A method for constructing and applying an electrochemiluminescence antifouling sensor based on zwitterionic hydrogels.

Abstract

The application discloses a construction method and application of an electrochemiluminescence (ECL) antifouling sensor based on a zwitterionic hydrogel, and belongs to the fields of photoelectric sensing analysis, interface antifouling and environmental science and technology. In the application, a hydrogel based on a methacrylic acid sulfobetaine zwitterion and bovine serum albumin is used as an antifouling coating, so that the adsorption of biological proteins and bacteria is effectively hindered, and the service life of the sensor is prolonged. Meanwhile, an internal standard Au luminol is encapsulated in the antifouling hydrogel, so that the integration of interface antifouling and ratio detection is realized, the detection accuracy is improved, and the construction process of the ECL sensor is simplified. The constructed ECL antifouling sensor has high sensitivity for chloramphenicol detection, and the detection limit is 0.39 pM, so that the ECL antifouling sensor is suitable for trace detection of antibiotics in seawater.

Description

Technical Field

[0001] This invention discloses a method for constructing an electrochemiluminescence antifouling sensor based on zwitterionic hydrogel and its application in chloramphenicol detection, belonging to the fields of photoelectric sensing analysis, interface antifouling, and environmental science technology. Background Technology

[0002] Chloramphenicol (CAP), a common broad-spectrum antibiotic, is widely used to treat certain infectious diseases. However, overuse of CAP can lead to water pollution, thus impacting human health. Electrochemiluminescence (ECL) shows promising application prospects in sensing analysis due to its advantages such as low background signal, simple operation mode, and high sensitivity. Therefore, there is an urgent need to construct a sensitive ECL sensing analysis platform for trace detection of CAP in the aquatic environment.

[0003] In complex aquatic environments, besides the target analyte, other interfering substances can affect the sensor's detection performance and lifespan through non-specific adsorption. To address this issue, bovine serum albumin (BSA) is often modified onto the sensing interface to resist non-specific adsorption. While BSA coatings offer good antifouling properties and biocompatibility, their inherent poor stability makes it difficult to maintain reliable antifouling capabilities in complex detection media. With the development of antifouling systems, many materials, such as polyethylene glycol, peptides, hydrogels, and zwitterions, have been found to possess antifouling properties. Among these, zwitterions exhibit strong hydration capacity and high stability, thus demonstrating good inertness to changes in pH and salinity in aqueous solutions. Meanwhile, superhydrophilic hydrogels are also a reasonable choice for antifouling materials. Therefore, designing an efficient and stable antifouling interface with these excellent properties is crucial.

[0004] Based on this, this invention grafts sulfobetaine methacrylate (SBMA) onto BSA via a thiol-ene click reaction, developing a zwitterionic hydrogel as an antifouling interface. This hydrogel combines the high hydrophilicity of the hydrogel with the strong hydration of zwitterions, exhibiting highly efficient antifouling and antibacterial capabilities. Compared to BSA, the developed zwitterionic hydrogel is more stable and suitable for sensitive detection of pollutants in complex aquatic environments. Furthermore, Au@luminol is encapsulated as an internal standard within the zwitterionic antifouling hydrogel, integrating interfacial antifouling and internal calibration, and providing numerous active sites for subsequent biomolecule binding. With the introduction of antifouling and ratiometric strategies, the sensor constructed in this invention exhibits high detection sensitivity and accuracy, as well as high stability and long lifespan in complex aquatic environments, thereby achieving trace detection of CAP with a detection limit as low as 0.39 pM, contributing to the effective monitoring of antibiotic-like environmental pollutants in seawater. Summary of the Invention

[0005] One of the technical tasks of this invention is to overcome the shortcomings of the prior art by preparing an amphoteric hydrogel as an antifouling interface for ECL sensing analysis, thereby improving the service life and specificity of the ECL sensor.

[0006] The second technical objective of this invention is to construct an ECL antifouling sensor based on zwitterionic hydrogel according to the interface antifouling and ratio detection strategy. This sensor has high detection sensitivity and long service life, and uses low-cost raw materials, has a simple preparation process, and is safe to operate.

