Construction method and application of an electrochemiluminescence biosensor based on cell membrane hydrogel antifouling interface

Abstract

The application discloses a construction method and application of an electrochemiluminescence (ECL) biosensor based on a cell membrane hydrogel anti-fouling interface, and belongs to the technical field of photo / electrochemical analysis, interface anti-fouling and biosensing. In the application, the cell membrane hydrogel is used as an efficient anti-fouling interface to hinder the non-specific adsorption of interfering proteins in a sample to be measured and prolong the service life of the biosensor. Meanwhile, the luminophore copper nanosheet and the quencher MnO2 nanosheet are both introduced into the cell membrane hydrogel. In the presence of alkaline phosphatase (ALP), L-ascorbic acid-2-phosphoric acid trisodium salt is catalytically hydrolyzed into ascorbic acid, and then MnO2 is reduced to restore the ECL emission of the copper nanosheet. Therefore, the ECL signal of the biosensor shows an upward trend with the increase of the ALP concentration. Based on this, the constructed ECL biosensor realizes sensitive detection of ALP, and the linear range is 10 ‑5 ~ 10 4 U / L, and the detection limit is as low as 3.4x10 ‑6 U / L, indicating the potential application in clinical diagnosis.

Description

Technical Field

[0001] This invention discloses a method for constructing an electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface and its application in alkaline phosphatase detection, belonging to the fields of photo / electrochemical analysis, interface antifouling, and biosensing technology. Background Technology

[0002] Alkaline phosphatase (ALP) is a zinc-containing metalloprotein widely distributed in the liver, tissues, and blood of mammals. Abnormal ALP levels are closely associated with bone and liver diseases. The normal range for serum ALP is typically 40–150 U / L, but it increases significantly under pathological conditions. Electrochemiluminescence (ECL) technology offers advantages such as low background, high sensitivity, and ease of operation, making it an ideal choice for trace ALP detection. However, in clinical diagnostics, the detection results of ECL biosensors can be affected by interfacial adsorption of interfering biomolecules, thereby reducing the sensor's detection sensitivity and lifespan. Therefore, developing effective antifouling materials to resist such nonspecific adsorption is crucial for the trace detection of biological targets in clinical diagnostics.

[0003] Hydrogels are ideal for interfacial antifouling due to their excellent hydrophilicity. In our previous work, we developed a bovine serum albumin hydrogel to construct an ECL biosensor for sensitive and accurate detection of the p53 gene. Furthermore, natural cell membranes, with their high biocompatibility, can also form hydration layers to prevent the non-specific adsorption of interfering proteins, showing broad application prospects in antifouling systems. Red blood cell membranes, in particular, have fewer membrane proteins than other cell membranes, meaning fewer binding sites for interfering proteins, making them more suitable for antifouling applications. Despite the unique advantages of red blood cell membranes in antifouling, their poor conductivity limits their application in electrochemical analysis.

[0004] Building upon this foundation, we developed an ECL biosensor using erythrocyte membrane hydrogel as an antifouling interface for the precise trace detection of ALP in serum and cell lysates. First, the unique network-porous structure of the erythrocyte membrane hydrogel increases the electrochemically active surface area and electron transfer efficiency of the sensing interface, overcoming the limitations of erythrocyte membranes in electrochemical analysis. Second, disordered copper nanoclusters can self-assemble into copper nanosheets with high ECL emission, and the introduced MnO2 nanosheets can quench the ECL signal of the copper nanosheets through a resonant energy transfer mechanism. Third, both the copper and MnO2 nanosheets are encapsulated within the erythrocyte membrane hydrogel, which not only shortens the distance between them but also enables one-step construction of the ECL biosensor. When the target ALP is present, L-ascorbic acid-2-trisodium phosphate is catalyzed to generate L-ascorbic acid, which then further reduces MnO2 to restore the quenched ECL signal. Based on this, the constructed ECL biosensor enables trace analysis of ALP with a detection limit as low as 3.4 × 10⁻⁶. -6 U / L improves the sensitivity and accuracy of ALP detection in complex biological systems. Summary of the Invention

[0005] One of the technical objectives of this invention is to overcome the shortcomings of the prior art by preparing a cell membrane hydrogel as an antifouling interface for ECL analysis, thereby improving the sensitivity and lifespan of the ECL biosensor.

