Electrochemical biosensor positively coated with erythrocyte membrane as well as preparation method and application of electrochemical biosensor

Abstract

The invention discloses an erythrocyte membrane forward coated electrochemical biosensor (ROCMCBs) as well as a preparation method and application thereof, and belongs to the technical field of biosensors. The ROCMCBs is specifically combined with an antibody modified on the surface of an electrochemical biosensor based on an extracellular binding region of a CD47 receptor on a cell membrane, and positive assembly of the red cell membrane (RBCMs) on the surface of a glassy carbon electrode (GCEs) is realized through immunoaffinity interaction between the antibodies; the positive orientation enables the cell membrane to be more firmly combined with the electrochemical biosensor and the conformation and the catalytic activity of the peripheral membrane anchoring protein AChE are exposed to the maximum extent, so that the biological activity of the acetylcholin esterase (AChE) is accurately measured, and the cell membrane can be used as a platform for screening anti-AD drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular, relates to an electrochemical biosensor, and also to a red blood cell membrane positive coating electrochemical biosensor (ROCMCBs) and a preparation method and application thereof. Background Art

[0002] The effective ingredients of traditional Chinese medicine are diverse in structure, with huge chemical diversity and multi-target characteristics. The screening of effective ingredients in complex traditional Chinese medicine is a bridge between the efficacy research and the mechanism research of traditional Chinese medicine. The existing methods for screening potential active ingredients of traditional Chinese medicine mainly include molecular docking technology, high-throughput screening technology, cell membrane technology, etc. They use the specific binding between small molecule drug ligands and receptors for screening, and have the characteristics of significant pharmacological activity, clear targeting, and clear mechanism of action. However, these methods also have the limitations of complex operation, short column life, poor sensitivity, and time-consuming and labor-intensive. Although promising virtual screening has become an attractive option, due to the multi-component and multi-target characteristics of traditional Chinese medicine, the field of traditional Chinese medicine is still in urgent need of an ideal strategy.

[0003] Designing cell membrane-based natural product screening methods by utilizing the drug-specific targeting of cell membranes has become a research hotspot. In recent decades, many methods for screening and separating target compounds have been proposed and improved. However, the wide application of these methods is limited by reasons such as long analysis time, poor peak shape, and short lifetime. Therefore, improving analytical methods with long analysis time and poor quality is crucial for developing more effective analytical methods for traditional Chinese medicine.

[0004] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by central cognitive dysfunction, memory loss, and intellectual decline, and is a major public health problem. The mechanism of AD development is elusive, and the cholinergic hypothesis explains it best. According to the cholinergic hypothesis of AD, the loss of cholinergic neurons in AD leads to decreased levels of thiocholine (ACh), which in turn induces cognitive and memory deficits.

[0005] Acetylcholinesterase (AChE) is a serine protease with aminopeptidase and carboxypeptidase activities. It is mainly present in the postsynaptic membrane of the motor nerve end plate and the synapses in the brain. Its main function is to hydrolyze acetylcholine as a neurotransmitter to terminate nerve conduction. For patients with mild to moderate AD, acetylcholinesterase inhibitors (AChE-I) are usually used to improve cognitive function. They increase the content of ACh in neuronal synapses. To date, AChE-I is still the gold standard treatment for Alzheimer's disease. Currently, the main inhibitors in clinical practice are donepezil (aricept), rivastine (exelon), galantamine (razadyne) and huperzine A. Among them, huperzine A is a plant alkaloid isolated from Huperzella serrata. It is a reversible and selective inhibitor of acetylcholinesterase and has a neuroprotective effect. It is approved for the treatment of mild to moderate AD.

[0006] Quantitative detection of AChE levels and the effects of drugs on AChE have important clinical significance for the early diagnosis and treatment of AD. By combining the specificity and sensitivity of biological systems with the accuracy and speed of physical transduction, biosensors provide a promising alternative tool for the quantification of target analytes in complex biological samples. Electrochemical biosensors have the characteristics of miniaturization and high selectivity of biosensors, and also embody the advantages of rapid and high sensitivity of electrochemical analysis. They have positive significance in drug design, drug testing, molecular screening, etc. AChE-based biosensors use the substrate thioacetylcholine to obtain the product TCh under the catalysis of AChE. The concentration of TCh is proportional to the oxidation current measured. In the presence of AChE-I drugs, the oxidation current of TCh will also change accordingly. The resulting electrical signal is captured by the enzyme sensor, which can be used as a general detection principle for AChE inhibitor drugs.

