High-stability cell membrane coated electrochemical biosensor as well as preparation method and application thereof

Abstract

The invention discloses a high-stability cell membrane coating electrochemical biosensor and a preparation method and application thereof, the method comprises the following steps: 1, adding RBCMs into a PBS solution, and uniformly stirring to obtain an RBCMs suspension; 2, mixing Chi with an acetic acid solution, stirring at room temperature to obtain a Chi solution, adding cMWCNTs into the Chi solution, and carrying out ultrasonic treatment to obtain cMWCNTs-Chi; and 3, dispensing the cMWCNTs-Chi on the surface of the GCE, carrying out air drying so as to obtain cMWCNTs-Chi / GCE, dispensing the RBCNTs suspension on the surface of the cMWCNTs-Chi / GCE, carrying out incubation so as to obtain CM-cMWCNTs-Chi / GCE, dispensing a BSA solution on the CM-cMWCNTs-Chi / GCE, carrying out incubation so as to obtain BSA-CM-cMWCNTs-Chi / GCE, washing the BSA-CM-cMWCNTs-Chi / GCE with PBS, and carrying out preservation at 4 DEG C so as to obtain the cell membrane coated electrochemical biosensor. The cell membrane coated electrochemical biosensor has high stability and high electrochemical responsiveness, and can

Description

Technical Field

[0001] The invention relates to an electrochemical biosensor, in particular to a highly stable cell membrane coated electrochemical biosensor and a preparation method and application thereof. Background Art

[0002] In modern agricultural production, organophosphorus pesticides have become the most widely used pesticides due to their high toxicity. However, as mentioned in Singh, BK, Organophosphorus-degrading bacteria: ecology and industrial applications. Nature Reviews Microbiology 2009, 7(2), 156-164, excessive use of pesticides has caused excessive pesticide residues in the environment and agricultural products, causing serious food safety and environmental pollution problems. In particular, pesticide residues in food can seriously affect human health.

[0003] The main mechanism of organophosphorus pesticide poisoning is to inhibit the activity of cholinesterase, leading to peripheral neuropathy. As mentioned in Chapalamadugu, S.; Chaudhry, G.R., Microbiological and Biotechnological Aspects of Metabolism of Carbamates and Organophosphates. Critical Reviews in Biotechnology 1992, 12(5-6), 357-389 and Sidhu, G.K.; Singh, S.; Kumar, V.; Dhanjal, D.S.; Datta, S.; Singh, J., Toxicity, monitoring and biodegradation of organophosphate pesticides: A review. Critical Reviews in Environmental Science and Technology 2019, 49(13), 1135-1187, cholinesterase is a glycoprotein that exists in the body in the form of multiple isozymes. Among them, true cholinesterase, also known as acetylcholinesterase (AChE), is mainly present in the synaptic cleft of cholinergic nerve endings, and also exists in cholinergic neurons and on the erythrocyte membrane. Based on this, Pereira, B.V.R.; Silva-Zacarin, E.C.M.; Costa, M.J.; Dos Santos, A.C.A.; do Carmo, J.B.; Nunes, B., Cholinesterases characterization of three tropical fish species, and their sensitivity towards specific contaminants. Ecotoxicology and Environmental Safety 2019, 173, 482-493. mentioned that the targeted evaluation and accurate measurement of AChE activity can lay the foundation for the precise and rapid detection of pesticide residues.

[0004] Nowadays, a variety of techniques for AChE analysis have been developed and studied, such as the ultraviolet-visible spectrophotometry mentioned in Satnami, M.L.; Korram, J.; Nagwanshi, R.; Vaishanav, S.K.; Karbhal, I.; Dewangan, H.K.; Ghosh, K.K., Gold nanoprobe for inhibition and reactivation of acetylcholinesterase: An application to detection of organophosphorus pesticides. Sensors and Actuators B: Chemical 2018, 267, 155-164; the fluorescence method mentioned in Zhao, C.; Zhou, F.; Lu, K.; Yang, S.; Tan, B.; Sun, W.; Shangguan, L.; Wang, H.-Y.; Liu, Y., Near-infrared fluorescent probe for in vivo monitoring acetylcholinesterase activity. Sensors and Actuators B: Chemical 2022, 360, 131647 and Mathew, M.S.; Baksi, A.; Pradeep, T.; Joseph, K., Choline-induced selective fluorescence quenching of acetylcholinesterase conjugated Au@BSA clusters. Biosensors and Bioelectronics 2016, 81, 68-74; and the colorimetric method mentioned in Liu, D.-M.; Xu, B.; Dong, C., Recent advances in colorimetric strategies for acetylcholinesterase assay and their applications. TrAC Trends in Analytical Chemistry 2021, 142, 116320.However, under physiological conditions, the main functional form of AChE is an amphiphilic tetramer anchored to the cell membrane. Once dissociated from the cell membrane, AChE undergoes conformational changes, leading to altered function, which in turn limits the ability of traditional methods to quantitatively evaluate AChE expression under conditions that reflect its true biological activity. See Herz, F.; Kaplan, E., A Review: Human Erythrocyte Acetylcholinesterase. Pediatric Research 1973, 7(4), 204 - 214 and Ott, P.; Lustig, A.; Brodbeck, U.; Rosenbusch, J.P., Acetylcholinesterase from human erythrocyte membranes: dimers as functional units. FEBS Letters 1982, 138(2), 187 - 189. Therefore, there is an urgent need to develop an accurate and rapid method for detecting pesticide residues based on the true biological activity of AChE.

[0005] In view of this, electrochemical biosensors with fast response, simple equipment, high sensitivity, low cost and suitable for on-site detection have attracted the attention of researchers. For example, Bakirhan, N.K.; Topal, B.D.; Ozcelikay, G.; Karadurmus, L.; Ozkan, S.A., Current Advances in Electrochemical Biosensors and Nanobiosensors. Critical Reviews in Analytical Chemistry 2022, 52(3), 519-534 mentioned that the cell membrane-coated electrochemical biosensors have both the characteristics of miniaturization and high selectivity of biosensors, and also reflect the advantages of fast and highly sensitive electrochemical analysis methods, which are of positive significance in drug design, drug detection, molecular screening and other aspects.However, referring to Miao, Y.; Yang, Y.; Guo, L.; Chen, M.; Zhou, X.; Zhao, Y.; Nie, D.; Gan, Y.; Zhang, X., Cell Membrane-Camouflaged Nanocarriers with Biomimetic Deformability of Erythrocytes for Ultralong Circulation and Enhanced Cancer Therapy. ACS Nano 2022, 16(4), 6527-6540 and Yang, X.; Chen, M.; Weng, C.; Zhuge, D.; Jin, F.; Xiao, Y.; Tian, D.; Yin, Q.; Li, L.; Zhang, X.; Shi, G.; Lu, X.; Yan, L.; Wang, L.; Wen, B.; Zhao, Y.; Lin, J.; Wang, F.; Zhang, W.; Chen, Y., Red Blood Cell Membrane-Coated Nanoparticles Enable Incompatible Blood Transfusions. Advanced Science 2024, 11(29), 2310230, it can be seen that cell membrane-coated electrochemical biosensors still face some challenges in practical applications. For example, traditional methods may cause the cell membrane to detach from the surface of the biosensor, resulting in the inability of drugs to effectively bind to specific sites and the failure to achieve the best activity in practical applications. Moreover, simple cell membrane coating may lead to weak electrical response due to poor conductivity. Therefore, developing cell membrane-coated electrochemical biosensors with high stability and high electrochemical response is of great significance for the detection of organophosphorus pesticide residues.