[0006] The second technical objective of this invention is to construct an ECL biosensor based on a cell membrane hydrogel antifouling interface, based on resonant energy transfer and interface antifouling strategies. This biosensor has high detection sensitivity and specificity, and its preparation process is simple and safe to operate.

[0007] The third technical objective of this invention is to provide the application of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed by the aforementioned construction method, namely, for the sensitive detection of ALP in serum and cell lysate; the ECL biosensor constructed by this invention exhibits a wide linear range and a low detection limit for ALP detection, 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 ECL biosensor based on a cell membrane hydrogel antifouling interface.

[0010] A glassy carbon electrode was polished with 0.30 μm alumina powder and ultrasonically cleaned sequentially with ultrapure water and ethanol to obtain a mirror-like surface. 3–7 μL of cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was applied to the polished glassy carbon electrode surface and dried overnight at room temperature. The modified electrode was then immersed in Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP for 3 min to construct an ECL biosensor based on the cell membrane hydrogel antifouling interface.

[0011] The copper nanosheets were prepared by mixing 5 mL of an ethanol solution containing 25 mM Cu(NO3)2 and 5 mL of an ethanol solution containing 40 mM 4,6-dimethyl-2-mercaptopyrimidine at room temperature and stirring for 10 min; centrifugation was performed to obtain copper nanoclusters, which were then washed three times with ethanol; the obtained copper nanoclusters were redispersed in ultrapure water under magnetic stirring and reacted for 1 h to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0012] The MnO2 nanosheets were obtained by adding 2 mL of 30% H2O2 to 18 mL of 1.33 M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of 0.3 M MnCl2·4H2O solution, causing the solution to turn brown. The solution was stirred at room temperature for 1 day, centrifuged, and washed sequentially with ultrapure water and ethanol to obtain the MnO2 nanosheets.

[0013] The cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was prepared by dispersing (1,2-distearate-sn-glycerol-3-phosphoethanolamine)-polyvinyl-acrylamide in a cell membrane solution and incubating it with shaking at 37°C for 30 min to obtain an acrylamide-embedded cell membrane solution. Then, 100 μL of the acrylamide-embedded cell membrane solution, 400 μL of a 4 mg / mL copper nanosheet solution, 400 μL of a 1 mg / mL MnO2 nanosheet solution, 280 mg of acrylamide, 5‰ ammonium persulfate, and 5‰ N,N,N',N'-tetramethylethylenediamine were sequentially added to a polytetrafluoroethylene mold to obtain the cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets.

[0014] The Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP is prepared by dispersing 80 mg of L-ascorbic acid-2-phosphate trisodium salt in 10 mL of Tris-HCl buffer, followed by the addition of ALP, so that the ALP concentrations are successively 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L.

[0015] 2. The application of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed by the aforementioned method is for the detection of ALP in serum and cell lysate. A phosphate buffer solution containing 75 mM triethylamine and with a pH of 7.0–8.0 is used as the detection solution. The constructed ECL biosensor based on the cell membrane hydrogel antifouling interface is immersed in the solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode is used for signal testing. The applied voltage range during the experiment is 0.2–1.1 V, the photomultiplier tube high voltage is 600 V, and the scan rate is 0.1 V / s. Based on the ECL response, a working curve is plotted, and the detection range of the biosensor is found to be 10. -5 ~10 4 U / L, detection limit as low as 3.4×10 -6 U / L; the results showed that the constructed ECL biosensor has high stability, specificity, and reproducibility, and is suitable for sensitive detection of ALP in serum and cell lysate.

[0016] The beneficial technical effects of the present invention are as follows:

[0017] 1. This invention prepares a cell membrane hydrogel as an antifouling interface for ECL analysis. The unique network porous structure of the cell membrane hydrogel can significantly increase the electrochemically active surface area and electron transfer efficiency of the sensing interface, thereby broadening the application of cell membranes in electrochemical analysis. Based on the stable antifouling properties of the cell membrane hydrogel, the constructed sensing interface can resist the non-specific adsorption of interfering biomolecules in complex media, enabling the direct detection of ALP in serum and cell lysate without pretreatment.

