8 μL of NaBH4 to the mixture and reacting at 4 °C for 3 h 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 h; adding 100 μL of 10 μg / mL detection antibody and incubating at 4 °C with shaking for 1 h to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

[0041] Example 4 describes the application of a biosensor with dual functions of self-catalytic enhancement and interfacial antifouling, constructed using the methods described in Examples 1, 2, and 3, for the detection of NSE in a serum environment. A phosphate buffer solution containing 80 mM K₂S₂O₈ at pH 7.4 was used as the detection solution. The constructed ECL biosensor was immersed in the solution, and a three-electrode system including a working electrode, a reference electrode, and an auxiliary electrode was used for ECL signal testing. The applied scanning voltage range was -1.6 to 0 V, and the photomultiplier tube voltage was 600 V. A linear curve was plotted based on the measured ECL signal, revealing that 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 indicate that the constructed ECL biosensor has high stability, specificity, and reproducibility, as well as a long service life, making it suitable for trace detection of NSE in serum media.

[0042] Example 5 describes the application of a biosensor with dual functions of self-catalytic enhancement and interfacial antifouling, constructed using the methods described in Examples 1, 2, and 3, for the detection of NSE in a serum environment. A phosphate buffer solution containing 90 mM K₂S₂O₈ at pH 7.4 was used as the detection solution. The constructed ECL biosensor was immersed in the solution, and a three-electrode system including a working electrode, a reference electrode, and an auxiliary electrode was used for ECL signal testing. The applied scanning voltage range was -1.6 to 0 V, and the photomultiplier tube voltage was 600 V. A linear curve was plotted based on the measured ECL signal, revealing that 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 indicate that the constructed ECL biosensor has high stability, specificity, and reproducibility, as well as a long service life, making it suitable for trace detection of NSE in serum media.

[0043] Example 6 describes the application of a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling, constructed using the methods described in Examples 1, 2, and 3, for the detection of NSE in a serum environment.

[0044] The constructed ECL biosensor was immersed in a phosphate buffer solution containing 100 mM K₂S₂O₈ at pH 7.4 as the detection solution. A three-electrode system comprising a working electrode, a reference electrode, and an auxiliary electrode was used to measure the ECL signal. The applied scanning voltage range was -1.6 to 0 V, and the photomultiplier tube voltage was 600 V. A linear curve was plotted based on the measured ECL signal, revealing that 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. These results indicate that the constructed ECL biosensor possesses high stability, specificity, and reproducibility, as well as a long lifespan, making it suitable for trace detection of NSE in serum media. Attached Figure Description

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

[0046] Figure 2 The images show (A) the fluorescence excitation and emission spectra of silver nanoclusters (Ag NCs) and (B) the ultraviolet absorption spectra.

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

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

[0049] Figure 5 The X-ray photoelectron spectrum of Cu2O is shown below, where (A) is the full-area X-ray photoelectron spectrum; (B) is the high-resolution X-ray photoelectron spectrum of the Cu 2p region; and (C) is the high-resolution X-ray photoelectron spectrum of the O 1s region.

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

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

[0052] Figure 8 This is a transmission electron microscope image of MXene NSs.

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

[0054] Figure 10 The image shows a scanning electron microscope (SEM) image and the corresponding elemental mapping of the Cu2O-MXene-PAM hydrogel.

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

[0056] Figure 12 The cyclic voltammetry plots are for (a) Ag NCs / GCE and (b) AgNCs / Cu2O / GCE in phosphate buffer solutions 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 for (a) GCE and (b) Cu2O-MXene-PAM hydrogel / GCE.

[0059] Figure 15 ECL intensity plots for (A) biosensors incubated in modified hydrogels (pink curves) and unmodified hydrogels (blue curves) at different concentrations of fetal bovine serum, and (B) the corresponding signal attenuation rates.

[0060] Figure 16 Fluorescence imaging of ITO and Cu2O-MXene-PAM hydrogel / ITO after incubation in a fluorescein isothiocyanate-conjugated bovine serum albumin solution (Indium Tin Oxide: ITO).

[0061] Figure 17 This is a characterization diagram of the construction process of a biosensor with dual functions of autocatalytic enhancement and interfacial antifouling, based on (A) cyclic voltammetry and (B) AC impedance testing.

[0062] Figure 18 The diagram shows the optimization results of a biosensor based on dual functions of autocatalytic enhancement and interfacial antifouling. (A) Optimization results of K2S2O8 concentration; (B) Optimization results of test solution pH; (C) Optimization results of MXene NSs concentration; (D) Optimization results of Cu2O concentration.