[0006] Carbon nanotubes (CNTs) have characteristics such as a high surface / volume ratio, excellent electrical conductivity, semiconductor properties, easy film formation, anti-aging, and high biocompatibility. These properties have led to extensive research in recent years on experiments using various CNTs to modify the surface of glassy carbon electrodes (GCEs) to enhance their electrochemical response. However, as can be seen from Gupta, N.; Gupta, S.M.; Sharma, S.K., Carbon nanotubes: synthesis, properties and engineering applications. Carbon Letters 2019, 29(5), 419 - 447 and Uwimbabazi, E.; Mukasekuru, M.R.; Sun, X., Glucose Biosensor Based on a Glassy Carbon Electrode Modified with Multi-Walled Carbon Nanotubes-Chitosan for the Determination of Beef Freshness. Food Analytical Methods 2017, 10(8), 2667 - 2676, due to the strong π-π interactions and van der Waals forces between CNTs, they exhibit chemical inertness. CNTs easily agglomerate in solutions and coating matrices in a short time, and it is difficult to obtain uniformly dispersed CNTs for uniformly coating GCEs. Wang, R.K.; Park, H.O.; Chen, W.C.; Silvera-Batista, C.; Reeves, R.D.; Butler, J.E.; Ziegler, K.J., Improving the effectiveness of interfacial trapping in removing single-walled carbon nanotube bundles. Journal of the American Chemical Society 2008, 130(44), 14721 - 14728 mentioned that covalent modification of CNTs for functionalization can improve the solubility and dispersibility of CNTs.

[0007] A large number of experiments have found that chitosan (Chi) and CNTs can form stable complexes through non-covalent binding. See Gao, C.; Guo, M.; Liu, Y.; Zhang, D.; Gao, F.; Sun, L.; Li, J.; Chen, X.; Terrones, M.; Wang, Y., Surface modification methods and mechanisms in carbon nanotubes dispersion. Carbon 2023, 212, 118133 and Pauliukaite, R.; Ghica, M.E.; Fatibello-Filho, O.; Brett, C.M.A., Electrochemical impedance studies of chitosan-modified electrodes for application in electrochemical sensors and biosensors. Electrochimica Acta 2010, 55(21), 6239-6247; Meanwhile, see Tang, C.; Zhou, T.; Yang, J.; Zhang, Q.; Chen, F.; Fu, Q.; Yang, L., Wet-grinding assisted ultrasonic dispersion of pristine multi-walled carbon nanotubes (MWCNTs) in chitosan solution. Colloids and Surfaces B: Biointerfaces 2011, 86(1), 189-197. Chi has many advantages such as non-toxicity, good mechanical strength, excellent film-forming ability, good biocompatibility, biodegradability, antibacterial ability, and high adsorption. Based on Chi, chemical sensors and biosensors have been rapidly developed. See Friedman, M.; Juneja, V.K., Review of Antimicrobial and Antioxidative Activities of Chitosans in Food. Journal of Food Protection 2010, 73(9), 1737-1761 and Salehi, E.; Daraei, P.; Arabi Shamsabadi, A., A review on chitosan-based adsorptive membranes.Carbohydrate Polymers 2016, 152, 419 - 432 laid a foundation for us to design a highly stable and highly electrochemically responsive cell membrane-coated electrochemical biosensor for detecting organophosphorus pesticide residues.

[0008] In addition, referring to Lian, M.; Shi, Y.; Chen, L.; in, Y.; Zhang, W.; Zhao, J.; Chen, D., Cell Membrane and V2C MXene-Based Electrochemical Immunosensor with Enhanced Antifouling Capability for Detection of CD44. ACS Sensors 2022, 7(9), 2701 - 2709 and Shi, R.; Fu, S.; Xu, Y.; Miao, P., Cell membranes cloaked magnetic nanoparticles for target recycling detection of nucleic acid. Chemical Engineering Journal 2023, 475, 146170, it can be known that red blood cells are the most abundant type of blood cells, lacking a nucleus and most organelles, and their plasma membrane (i.e., red blood cell membrane, RBCMs) has high biocompatibility and is a good source of cell membranes.

[0009] In summary, it is expected to provide a highly stable cell membrane-coated electrochemical biosensor based on Chi-modified CNTs modified GCE. Summary of the Invention

[0010] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a highly stable cell membrane-coated electrochemical biosensor and its preparation method and application, which have high stability and high electrochemical responsiveness and can quickly and accurately detect organophosphorus pesticide residues in agricultural products.

[0011] To achieve the above purpose, the present invention adopts the following technical solutions to implement:

[0012] A preparation method of a highly stable cell membrane-coated electrochemical biosensor, comprising the following steps:

[0013] Step 1: Prepare an RBCMs suspension

[0014] First, collect RBCMs, and then add the RBCMs to a 1×PBS solution with a pH value of 7.4 and stir evenly to obtain an RBCMs suspension with a concentration of 2.0 mg / mL;

[0015] Step 2: Preparation of cMWCNT-Chi

[0016] Step 2.1: Mix 2.0 - 3.0 mg of Chi with 20 mL of 2% acetic acid solution, and stir magnetically at room temperature to obtain a Chi solution;

[0017] Step 2.2: Add 10 - 30 mg of cMWCNTs to the Chi solution and sonicate to obtain a black solution, denoted as cMWCNTs-Chi;

[0018] Step 3: Preparation of cell membrane-coated electrochemical biosensor

[0019] Step 3.1: Pretreat the GCE;

[0020] Step 3.2: Drop-coat 8 - 12 μL of cMWCNTs-Chi onto the surface of a pretreated circular GCE with a diameter of 3 mm, and air-dry at room temperature to obtain cMWCNTs-Chi / GCE;

[0021] Step 3.3: Drop-coat 8 - 12 μL of RBCMs suspension onto the surface of cMWCNTs-Chi / GCE, and incubate at 25 - 37 °C for 1 - 2 h to obtain CM-cMWCNTs-Chi / GCE;

[0022] Step 3.4: First, dissolve 0.01 - 0.03 mg of BSA in 1 mL of 1×PBS with a pH of 7.4 to obtain a BSA solution with a concentration of 0.01% - 0.03%. Then, drop-coat 8 - 12 μL of the BSA solution onto the surface of CM-cMWCNTs-Chi / GCE, and incubate at 4 °C for 30 - 60 min to obtain BSA-CM-cMWCNTs-Chi / GCE;

[0023] Step 3.5: Wash BSA-CM-cMWCNTs-Chi / GCE several times with 1×PBS, and store at 4 °C to obtain a cell membrane-coated electrochemical biosensor.