[0007] The third technical objective of this invention is to provide the application of the ECL antifouling sensor based on zwitterionic hydrogel constructed by the aforementioned construction method, namely, for ultrasensitive detection of CAP in marine environments. The ECL antifouling sensor constructed by this invention has a wide linear range, low detection limit and good specificity for CEA detection, and can be used for effective monitoring of antibiotic-like environmental pollutants in seawater, and has certain industrialization prospects.

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

[0009] 1. A method for constructing an electrochemiluminescence antifouling sensor based on zwitterionic hydrogels.

[0010] A glassy carbon electrode was polished with Al2O3 powder to obtain a mirror-like surface; 4–6 μL of Au@luminol-SBMA-BSA hydrogel was applied to the pretreated glassy carbon electrode surface; the treated electrode was then immersed in a solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide for 2 h for crosslinking; next, the electrode was incubated in 10 μM H1 solution at 4 °C for 2 h; the modified electrode was further incubated in 10 μM H2-CdS solution for 2 h; finally, the resulting electrode was incubated in CAP solutions of different concentrations for 50 min to construct an ECL antifouling sensor based on zwitterionic hydrogel.

[0011] The Au@luminol-SBMA-BSA hydrogel was prepared by dissolving 80 mg of BSA in 20 mL of phosphate buffer solution, then adding 37.8 mg of NaBH4 and stirring for 30 min to reduce the disulfide bonds to thiol groups; then adding 2.24 g of SBMA monomer and irradiating under 365 nm ultraviolet light for 2 h to induce a thiol-ene click reaction; the resulting solution was stirred overnight for polymerization, and the polymerization product was dialyzed against a 14 kDa dialysis bag for 3 days and freeze-dried to obtain SBMA-BSA; 8 mg of SBMA-BSA was dissolved in 100 μL of ultrapure water, and 20 μL of the prepared Au@luminol solution was added; finally, 5 μL of glutaraldehyde was added for crosslinking to obtain the Au@luminol-SBMA-BSA hydrogel.

[0012] The Au@luminol solution was prepared by heating 100 mL of HAuCl4 solution to boiling with stirring; rapidly adding 1 mL of luminol to the solution and maintaining boiling for 30 min in the dark until the solution color changed from yellow to wine red; centrifuging the cooled solution at 10000 rpm and washing it three times; and redispersing the resulting precipitate in ultrapure water to obtain the Au@luminol solution.

[0013] The H2-CdS solution was prepared by dispersing 86 μL of mercaptopropionic acid in 20 mL of CdCl2 solution; adjusting the pH of the mixture to 10 with 1 M NaOH, adding 20 mL of thioacetamide solution and stirring for 30 min; refluxing the mixture at 80 °C for 10 h and dialyzing overnight in ultrapure water; adding a mixed solution containing 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.01 M N-hydroxysuccinimide and shaking for 1 h to activate the carboxyl groups of CdS quantum dots; adding CAP aptamer H2 and incubating at 37 °C for 4 h to obtain the H2-CdS solution.

[0014] The CAP solutions of different concentrations are obtained by uniformly dispersing CAP at concentrations of 0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM in a phosphate buffer solution with a pH of 7.4.

[0015] In each step of the electrode modification process, the modified electrode surface is gently rinsed with ultrapure water to remove any incompletely bound reagents.

[0016] 2. The application of the electrochemiluminescence antifouling sensor based on zwitterionic hydrogel constructed by the aforementioned method is for the detection of broad-spectrum antibiotics (CAP) in marine environments. A phosphate buffer solution containing 0.1 M K₂S₂O₈ and 6.5 mM H₂O₂ with a pH of 6.0–8.0 was used as the test solution. The constructed ECL sensor was immersed in the detection solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for ECL signal testing. The applied voltage range during the experiment was -1.6–0.8 V, the amplification stage was 3, and the photomultiplier tube voltage was 600 V. Based on the linear curve plotted from the ECL response, the detection range of the constructed ECL sensor was found to be 1 pM–100 nM, with a detection limit as low as 0.39 pM. The results show that the constructed ECL sensor has high stability, specificity, and reproducibility, and is suitable for ultrasensitive detection of CAP in seawater.

