Construction method and application of electrochemical luminescence biosensor based on cell membrane hydrogel antifouling interface

Abstract

The invention discloses a construction method and application of an electrochemical luminescence (ECL) biosensor based on a cell membrane hydrogel antifouling interface, and belongs to the technical fields of photo / electrochemical analysis, interface antifouling and biosensing. According to the invention, the cell membrane hydrogel is used as an efficient antifouling interface, so that non-specific adsorption of interfering proteins in a sample to be detected is hindered, and the service life of the biosensor is prolonged. Meanwhile, a luminophor copper nanosheet and a quencher MnO2 nanosheet are both introduced into the cell membrane hydrogel, in the presence of alkaline phosphatase (ALP), L-ascorbic acid-2-trisodium phosphate is catalyzed and hydrolyzed into ascorbic acid, and then MnO2 is reduced to recover ECL emission of the copper nanosheet. Therefore, the ECL signal of the biosensor is in a rising trend along with the rising of the ALP concentration. Based on this, the constructed ECL biosensor realizes sensitive detection of ALP, the linear range is 10 <-5 >-10 < 4 > U / L, and the detection limit is as low as 3.4 * 10 <-6 > U / L, which indicates that the ECL biosensor has potential application in clinical diagnosis.

Description

Technical Field

[0001] The invention discloses a construction method of an electrochemiluminescence biosensor based on a cell membrane hydrogel antifouling interface and its application to alkaline phosphatase detection, belonging to the technical fields of light / electrochemical analysis, interface antifouling and biosensor. Background Art

[0002] Alkaline phosphatase (ALP) is a zinc-containing metalloprotein that is widely distributed in the liver, tissues, and blood of mammals. Its abnormal levels are closely related to bone and liver diseases. The normal range of ALP in serum is usually 40-150 U / L, which can increase significantly under pathological conditions. Electrochemiluminescence (ECL) technology has the advantages of low background, high sensitivity, and easy operation, making it an ideal choice for trace detection of ALP. However, in clinical diagnosis, the detection results of ECL biosensors can be affected by the interfacial adsorption of interfering biomolecules, thereby reducing the detection sensitivity and service life of the sensor. Therefore, the development of effective antifouling materials to resist the above-mentioned nonspecific adsorption is crucial for the trace detection of biological targets in clinical diagnosis.

[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. In addition, natural cell membranes with high biocompatibility can also form a hydration layer to prevent the nonspecific adsorption of interfering proteins, which has broad application prospects in antifouling systems. Among them, red blood cell membranes have fewer membrane proteins than other cell membranes, which means there are fewer binding sites for interfering proteins, making them more suitable for antifouling applications. Although red blood cell membranes have unique advantages in antifouling, their poor electrical conductivity limits their application in electrochemical analysis.

[0004] Based on this, we developed an ECL biosensor using an erythrocyte membrane hydrogel as an antifouling interface for the precise trace detection of ALP in serum and cell lysates. First, the unique reticular porous structure of the erythrocyte membrane hydrogel increases the electrochemically active surface area and electron transfer efficiency of the sensing interface, addressing the limitations of erythrocyte membranes in electrochemical analysis. Second, disordered copper nanoclusters self-assemble into copper nanosheets with high ECL emission, and the introduced MnO2 nanosheets quench the ECL signal of the copper nanosheets via a resonance energy transfer mechanism. Third, both the copper nanosheets and the MnO2 nanosheets are encapsulated in the erythrocyte membrane hydrogel, which not only shortens the distance between them but also enables the one-step construction of the ECL biosensor. In the presence of the target ALP, L-ascorbic acid-2-phosphate trisodium salt 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 achieved trace analysis of ALP with a detection limit as low as 3.4×10 -6 U / L, which improves the sensitivity and accuracy of ALP detection in complex biological systems. Summary of the Invention

[0005] One of the technical tasks of the present invention is to make up for the shortcomings of the existing technology and prepare a cell membrane hydrogel as an antifouling interface for ECL analysis, thereby improving the sensitivity and service life of the ECL biosensor;

[0006] The second technical task of the present invention is to construct an ECL biosensor based on the cell membrane hydrogel antifouling interface based on the resonance energy transfer and interface antifouling strategy, which has high detection sensitivity and specificity, simple preparation process and safe operation;

[0007] The third technical task of the present invention is to provide the use of the ECL biosensor based on the cell membrane hydrogel anti-fouling interface constructed by the construction method, namely, for the sensitive detection of ALP in serum and cell lysate; the ECL biosensor constructed by the present invention exhibits a wide linear range and low detection limit for ALP detection, and has certain industrial prospects.