[0024] Furthermore, the process of collecting RBCMs in Step 1 is as follows:

[0025] At 4 °C, first centrifuge 1000 g of whole blood for 10 min, collect the precipitate, then centrifuge and wash the precipitate several times with 1×PBS solution with a pH of 7.4 to separate pure red blood cells. Then, suspend the pure red blood cells in 0.25×PBS solution to obtain a suspension. Then, centrifuge the suspension at a centrifugal force of not less than 12000 g for 20 min, collect the precipitate, and wash the precipitate several times with 1×PBS solution to obtain RBCMs. Store at 4 °C for at least 12 h for standby.

[0026] Further, the number of times of washing in step 1 is 3 - 5 times.

[0027] Further, the time of ultrasonic treatment in step 2.2 is 1 - 3 h.

[0028] Further, the process of pre - treating the GCE in step 3.1 is as follows:

[0029] First, use alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm to polish the GCE to mirror finish, and then place the polished GCE in dilute nitric acid, absolute ethanol, and ultrapure water in sequence for ultrasonic cleaning for 3 - 5 min each time. Then place the cleaned GCE in a nitrogen atmosphere for drying to obtain the pre - treated GCE.

[0030] A highly stable cell - membrane - coated electrochemical biosensor has a linear detection range for organophosphorus pesticides of 1×10 -13 M~1×10 -7 M.

[0031] Further, the limit of detection LOD of the highly stable cell - membrane - coated electrochemical biosensor for organophosphorus pesticides is 0.1 pmol / L.

[0032] Application of a highly stable cell - membrane - coated electrochemical biosensor in the detection of organophosphorus pesticide residues.

[0033] Compared with the prior art, the present invention has the following technical effects:

[0034] 1), The present invention prepares a nanocomposite using carboxyl - functionalized multi - walled carbon nanotubes (cMWCNTs) and chitosan (Chi) as raw materials, coats it on the surface of a glassy carbon electrode (GCE) to improve its electrical response, and adsorbs red blood cell membranes (RBCMs). Finally, bovine serum albumin (BSA) is used to protect and seal the red - blood - cell - membrane - coated electrochemical biosensor to obtain a highly stable red - blood - cell - membrane - coated electrochemical biosensor; compared with traditional cell - membrane - coated electrochemical biosensors, the cell - membrane - coated electrochemical biosensor based on the cMWCNTs - Chi hydrogel coating can effectively ensure that the cell membrane does not detach from the electrode surface during use, improving the durability of the biosensor; compared with traditional acetylcholinesterase (AChE) electrochemical biosensors, it ensures the conformation of AChE on the cell membrane, thus being able to truly restore its biological activity, not only improving the electrochemical response of AChE, but also protecting the structure of the electrode, showing good stability at 4°C; in addition, the red - blood - cell - membrane - coated electrochemical biosensor of the present invention has a sensitive detection limit for the organophosphorus pesticide methyl parathion as low as 0.1 pmol / L, and has a range of 1×10-13 M~1×10 -7 The linear detection range of M.

[0035] 2), The inhibition rate of the activity of AChE on the cell membrane determined by the erythrocyte membrane-coated electrochemical biosensor constructed in the present invention for the organophosphorus pesticide residue on apples is 14.8%. After calculation, the pesticide residue is 1×10 -17 mol, with high precision. It can be seen that the present invention expands the application of cell membrane biomimetic technology in electrochemical biosensors and provides new insights and ideas for evaluating the residues of organophosphorus pesticides in agricultural products.

[0036] 3), The erythrocyte membrane-coated electrochemical biosensor constructed in the present invention has the characteristics of miniaturization and high selectivity, and can quickly and sensitively measure the activity of AChE to detect organophosphorus pesticides, and can be effectively used for food quality monitoring. Description of the Drawings

[0037] Figure 1 : The concentration-peak current curve of the RBCMs suspension constructed in the present invention;

[0038] Figure 2 : The incubation time-peak current curve constructed in the present invention;

[0039] Figure 3 : The pH-peak current curve of the buffer solution PBS constructed in the present invention;

[0040] Figure 4 : The incubation temperature-peak current curve constructed in the present invention;

[0041] Figure 5 : The CV and EIS results of different electrochemical biosensors in a solution containing 0.1 mol / L KCl and 5 mmol / L [Fe(CN) 6 3- / 4- ;

[0042] Figure 6 : The SEM images of different electrochemical biosensors;

[0043] Figure 7 : The CLSM results of the electrochemical biosensor prepared in Example 1 of the present invention;

[0044] Figure 8 : The electrochemical response curves of the electrochemical biosensor prepared in Example 1 of the present invention in methyl parathion solutions with different concentrations;

[0045] Figure 9 : The electrochemical response curves of the electrochemical biosensor prepared in Example 1 of the present invention in butylcarbamate solutions with different concentrations; ​

[0046] Figure 10 : The present invention uses the DPV method to measure the peak current of the BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 and the BSA-CM / GCE prepared in Comparative Example 1;

[0047] Figure 11 : The DPV results of organophosphorus pesticides in real apple samples detected by the electrochemical biosensor prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The specific contents of the present invention are further explained in detail below in conjunction with embodiments.

[0049] 1. Experimental Materials and Methods

[0050] 1.1 Experimental instruments and reagents

[0051] 1.1.1 Test Instrument

[0052] The images were acquired using a CHI-660E electrochemical workstation (Shanghai CH Instrument Co., Ltd.), a scanning electron microscope (Gemini SEM500, Carl Zeiss; Baden-Württemberg, Germany), and a confocal laser scanning microscope (CLSM; TCS SP8 STED 3X, Leica; Solms, Germany).