[0017] The beneficial technical effects of the present invention are as follows:

[0018] 1. This invention prepares a zwitterionic hydrogel as an antifouling interface for ECL sensing analysis. By grafting SBMA onto BSA using a thiol-ene click reaction, the prepared antifouling sensing interface combines the high hydrophilicity of the hydrogel with the strong hydration of zwitterions, exhibiting highly efficient antifouling and antibacterial capabilities. Compared to BSA, the prepared zwitterionic antifouling gel is more stable and suitable for applications requiring sensitive detection of pollutants in complex marine environments.

[0019] 2. This invention constructs an ECL antifouling sensor based on zwitterionic hydrogel. Through the application of interface antifouling and ratio detection strategies, the constructed ECL sensor has high detection sensitivity and precision, as well as long working life. Moreover, the raw materials used are low-cost, the preparation process is simple, and the operation is safe.

[0020] 3. The ECL antifouling sensor based on zwitterionic hydrogel constructed in this invention exhibits a low detection limit and wide linear range, long working life, and high stability, specificity, and reproducibility for the detection of CAP in seawater. It can be used for the effective monitoring of antibiotic pollutants in complex marine environments and has certain prospects for industrial application. Detailed Implementation

[0021] 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.

[0022] Example 1: A method for constructing an electrochemiluminescence antifouling sensor based on zwitterionic hydrogel. A glassy carbon electrode was polished with Al2O3 powder to obtain a mirror-like surface; 4 μL of Au@luminol-SBMA-BSA hydrogel was applied to the pretreated glassy carbon electrode surface; the treated electrode was then immersed in a solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide for crosslinking for 2 h; next, the electrode was incubated in 10 μM H1 solution at 4 °C for 2 h; the modified electrode was further incubated in 10 μM H2-CdS solution for 2 h; finally, the resulting electrode was incubated in CAP solutions of different concentrations for 50 min, thus constructing an ECL antifouling sensor based on zwitterionic hydrogel.

[0023] The Au@luminol-SBMA-BSA hydrogel was prepared by dissolving 80 mg of BSA in 20 mL of phosphate buffer solution, then adding 37.8 mg of NaBH4 and stirring for 30 min to reduce the disulfide bonds to thiol groups; then adding 2.24 g of SBMA monomer and irradiating under 365 nm ultraviolet light for 2 h to induce a thiol-ene click reaction; the resulting solution was stirred overnight for polymerization, and the polymerization product was dialyzed against a 14 kDa dialysis bag for 3 days and freeze-dried to obtain SBMA-BSA; 8 mg of SBMA-BSA was dissolved in 100 μL of ultrapure water, and 20 μL of the prepared Au@luminol solution was added; finally, 5 μL of glutaraldehyde was added for crosslinking to obtain the Au@luminol-SBMA-BSA hydrogel.

[0024] The Au@luminol solution was prepared by heating 100 mL of HAuCl4 solution to boiling with stirring; rapidly adding 1 mL of luminol to the solution and maintaining boiling for 30 min in the dark until the solution color changed from yellow to wine red; centrifuging the cooled solution at 10000 rpm and washing it three times; and redispersing the resulting precipitate in ultrapure water to obtain the Au@luminol solution.

[0025] The H2-CdS solution was prepared by dispersing 86 μL of mercaptopropionic acid in 20 mL of CdCl2 solution; adjusting the pH of the mixture to 10 with 1 M NaOH, adding 20 mL of thioacetamide solution and stirring for 30 min; refluxing the mixture at 80 °C for 10 h and dialyzing overnight in ultrapure water; adding a mixed solution containing 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.01 M N-hydroxysuccinimide and shaking for 1 h to activate the carboxyl groups of CdS quantum dots; adding CAP aptamer H2 and incubating at 37 °C for 4 h to obtain the H2-CdS solution.

[0026] The CAP solutions of different concentrations are obtained by uniformly dispersing CAP at concentrations of 0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM in a phosphate buffer solution with a pH of 7.4.

