Construction method and application of an ultrasensitive self-enhanced electrochemiluminescence biosensor

Abstract

The present invention discloses a construction method and application of a super-sensitive self-enhanced electrochemiluminescence biosensor, belonging to the fields of biochemical analysis, nanomaterials, and life science and technology. In the present invention, Cu 2+ is introduced into the luminescent ZrEu-MOF, and it is used to catalyze the coreactant K2S2O8 to generate more active free radicals to prepare ZrEuCu-MOF with self-enhanced function; further, a resorcinol-formaldehyde resin with a yolk structure is synthesized as a quenching probe, and based on the electrochemiluminescence (ECL) resonance energy transfer between it and ZrEuCu-MOF, the effective quenching of the luminescence signal is realized, thereby improving the detection sensitivity. The constructed ECL biosensor realizes the highly sensitive detection of neuron-specific enolase, has a low detection limit, a wide detection range, as well as good stability and industrial application prospects.

Description

Technical Field

[0001] The invention relates to a construction method of an ultrasensitive self-enhanced electrochemiluminescence biosensor and its application in detecting neuron-specific enolase, belonging to the technical fields of biochemical analysis, nanomaterials and life sciences. Background Art

[0002] At present, clinical diagnosis of cancer still mainly relies on imaging, which is expensive and can only be monitored when the tumor develops to a certain extent, making it difficult to achieve early diagnosis of cancer. With the rapid development of molecular biology and proteomics, a large number of cancer-related biomarkers have been discovered, reflecting the occurrence and progression of tumors. Therefore, sensitive and accurate detection of specific biomarkers is of great significance in early warning and clinical diagnosis of cancer. Lung cancer is a malignant tumor originating from bronchial mucosa and glands, with the fastest growing incidence and mortality, posing a huge threat to people's life and health. Neuron-specific enolase (NSE) is a highly specific serological biomarker for the diagnosis of small cell lung cancer and can be used as an effective indicator for the diagnosis of small cell lung cancer. Therefore, it is particularly important to develop effective detection methods to achieve trace detection of NSE. Electrochemiluminescence (ECL) is a chemiluminescence phenomenon caused by high-energy electron transfer caused by electrochemical reaction. It has the advantages of high sensitivity, low background, and rapid signal response, and is highly favored in the field of biosensor analysis. Therefore, the construction of high-performance ECL biosensors is of great significance for the sensitive detection of NSE and the early diagnosis of small cell lung cancer.

[0003] The development of efficient luminophores is crucial to improving the sensitivity of ECL biosensors. Metal-organic frameworks (MOFs) are an emerging class of porous crystalline materials constructed from metal ions / clusters and organic linkers. They have attracted widespread interest in the field of biochemical analysis due to their large surface area, adjustable structure and pore size. Among them, Ln-MOFs have unique photochemical properties, including large Stokes shift, sharp characteristic emission peaks, high color purity, non-overlapping spectra and relatively long luminescence lifetime. Therefore, Ln-MOFs are considered to be an excellent class of luminescent materials.

[0004] In the ternary ECL system, the co-reactant promoter can catalyze the co-reactant to generate more active free radicals to achieve signal amplification. However, the luminophore and the co-reactant promoter belong to different functional components. As the sensing interface is modified layer by layer, the distance between the two becomes larger, and the contact time between the luminophore and the catalytically generated co-reactant free radicals becomes longer, thereby affecting the output signal of the ECL system. In order to solve the above problems, the present invention proposes a strategy of integrating the luminophore and the catalyst, which integrates the catalytic component Cu 2+The introduction of luminescent ZrEu-MOF promotes the synergistic effect between the luminophore, co-reactant, and co-reactant promoter, achieving signal self-enhancement. In addition, a quencher is introduced into the ECL sensing system to produce a significant signal change. The efficient ECL resonance energy transfer between the prepared self-enhanced luminophore and the quencher greatly improves the detection sensitivity of the ECL biosensor, thereby better meeting the needs of clinical precision detection of NSE. Summary of the invention

[0005] One of the technical tasks of the present invention is to make up for the deficiencies of the prior art and prepare a highly efficient self-enhanced luminescent ZrEuCu-MOF that integrates the luminescent function and the catalytic function of a co-reaction promoter.