[0053] 1.1.2 Reagents

[0054] Glacial acetic acid was purchased from Tianjin Damao Chemical Reagent Factory; anhydrous ethanol was purchased from Tianjin Tianli Chemical Reagent Co., Ltd.; dilute nitric acid was purchased from Shanghai Anpu Experimental Technology Co., Ltd.; chitosan (Chi) and carboxylated multi-walled carbon nanotubes (cMWCNTs) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 1,1'-octadecyl-3,3,3',3'-11-tetramethylindocyanine perchlorate (DiI) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., China; Alexa Fluor 488-labeled goat anti-rabbit SA was purchased from Beijing Hesheng Biotechnology Co., Ltd.; sodium dihydrogen phosphate dihydrate, sodium monohydrogen phosphate dihydrate, potassium chloride, potassium ferrocyanide and potassium ferrocyanide were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; bovine serum albumin (BSA) was purchased from Shanghai Diyi Biotechnology Co., Ltd.; acetylcholine chloride (ATCl) was purchased from Merck Group, Germany. All the above chemicals were of analytical grade or higher purity;

[0055] Ultrapure water (resistivity of 18.2 MΩ·cm) produced by Feixiang Technology Co., Ltd. was used to prepare buffer solutions. Alumina powders of 1.0, 0.3, and 0.05 μm were purchased from Wuhan Gaoshi Ruilian Technology Co., Ltd., and methyl parathion and carbosulfan standard solutions were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0056] 1.2. Collect RBCMs and prepare an RBCM suspension

[0057] 1.2.1. Collect RBCMs

[0058] See Gao, M.; Liang, C.; Song, X.; Chen, Q.; Jin, Q.; Wang, C.; Liu, Z., Erythrocyte-Membrane-Enveloped Perfluorocarbon as Nanoscale Artificial Red Blood Cells to Relieve Tumor Hypoxia and Enhance Cancer Radiotherapy. Advanced Materials 2017, 29(35), 1701429; at 4 °C, first centrifuge 1000 g of whole blood for 10 min, collect the precipitate, then centrifuge and wash the precipitate 3 - 5 times with 1×PBS solution with a pH of 7.4 to separate pure red blood cells. Then suspend the pure red blood cells in 0.25×PBS solution to obtain a suspension. Then, centrifuge the suspension at a centrifugal force of not less than 12000 g for 20 min, collect the precipitate, and wash the precipitate 3 - 5 times with 1×PBS solution to obtain RBCMs. Store at 4 °C for at least 12 h for later use;

[0059] 1.2.2. Prepare an RBCM suspension

[0060] Add RBCMs to 1×PBS solution with a pH of 7.4 and stir evenly to obtain an RBCM suspension with a concentration of 2.0 mg / mL.

[0061] 1.3. Prepare cMWCNT-Chi

[0062] First, mix 2.0 - 3.0 mg of Chi and 20 mL of 2% acetic acid solution, and stir magnetically at room temperature to obtain a Chi solution. Then add 10 - 30 mg of cMWCNTs to the Chi solution and sonicate for 1 - 3 h to obtain a black solution, denoted as cMWCNTs-Chi.

[0063] 1.4. Prepare a cell membrane-coated electrochemical biosensor

[0064] 1.4.1. Pretreat the GCE

[0065] First, use alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm to polish the GCE to a mirror finish. Then, place the polished GCE successively in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 3 - 5 minutes each time. Then, place the cleaned GCE in a nitrogen atmosphere and dry it to obtain the pretreated GCE.

[0066] 1.4.2. Preparation of Electrochemical Biosensor

[0067] First, drop 8 - 12 μL of cMWCNTs - Chi onto the surface of the pretreated circular GCE with a diameter of 3 mm and air - dry it at room temperature to obtain cMWCNTs - Chi / GCE. Then, drop 8 - 12 μL of the RBCMs suspension onto the surface of cMWCNTs - Chi / GCE and incubate it at 25 - 37 °C for 1 - 2 h to obtain CM - cMWCNTs - Chi / GCE. Then, dissolve 0.01 - 0.03 mg of BSA in 1 mL of 1×PBS with a pH value of 7.4 to obtain a BSA solution with a concentration of 0.01% - 0.03%. Then, drop 8 - 12 μL of the BSA solution onto the surface of CM - cMWCNTs - Chi / GCE and incubate it at 4 °C for 30 - 60 min to obtain BSA - CM - cMWCNTs - Chi / GCE. Then, wash BSA - CM - cMWCNTs - Chi / GCE several times with 1×PBS and store it at 4 °C to obtain the cell - membrane - coated electrochemical biosensor.

[0068] For the specific experimental procedures and methods involved in the above 1.2 - 1.4, refer to Examples 1 - 3:

[0069] Example 1

[0070] Step 1: Add RBCMs to a 1×PBS solution with a pH value of 7.4 and stir evenly to obtain an RBCMs suspension with a concentration of 2.0 mg / mL.

[0071] Step 2: First, mix 2.0 mg of Chi and 20 mL of a 2% acetic acid solution and stir magnetically at room temperature to obtain a Chi solution. Then, add 10 mg of cMWCNTs to the Chi solution and ultrasonically treat it for 2 h to obtain a black solution, denoted as cMWCNTs - Chi.

[0072] Step 3: First, use alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm to polish the GCE to a mirror finish. Then, place the polished GCE successively in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 3 minutes each time. Then, place the cleaned GCE in a nitrogen atmosphere and dry it to obtain the pretreated GCE.

[0073] Step 4: First, 8 μL of cMWCNTs-Chi was drop-coated on the surface of a pre-treated circular GCE with a diameter of 3 mm and air-dried at room temperature to obtain cMWCNTs-Chi / GCE. Then, 8 μL of the RBCMs suspension was drop-coated on the surface of cMWCNTs-Chi / GCE and incubated at 37 °C for 1 h to obtain CM-cMWCNTs-Chi / GCE. Subsequently, 0.01 mg of BSA was dissolved in 1 mL of 1×PBS with a pH value of 7.4 to obtain a BSA solution with a concentration of 0.01%. Then, 8 μL of the BSA solution was drop-coated on the surface of CM-cMWCNTs-Chi / GCE and incubated at 4 °C for 30 min to obtain BSA-CM-cMWCNTs-Chi / GCE. Then, BSA-CM-cMWCNTs-Chi / GCE was washed three times with 1×PBS and stored at 4 °C to obtain a cell membrane-coated electrochemical biosensor.

[0074] Example 2

[0075] Step 1: RBCMs were added to a 1×PBS solution with a pH value of 7.4 and stirred evenly to obtain an RBCMs suspension with a concentration of 2.0 mg / mL;

[0076] Step 2: First, 2.5 mg of Chi was mixed with 20 mL of a 2% acetic acid solution and magnetically stirred at room temperature to obtain a Chi solution. Then, 20 mg of cMWCNTs was added to the Chi solution and ultrasonically treated for 1 h to obtain a black solution, denoted as cMWCNTs-Chi;

[0077] Step 3: First, the GCE was polished to a mirror finish using alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm. Then, the polished GCE was successively placed in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 4 min each time. Then, the cleaned GCE was placed in a nitrogen atmosphere and dried to obtain a pre-treated GCE;

[0078] Step 4: First, 10 μL of cMWCNTs-Chi was drop-coated onto the surface of a pre-treated glassy carbon electrode (GCE) with a diameter of 3 mm, and air-dried at room temperature to obtain cMWCNTs-Chi / GCE. Then, 10 μL of the RBCMs suspension was drop-coated onto the surface of cMWCNTs-Chi / GCE and incubated at 30 °C for 1.5 h to obtain CM-cMWCNTs-Chi / GCE. Subsequently, 0.02 mg of BSA was dissolved in 1 mL of 1×PBS with a pH value of 7.4 to obtain a BSA solution with a concentration of 0.02%. Then, 10 μL of the BSA solution was drop-coated onto the surface of CM-cMWCNTs-Chi / GCE and incubated at 4 °C for 45 min to obtain BSA-CM-cMWCNTs-Chi / GCE. Then, BSA-CM-cMWCNTs-Chi / GCE was washed 4 times with 1×PBS and stored at 4 °C to obtain a cell membrane-coated electrochemical biosensor.