[0027] In each step of the electrode modification process, the modified electrode surface is gently rinsed with ultrapure water to remove any incompletely bound reagents.

[0028] Example 2: A method for constructing an electrochemiluminescence antifouling sensor based on zwitterionic hydrogel. A glassy carbon electrode was polished with Al2O3 powder to obtain a mirror-like surface; 5 μL of Au@luminol-SBMA-BSA hydrogel was applied to the pretreated glassy carbon electrode surface; the treated electrode was then immersed in a solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide for crosslinking for 2 h; next, the electrode was incubated in 10 μM H1 solution at 4 °C for 2 h; the modified electrode was further incubated in 10 μM H2-CdS solution for 2 h; finally, the resulting electrode was incubated in CAP solutions of different concentrations for 50 min, thus constructing an ECL antifouling sensor based on zwitterionic hydrogel.

[0029] The Au@luminol-SBMA-BSA hydrogel was prepared by dissolving 80 mg of BSA in 20 mL of phosphate buffer solution, then adding 37.8 mg of NaBH4 and stirring for 30 min to reduce the disulfide bonds to thiol groups; then adding 2.24 g of SBMA monomer and irradiating under 365 nm ultraviolet light for 2 h to induce a thiol-ene click reaction; the resulting solution was stirred overnight for polymerization, and the polymerization product was dialyzed against a 14 kDa dialysis bag for 3 days and freeze-dried to obtain SBMA-BSA; 8 mg of SBMA-BSA was dissolved in 100 μL of ultrapure water, and 20 μL of the prepared Au@luminol solution was added; finally, 5 μL of glutaraldehyde was added for crosslinking to obtain the Au@luminol-SBMA-BSA hydrogel.

[0030] The Au@luminol solution was prepared by heating 100 mL of HAuCl4 solution to boiling with stirring; rapidly adding 1 mL of luminol to the solution and maintaining boiling for 30 min in the dark until the solution color changed from yellow to wine red; centrifuging the cooled solution at 10000 rpm and washing it three times; and redispersing the resulting precipitate in ultrapure water to obtain the Au@luminol solution.

[0031] The H2-CdS solution was prepared by dispersing 86 μL of mercaptopropionic acid in 20 mL of CdCl2 solution; adjusting the pH of the mixture to 10 with 1 M NaOH, adding 20 mL of thioacetamide solution and stirring for 30 min; refluxing the mixture at 80 °C for 10 h and dialyzing overnight in ultrapure water; adding a mixed solution containing 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.01 M N-hydroxysuccinimide and shaking for 1 h to activate the carboxyl groups of CdS quantum dots; adding CAP aptamer H2 and incubating at 37 °C for 4 h to obtain the H2-CdS solution.

[0032] The CAP solutions of different concentrations are obtained by uniformly dispersing CAP at concentrations of 0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM in a phosphate buffer solution with a pH of 7.4.

[0033] In each step of the electrode modification process, the modified electrode surface is gently rinsed with ultrapure water to remove any incompletely bound reagents.

[0034] Example 3: A method for constructing an electrochemiluminescence antifouling sensor based on zwitterionic hydrogel. A glassy carbon electrode was polished with Al2O3 powder to obtain a mirror-like surface; 6 μL of Au@luminol-SBMA-BSA hydrogel was applied to the pretreated glassy carbon electrode surface; the treated electrode was then immersed in a solution of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide for crosslinking for 2 h; next, the electrode was incubated in 10 μM H1 solution at 4 °C for 2 h; the modified electrode was further incubated in 10 μM H2-CdS solution for 2 h; finally, the resulting electrode was incubated in CAP solutions of different concentrations for 50 min, thus constructing an ECL antifouling sensor based on zwitterionic hydrogel.