[0006] The second technical task of the present invention is to construct an ultra-sensitive self-enhanced electrochemiluminescence biosensor based on the high-efficiency self-enhanced luminescent ZrEuCu-MOF and resorcinol-formaldehyde resin-gold nanoparticles-detection antibody quenching probe, which has low raw material cost, simple preparation process and low operation risk.

[0007] The third technical task of the present invention is to provide the use of the ultrasensitive self-enhanced electrochemiluminescence biosensor constructed by the construction method, namely, for the trace detection of neuron-specific enolase. The constructed biosensor has a low detection limit, a wide detection range and good stability for NSE detection, can be used for the early diagnosis of small cell lung cancer, and has certain industrial application prospects.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] 1. Construction method of an ultrasensitive self-enhanced electrochemiluminescence biosensor

[0010] 10 μL of 6-10 mg / mL ZrEuCu-MOF solution, 8 μL of 1 μg / mL capture antibody solution, 5 μL of 1% bovine serum albumin solution, 5 μL of NSE solution, and 8 μL of egg yolk structure resorcinol-formaldehyde resin-gold nanoparticles-detection antibody composite solution were sequentially dripped onto the electrode surface and incubated at 4 °C to construct an ultrasensitive self-enhanced electrochemiluminescence biosensor.

[0011] The ZrEuCu-MOF solution is prepared by dissolving 2.34 g of ZrCl4 and 4.33 g of 1, 2, 4, 5-benzenetetracarboxylic acid in 70 mL of ultrapure water and stirring at room temperature for 30 min; after refluxing for 24 h, centrifuging the obtained mixture at 9000 rpm and washing it three times with ultrapure water; dispersing the solid obtained by centrifugation in ultrapure water and refluxing for 16 h, washing it three times with ethanol by centrifugation, and vacuum drying it at 60°C to obtain Zr-MOF; dissolving 0.034 g of EuCl3·6H2O in 10 mL of ethanol, adding 100 mg of the prepared Zr-MOF; stirring the obtained mixture at room temperature for 20 h, centrifuging it three times with ethanol, and vacuum drying it at 60°C to obtain ZrEu-MOF; dispersing 79 mg of the prepared ZrEu-MOF in a 10 mM CuCl2 solution and stirring it for 1 h later, the obtained solution was washed three times by centrifugation with ultrapure water, and vacuum dried at 60 °C to obtain ZrEuCu-MOF; the obtained ZrEuCu-MOF was dispersed in ultrapure water to obtain a ZrEuCu-MOF solution;

[0012] The capture antibody solution is obtained by uniformly dispersing the capture antibody in a phosphate buffered saline solution having a concentration of 0.1 M and a pH of 7.4;

[0013] The NSE solution is prepared by uniformly dispersing NSE at concentrations of 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL in a 0.1 M phosphate buffered saline solution having a pH of 7.4 to obtain an NSE solution;

[0014] The egg yolk structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution is prepared by adding 0.2 g of resorcinol to a mixed solution containing 8 mL of ethanol and 20 mL of ultrapure water, and mixing by ultrasonication; adding 0.54 mL of formaldehyde solution to the mixed solution, stirring for 5 min, placing the obtained mixed solution in an ice bath, slowly adding 0.3 mL of NH3·H2O, and continuously stirring for 24 h; then, washing the obtained suspension with ultrapure water and ethanol alternately for 3 times, freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin; adding 0.05 g of the egg yolk structured resorcinol-formaldehyde resin to 50 mL of the gold nanoparticle solution, mixing by ultrasonication for 30 min, and storing overnight; centrifuging the obtained mixed solution with ultrapure water for 3 times, and then freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles; dissolving 2 mg of the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles in 1 mL of phosphate buffered saline with a pH of 7.4, and then shaken with 1 μg / mL of detection antibody at 4 °C for 8 h to prepare a yolk-structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody complex solution;

[0015] Each step of solution drop coating is performed after the solution drop coating of the previous step is completed and dried to form a film.