[0079] Example 3

[0080] Step 1: RBCMs were added to 1×PBS solution with a pH value of 7.4 and stirred evenly to obtain an RBCMs suspension with a concentration of 2.0 mg / mL.

[0081] Step 2: First, 3 mg of Chi was mixed with 20 mL of 2% acetic acid solution and magnetically stirred at room temperature to obtain a Chi solution. Then, 30 mg of cMWCNTs was added to the Chi solution and ultrasonicated for 3 h to obtain a black solution, denoted as cMWCNTs-Chi.

[0082] Step 3: First, the GCE was polished to mirror finish using alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm. Then, the polished GCE was successively placed in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 5 min each time. Then, the cleaned GCE was placed in a nitrogen atmosphere and dried to obtain a pre-treated GCE.

[0083] Step 4: First, 12 μL of cMWCNTs-Chi was drop-coated onto the surface of a pre-treated circular GCE with a diameter of 3 mm, and air-dried at room temperature to obtain cMWCNTs-Chi / GCE. Then, 12 μL of the RBCMs suspension was drop-coated onto the surface of cMWCNTs-Chi / GCE and incubated at 25 °C for 2 h to obtain CM-cMWCNTs-Chi / GCE. Next, 0.03 mg of BSA was dissolved in 1 mL of 1×PBS with a pH value of 7.4 to obtain a BSA solution with a concentration of 0.03%. Then, 12 μL of the BSA solution was drop-coated onto the surface of CM-cMWCNTs-Chi / GCE and incubated at 4 °C for 60 min to obtain BSA-CM-cMWCNTs-Chi / GCE. Then, BSA-CM-cMWCNTs-Chi / GCE was washed 5 times with 1×PBS and stored at 4 °C to obtain a cell membrane-coated electrochemical biosensor.

[0084] In addition, for comparative experiments, BSA-CM / GCE, cMWCNTs-Chi / GCE, and CM-cMWCNTs-Chi / GCE were also prepared respectively according to the above method for comparative experiments. The specific experimental procedures are shown in Comparative Examples 1 to 3:

[0085] Comparative Example 1

[0086] Step 1: RBCMs were added to a 1×PBS solution with a pH value of 7.4 and stirred evenly to obtain an RBCMs suspension with a concentration of 2.0 mg / mL.

[0087] Step 2: First, alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm were used to polish the GCE to a mirror finish. Then, the polished GCE was successively placed in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 3 min each time. Then, the cleaned GCE was placed in a nitrogen atmosphere and dried to obtain a pre-treated GCE.

[0088] Step 3: First, 8 μL of the RBCMs suspension was drop-coated onto the surface of a pre-treated circular GCE with a diameter of 3 mm and incubated at 37 °C for 1 h to obtain CM / GCE. Then, 0.01 mg of BSA was dissolved in 1 mL of 1×PBS with a pH value of 7.4 to obtain a BSA solution with a concentration of 0.01%. Then, 8 μL of the BSA solution with a concentration of 0.01% was drop-coated onto the surface of CM / GCE and incubated at 4 °C for 30 min to obtain BSA-CM / GCE. BSA-CM / GCE was washed 3 times with 1×PBS and stored at 4 °C for standby.

[0089] Comparative Example 2

[0090] Step 1: First, mix 2.0 mg of Chi with 20 mL of acetic acid solution with a concentration of 2%, and stir magnetically at room temperature to obtain a Chi solution. Then, add 10 mg of cMWCNTs to the Chi solution and ultrasonically treat it to obtain a black solution, denoted as cMWCNTs-Chi;

[0091] Step 2: First, use alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm to polish the GCE to a mirror finish. Then, place the polished GCE successively in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 3 min each time. Then, place the cleaned GCE in a nitrogen atmosphere and dry it to obtain a pretreated GCE;

[0092] Step 3: Drop 8 μL of cMWCNTs-Chi onto the surface of a pretreated circular GCE with a diameter of 3 mm, and air-dry it at room temperature to obtain cMWCNTs-Chi / GCE. Wash cMWCNTs-Chi / GCE three times with 1×PBS and store it at 4 °C for later use.

[0093] Comparative Example 3

[0094] Step 1: Add RBCMs to a 1×PBS solution with a pH of 7.4 and stir evenly to obtain an RBCMs suspension with a concentration of 2.0 mg / mL;

[0095] Step 2: First, mix 2.0 mg of Chi with 20 mL of acetic acid solution with a concentration of 2%, and stir magnetically at room temperature to obtain a Chi solution. Then, add 10 mg of cMWCNTs to the Chi solution and ultrasonically treat it to obtain a black solution, denoted as cMWCNTs-Chi;

[0096] Step 3: First, use alumina powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm to polish the GCE to a mirror finish. Then, place the polished GCE successively in dilute nitric acid, absolute ethanol, and ultrapure water for ultrasonic cleaning for 3 min each time. Then, place the cleaned GCE in a nitrogen atmosphere and dry it to obtain a pretreated GCE;

[0097] Step 4: First, drop 8 μL of cMWCNTs-Chi onto the surface of a pretreated circular GCE with a diameter of 3 mm, and air-dry it at room temperature to obtain cMWCNTs-Chi / GCE. Then, drop 8 μL of the RBCMs suspension onto the surface of cMWCNTs-Chi / GCE and incubate it at 37 °C for 1 h to obtain CM-cMWCNTs-Chi / GCE. Wash CM-cMWCNTs-Chi / GCE three times with 1×PBS and store it at 4 °C for later use.