[0018] 2. This invention constructs an ECL biosensor based on a cell membrane hydrogel antifouling interface. Through resonant energy transfer and interface antifouling strategies, the constructed ECL biosensor possesses high detection sensitivity and precision, as well as a long service life. Furthermore, the fabrication process is simple and the operation is safe.

[0019] 3. The ECL biosensor based on the cell membrane hydrogel antifouling interface constructed in this invention exhibits low detection limit and wide linear range, long service life, and high stability, specificity and reproducibility for the detection of ALP in serum and cell lysates. It can be used for the effective detection of biological enzymes in complex biological environments such as serum and cell lysates, and has certain industrial application prospects. Detailed Implementation

[0020] 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.

[0021] Example 1: Construction method of ECL biosensor based on cell membrane hydrogel antifouling interface. A glassy carbon electrode was polished with 0.30 μm alumina powder and ultrasonically cleaned with ultrapure water and ethanol in sequence to obtain a mirror-like surface. 3 μL of cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was modified on the polished glassy carbon electrode surface and dried overnight at room temperature. The modified electrode was immersed in Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP for 3 min to construct an ECL biosensor based on cell membrane hydrogel antifouling interface.

[0022] The copper nanosheets were prepared by mixing 5 mL of an ethanol solution containing 25 mM Cu(NO3)2 and 5 mL of an ethanol solution containing 40 mM 4,6-dimethyl-2-mercaptopyrimidine at room temperature and stirring for 10 min; centrifugation was performed to obtain copper nanoclusters, which were then washed three times with ethanol; the obtained copper nanoclusters were redispersed in ultrapure water under magnetic stirring and reacted for 1 h to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0023] The MnO2 nanosheets were obtained by adding 2 mL of 30% H2O2 to 18 mL of 1.33 M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of 0.3 M MnCl2·4H2O solution, causing the solution to turn brown. The solution was stirred at room temperature for 1 day, centrifuged, and washed sequentially with ultrapure water and ethanol to obtain the MnO2 nanosheets.

[0024] The cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was prepared by dispersing (1,2-distearate-sn-glycerol-3-phosphoethanolamine)-polyvinyl-acrylamide in a cell membrane solution and incubating it with shaking at 37°C for 30 min to obtain an acrylamide-embedded cell membrane solution. Then, 100 μL of the acrylamide-embedded cell membrane solution, 400 μL of a 4 mg / mL copper nanosheet solution, 400 μL of a 1 mg / mL MnO2 nanosheet solution, 280 mg of acrylamide, 5‰ ammonium persulfate, and 5‰ N,N,N',N'-tetramethylethylenediamine were sequentially added to a polytetrafluoroethylene mold to obtain the cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets.

[0025] The Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP is prepared by dispersing 80 mg of L-ascorbic acid-2-phosphate trisodium salt in 10 mL of Tris-HCl buffer, followed by the addition of ALP, so that the ALP concentrations are successively 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L.

[0026] Example 2: A method for constructing an ECL biosensor based on a cell membrane hydrogel antifouling interface. A glassy carbon electrode was polished with 0.30 μm alumina powder and ultrasonically cleaned with ultrapure water and ethanol sequentially to obtain a mirror-like surface. 5 μL of cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was applied to the polished glassy carbon electrode surface and dried overnight at room temperature. The modified electrode was then immersed in Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP for 3 min to construct an ECL biosensor based on a cell membrane hydrogel antifouling interface.

[0027] The copper nanosheets were prepared by mixing 5 mL of an ethanol solution containing 25 mM Cu(NO3)2 and 5 mL of an ethanol solution containing 40 mM 4,6-dimethyl-2-mercaptopyrimidine at room temperature and stirring for 10 min; centrifugation was performed to obtain copper nanoclusters, which were then washed three times with ethanol; the obtained copper nanoclusters were redispersed in ultrapure water under magnetic stirring and reacted for 1 h to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0028] The MnO2 nanosheets were obtained by adding 2 mL of 30% H2O2 to 18 mL of 1.33 M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of 0.3 M MnCl2·4H2O solution, causing the solution to turn brown. The solution was stirred at room temperature for 1 day, centrifuged, and washed sequentially with ultrapure water and ethanol to obtain the MnO2 nanosheets.