[0016] 2. Application of the ultrasensitive self-enhanced electrochemiluminescence biosensor constructed by the construction method for detecting the small cell lung cancer marker NSE

[0017] The modified glassy carbon electrode was used as the working electrode, the platinum wire electrode was used as the counter electrode, and the silver / silver chloride electrode was used as the reference electrode. Based on the three-electrode detection system, the ECL performance was tested in 10 mL of phosphate buffer solution containing 80 ~ 120 mM K2S2O8 and pH 6.5; the scanning potential range applied in the experiment was -1.8 ~ 0 V, the photomultiplier tube high voltage was 800 V, and the scanning rate was 150 mV / s; according to the ECL test results, a linear relationship curve was drawn, and the applicable detection range of the constructed biosensor was 1 fg / mL ~ 100 ng / mL, which had efficient analytical detection capabilities; according to the signal test results of various interfering substances, it was shown that the constructed biosensor had high specificity; according to the results of multiple cycle tests, it was found that the constructed biosensor had good stability; according to the signal responses of different batches of biosensors, it was found that the constructed biosensor had good reproducibility.

[0018] Beneficial technical effects of the present invention:

[0019] 1. The present invention has prepared a highly efficient self-enhanced luminescent ZrEuCu-MOF integrating luminescence and catalytic functions; the integration of the luminophore and the co-reactant promoter promotes the generation of more free radicals on the electrode surface, shortens the contact time between the luminophore and the co-reactant free radicals, enhances the synergistic effect between the luminophore, the co-reactant and the co-reactant promoter, and realizes the effective amplification of the output signal.

[0020] 2. The present invention constructs an ultrasensitive self-enhanced electrochemiluminescence biosensor based on the self-enhanced luminescent ZrEuCu-MOF and the efficient resorcinol-formaldehyde resin-gold nanoparticles-detection antibody quenching probe, which produces obvious signal "on-off" changes and greatly improves the detection sensitivity of the ECL biosensor.

[0021] 3. The ultrasensitive self-enhanced electrochemiluminescence biosensor constructed in the present invention has a low detection limit, a wide linear range, and excellent stability, specificity and reproducibility for NSE detection, and can be used for early warning and timely diagnosis of small cell lung cancer; the ECL biosensor has a simple preparation process and low cost, and has a certain prospect for industrial application. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto, and all 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 an ultrasensitive self-enhanced electrochemiluminescence biosensor

[0024] 10 μL of 6 mg / mL ZrEuCu-MOF solution, 8 μL of 1 μg / mL capture antibody solution, 5 μL of 1% bovine serum albumin solution, 5 μL of NSE solution, and 8 μL of egg yolk structure resorcinol-formaldehyde resin-gold nanoparticles-detection antibody composite solution were sequentially dripped onto the electrode surface and incubated at 4 ℃ to construct an ultrasensitive self-enhanced electrochemiluminescence biosensor.

[0025] The ZrEuCu-MOF solution is prepared by dissolving 2.34 g of ZrCl4 and 4.33 g of 1, 2, 4, 5-benzenetetracarboxylic acid in 70 mL of ultrapure water and stirring at room temperature for 30 min; after refluxing for 24 h, centrifuging the obtained mixture at 9000 rpm and washing it three times with ultrapure water; dispersing the solid obtained by centrifugation in ultrapure water and refluxing for 16 h, washing it three times with ethanol by centrifugation, and vacuum drying it at 60°C to obtain Zr-MOF; dissolving 0.034 g of EuCl3·6H2O in 10 mL of ethanol, adding 100 mg of the prepared Zr-MOF; stirring the obtained mixture at room temperature for 20 h, centrifuging it three times with ethanol, and vacuum drying it at 60°C to obtain ZrEu-MOF; dispersing 79 mg of the prepared ZrEu-MOF in a 10 mM CuCl2 solution and stirring it for 1 h later, the obtained solution was washed three times by centrifugation with ultrapure water, and vacuum dried at 60 °C to obtain ZrEuCu-MOF; the obtained ZrEuCu-MOF was dispersed in ultrapure water to obtain a ZrEuCu-MOF solution;

[0026] The capture antibody solution is obtained by uniformly dispersing the capture antibody in a phosphate buffered saline solution having a concentration of 0.1 M and a pH of 7.4;

[0027] The NSE solution is prepared by uniformly dispersing NSE at concentrations of 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL in a 0.1 M phosphate buffered saline solution having a pH of 7.4 to obtain an NSE solution;