[0008] To achieve the above object, 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. The polished glassy carbon electrode surface was modified with 3–7 μL of cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets and dried overnight at room temperature. The modified electrode was immersed in Tris-HCl buffer containing trisodium L-ascorbic acid-2-phosphate and ALP for 3 minutes to construct an ECL biosensor based on the cell membrane hydrogel antifouling interface.

[0011] The copper nanosheets are prepared by thoroughly 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 minutes; centrifuging to obtain copper nanoclusters, which are then washed three times with ethanol; and redispersing the obtained copper nanoclusters in ultrapure water under magnetic stirring and reacting for 1 hour to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0012] The MnO2 nanosheets are prepared by adding 2 mL of 30% H2O2 to 18 mL of a 1.33M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of a 0.3M MnCl2·4H2O solution until the solution changes color to brown. The resulting solution is stirred at room temperature for one 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 is prepared by dispersing (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-polyethylene-acrylamide in a cell membrane solution and incubating the solution with shaking at 37°C for 30 minutes to obtain an acrylamide-embedded cell membrane solution; 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 are sequentially added into a polytetrafluoroethylene mold to obtain a 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, and then adding ALP to adjust the solubility of ALP to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L, respectively.

[0015] 2. The use of an ECL biosensor based on a cell membrane hydrogel antifouling interface constructed by the construction method is used for the detection of ALP in serum and cell lysates. A phosphate buffered saline solution containing 75mM triethylamine and 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 signal test is performed using a three-electrode system comprising a working electrode, an auxiliary electrode, and a reference electrode. During the experiment, the applied voltage range is 0.2-1.1V, the photomultiplier tube high voltage is 600V, and the scanning rate is 0.1V / s. A working curve is drawn based on the ECL response, and the detection range of the biosensor is 10 -5 ~10 4 U / L, the detection limit is 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 the sensitive detection of ALP in serum and cell lysate.

[0016] Beneficial technical effects of the present invention:

[0017] 1. This invention prepares a cell membrane hydrogel as an antifouling interface for ECL analysis. The unique reticular porous structure of the cell membrane hydrogel significantly increases the electrochemically active surface area and electron transfer efficiency of the sensing interface, thereby expanding 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 nonspecific adsorption of interfering biomolecules in complex media, enabling direct detection of ALP in serum and cell lysates without pretreatment.

[0018] 2. This paper constructs an ECL biosensor based on a cell membrane hydrogel antifouling interface. Through resonance energy transfer and interfacial antifouling strategies, the constructed ECL biosensor has high detection sensitivity and precision, as well as a long service life. The preparation process is simple and the operation is safe.

[0019] 3. The ECL biosensor constructed in the present invention based on the cell membrane hydrogel anti-fouling interface exhibits a low detection limit and a wide linear range, a long service life, as well as high stability, specificity and reproducibility for the detection of ALP in serum and cell lysate. It can be used for the effective detection of biological enzymes in complex biological environments such as serum and cell lysate, and has certain prospects for industrial application. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Any changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0021] Example 1 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 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 at room temperature overnight. The modified electrode was immersed in Tris-HCl buffer containing trisodium L-ascorbic acid-2-phosphate and ALP for 3 minutes to construct an ECL biosensor based on the cell membrane hydrogel antifouling interface.

[0022] The copper nanosheets are prepared by thoroughly 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 minutes; centrifuging to obtain copper nanoclusters, which are then washed three times with ethanol; and redispersing the obtained copper nanoclusters in ultrapure water under magnetic stirring and reacting for 1 hour to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0023] The MnO2 nanosheets are prepared by adding 2 mL of 30% H2O2 to 18 mL of a 1.33M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of a 0.3M MnCl2·4H2O solution until the solution changes color to brown. The resulting solution is stirred at room temperature for one 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 is prepared by dispersing (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-polyethylene-acrylamide in a cell membrane solution and incubating the solution with shaking at 37°C for 30 minutes to obtain an acrylamide-embedded cell membrane solution; 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 are sequentially added into a polytetrafluoroethylene mold to obtain a 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, and then adding ALP to adjust the solubility of ALP to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L, respectively.