[0007] Traditional Chinese medicine has the characteristics of multi-component, multi-pathway and multi-target. In recent years, the design of cell membrane-based natural product screening methods has become a hot topic of research by utilizing the biocompatibility and drug-specific targeting of cell membranes. Cell membrane-coated electrochemical biosensors have the characteristics of miniaturization and high selectivity of biosensors, and also embody the advantages of rapid and high sensitivity of electrochemical analysis methods. They have positive significance in drug design, drug detection, molecular screening and other aspects. However, cell membrane-coated electrochemical biosensors still face some challenges in practical applications. For example, the random orientation used in traditional methods may cause the cell membrane to detach from the biosensor surface, resulting in the inability of drugs to effectively bind to specific sites; in addition, only correctly oriented cell membranes can maintain the natural conformation and function of membrane proteins or receptors and achieve optimal activity during drug screening; therefore, the development of more robust and correctly oriented cell membrane-coated electrochemical biosensors is an important part of current research. Summary of the invention

[0008] In view of the problems of easy shedding of cell membranes, conformational changes of membrane enzymes, changes in activity and the possibility of enzyme loss in practical applications of cell membrane-coated electrochemical biosensors, the object of the present invention is to provide a red blood cell membrane positively coated electrochemical biosensor (ROCMCBs) and its preparation method and application.

[0009] In order to achieve the above-mentioned purpose, the present invention designs and prepares a red blood cell membrane forward coating electrochemical biosensor, which is based on the extracellular binding region of the CD47 receptor on the cell membrane specifically binding to the antibody modified on the surface of the electrochemical biosensor, and realizes the forward assembly of red blood cell membranes (RBCMs) on the surface of glassy carbon electrodes (GCEs) through the immunoaffinity interaction between the antibodies. The forward orientation makes the cell membrane more firmly bound to the electrochemical biosensor and maximizes the exposure of the conformation and catalytic activity of the peripheral membrane anchored protein AChE, so as to accurately measure the biological activity of AChE, thereby serving as a platform for anti-AD drug screening.

[0010] Specifically, the technical solution to achieve the purpose of the present invention is as follows: a method for preparing a red blood cell membrane positive coating electrochemical biosensor, the method comprising the following steps:

[0011] (1) GCEs pretreatment: GCEs are taken and polished to a mirror effect;

[0012] (2) Preparation of SABG / GCEs: dilute the gold-conjugated anti-rabbit IgG secondary antibody with a BSA (bovine serum albumin) solution at a ratio of (10-40):1, then add glutaraldehyde, and mix thoroughly to obtain SABG; drop SABG on the surface of GCEs and dry at room temperature to obtain SABG / GCEs;

[0013] (3) Preparation of CIPA / RBCMs: CIPA (N-term CD47 antibody) solution and RBCMs solution were fully mixed and allowed to stand at 36.5-37.5°C for 0.5-2 hours to allow CIPA to fully bind to the CD47 receptor on the surface of RBCMs. Then, BSA solution was added and placed in a centrifuge for centrifugation at 11,000-13,000 r / min and 4°C for 10-20 minutes. The supernatant was discarded and the CIPA / RBCMs were resuspended in PBS.

[0014] (4) Preparation of ROCMCBs: The CIPA / RBCMs prepared in step (3) were dropped onto the SABG / GCEs prepared in step (2), and allowed to stand at room temperature for 1.5 to 2.5 hours. The mixture was rinsed with PBS for 1 to 5 times to remove the unbound cell membrane, thereby obtaining the red blood cell membrane positively coated electrochemical biosensor ROCMCBs.

[0015] Further preferably, in the method for preparing the red blood cell membrane forward coated electrochemical biosensor as described above, the specific method of the step (1) GCEs pretreatment is: polishing the exposed GCEs with 1.0, 0.3 and 0.05 μm alumina powders in sequence, and after each polishing, ultrasonically treating them in dilute nitric acid, ethanol and double distilled water for 1 to 5 minutes respectively to wash away the alumina powder adsorbed on the surface of the GCEs, and then drying the GCEs in a nitrogen environment.