[0029] The cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was prepared by dispersing (1,2-distearate-sn-glycerol-3-phosphoethanolamine)-polyvinyl-acrylamide in a cell membrane solution and incubating it with shaking at 37°C for 30 min to obtain an acrylamide-embedded cell membrane solution. Then, 100 μL of the acrylamide-embedded cell membrane solution, 400 μL of a 4 mg / mL copper nanosheet solution, 400 μL of a 1 mg / mL MnO2 nanosheet solution, 280 mg of acrylamide, 5‰ ammonium persulfate, and 5‰ N,N,N',N'-tetramethylethylenediamine were sequentially added to a polytetrafluoroethylene mold to obtain the cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets.

[0030] The Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP is prepared by dispersing 80 mg of L-ascorbic acid-2-phosphate trisodium salt in 10 mL of Tris-HCl buffer, followed by the addition of ALP, so that the ALP concentrations are successively 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L.

[0031] Example 3: Construction method of ECL biosensor based on cell membrane hydrogel antifouling interface. A glassy carbon electrode was polished with 0.30 μm alumina powder and ultrasonically cleaned with ultrapure water and ethanol in sequence to obtain a mirror-like surface. 7 μL of cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was modified on the polished glassy carbon electrode surface and dried overnight at room temperature. The modified electrode was immersed in Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP for 3 min to construct an ECL biosensor based on cell membrane hydrogel antifouling interface.

[0032] The copper nanosheets were prepared by mixing 5 mL of an ethanol solution containing 25 mM Cu(NO3)2 and 5 mL of an ethanol solution containing 40 mM 4,6-dimethyl-2-mercaptopyrimidine at room temperature and stirring for 10 min; centrifugation was performed to obtain copper nanoclusters, which were then washed three times with ethanol; the obtained copper nanoclusters were redispersed in ultrapure water under magnetic stirring and reacted for 1 h to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0033] The MnO2 nanosheets were obtained by adding 2 mL of 30% H2O2 to 18 mL of 1.33 M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of 0.3 M MnCl2·4H2O solution, causing the solution to turn brown. The solution was stirred at room temperature for 1 day, centrifuged, and washed sequentially with ultrapure water and ethanol to obtain the MnO2 nanosheets.

[0034] The cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was prepared by dispersing (1,2-distearate-sn-glycerol-3-phosphoethanolamine)-polyvinyl-acrylamide in a cell membrane solution and incubating it with shaking at 37°C for 30 min to obtain an acrylamide-embedded cell membrane solution. Then, 100 μL of the acrylamide-embedded cell membrane solution, 400 μL of a 4 mg / mL copper nanosheet solution, 400 μL of a 1 mg / mL MnO2 nanosheet solution, 280 mg of acrylamide, 5‰ ammonium persulfate, and 5‰ N,N,N',N'-tetramethylethylenediamine were sequentially added to a polytetrafluoroethylene mold to obtain the cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets.

[0035] The Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and ALP is prepared by dispersing 80 mg of L-ascorbic acid-2-phosphate trisodium salt in 10 mL of Tris-HCl buffer, followed by the addition of ALP, so that the ALP concentrations are successively 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L.

[0036] Example 4 describes the application of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed using the methods described in Examples 1, 2, and 3. It is used for the detection of bioenzymes in complex biological environments such as serum and cell lysates. A phosphate buffer solution containing 75 mM triethylamine and with a pH of 7.0 was used as the detection solution. The constructed ECL biosensor based on the cell membrane hydrogel antifouling interface was immersed in the solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for signal testing. The applied voltage range during the experiment was 0.2–1.1 V, the photomultiplier tube voltage was 600 V, and the scan rate was 0.1 V / s. Based on the ECL response curve, the detection range of the biosensor was found to be 10... -5 ~10 4 U / L, detection limit as low as 3.4×10 -6U / L; the results showed that the constructed ECL biosensor has high stability, specificity, and reproducibility, and is suitable for sensitive detection of ALP in serum and cell lysate.