[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 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 modified on the polished glassy carbon electrode surface and dried at room temperature overnight. The modified electrode was immersed in Tris-HCl buffer containing trisodium L-ascorbic acid-2-phosphate and ALP for 3 minutes to construct an ECL biosensor based on the cell membrane hydrogel antifouling interface.

[0027] The copper nanosheets are prepared by thoroughly 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 minutes; centrifuging to obtain copper nanoclusters, which are then washed three times with ethanol; and redispersing the obtained copper nanoclusters in ultrapure water under magnetic stirring and reacting for 1 hour to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0028] The MnO2 nanosheets are prepared by adding 2 mL of 30% H2O2 to 18 mL of a 1.33M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of a 0.3M MnCl2·4H2O solution until the solution changes color to brown. The resulting solution is stirred at room temperature for one 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 is prepared by dispersing (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-polyethylene-acrylamide in a cell membrane solution and incubating the solution with shaking at 37°C for 30 minutes to obtain an acrylamide-embedded cell membrane solution; 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 are sequentially added into a polytetrafluoroethylene mold to obtain a 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, and then adding ALP to adjust the solubility of ALP to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L, respectively.

[0031] Example 3: 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 sequentially with ultrapure water and ethanol 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 trisodium L-ascorbic acid-2-phosphate and ALP for 3 minutes to construct an ECL biosensor based on the cell membrane hydrogel antifouling interface.

[0032] The copper nanosheets are prepared by thoroughly 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 minutes; centrifuging to obtain copper nanoclusters, which are then washed three times with ethanol; and redispersing the obtained copper nanoclusters in ultrapure water under magnetic stirring and reacting for 1 hour to obtain copper nanosheets self-assembled from the copper nanoclusters.

[0033] The MnO2 nanosheets are prepared by adding 2 mL of 30% H2O2 to 18 mL of a 1.33M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of a 0.3M MnCl2·4H2O solution until the solution changes color to brown. The resulting solution is stirred at room temperature for one 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 is prepared by dispersing (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-polyethylene-acrylamide in a cell membrane solution and incubating the solution with shaking at 37°C for 30 minutes to obtain an acrylamide-embedded cell membrane solution; 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 are sequentially added into a polytetrafluoroethylene mold to obtain a 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, and then adding ALP to adjust the solubility of ALP to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L, respectively.

[0036] Example 4 The use of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed by the construction method described in Example 1, Example 2 and Example 3 is applied to the application of biological enzyme detection in complex biological environments such as serum and cell lysate. A phosphate buffer solution containing 75mM triethylamine and a pH of 7.0 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 signal test is performed using a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode. During the experiment, the applied voltage range is 0.2-1.1V, the photomultiplier tube high voltage is 600V, and the scanning rate is 0.1V / s. The working curve is drawn according to the ECL response, and the detection range of the biosensor is 10 -5 ~10 4 U / L, the detection limit is 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 the sensitive detection of ALP in serum and cell lysate.

[0037] Example 5 The use of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed by the construction method described in Example 1, Example 2 and Example 3 is applied to the detection of biological enzymes in complex biological environments such as serum and cell lysate. A phosphate buffer solution containing 75mM triethylamine and a pH of 7.4 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 signal test is performed using a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode. During the experiment, the applied voltage range is 0.2 to 1.1V, the photomultiplier tube high voltage is 600V, and the scanning rate is 0.1V / s. The working curve is drawn according to the ECL response, and the detection range of the biosensor is 10 -5 ~10 4 U / L, the detection limit is 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 the sensitive detection of ALP in serum and cell lysate.