[0016] Further preferably, in the method for preparing the red blood cell membrane forward coated electrochemical biosensor as described above, the concentration of the BSA solution in step (2) is 0.3-0.8%.

[0017] Further preferably, in the method for preparing the red blood cell membrane forward coated electrochemical biosensor as described above, the amount of SABG dropped on the surface of GCEs in step (2) is 5-10 μL.

[0018] Further preferably, in the method for preparing the red blood cell membrane forward coated electrochemical biosensor as described above, the CIPA solution in step (3) is prepared by diluting CIPA with 0.8-1.5% BSA in a multiple of (25-100):1.

[0019] Further preferably, in the method for preparing the red blood cell membrane forward coated electrochemical biosensor as described above, the RBCMs solution described in step (3) is prepared as follows: fresh blood is drawn and injected into a centrifuge tube containing an anticoagulant, and the whole blood is centrifuged at 4°C to separate red blood cells and plasma by low-speed centrifugation, and then the precipitate is collected and washed three times with pH7.4 PBS precooled at 4°C to obtain pure red blood cells, and the washed red blood cells are completely suspended in pH7.4 PBS and placed in a refrigerator at 4°C. Due to the osmotic pressure acting on the plasma membrane, the red blood cells are fully expanded and hemolyzed, and then the suspension is centrifuged at 11000-13000r and 4°C for 15-25 minutes, and the supernatant is removed to obtain RBCMs.

[0020] In addition, the present invention also provides an erythrocyte membrane forward coated electrochemical biosensor obtained according to the above preparation method. Moreover, the purpose of designing the erythrocyte membrane forward coated electrochemical biosensor in the present invention is to explore its ability to screen potential active ingredients against Alzheimer's disease (AD), so the present invention also provides the use of the above erythrocyte membrane forward coated electrochemical biosensor in screening potential active ingredients against Alzheimer's disease. Further preferably, the active ingredient is an effective part of traditional Chinese medicine or a monomer of traditional Chinese medicine.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) CLSM results showed that RBCMs were positively coated on the surface of RORCMBs, and AChE anchored on the surface of RBCMs could be used for subsequent drug screening; FTIR, XPS, and EDS results showed that RORCMBs retained the characteristic elements and characteristic absorption peaks of RBCMs; contact angle measurement results showed that RORCMBs had a smaller water contact angle; SEM, EIS, and DPV results showed that compared with randomly coated CMCBs, RORCMBs had a more uniform film coating and better film coating stability.

[0023] 2) Under the optimized experimental conditions, at 4.1×10 -6 -4.1×10 -12 mol / L, the inhibitory effect of Huperzine A on AChE is proportional to its concentration. Inhibition rate (%) = 10.32logC + 136.99 (%), correlation coefficient (R 2) was 0.9986. The limit of detection (LOD) was about 0.41pmol / L; the results of the interfering drug experiment showed that ROCMCBs had good selective recognition ability; the relative standard deviation (RSD) of the DPV test results of different ROCMCBs (n=5) was 3.39%, indicating that the prepared ROCMCBs had acceptable reproducibility; after storage for 7 days, the average inhibition rate of RORCMBs remained at about 72% of the initial value, indicating that ROCMCBs had good storage stability.

[0024] 3) Six potential anti-AD active ingredients were screened, including berberine, baicalin, gardenia jasminoides, gastrodin, ligusticum chuanxiong lactone A and rhynchophylline. Their IC 50 The values ​​were 4.7, 12.2, 8.3, 3.2, 86.6, and 66.3 μmol / L μmol / L, respectively. The molecular docking results were consistent with the DPV results.

[0025] 4) Successfully prepared cell membrane positive coating electrochemical biosensors and established an efficient and rapid drug screening platform; the application of cell membrane positive coating electrochemical biosensors is suitable for drug screening processes, expanding new perspectives and methods for drug lead discovery. This innovative method is expected to lay a solid foundation for personalized and precise disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :Schematic diagram of the preparation of cell membrane-coated electrochemical biosensor and its application in screening of active ingredients in traditional Chinese medicine.