[0098] 1.5. Detection procedure of the electrochemical biosensor

[0099] Using a CHI-660E electrochemical workstation, a three-electrode system was constructed with 3.0 mL of PBS electrolyte solution with a concentration of 100 mM and a pH value of 7.4. A GCE with a diameter of 3 mm was used as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode;

[0100] The electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV) were respectively used to test the electrochemical response of the electrochemical biosensor prepared in Example 1. Among them: for the EIS measurement, in 0.1 mol / L PBS (pH 7.4) containing 5.0 mmol / L [Fe(CN) 6 3- / 4- and 0.1 mol / L KCl, at a constant potential of 0 V, an alternating current impedance with an amplitude of 50 mV and different frequencies from 10 to 1,000,000 Hz was applied; the CV measurement was carried out in the same solution as the EIS measurement, and the scanning potential was from -0.2 to 0.6 V. All experiments used 3.0 mL of electrolyte solution;

[0101] Acetylcholine chloride (ATCl) was added to 0.1 mol / L PBS (pH 7.4) solution to prepare an incubation solution with an optimal concentration of 5.0 mmol / L. Due to the redox reaction between the substrate ATCl and AChE, an obvious oxidation peak was detected at 650 mV by the DPV method. The reaction equation based on ATCl is shown in Equation (1). First, ATCl is hydrolyzed by acetylcholinesterase (AChE) into thiocholine (TCh) and acetic acid (HA). Subsequently, according to Equation (2), TCh is further oxidized on the electrode surface to 2-hydroxy-N,N,N-trimethylethanaminium (choline):

[0102]

[0103] TCh(red)→TCh(ox)+ 2H + +2e - (2)

[0104] 1.6. Electrochemical response of the electrochemical biosensor

[0105] Using the commonly used organophosphorus pesticides parathion-methyl and carbosulfan as inhibitors of AChE, the parathion-methyl stock solution and carbosulfan were respectively added to the incubation solution. The parathion-methyl stock solution and carbosulfan were diluted to concentrations of 1×10 -7 mol / L and 1×10 -8 ​mol / L, 1×10 -9 mol / L, 1×10 -10 mol / L, 1×10 11 mol / L, 1×10 -12 mol / L and 1×10 -13 mol / L solutions were used to test the inhibitory effect of parathion-methyl at different concentrations on the current response of the electrochemical biosensor prepared in Example 1, and the inhibition rate was calculated according to Equation (3):

[0106]

[0107] In the formula, I max is the steady-state current measured in the incubation solution, and I min is the steady-state current measured in the parathion-methyl solution.

[0108] 1.7. Optimization of experimental conditions

[0109] To optimize the concentration of RBCMs suspension, RBCMs suspensions with concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL and 3.5 mg / mL were prepared respectively. The absorbance of each suspension was measured with a spectrophotometer to ensure uniformity and appropriate transmittance. The peak currents at different concentrations were compared by DPV method. Then, a concentration-peak current curve as shown in Figure 1 was constructed to determine the optimal cell membrane concentration. Subsequently, the incubation time of ATCl on the electrochemical biosensor was optimized and the peak current of the biosensor was tested over time. A time-peak current curve as shown in Figure 2 was constructed to determine the optimal incubation time;

[0110] In addition, the buffer solution PBS was precisely adjusted to pH values of 6.0, 6.5, 7.0, 7.4 and 8.0 in turn. The peak currents of the biosensor at different pH values were compared, and a pH-peak current curve as shown in Figure 3 was constructed to determine the optimal solution pH value; Meanwhile, the peak currents of the biosensor were evaluated at different temperatures of 4 °C, 25 °C, 37 °C and 50 °C, and a temperature-peak current curve as shown in Figure 4 was established to determine the optimal culture temperature;

[0111] To ensure the reliability of the results, each experiment was carried out three times.

[0112] 1.8. Verification of the stability and repeatability of the electrochemical biosensor

[0113] To evaluate the stability of the biosensor, the peak currents of BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 and BSA-CM / GCE prepared in Comparative Example 1 stored at 4 °C for standby were measured by DPV method at 1 d, 3 d, 1 w, 3 w and 5 w respectively over a period of 1 month, and the experiment was repeated three times.

[0114] 1.9. Practical applications of electrochemical biosensors

[0115] To evaluate the practicality of the electrochemical biosensor, the apple peel was air-dried and crushed, 5 g of the peel powder was mixed and added to 5 mL of 0.1 mol / L PBS (pH 7.4) containing 5.0 mmol / L ATCl, sonicated for 1 - 3 h, the sonicated liquid was centrifuged at 10,000 rpm for 10 min, the supernatant was collected, labeled as the real sample, and the inhibitory effect of the real sample on the current response of the biosensor was tested to detect its organophosphorus pesticide content.

[0116] 1.10. Statistical analysis

[0117] The experimental data were analyzed using Origin 2024b (OriginLab Corp., Massachusetts, USA) and GraphPad Prism Software 9.5 (GraphPad Software, Inc., California, USA), and were expressed as mean ± standard error (SD). One-way analysis of variance (ANOVA) was used for multiple comparisons. A P value < 0.05 was considered statistically significant (ns, not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001).

[0118] 2. Experimental results

[0119] 2.1. To prove the successful preparation of the electrochemical biosensor, in a solution containing 0.1 mol / L KCl and 5 mmol / L [Fe(CN) 6 3- / 4- the stepwise construction of the biosensors in Comparative Example 2, Comparative Example 3 and Example 1 was analyzed by EIS and CV, and the results are as Figure 5 shown, where: Figure 5 ​(a) shows the CV results. In the figure, a, b, c, and d correspond to bare GCE, cMWCNTs-Chi / GCE, CM-cMWCNTs-Chi / GCE, and BSA-CM-cMWCNTs-Chi / GCE respectively, indicating that compared with the bare GCE (curve a), the peak current and reversibility of cMWCNTs-Chi / GCE (curve b) prepared in Comparative Example 2 increased, indicating an increase in the electrode conductivity. By observing CM-cMWCNTs-Chi / GCE (curve c) prepared in Comparative Example 3 and BSA-CM-cMWCNTs-Chi / GCE (curve d) prepared in Example 1, it can be seen that with the modification of RBCMs and BSA, both the peak current and reversibility decreased; Figure 5 (b) shows the EIS results. In the figure, a, b, c, and d correspond to the Nyquist curves of bare GCE, cMWCNTs-Chi / GCE, CM-cMWCNTs-Chi / GCE, and BSA-CM-cMWCNTs-Chi / GCE respectively. The charge transfer resistance (Rct) was calculated based on the diameter of the semicircle. The bare GCE (curve a) showed a higher Rct and was almost linear in the EIS analysis. On the contrary, when cMWCNTs-Chi was used to modify the GCE surface in Comparative Example 2, due to the high conductivity of cMWCNTs, a significant reduction in impedance coupling was observed (curve b). After using cMWCNTs-Chi and RBCMs coating in Comparative Example 3, the impedance increased compared with before (curve c). After using RBCMs and BSA coating in Example 1, an increase in impedance was observed again compared with before (curve d); Figure 5 The CV of (a) and Figure 5 the EIS results of (b) are basically consistent, indicating that the biosensor was successfully constructed step by step.