[0028] The egg yolk structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution is prepared by adding 0.2 g of resorcinol to a mixed solution containing 8 mL of ethanol and 20 mL of ultrapure water, and mixing by ultrasonication; adding 0.54 mL of formaldehyde solution to the mixed solution, stirring for 5 min, placing the obtained mixed solution in an ice bath, slowly adding 0.3 mL of NH3·H2O, and continuously stirring for 24 h; then, washing the obtained suspension with ultrapure water and ethanol alternately for 3 times, freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin; adding 0.05 g of the egg yolk structured resorcinol-formaldehyde resin to 50 mL of the gold nanoparticle solution, mixing by ultrasonication for 30 min, and storing overnight; centrifuging the obtained mixed solution with ultrapure water for 3 times, and then freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles; dissolving 2 mg of the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles in 1 mL of phosphate buffered saline with a pH of 7.4, and then shaken with 1 μg / mL of detection antibody at 4 °C for 8 h to prepare a yolk-structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody complex solution;

[0029] Each step of solution drop coating is performed after the solution drop coating of the previous step is completed and dried to form a film.

[0030] Example 2 A method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor

[0031] 10 μL of 8 mg / mL ZrEuCu-MOF solution, 8 μL of 1 μg / mL capture antibody solution, 5 μL of 1% bovine serum albumin solution, 5 μL of NSE solution, and 8 μL of egg yolk structure resorcinol-formaldehyde resin-gold nanoparticles-detection antibody complex solution were sequentially dripped onto the electrode surface and incubated at 4 °C to construct an ultrasensitive self-enhanced electrochemiluminescence biosensor.

[0032] The ZrEuCu-MOF solution is prepared by dissolving 2.34 g of ZrCl4 and 4.33 g of 1, 2, 4, 5-benzenetetracarboxylic acid in 70 mL of ultrapure water and stirring at room temperature for 30 min; after refluxing for 24 h, centrifuging the obtained mixture at 9000 rpm and washing it three times with ultrapure water; dispersing the solid obtained by centrifugation in ultrapure water and refluxing for 16 h, washing it three times with ethanol by centrifugation, and vacuum drying it at 60°C to obtain Zr-MOF; dissolving 0.034 g of EuCl3·6H2O in 10 mL of ethanol, adding 100 mg of the prepared Zr-MOF; stirring the obtained mixture at room temperature for 20 h, centrifuging it three times with ethanol, and vacuum drying it at 60°C to obtain ZrEu-MOF; dispersing 79 mg of the prepared ZrEu-MOF in a 10 mM CuCl2 solution and stirring it for 1 h later, the obtained solution was washed three times by centrifugation with ultrapure water, and vacuum dried at 60 °C to obtain ZrEuCu-MOF; the obtained ZrEuCu-MOF was dispersed in ultrapure water to obtain a ZrEuCu-MOF solution;

[0033] The capture antibody solution is obtained by uniformly dispersing the capture antibody in a phosphate buffered saline solution having a concentration of 0.1 M and a pH of 7.4;

[0034] The NSE solution is prepared by uniformly dispersing NSE at concentrations of 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL in a 0.1 M phosphate buffered saline solution having a pH of 7.4 to obtain an NSE solution;

[0035] The egg yolk structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution is prepared by adding 0.2 g of resorcinol to a mixed solution containing 8 mL of ethanol and 20 mL of ultrapure water, and mixing by ultrasonication; adding 0.54 mL of formaldehyde solution to the mixed solution, stirring for 5 min, placing the obtained mixed solution in an ice bath, slowly adding 0.3 mL of NH3·H2O, and continuously stirring for 24 h; then, washing the obtained suspension with ultrapure water and ethanol alternately for 3 times, freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin; adding 0.05 g of the egg yolk structured resorcinol-formaldehyde resin to 50 mL of the gold nanoparticle solution, mixing by ultrasonication for 30 min, and storing overnight; centrifuging the obtained mixed solution with ultrapure water for 3 times, and then freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles; dissolving 2 mg of the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles in 1 mL of phosphate buffered saline with a pH of 7.4, and then shaken with 1 μg / mL of detection antibody at 4 °C for 8 h to prepare a yolk-structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody complex solution;

[0036] Each step of solution drop coating is performed after the solution drop coating of the previous step is completed and dried to form a film.