[0037] Example 5 describes the application of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed using the methods described in Examples 1, 2, and 3. It is used for the detection of bioenzymes in complex biological environments such as serum and cell lysates. A phosphate buffer solution containing 75 mM triethylamine and with a pH of 7.4 was used as the detection solution. The constructed ECL biosensor based on the cell membrane hydrogel antifouling interface was immersed in the solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for signal testing. The applied voltage range during the experiment was 0.2–1.1 V, the photomultiplier tube voltage was 600 V, and the scan rate was 0.1 V / s. Based on the ECL response curve, the detection range of the biosensor was found to be 10... -5 ~10 4 U / L, detection limit as low as 3.4×10 -6 U / L; the results showed that the constructed ECL biosensor has high stability, specificity, and reproducibility, and is suitable for sensitive detection of ALP in serum and cell lysate.

[0038] Example 6 describes the application of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed using the methods described in Examples 1, 2, and 3. It is used for the detection of bioenzymes in complex biological environments such as serum and cell lysates. A phosphate buffer solution containing 75 mM triethylamine and with a pH of 8.0 was used as the detection solution. The constructed ECL biosensor based on the cell membrane hydrogel antifouling interface was immersed in the solution, and a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode was used for signal testing. The applied voltage range during the experiment was 0.2–1.1 V, the photomultiplier tube voltage was 600 V, and the scan rate was 0.1 V / s. Based on the ECL response curve, the detection range of the biosensor was found to be 10... -5 ~10 4 U / L, detection limit as low as 3.4×10 -6 U / L; the results showed that the constructed ECL biosensor has high stability, specificity, and reproducibility, and is suitable for sensitive detection of ALP in serum and cell lysate. Attached Figure Description

[0039] Figure 1The flowchart shows the construction method of ECL biosensor based on cell membrane hydrogel antifouling interface (copper nanoclusters: Cu NCs; copper nanosheets: Cu NSs; 4,6-dimethyl-2-mercaptopyrimidine: DMMP; erythrocyte membrane: RBCM; (1,2-distearyl-sn-glycerol-3-phosphoethanolamine)-polyethylene-acrylamide: DSPE-PEG-AM; L-ascorbic acid: AA; L-ascorbic acid-2-phosphate trisodium salt: AAP).

[0040] Figure 2 Transmission electron microscopy images of (A) Cu NCs, (B) Cu NSs and (C) MnO2 NSs.

[0041] Figure 3 The images show scanning electron microscope (SEM) images of (A) RBCM / GCE and (B) RBCM-AM / GCE, and the SEM image and corresponding elemental mapping of (C) RBCM hydrogel (glassy carbon electrode: GCE).

[0042] Figure 4 Transmission electron micrographs of (A) RBCM vesicles and (B) RBCM-AM vesicles.

[0043] Figure 5 The particle size distribution diagrams are for RBCM vesicles and RBCM-AM vesicles.

[0044] Figure 6 The energy dispersive X-ray spectra of (A) Cu NSs and (B) MnO2 NSs are shown.

[0045] Figure 7 X-ray photoelectron spectra of Cu NSs and MnO2 NSs are shown, where (A) is the full-area X-ray photoelectron spectrum of Cu NSs and (B) is the high-resolution X-ray photoelectron spectrum of the Cu 2p region, and (C) is the full-area X-ray photoelectron spectrum of MnO2 NSs and (D) is the high-resolution X-ray photoelectron spectrum of the Mn 2p region.

[0046] Figure 8 Static water contact angle diagrams for (A) GCE, (B) MBAA hydrogel and (C) RBCM hydrogel (N,N'-methylenebisacrylamide:MBAA).

[0047] Figure 9 Fluorescence imaging images of (A) ITO, (B) MBAA hydrogel and (C) RBCM hydrogel after incubation in fluorescein-conjugated bovine serum albumin (Indium Tin Oxide: ITO).

[0048] Figure 10Differential pulse voltammetry curves of (A) GCE, (B) MBAA hydrogel / GCE and (C) RBCM hydrogel / GCE incubated in fetal bovine serum at different concentrations.