[0027] Figure 2 : CLSM images of ROCMCB. (a) DiI-labeled RBCMs; (b) AChE labeled with goat anti-rabbit IgG H&L / AF488 antibody; (c) colocalization image. Scale bar: 5 μm.

[0028] Figure 3 : CLSM images of bare GCEs (a), SABG / GCEs (b), CMCBs (c) and ROCMCBs (d) after immersion in FITC-labeled BSA solution. Scale bar: 50 μm. (***P<0.001).

[0029] Figure 4 : Water contact angle images of bare GCE (a), SABG / GCEs (b), CMCBs (c), and ROCMCBs (d).

[0030] Figure 5 : Wavelength is 4000-1000cm -1 (A) and wavelength 1800-1000cm -1(B) Fourier transform infrared spectroscopy image.

[0031] Figure 6 : EDS images of ROCMCBs (A), SABG / GCEs (B), and RBCMs (C).

[0032] Figure 7 : Atomic force microscopy images of SABG (A) and ROCMCBs (B).

[0033] Figure 8 : XPS images of SABG (a), CIPA / RBCMs (b) and ROCMCBs (c).

[0034] Fig. 9 : Schematic representation of two series of immunofluorescence assays.

[0035] Fig.10 : CLSM images and fluorescence intensity quantification of ROCMCBs (A); CLSM images and fluorescence intensity quantification of CMCBs (B). Scale bar = 5 μm. (ns, no significant difference; ***P<0.001).

[0036] Fig.11 : SEM images of (a) bare GCEs, (b) SABG / GCEs, (c) CMCBs, and (d) RORCMCBs. Scale bar: 2 μm.

[0037] Fig.12 : EIS images under different incubation methods, including bare GCE (a); RBCMs droplets incubated with GCE for 2 hours, then washed with PBS solution and dried under nitrogen environment (b); RBCMs droplets incubated with GCE overnight at 4°C and humid conditions to avoid complete evaporation of the sample (c); RBCM droplets incubated at room temperature overnight to allow the sample to completely dry (d); RBCM droplets incubated with SABG / GCEs at room temperature overnight to allow the sample to completely dry (e). RBCM droplets incubated with SABG / GCEs for 2 hours, then washed with PBS solution and dried under nitrogen environment (f).

[0038] Fig.13 : ROCMCBs (purple curve) and CMCBs (yellow curve) in 5.0mmol / L ATCl (a, c) or 5.0mmol / L ATCl and 4.2×10 -8 Quantification of DPV signal and current signal inhibition rate in 1 mol / L Huperzine A (b, d). (***P<0.001).

[0039] Fig.14: CV images (A) and EIS images (B) of bare GCE (a), SABG / GCE (b) and ROCMCB (c); (C) DPV images of ROCMCBs in 5.0 mmol / L ATCl (a) or 0.1 mol / L PBS (b).

[0040] Fig.15 : CV images of bare GCEs (a), SABG / GCEs (b) and ROCMCBs (c) in the absence (A) and presence (B) of 5.0 mmol / L ATCl electrolyte.

[0041] Fig.16 : CV curves of RORCMBs at different scan rates (A), scan rate: 10-100 mV; linear relationship between peak current and scan rate (B), (n=3).

[0042] Fig.17 : Effects of RBCMs concentration (A, B), huperzine A incubation time (C, D), pH value (E, F) and temperature (G, H) on the amperometric response.

[0043] Fig.18 :The linear relationship between the concentration of Huperzine A and the peak current; the concentration of Huperzine A was 4.1×10 -6 , 8.2×10 -7 4.1×10 -7 , 8.2×10 -8 4.1×10 -8 4.1×10 -9 4.1×10 -10 4.1×10 -11 4.1×10 - 12 mol / L(n=3).

[0044] Fig.19 : It curve of ROCMCBs with continuous addition of ATCl under stirring at an applied potential of 650 mV. The inset is the calibration diagram for the determination of ATCl.

[0045] Fig. 20 : Selectivity (A), reproducibility (B) and stability (C) of RORCMBs (n=5).

[0046] Fig.21 :(A) berberine, baicalin; (B) gardenia glycoside, gastrodin; (C) rhynchophylline, ligusticum chuanxiong lactone A; (D) relationship between the concentration of tanshinone IIA, echinacoside and RORCMBs inhibition rate (n=3).