[0037] Example 3 A method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor

[0038] 10 μL of 10 mg / mL ZrEuCu-MOF solution, 8 μL of 1 μg / mL capture antibody solution, 5 μL of 1% bovine serum albumin solution, 5 μL of NSE solution, and 8 μL of egg yolk structure resorcinol-formaldehyde resin-gold nanoparticles-detection antibody composite solution were sequentially dripped onto the electrode surface and incubated at 4 °C to construct an ultrasensitive self-enhanced electrochemiluminescence biosensor.

[0039] The ZrEuCu-MOF solution is prepared by dissolving 2.34 g of ZrCl4 and 4.33 g of 1, 2, 4, 5-benzenetetracarboxylic acid in 70 mL of ultrapure water and stirring at room temperature for 30 min; after refluxing for 24 h, centrifuging the obtained mixture at 9000 rpm and washing it three times with ultrapure water; dispersing the solid obtained by centrifugation in ultrapure water and refluxing for 16 h, washing it three times with ethanol by centrifugation, and vacuum drying it at 60°C to obtain Zr-MOF; dissolving 0.034 g of EuCl3·6H2O in 10 mL of ethanol, adding 100 mg of the prepared Zr-MOF; stirring the obtained mixture at room temperature for 20 h, centrifuging it three times with ethanol, and vacuum drying it at 60°C to obtain ZrEu-MOF; dispersing 79 mg of the prepared ZrEu-MOF in a 10 mM CuCl2 solution and stirring it for 1 h later, the obtained solution was washed three times by centrifugation with ultrapure water, and vacuum dried at 60 °C to obtain ZrEuCu-MOF; the obtained ZrEuCu-MOF was dispersed in ultrapure water to obtain a ZrEuCu-MOF solution;

[0040] The capture antibody solution is obtained by uniformly dispersing the capture antibody in a phosphate buffered saline solution having a concentration of 0.1 M and a pH of 7.4;

[0041] The NSE solution is prepared by uniformly dispersing NSE at concentrations of 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL in a 0.1 M phosphate buffered saline solution having a pH of 7.4 to obtain an NSE solution;

[0042] The egg yolk structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution is prepared by adding 0.2 g of resorcinol to a mixed solution containing 8 mL of ethanol and 20 mL of ultrapure water, and mixing by ultrasonication; adding 0.54 mL of formaldehyde solution to the mixed solution, stirring for 5 min, placing the obtained mixed solution in an ice bath, slowly adding 0.3 mL of NH3·H2O, and continuously stirring for 24 h; then, washing the obtained suspension with ultrapure water and ethanol alternately for 3 times, freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin; adding 0.05 g of the egg yolk structured resorcinol-formaldehyde resin to 50 mL of the gold nanoparticle solution, mixing by ultrasonication for 30 min, and storing overnight; centrifuging the obtained mixed solution with ultrapure water for 3 times, and then freeze-drying for 12 h, and obtaining the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles; dissolving 2 mg of the egg yolk structured resorcinol-formaldehyde resin-gold nanoparticles in 1 mL of phosphate buffered saline with a pH of 7.4, and then shaken with 1 μg / mL of detection antibody at 4 °C for 8 h to prepare a yolk-structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody complex solution;

[0043] Each step of solution drop coating is performed after the solution drop coating of the previous step is completed and dried to form a film.

[0044] Example 4 Application of the ultrasensitive self-enhanced electrochemiluminescence biosensor constructed by the construction methods described in Examples 1, 2 and 3 for the detection of small cell lung cancer marker NSE

[0045] Using the modified glassy carbon electrode as the working electrode, the platinum wire electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode, based on a three-electrode detection system, its ECL performance was tested in 10 mL of phosphate buffer solution containing 80 mM K2S2O8 and a pH of 6.5; the scanning potential range applied in the experiment was -1.8 ~ 0 V, the photomultiplier tube high voltage was 800 V, and the scanning rate was 150 mV / s; a linear relationship curve was drawn based on the ECL test results, and the applicable detection range of the constructed biosensor was 1 fg / mL ~ 100 ng / mL, with efficient analytical detection capabilities; according to the signal test results of various interfering substances, it was shown that the constructed biosensor had high specificity; according to the results of multiple cycle tests, the constructed biosensor had good stability; according to the signal responses of different batches of biosensors, the constructed biosensor had good reproducibility. Example 5 The use of the ultrasensitive self-enhanced electrochemiluminescence biosensor constructed by the construction method described in Example 1, Example 2 and Example 3 for the detection of small cell lung cancer marker NSE.