[0049] Figure 11 Differential pulse voltammetry curves of (A) GCE, (B) MBAA hydrogel / GCE and (C) RBCM hydrogel / GCE incubated in human serum at different concentrations.

[0050] Figure 12 The diagram shows the optimization of conditions for an ECL biosensor based on a cell membrane hydrogel antifouling interface, where (A) is the optimized result of MnO2 nanosheet concentration; (B) is the optimized result of AAP incubation time; and (C) is the optimized result of concentration.

[0051] Figure 13 This is a characterization diagram of the construction process of an ECL biosensor based on a cell membrane hydrogel antifouling interface, based on (A) differential pulse voltammetry and (B) AC impedance testing.

[0052] Figure 14 ECL response diagrams for Cu NCs / GCE, Cu NSs / GCE, and MnO2 NSs / Cu NSs / GCE in phosphate buffer solution containing 75 mM triethylamine.

[0053] Figure 15 The UV-Vis absorption spectrum of MnO2 NSs and the fluorescence emission spectrum of Cu NSs are shown.

[0054] Figure 16 (A) ECL curve and (B) corresponding calibration curve are shown for the ECL biosensor based on the cell membrane hydrogel antifouling interface after incubation with different concentrations of ALP; where the concentration gradient of ALP is 0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 U / L.

[0055] Figure 17 To evaluate the (A) specificity, (B) stability, and (C) reproducibility of ECL biosensors based on cell membrane hydrogel antifouling interfaces.

Claims

1. A method for constructing an electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface, characterized in that, A glassy carbon electrode was polished with 0.30 μm alumina powder and ultrasonically cleaned sequentially with ultrapure water and ethanol to obtain a mirror-like surface. 3–7 μL of cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was applied to the polished glassy carbon electrode surface and dried overnight at room temperature. The modified electrode was then immersed in Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and alkaline phosphatase for 3 min to construct an electrochemiluminescent biosensor based on the antifouling interface of the cell membrane hydrogel. The cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets was obtained by dispersing (1,2-distearate-sn-glycerol-3-phosphoethanolamine)-polyvinyl-acrylamide in a cell membrane solution and incubating with shaking at 37 °C for 30 min to obtain an acrylamide-intercalated cell membrane solution. A mixture of 100 μL acrylamide-embedded cell membrane solution, 400 μL copper nanosheet solution (4 mg / mL), 400 μL MnO2 nanosheet solution (1 mg / mL), 280 mg acrylamide, 5‰ ammonium persulfate, and 5‰... N, N, N', N' -Tetramethylethylenediamine was added sequentially into a polytetrafluoroethylene mold to obtain a cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets.

2. The method for constructing an electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface as described in claim 1, characterized in that, The copper nanosheets were prepared by mixing 5 mL of an ethanol solution containing 25 mM Cu(NO3)2 and 5 mL of an ethanol solution containing 40 mM 4,6-dimethyl-2-mercaptopyrimidine at room temperature and stirring for 10 min; centrifuging to obtain copper nanoclusters, and washing three times with ethanol; redispersing the obtained copper nanoclusters in ultrapure water under magnetic stirring and reacting for 1 h to obtain copper nanosheets self-assembled from copper nanoclusters.

3. The method for constructing an electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface as described in claim 1, characterized in that, The MnO2 nanosheets were obtained by adding 2 mL of 30% H2O2 to 18 mL of 1.33 M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of 0.3 M M MnCl2·4H2O solution, causing the solution to turn brown. The solution was stirred at room temperature for 1 day, centrifuged, and washed sequentially with ultrapure water and ethanol to obtain the MnO2 nanosheets.

4. The method for constructing an electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface as described in claim 1, characterized in that, The Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and alkaline phosphatase is prepared by dispersing 80 mg of L-ascorbic acid-2-phosphate trisodium salt in 10 mL of Tris-HCl buffer, followed by the addition of alkaline phosphatase to achieve concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L, respectively.

5. The use of the electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface constructed by the method described in claim 1, characterized in that, Applications for alkaline phosphatase detection.

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