[0035] The Au@luminol-SBMA-BSA hydrogel was prepared by dissolving 80 mg of BSA in 20 mL of phosphate buffer solution, then adding 37.8 mg of NaBH4 and stirring for 30 min to reduce the disulfide bonds to thiol groups; then adding 2.24 g of SBMA monomer and irradiating under 365 nm ultraviolet light for 2 h to induce a thiol-ene click reaction; the resulting solution was stirred overnight for polymerization, and the polymerization product was dialyzed against a 14 kDa dialysis bag for 3 days and freeze-dried to obtain SBMA-BSA; 8 mg of SBMA-BSA was dissolved in 100 μL of ultrapure water, and 20 μL of the prepared Au@luminol solution was added; finally, 5 μL of glutaraldehyde was added for crosslinking to obtain the Au@luminol-SBMA-BSA hydrogel.

[0036] The Au@luminol solution was prepared by heating 100 mL of HAuCl4 solution to boiling with stirring; rapidly adding 1 mL of luminol to the solution and maintaining boiling for 30 min in the dark until the solution color changed from yellow to wine red; centrifuging the cooled solution at 10000 rpm and washing it three times; and redispersing the resulting precipitate in ultrapure water to obtain the Au@luminol solution.

[0037] The H2-CdS solution was prepared by dispersing 86 μL of mercaptopropionic acid in 20 mL of CdCl2 solution; adjusting the pH of the mixture to 10 with 1 M NaOH, adding 20 mL of thioacetamide solution and stirring for 30 min; refluxing the mixture at 80 °C for 10 h and dialyzing overnight in ultrapure water; adding a mixed solution containing 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.01 M N-hydroxysuccinimide and shaking for 1 h to activate the carboxyl groups of CdS quantum dots; adding CAP aptamer H2 and incubating at 37 °C for 4 h to obtain the H2-CdS solution.

[0038] The CAP solutions of different concentrations are obtained by uniformly dispersing CAP at concentrations of 0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM in a phosphate buffer solution with a pH of 7.4.

[0039] In each step of the electrode modification process, the modified electrode surface is gently rinsed with ultrapure water to remove any incompletely bound reagents.

[0040] Example 4 describes the application of the zwitterionic hydrogel-based electrochemiluminescence antifouling sensor constructed using the methods described in Examples 1, 2, and 3, for the detection of broad-spectrum antibiotics (CAP) in marine environments. A phosphate buffer solution containing 0.1 M K₂S₂O₈ and 6.5 mM H₂O₂ at pH 6.0 was used as the test solution. The constructed ECL sensor was immersed in the detection solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for ECL signal testing. The applied voltage range during the experiment was -1.6 to 0.8 V, the amplification stage was 3, and the photomultiplier tube voltage was 600 V. Based on the linear curve plotted from the ECL response, the detection range of the constructed ECL sensor was found to be 1 pM to 100 nM, with a detection limit as low as 0.39 pM. The results indicate that the constructed ECL sensor has high stability, specificity, and reproducibility, making it suitable for ultrasensitive detection of CAP in seawater.

[0041] Example 5 describes the application of the zwitterionic hydrogel-based electrochemiluminescence antifouling sensor constructed using the methods described in Examples 1, 2, and 3, for the detection of broad-spectrum antibiotics (CAP) in marine environments. A phosphate buffer solution containing 0.1 M K₂S₂O₈ and 6.5 mM H₂O₂ at pH 7.0 was used as the test solution. The constructed ECL sensor was immersed in the detection solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for ECL signal testing. The applied voltage range during the experiment was -1.6 to 0.8 V, the amplification stage was 3, and the photomultiplier tube voltage was 600 V. Based on the linear curve plotted from the ECL response, the detection range of the constructed ECL sensor was found to be 1 pM to 100 nM, with a detection limit as low as 0.39 pM. The results indicate that the constructed ECL sensor has high stability, specificity, and reproducibility, making it suitable for ultrasensitive detection of CAP in seawater.