[0063] Figure 19(A) ECL intensity curve and (B) corresponding calibration curve are shown for a biosensor based on autocatalytic enhancement and interfacial antifouling dual functions after incubation with different concentrations of NSE; where a to h represent 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 To evaluate the (A) stability, (B) specificity, and (C) reproducibility of a biosensor based on a dual function of autocatalytic enhancement and interfacial antifouling.

Claims

1. A method of constructing a biosensor with dual functions of self-catalysis enhancement and interface anti-fouling, characterized in that, The glassy carbon electrode is polished to a mirror surface by using alumina powder and is washed with ultrapure water and ethanol in sequence; 6-10 muL of Cu2O-MXene-polyacrylamide hydrogel is modified on the polished glassy carbon electrode as an antifouling interface; 5 muL of short peptide ligand KHRFNKDC with a concentration of 50 ng / mL is added dropwise for targeted fixation of the antibody; 8 muL of capture antibody solution with a concentration of 10 mu g / mL, 8 muL of neuron-specific enolase solution, and 8 muL of silver nanocluster-KHRFNKDC-detection antibody solution with a concentration of 10 mu g / mL are added dropwise on the electrode in sequence, and are sequentially incubated at 4 DEG C for 2 h, so as to construct a biosensor with self-catalytic enhancement and interface antifouling dual functions; the silver nanocluster-KHRFNKDC-detection antibody solution is obtained by mixing 8 muL of AgNO3 with a concentration of 1 mM and 15 muL of DNA ligand with a concentration of 0.1 mM, stirring at 0 DEG C for 30 min; 8 muL of NaBH4 is added dropwise into the above mixture, and is reacted at 4 DEG C for 3 h; 200 muL of short peptide ligand KHRFNKDC is dispersed in 600 muL of silver nanocluster solution, and is oscillated at 4 DEG C for 1 h; 100 muL of detection antibody with a concentration of 10 mu g / mL is added, and is oscillated and incubated at 4 DEG C for 1 h, so as to obtain the silver nanocluster-KHRFNKDC-detection antibody solution.

2. A method of constructing a biosensor with dual functions of self-catalytic enhancement and interface anti-fouling as claimed in claim 1, wherein, The Cu2O is obtained by dissolving 8.8 mL of CuCl2 with a concentration of 0.1 M in 300 mL of ultrapure water, fully stirring, adding 7.7 g of sodium dodecyl sulfate, and stirring until completely dissolved; 17.7 mL of NaOH with a concentration of 0.1 M and 54 mL of hydroxylamine hydrochloride with a concentration of 0.2 M are dispersed in the obtained solution in sequence, and are stirred for 20 min; after the mixed solution is placed for 5 h, centrifugation and ethanol washing are performed for 3 times, so as to obtain Cu2O.

3. A method of constructing a biosensor with dual functions of self-catalytic enhancement and interface anti-fouling as claimed in claim 1, wherein, The MXene is obtained by adding 1 g of Ti3AlC2 powder into 10 mL of etching solution containing 6 mL of HCl and 1 mL of HF, stirring at 41 DEG C for 15 h; the pH of the obtained solution is adjusted to 6 by using ultrapure water; 1.5 g of LiCl is dissolved in 25 mL of ultrapure water, and is then poured into the above solution and stirred for 2 h; the obtained mixed solution is centrifuged, and is washed with ultrapure water for 3 times, so as to obtain MXene.

4. The method of constructing a biosensor with dual functions of self-catalytic enhancement and interface anti-fouling as claimed in claim 1, wherein, The Cu2O-MXene-polyacrylamide hydrogel is prepared by dispersing 100 mg of Cu2O in 50 mL of ethanol, then slowly dropping 1 mL of 3-aminopropyltriethoxysilane into the mixed solution, stirring at room temperature for 48 h to obtain aminated Cu2O; dissolving 20 mg of MXene in 12 mL of ultrapure water, then adding 2.6 g of ClCH2COOH, and reacting under nitrogen protection for 40 min; continuing to add 9.6 mL of NaOH, stirring at 60 DEG C for 3 h to obtain carboxylated MXene; dispersing the carboxylated MXene in 2 mL of ultrapure water, adding 50 μL of a mixture containing 40 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mM of N-hydroxysuccinimide to activate the carboxyl group thereof; adding the aminated Cu2O to the above mixed solution to perform acylation reaction; adding acrylamide and bisacrylamide in sequence, and stirring for 10 min; under the irradiation of ultraviolet light at 365 nm, adding 2-hydroxy-2-methylpropiophenone to form a gel to obtain the Cu2O-MXene-polyacrylamide hydrogel.

5. The method of constructing a biosensor with dual functions of self-catalytic enhancement and interface anti-fouling as claimed in claim 1, wherein, The neuron-specific enolase solution is prepared by uniformly dispersing neuron-specific enolase in a phosphate buffer solution with a pH of 7.4 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, respectively, to obtain a neuron-specific enolase solution.

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