[0120] In addition, scanning electron microscopy (SEM) was used to observe the step-by-step construction of the biosensor. The results are as Figure 6 shown. The scale bar is 1 μm. In the figure, a, b, c, and d correspond to bare GCE, cMWCNTs-Chi / GCE, CM-cMWCNTs-Chi / GCE, and BSA-CM-cMWCNTs-Chi / GCE respectively. It can be seen that there are microscopic scratches on the surface of the uncoated bare GCE electrode. The surface of cMWCNTs-Chi / GCE prepared in Comparative Example 2 presents a typical CNTs coating. The CM-cMWCNTs-Chi / GCE prepared in Comparative Example 3 presents a relatively flat RBCMs coverage. The surface of BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 presents an overall coating with slightly protrusions. The observation results emphasize the successful step-by-step construction of the biosensor.

[0121] To study the stable presence of AChE on the biosensor prepared in Example 1, AChE on RBCMs was stained with a specific primary antibody (AChE monoclonal antibody MA3-042), and then stained with SA compatible with immunofluorescence analysis (goat anti-rabbit labeled with AlexaFluor 488). To stain RBCMs, the sample was incubated in the DiI staining reagent in the dark. The CLSM results are as Figure 7 shown. The scale bar is 5 μm. The red fluorescence labeled with DiI indicates the presence of RBCMs (Figure a), and the green fluorescence labeled with goat anti-rabbit labeled with Alexa Fluor 488 indicates the presence of AChE (Figure b). Figure (c) is the co-localization map, with obvious red and green signals, confirming the presence of AChE on RBCM, which lays the foundation for the detection of organophosphorus pesticides by the electrochemical biosensor.

[0122] 2.2. Results of experimental condition optimization

[0123] During the preparation of the biosensor, the content of AChE is a key factor. To study this, the effects of RBCMs concentration (1.0 - 3.5 mg / mL) were studied in 0.1 mol / L PBS (pH 7.4) and 5.0 mmol / L ATCl respectively. The results are as Figure 1 shown, where Figure 1 (a) a, b, c, d, e, f represent the current responses corresponding to RBCMs suspensions with concentrations of 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL and 3.5 mg / mL respectively. It can be seen that the current response increases with the increase of RBCMs concentration and reaches the maximum value at 2.0 mg / mL. From Figure 1 (b), it can be intuitively seen that with the further increase of RBCMs concentration, the current response shows an obvious downward trend, which is due to the thicker RBCMs layer increasing the resistance of the electrochemical process. Therefore, 2.0 mg / mL was selected as the ideal concentration of the RBCMs suspension;

[0124] The incubation time in the ATCl solution is also one of the most important parameters affecting the response of the electrochemical biosensor. In 0.1 mol / L PBS (pH 7.4) containing 5.0 mmol / L ATCl, the activation effect of different incubation times (3 - 24 h) on AChE activity was detected respectively. The dependence of the current difference on the incubation time is as Figure 2 shown, where Figure 2In (a), a, b, c, d, and e represent the responses of the current at 3 h, 6 h, 8 h, 12 h, and 24 h respectively. The results show that as the incubation time increases, the activation effect of ATCl on AChE becomes better and better, and the current response reaches the maximum value at 6 h. From Figure 2 It can be intuitively seen from (b) that as the incubation time further increases, the current response shows an obvious downward trend, which is caused by the deformation of the electrochemically biosensor coating in the solution. Therefore, 6 h is selected as the optimal parameter for the incubation time of the electrochemically biosensor in the ATCl solution for the detection experiment;

[0125] In addition, scientists generally believe that the biological activity of immobilized AChE is affected by the solution pH and temperature. Therefore, the relationship between the catalytic peak current of AChE and the solution pH and temperature in the ATCl reaction was studied. The results are as Figure 3 and Figure 4 shown. Figure 3 In (a), a, b, c, d, and e represent the current responses corresponding to pH values of 6.0, 6.5, 7.0, 7.4, and 8.0 respectively. Figure 4 In (a), a, b, c, and d represent the current responses at 4 °C, 25 °C, 37 °C, and 50 °C respectively. It should be noted that: from Figure 3 (a) and Figure 3 (b), it can be seen that within the range of pH 6.0 - 8.0, the current peak value is the largest at pH 7.4; from Figure 4 (a) and Figure 4 (b), it can be seen that at the test temperatures of 4 °C, 25 °C, 37 °C, and 50 °C, the current peak value is the highest at 25 °C. Therefore, pH 7.4 and temperature 25 °C are selected for the detection experiment.

[0126] 2.3 Electrochemical Behavior of Organophosphorus Pesticides

[0127] The mechanism of the redox reaction between AChE and its substrate ATCl is that ATCl is hydrolyzed by AChE into TCh and HA. Subsequently, TCh is oxidized to form TCh(ox). This special redox reaction requires the presence of ATCl and does not occur only in PBS. In addition, if the activity of AChE is inhibited by an inhibitor, the reaction also stops. Therefore, in order to evaluate the electrochemical response of the biosensor prepared in Example 1 to organophosphorus pesticides, we used the DPV method to observe the inhibitory effect of two organophosphorus pesticides at different concentrations on the AChE current, evaluate the relationship between the inhibition rate and the concentration of organophosphorus pesticides, and calculate its IC50 value. The results are as Figure 8 and Figure 9 shown, where Figure 8 is the DVP curve of the electrochemically biosensor prepared in Example 1 in different concentrations of parathion-methyl solution and the inhibition rate of the AChE current.Figure 9 DVP curves of the electrochemical biosensor prepared in Example 1 in carbosulfan solutions with different concentrations and the inhibition rates of AChE current Figure 8 (a) and Figure 9 (a), where a, b, c, d, e, f, g, and h respectively correspond to the incubation solutions without adding methyl parathion stock solution and carbosulfan, and the solutions with added methyl parathion stock solution or carbosulfan at concentrations of 1×10 -7 mol / L, 1×10 -8 mol / L, 1×10 -9 mol / L, 1×10 -10 mol / L, 1×10 11 mol / L, 1×10 -12 mol / L, and 1×10 -13 mol / L. It can be seen that as the concentrations of methyl parathion and carbosulfan in the ATCl solution increase, the generated current of AChE decreases. Under the optimized experimental conditions, the inhibitory effects of both on AChE are proportional to their concentrations (from 1×10 -13 mol / L to 1×10 -7 mol / L). The linear equations corresponding to methyl parathion and carbosulfan are as Figure 8 (b) and Figure 9 (b) shown, which are respectively: inhibition rate (%) = 12.0864logC + 173.9102, R 2 = 0.9845; inhibition rate (%) = 13.8208logC + 182.3347, R 2 = 0.9877. The detection limits (LOD) of methyl parathion and carbosulfan are 0.1 pmol / L (S / N = 3). The calculated IC50 values are 5.59×10-11 mol / L and 2.66×10-10 mol / L respectively;

[0128] Compared with the traditional cell membrane-coated electrochemical biosensor, the cell membrane-coated electrochemical biosensor proposed in this example not only maintains the true biological activity of AChE as a peripheral membrane-anchored protein, but also shows higher sensitivity (LOD = 0.1 pmol / L), indicating that the electrochemical biosensor prepared in this example can detect low-concentration organophosphorus pesticides and has good application prospects in ultrasensitive biomedical assays.