[0038] Example 6 The use of the ECL biosensor based on the cell membrane hydrogel antifouling interface constructed by the construction method described in Example 1, Example 2 and Example 3 is applied to the detection of biological enzymes in complex biological environments such as serum and cell lysate. A phosphate buffered saline solution containing 75mM triethylamine and a pH of 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 signal test is performed using a three-electrode system including a working electrode, an auxiliary electrode, and a reference electrode. During the experiment, the applied voltage range is 0.2 to 1.1V, the photomultiplier tube high voltage is 600V, and the scanning rate is 0.1V / s. The working curve is drawn according to the ECL response, and the detection range of the biosensor is 10 -5 ~10 4 U / L, the detection limit is 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 the sensitive detection of ALP in serum and cell lysate. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1Flowchart of 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; red blood cell membrane: RBCM; (1,2-distearoyl-sn-glycero-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 Scanning electron microscopy images of (A) RBCM / GCE and (B) RBCM-AM / GCE, and (C) scanning electron microscopy image and corresponding elemental mapping of RBCM hydrogel (glassy carbon electrode: GCE).

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

[0043] Figure 5 Figure 3 is the particle size distribution of RBCM vesicles and RBCM-AM vesicles.

[0044] Figure 6 Energy dispersive X-ray spectroscopy analysis diagrams of (A) Cu NSs and (B) MnO2 NSs.

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

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

[0047] Figure 9 Fluorescence imaging 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 different concentrations of fetal bovine serum.

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

[0050] Figure 12 Condition optimization diagram of the ECL biosensor based on the cell membrane hydrogel antifouling interface, including (A) MnO2 nanosheet concentration optimization result diagram; (B) AAP incubation time and (C) concentration optimization result diagram.

[0051] Figure 13 Figure 2 Characterization of the ECL biosensor construction process based on the cell membrane hydrogel antifouling interface according to (A) differential pulse voltammetry and (B) electrochemical impedance spectroscopy.

[0052] Figure 14 The ECL response graphs of Cu NCs / GCE, Cu NSs / GCE and MnO2 NSs / Cu NSs / GCE in phosphate buffered saline containing 75 mM triethylamine.

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

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

[0055] Figure 17 (A) Specificity, (B) stability, and (C) reproducibility of the ECL biosensor based on the cell membrane hydrogel antifouling interface.

Claims

1. A method for constructing an electrochemiluminescent biosensor based on a cell membrane hydrogel antifouling interface, characterized in that: The 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 to 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 at room temperature overnight; the modified electrode was immersed in Tris-HCl buffer containing L-ascorbic acid-2-phosphate trisodium salt and alkaline phosphatase for 3 minutes to construct an electrochemiluminescent biosensor based on the anti-fouling interface of the cell membrane hydrogel.

2. The method for constructing an electrochemiluminescent biosensor based on a cell membrane hydrogel antifouling interface according to claim 1, wherein: The copper nanosheets are prepared by fully 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 minutes; centrifuging to obtain copper nanoclusters, which are then washed three times with ethanol; and redispersing the obtained copper nanoclusters in ultrapure water under magnetic stirring and reacting for 1 hour to obtain copper nanosheets self-assembled from the copper nanoclusters.

3. The method for constructing an electrochemiluminescent biosensor based on a cell membrane hydrogel antifouling interface according to claim 1, wherein: The MnO2 nanosheets are prepared by adding 2 mL of 30% H2O2 to 18 mL of a 1.33M tetramethylammonium hydroxide solution, then quickly pouring the resulting solution into 10 mL of a 0.3M MnCl2·4H2O solution until the solution changes color to brown. The resulting solution is stirred at room temperature for one day, centrifuged, and washed sequentially with ultrapure water and ethanol to obtain the MnO2 nanosheets.

4. The method for constructing an electrochemiluminescent biosensor based on a cell membrane hydrogel antifouling interface according to claim 1, wherein: The cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets is prepared by dispersing (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-polyethylene-acrylamide in a cell membrane solution and incubating the solution with shaking at 37°C for 30 minutes to obtain an acrylamide-embedded cell membrane solution; 100 μL of 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 are sequentially added into a polytetrafluoroethylene mold to obtain a cell membrane hydrogel containing 4 mg / mL copper nanosheets and 1 mg / mL MnO2 nanosheets.

5. The method for constructing an electrochemiluminescent biosensor based on a cell membrane hydrogel antifouling interface according to claim 1, wherein: 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, and then adding alkaline phosphatase to adjust the solubility of alkaline phosphatase to 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 U / L, respectively.

6. Use of the electrochemiluminescent biosensor based on the cell membrane hydrogel antifouling interface constructed by the construction method according to claim 1, characterized in that: Application for alkaline phosphatase detection.

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