[0042] Example 6 describes the application of the zwitterionic hydrogel-based electrochemiluminescence antifouling sensor constructed using the methods described in Examples 1, 2, and 3, for the detection of broad-spectrum antibiotics (CAP) in marine environments. A phosphate buffer solution containing 0.1 M K₂S₂O₈ and 6.5 mM H₂O₂ at pH 8.0 was used as the test solution. The constructed ECL sensor was immersed in the detection solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for ECL signal testing. The applied voltage range during the experiment was -1.6 to 0.8 V, the amplification stage was 3, and the photomultiplier tube voltage was 600 V. Based on the linear curve plotted from the ECL response, the detection range of the constructed ECL sensor was found to be 1 pM to 100 nM, with a detection limit as low as 0.39 pM. The results indicate that the constructed ECL sensor has high stability, specificity, and reproducibility, making it suitable for ultrasensitive detection of CAP in seawater. Attached Figure Description

[0043] Figure 1 The flowchart shows the construction method of the ECL antifouling sensor based on zwitterionic hydrogel (glassy carbon electrode: GCE; reduced bovine serum albumin: re-BSA; glutaraldehyde: GA; complementary chain: H1; chloramphenicol aptamer chain: H2).

[0044] Figure 2 Infrared spectra of BSA, SBMA, and BSA@PSBMA.

[0045] Figure 3 The X-ray photoelectron spectra of BSA@PSBMA are shown below. (A) is the full-area X-ray photoelectron spectrum of BSA@PSBMA; (B) is the high-resolution X-ray photoelectron spectrum of the N 1s region; and (C) is the high-resolution X-ray photoelectron spectrum of the S2p region.

[0046] Figure 4 Transmission electron microscopy images of (A) Au@luminol and (B) CdS quantum dots.

[0047] Figure 5 The images show the UV-Vis absorption spectra of (A) Au NPs (black curve), luminol (red curve), and Au@luminol (blue curve), and (B) CdS quantum dots.

[0048] Figure 6 The image shows a scanning electron microscope (SEM) image and the corresponding elemental mapping of the Au@luminol-BSA@PSBMA hydrogel.

[0049] Figure 7 The figure shows the rheological measurement results of the Au@luminol-BSA@PSBMA hydrogel.

[0050] Figure 8 Static water contact angle diagrams for (A) GCE, (B) BSA hydrogel / GCE, and (C) Au@luminol-BSA@PSBMA hydrogel / GCE.

[0051] Figure 9 Histograms of static water contact angles for BSA hydrogel / GCE and Au@luminol-BSA@PSBMA hydrogel / GCE in different (A) pH and (B) ionic salts.

[0052] Figure 10 The diagram shows the optimization of conditions for an ECL antifouling sensor based on zwitterionic hydrogel, where (A) is the optimized contact angle diagram and (B) is the optimized CAP incubation time diagram.

[0053] Figure 11 Differential pulse voltammetry curves of (A) GCE and (B) Au@luminol-BSA@PSBMA hydrogel / GCE incubated in fetal bovine serum at different concentrations.

[0054] Figure 12 (A) Fluorescence imaging of ITO, BSA hydrogel / ITO and Au@luminol-BSA@PSBMA hydrogel / ITO after incubation in fluorescein-conjugated BSA solution and (B) corresponding quantitative fluorescence analysis.

[0055] Figure 13 The images show (A) fluorescence imaging of ITO, BSA hydrogel / ITO and Au@luminol-BSA@PSBMA hydrogel / ITO after incubation in E. coli solution and (B) the corresponding quantitative fluorescence analysis.

[0056] Figure 14Characterization diagram of the construction process of the ECL antifouling sensor based on zwitterionic hydrogel according to (A) differential pulse voltammetry and (B) AC impedance testing.

[0057] Figure 15 GCE, Au@luminol-BSA@PSBMA / GCE, H2-CdS / H1 / Au@luminol-BSA@PSBMA / GCE, and CAP / H2-CdS / H1 / Au@luminol-BSA@PSBMA / GCE in a solution containing 0.1 M S2O8 2- ECL curves in phosphate buffer solution with 6.5 mM H2O2.

[0058] Figure 16 (A) ECL curve and (B) corresponding calibration curve of the ECL antifouling sensor based on zwitterionic hydrogel after incubation with different concentrations of CAP; where a to g represent concentrations of 0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM, respectively.

[0059] Figure 17 To evaluate the (A) specificity, (B) signal stability, (C) reproducibility, and (D) storage stability of the ECL antifouling sensor based on zwitterionic hydrogels.