[0129] 2.4. Stability of the electrochemical biosensor

[0130] To determine the durability of the electrochemical biosensor during storage, the peak currents of BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 and BSA-CM / GCE prepared in Comparative Example 1 stored at 4 °C were measured by DPV method at 1 d, 3 d, 1 w, 3 w, and 5 w after preparation, respectively. The results are shown as Figure 10 (a) and Figure 10 (b). In the figures, a, b, c, d, and e correspond to 1 d, 3 d, 1 w, 3 w, and 5 w, respectively. It can be seen that after 3 w of storage, the peak current of BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 is still relatively stable, which is 67.5% of the initial value. At the same time, the peak current of BSA-CM / GCE prepared in Comparative Example 1 has lost stability at 1 w. This indicates that compared with BSA-CM / GCE prepared in Comparative Example 1, BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 has significantly better storage stability and can maintain consistent and reliable results during extended storage time.

[0131] 2.5 Detection of real samples

[0132] The DPV results obtained by using BSA-CM-cMWCNTs-Chi / GCE prepared in Example 1 to detect organophosphorus pesticides in real apple samples are shown as Figure 11 . In the figure, a and b represent the DPV results of the biosensor without incubation with real samples and the DPV results of the biosensor after incubation with real samples, respectively. Through analysis, it can be known that the inhibition rate of the generated current of AChE by real samples is 14.8%. After calculation, it is obtained that 1 g of real sample contains approximately 1×10 -17 mol of organophosphorus pesticides, which is much less than the national standard, indicating that the electrochemical biosensor prepared in this example is suitable for the analysis of real samples.

[0133] 3. Conclusion

[0134] The BSA-CM-cMWCNTs-Chi / GCE biosensor prepared in Example 1 exhibits excellent sensing performance for organophosphorus pesticides, has a wide linear detection range (1×10 -13 M~1×10 -7 M) and a low detection limit (LOD = 0.1 pmol / L), and can be applied to the detection of organophosphorus pesticides in real apple samples, and can be effectively used for food quality monitoring. It can be seen that this example expands the application of cell membrane biomimetic technology in electrochemical biosensors and provides new insights and ideas for evaluating the residues of organophosphorus pesticides in agricultural products.

Claims

1. A method for preparing a highly stable cell membrane coated electrochemical biosensor, characterized in that: The steps include: Step 1: Prepare RBCMs suspension First, RBCMs were collected, and then added to a 1× PBS solution with a pH value of 7.4 and stirred evenly to obtain a RBCMs suspension with a concentration of 2.0 mg / mL; Step 2: Preparation of cMWCNT-Chi Step 2.1, 2.0-3.0 mg Chi and 20 mL 2% acetic acid solution were mixed, and magnetic stirring was performed at room temperature to obtain a Chi solution; Step 2.2, 10-30 mg of cMWCNTs were added to the Chi solution and ultrasonicated to obtain a black solution, which was recorded as cMWCNTs-Chi; Step 3: Preparation of cell membrane-coated electrochemical biosensor Step 3.1, preprocessing GCE; Step 3.2, 8-12 μL cMWCNTs-Chi was dropped onto the pretreated circular GCE surface with a diameter of 3 mm, and air-dried at room temperature to obtain cMWCNTs-Chi / GCE; Step 3.3, drop 8-12 μL of RBCMs suspension onto the surface of cMWCNTs-Chi / GCE and incubate at 25-37°C for 1-2 h to obtain CM-cMWCNTs-Chi / GCE; Step 3.4, first dissolve 0.01-0.03 mg of BSA in 1 mL of 1× PBS with a pH value of 7.4 to obtain a BSA solution with a concentration of 0.01%-0.03%, then drop 8-12 μL of the BSA solution on the surface of CM-cMWCNTs-Chi / GCE, incubate at 4°C for 30-60 min, and obtain BSA-CM-cMWCNTs-Chi / GCE; Step 3.5: Wash BSA-CM-cMWCNTs-Chi / GCE with 1×PBS for several times and store at 4°C to obtain a cell membrane-coated electrochemical biosensor.

2. The method for preparing a highly stable cell membrane coated electrochemical biosensor according to claim 1, characterized in that: The process of collecting RBCMs in step 1 is as follows: At 4°C, first centrifuge 1000g whole blood for 10 min, collect the precipitate, then centrifuge and wash the precipitate several times with 1× PBS solution with a pH value of 7.4 to separate pure red blood cells, then suspend the pure red blood cells in 0.25× PBS solution to obtain a suspension, then centrifuge the suspension at a centrifugal force of not less than 12000g for 20 min, collect the precipitate, wash the precipitate several times with 1× PBS solution to obtain RBCMs, and refrigerate at 4°C for at least 12 h for use.

3. The method for preparing a highly stable cell membrane coated electrochemical biosensor according to claim 2, characterized in that: The number of washing in step 1 is 3-5 times.

4. The method for preparing a highly stable cell membrane coated electrochemical biosensor according to claim 2, characterized in that: The ultrasonic treatment time in step 2.2 is 1-3h.

5. The method for preparing a highly stable cell membrane coated electrochemical biosensor according to claim 2, characterized in that: The process of pre-processing GCE in step 3.1 is as follows: First, the GCE was polished to a mirror finish using alumina powders with particle sizes of 1.0 μm, 0.3 μm and 0.05 μm, and then the polished GCE was placed in dilute nitric acid, anhydrous ethanol and ultrapure water in turn for ultrasonic cleaning, each cleaning lasting 3-5 minutes, and then the cleaned GCE was placed in a nitrogen atmosphere and dried to obtain the pretreated GCE.

6. The method for preparing a highly stable cell membrane coated electrochemical biosensor according to claim 1, characterized in that: The washing times in step 3.5 are 3 to 5 times.

7. A highly stable cell membrane coated electrochemical biosensor prepared by the method according to any one of claims 1 to 6, characterized in that: The linear detection range for organophosphorus pesticides is 1×10 -13 M to 1×10 -7 M.

8. The highly stable cell membrane coated electrochemical biosensor according to claim 7, characterized in that: The detection limit LOD for organophosphorus pesticides is 0.1 pmol / L.

9. Use of the highly stable cell membrane coated electrochemical biosensor according to claim 7 in the detection of organophosphorus pesticide residues.

Images