[0047] Fig. 22: DPV curves of different concentrations of (A) berberine; (B) baicalin; (C) geniposide; (D) gastrodin; (E) ligusticum bisporum L. A; (F) rhynchophylline acting on RORCMBs.

[0048] Fig.23 :(A) berberine, (B) baicalin, (C) gardenia glycoside, (D) gastrodin, (E) ligusticum bisporum L. A, (F) rhynchophylline, (G) tanshinone IIA and (H) echinacoside docking with the AChE active site. (PDB ID: 1VOT). DETAILED DESCRIPTION

[0049] In this embodiment, a red blood cell membrane positive coating electrochemical biosensor (ROCMCBs) is designed. Figure 1 The constructed ROCMCBs were used to accurately measure the biological activity of AChE as a platform for anti-AD drug screening. The inhibition rates of eight active ingredients of traditional Chinese medicine (berberine, baicalin, gardenia glycoside, gastrodin, rhynchophylline, ligusticum chuanxiong lactone A, tanshinone IIA and echinacoside) on the activity of AChE on the cell membrane were determined, and the IC 50 The molecular docking of 8 compounds with AChE was performed by SYBYL-X 2.0 to infer the interaction between small molecule drugs and enzymes. The specific experimental methods are as follows:

[0050] 1 Materials and reagents

[0051] Table 1 Experimental reagents

[0052]

[0053]

[0054] 2 Experimental methods

[0055] 2.1 Extraction of RBCMs

[0056] Fresh blood was drawn and injected into a centrifuge tube containing an anticoagulant. Whole blood was centrifuged at 1000g (low speed) for 10 minutes at 4°C to separate red blood cells and plasma. The precipitate was then collected and washed three times with 1×PBS (pH 7.4) precooled at 4°C to obtain pure red blood cells. The washed red blood cells were completely suspended in 0.25×PBS and placed in a refrigerator at 4°C. Due to the osmotic pressure acting on the plasma membrane, the red blood cells were fully swollen and hemolyzed. The suspension was then centrifuged at 12000r (high speed) and 4°C for 20 minutes, and the supernatant was removed. RBCMs can be obtained through the above operation. Finally, the RBCMs precipitate was rinsed with 1×PBS and the above process was repeated. The obtained RBCMs can be temporarily stored at 4°C.

[0057] 2.2 Preparation of ROCMCBs and CMCBs

[0058] 1) GCEs pretreatment

[0059] Before electrode modification, the bare GCEs were polished to a mirror finish using 1.0, 0.3, and 0.05 μm alumina powders. After each polishing, the GCEs were ultrasonically treated in dilute nitric acid, ethanol, and double distilled water for 3 min to remove the materials adsorbed on the surface of the GCEs. Finally, the GCEs were dried in a nitrogen atmosphere.

[0060] 2) Preparation of SABG / GCEs

[0061] Dilute 10 μL of secondary antibody (anti-rabbit IgG secondary antibody conjugated to gold) with 0.5% BSA at a ratio of 20:1, and then add 5 μL of 25% glutaraldehyde to prepare SABG. After thorough mixing, drop 8 μL SABG on the surface of GCEs and dry at room temperature. Glutaraldehyde helps the secondary antibody to firmly attach to the surface of GCEs.

[0062] 3) Preparation of CIPA / RBCMs

[0063] CIPA was diluted with 1% BSA at a ratio of 50:1, 100 μL of the diluted CIPA was mixed with 100 μL of the extracted RBCMs, and placed at 37°C for 1 hour to allow CIPA to fully bind to the CD47 receptor on the surface of RBCMs. After adding 500 μL of 0.1% BSA (washing buffer), it was placed in a centrifuge at 12000r, centrifuged at 4°C for 15 minutes, the supernatant was discarded, and the obtained CIPA / RBCMs were resuspended with 50 μL of PBS.

[0064] 4) Preparation of ROCMCBs

[0065] 8 μL of CIPA / RBCMs was dropped on SABG / GCEs, left at room temperature for 2 hours, and rinsed with PBS three times to wash away the unbound cell membrane. Due to the immunoaffinity between antibodies, RBCMs were positively coated on the sensor surface in the right-outward direction, and ROCMCBs were successfully prepared.