Claims

1. A method for constructing an electrochemiluminescent zwitterionic hydrogel-based anti-fouling sensor, characterized in that, A glassy carbon electrode is polished with Al2O3 powder to obtain a mirror surface; 4-6 μL of Au@luminol-methacrylic acid sulfobetaine zwitterion-bovine serum albumin hydrogel is modified on the surface of the pretreated glassy carbon electrode; then the treated electrode is immersed in a 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt solution for crosslinking for 2 h; next, the electrode is incubated in a 10 μM complementary chain solution at 4 °C for 2 h; the modified electrode is further incubated in a 10 μM chloramphenicol aptamer chain-CdS solution for 2 h; finally, the obtained electrode is incubated in a chloramphenicol solution with different concentrations for 50 min, and an electrochemiluminescence anti-fouling sensor based on zwitterionic hydrogel is constructed.

2. A method of constructing a zwitterionic hydrogel-based electrochemiluminescent antifouling sensor as claimed in claim 1, wherein, The Au@luminol-methacrylic acid sulfobetaine zwitterion-bovine serum albumin hydrogel is prepared by dissolving 80 mg of bovine serum albumin in 20 mL of phosphate buffered saline solution, then adding 37.8 mg of NaBH4 and stirring for 30 min to reduce the disulfide bond to thiol; then 2.24 g of methacrylic acid sulfobetaine monomer is added and irradiated under 365 nm ultraviolet light for 2 h to induce thiol-alkene click reaction; the obtained solution is stirred overnight for polymerization, and the polymerization product is dialyzed with a 14 KDa dialysis bag for 3 days, and freeze-dried to obtain methacrylic acid sulfobetaine zwitterion-bovine serum albumin; 8 mg of methacrylic acid sulfobetaine zwitterion-bovine serum albumin is dissolved in 100 μL of ultrapure water, 20 μL of prepared Au@luminol solution is added; finally, 5 μL of glutaraldehyde is added for crosslinking to obtain Au@luminol-methacrylic acid sulfobetaine zwitterion-bovine serum albumin hydrogel.

3. A method of constructing a zwitterionic hydrogel-based electrochemiluminescent antifouling sensor as claimed in claim 2, wherein, The Au@luminol solution is prepared by heating 100 mL of HAuCl4 solution to boiling under stirring; 1 mL of luminol is quickly added to the above solution, and the boiling is maintained for 30 min in the dark until the solution color changes from yellow to wine red; the cooled solution is centrifuged at 10000 rpm and washed for 3 times; the obtained precipitate is redispersed in ultrapure water to obtain the Au@luminol solution.

4. A method of constructing a zwitterionic hydrogel-based electrochemiluminescent antifouling sensor as claimed in claim 1, wherein, The chloramphenicol aptamer chain-CdS solution is prepared by dispersing 86 μL of mercaptopropionic acid in 20 mL of CdCl2 solution; after adjusting the pH value of the mixed solution to 10 with 1 M NaOH, 20 mL of thioacetamide solution is added and stirred for 30 min; the mixture is refluxed at 80 ℃ for 10 h and dialyzed in ultrapure water overnight; a mixed solution containing 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.01 M N-hydroxysuccinimide is added, and shaken for 1 h to activate the carboxyl group of CdS quantum dots; chloramphenicol aptamer chain is added and incubated at 37 °C for 4 h to obtain the chloramphenicol aptamer chain-CdS solution.

5. A method of constructing a zwitterionic hydrogel-based electrochemiluminescent antifouling sensor as claimed in claim 1, wherein, The chloramphenicol solutions with different concentrations are obtained by uniformly dispersing chloramphenicol in a phosphate buffer solution with pH 7.4 at concentrations of 0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 100 nM, respectively.

6. A method of constructing a zwitterionic hydrogel-based electrochemiluminescent antifouling sensor as claimed in claim 1, wherein, The modified surface of each electrode is gently washed with ultrapure water to remove the reagents that are not completely combined.

7. Use of the zwitterionic hydrogel-based electrochemiluminescence antifouling sensor constructed by the construction method of claim 1 in the application of chloramphenicol detection.

Images