[0046] Using the modified glassy carbon electrode as the working electrode, the platinum wire electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode, based on a three-electrode detection system, its ECL performance was tested in 10 mL of phosphate buffer solution containing 100 mM K2S2O8 and a pH of 6.5; the scanning potential range applied in the experiment was -1.8 ~ 0 V, the photomultiplier tube high voltage was 800 V, and the scanning rate was 150 mV / s; a linear relationship curve was drawn based on the ECL test results, and the applicable detection range of the constructed biosensor was 1 fg / mL ~ 100 ng / mL, with efficient analytical detection capabilities; according to the signal test results of various interfering substances, it was shown that the constructed biosensor had high specificity; according to the results of multiple cycle tests, the constructed biosensor had good stability; according to the signal responses of different batches of biosensors, the constructed biosensor had good reproducibility. Example 6 Use of the ultrasensitive self-enhanced electrochemiluminescence biosensor constructed by the construction method described in Example 1, Example 2 and Example 3 for the detection of small cell lung cancer marker NSE.

[0047] Using the modified glassy carbon electrode as the working electrode, the platinum wire electrode as the counter electrode, and the silver / silver chloride electrode as the reference electrode, based on a three-electrode detection system, its ECL performance was tested in 10 mL of phosphate buffered saline solution containing 120 mM K2S2O8 and a pH of 6.5; the scanning potential range applied in the experiment was -1.8 ~ 0 V, the photomultiplier tube high voltage was 800 V, and the scanning rate was 150 mV / s; a linear relationship curve was drawn based on the ECL test results, and it was found that the applicable detection range of the constructed biosensor was 1 fg / mL ~ 100 ng / mL, and it had efficient analytical detection capabilities; based on the signal test results of a variety of interfering substances, it was shown that the constructed biosensor had high specificity; based on the results of multiple cycle tests, it was found that the constructed biosensor had good stability; based on the signal responses of different batches of biosensors, it was found that the constructed biosensor had good reproducibility. Description of the Figures

[0048] Figure 1 The flowchart is a method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor (egg yolk structure resorcinol-formaldehyde resin: YRF; capture antibody: Ab1; detection antibody: Ab2; bovine serum albumin: BSA).

[0049] Figure 2 (A) XRD spectrum and (B) infrared spectrum of ZrEuCu-MOF.

[0050] Figure 3X-ray photoelectron spectrum of ZrEuCu-MOF, including: (A) full-area X-ray photoelectron spectrum of ZrEuCu-MOF; (B) high-resolution X-ray photoelectron spectrum of O 1s region; (C) high-resolution X-ray photoelectron spectrum of Cu 2p region; (D) high-resolution X-ray photoelectron spectrum of Zr 3d region; (E) high-resolution X-ray photoelectron spectrum of Eu 3d region.

[0051] Figure 4 (A) Scanning electron microscope image of ZrEuCu-MOF; (B) Transmission electron microscope image; (C) elemental mapping image.

[0052] Figure 5 (A) XRD spectra and (B) UV spectra of resorcinol-formaldehyde resin with egg yolk structure and resorcinol-formaldehyde resin-gold nanoparticles with egg yolk structure; (C) transmission electron microscopy image of resorcinol-formaldehyde resin with egg yolk structure; (D) transmission electron microscopy image of resorcinol-formaldehyde resin-gold nanoparticles with egg yolk structure.

[0053] Figure 6 (A) Excitation and emission spectra of ZrEuCu-MOF; (B) Ligand H4btec to Eu 3+ (Left) and Cu 2+ To Eu 3 + (Right) Schematic diagram of the energy transfer process; (C) ECL intensity-potential curves under different conditions, where a and b refer to the ECL intensity-potential curves of ZrEuCu-MOF and ZrEu-MOF in K2S2O8 solution, respectively; c and d refer to the ECL intensity-potential curves of ZrEuCu-MOF and ZrEu-MOF in phosphate buffered saline solution, respectively; (D) Schematic diagram of the ECL mechanism of ZrEuCu-MOF.

[0054] Figure 7 (A) ECL emission spectrum of ZrEuCu-MOF and UV absorption spectrum of resorcinol-formaldehyde resin-gold nanoparticles; (B) Schematic diagram of ECL resonance energy transfer from ZrEuCu-MOF to resorcinol-formaldehyde resin-gold nanoparticles.

[0055] Figure 8 Diagram showing the construction process of an ultrasensitive self-enhanced electrochemiluminescence biosensor based on (A) cyclic voltammetry and (B) electrochemical impedance spectroscopy.

[0056] Fig. 9These are the condition optimization diagrams for the ultrasensitive self-enhanced electrochemiluminescence biosensor, including: (A) test solution pH value optimization result diagram; (B) K2S2O8 concentration optimization result diagram; (C) ZrEuCu-MOF concentration optimization result diagram; (D) resorcinol-formaldehyde resin-gold nanoparticle concentration optimization result diagram.

[0057] Fig.10 (A) ECL response of the ultrasensitive self-enhanced electrochemiluminescence biosensor after incubation with different concentrations of NSE and (B) the corresponding calibration curve;

[0058] Fig.11 Ultrasensitive self-enhanced electrochemiluminescence biosensor: (A) specificity; (B) stability; (C) reproducibility.

Claims

1. A method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor, characterized in that: 10 μL of 6-10 mg / mL ZrEuCu-MOF solution, 8 μL of 1 μg / mL capture antibody solution, 5 μL of 1% bovine serum albumin solution, 5 μL of neuron-specific enolase solution, and 8 μL of egg yolk-structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution were sequentially dripped onto the electrode surface and incubated at 4°C to construct an ultrasensitive self-enhanced electrochemiluminescence biosensor; the egg yolk-structured resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution was prepared by adding 0.2 g of resorcinol to a mixed solution containing 8 mL of ethanol and 20 mL of ultrapure water, and ultrasonically mixing; 0.54 mL of formaldehyde solution was added to the mixed solution, stirred for 5 minutes, and the resulting mixed solution was placed in an ice bath, and slowly stirred. 0.3 mL of NH3·H2O was added and stirred for 24 h. Subsequently, the obtained suspension was washed alternately with ultrapure water and ethanol for 3 times, and freeze-dried for 12 h to obtain the resorcinol-formaldehyde resin with egg yolk structure. 0.05 g of the resorcinol-formaldehyde resin with egg yolk structure was added to 50 mL of the gold nanoparticle solution, mixed by ultrasound for 30 min, and stored overnight. The obtained mixed solution was centrifuged with ultrapure water for 3 times, and then freeze-dried for 12 h to obtain the resorcinol-formaldehyde resin-gold nanoparticles with egg yolk structure. 2 mg of the resorcinol-formaldehyde resin-gold nanoparticles with egg yolk structure were dissolved in 1 mL of phosphate buffered saline with a pH of 7.4, and then shaken with 1 μg / mL of the detection antibody at 4°C for 8 h to obtain the resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution with egg yolk structure.

2. The method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor according to claim 1, characterized in that: The capture antibody solution is obtained by uniformly dispersing the capture antibody in a phosphate buffered saline solution with a concentration of 0.1 M and a pH of 7.

4.

3. The method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor according to claim 1, characterized in that: The neuron-specific enolase solution is prepared by uniformly dispersing neuron-specific enolase at concentrations of 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL in a 0.1 M phosphate buffered saline solution having a pH of 7.4 to obtain a neuron-specific enolase solution.

4. The method for constructing an ultrasensitive self-enhanced electrochemiluminescence biosensor according to claim 1, characterized in that: The capture antibody solution, bovine serum albumin solution, neuron-specific enolase solution, and egg yolk structure resorcinol-formaldehyde resin-gold nanoparticle-detection antibody composite solution are all dripped after the previous solution drips and is dried to form a film.

5. Use of the ultrasensitive self-enhanced electrochemiluminescence biosensor constructed by the construction method according to claim 1, characterized in that: Application for the detection of neuron-specific enolase.

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