[0066] 5) Preparation of CMCBs

[0067] Directly take 8 μL of RBCMs and drop them on GCEs, dry them at room temperature, and rinse them three times with PBS to wash away the unbound cell membrane to obtain CMCBs.

[0068] 2.3 Characterization of ROCMCBs

[0069] 1) Acetylcholinesterase on the cell membrane

[0070] To demonstrate that acetylcholinesterase is anchored on RBCMs, AChE was stained using a specific primary antibody (acetylcholinesterase monoclonal antibody (HR2), MA3-042) and a secondary antibody compatible with immunofluorescence analysis (goat anti-rabbit IgG H&L / AF488 antibody), and the samples were incubated with antibodies for 2 hours and 40 minutes at room temperature, respectively. After treatment, RBCMs were washed three times with PBS. Then, the samples were incubated in DiI staining reagent for 15 minutes and then washed three times with PBS to stain RBCMs.

[0071] 2) Contact angle test

[0072] Place GCEs, SABG / GCEs, RORCMBs, and CMCBs respectively. Inject ultrapure water droplets electrically, raise the sample platform to receive the droplets, and make the solid sample contact the droplets. Take photos and analyze to obtain the results.

[0073] 3) Fourier transform infrared spectroscopy (FT-IR)

[0074] Take 1 ml of RBCMs prepared in 2.1, 1 ml of SABG prepared in 2.2 and 1 ml of RBCMs / CIPA / SABG solution respectively, place them in a freeze dryer and dry them. Add 100 mg of potassium bromide (KBr) to the obtained freeze-dried powders, mix and grind them, place them under an infrared lamp to fully dry the powders, and then take an appropriate amount of powder and place them in a tableting mold for tableting (pressure 10 MPa, pressurization 1 min). Finally, place the sample in an FT-IR detector and observe at 4000-1000 cm -1 Detection within the wavenumber range was performed to analyze the changes in functional groups during the synthesis process and determine whether RORCMBs were successfully prepared.

[0075] 4) X-ray Photoelectron Spectroscopy (XPS)

[0076] Appropriate amounts of RBCMs, SABG and RBCMs / CIPA / SABG powders were fixed on the double-sided tape on the indium foil. After pressing, the valence distribution and content of the five elements C, O, N, S and P were detected in a vacuum environment to analyze the constituent elements of RORCMBs.

[0077] 5) Atomic force microscopy (AFM)

[0078] SABG solution and RBCMs / CIPA / SABG solution were drop-coated on mica sheets and imaged using non-contact mode to characterize the thickness and roughness of the biosensor surface.

[0079] 6) Scanning electron microscopy (SEM)

[0080] Take appropriate amount of RBCMs, SABG and SABG / CIPA / RBCMs powders and evenly coat them on the surface of the conductive glue. Use an ear bulb to blow away the residual powder. Spray gold to make the sample conductive. Observe and photograph the surface morphology of the sample under SEM, and scan the EDS energy spectrum to determine its elemental composition.

[0081] 2.4 Film orientation experiments of ROCMCBs and CMCBs

[0082] First, according to the preparation method in Section 2.2, two complete sets of RORCMBs and CMCBs were assembled with DiI-labeled RBCMs, each containing at least two identical sensors. Subsequently, CIPA and CEPA solutions were drop-coated on the surface of each set of sensors. The sensors were incubated at room temperature for 2 hours and then rinsed three times with PBS. Finally, secondary antibodies labeled with Abflo405 blue fluorophore were added to each set of sensors. To prevent potential fluorescence quenching, the sensors were incubated in the dark for 30 minutes. Then, they were rinsed three times with PBS to remove residual traces. The fluorescence on the sensor surface was observed using a laser confocal microscope and the fluorescence intensity was quantified using Image J. The experiment was repeated three times.

[0083] First, RBCM was labeled with the membrane dye DiI in red. Next, ROCMCBs and CMCBs were assembled according to the preparation method in Section 2.2, at least two of each type. 8 μL of CIPA and CEPA solution were applied to the surface of each set of sensors, respectively, and then incubated at room temperature for 2 hours and washed 3 times with PBS. Subsequently, 8 μL of Alexa Fluor405-labeled secondary antibody was drop-coated on the sensor surface and incubated in the dark for 30 minutes to prevent fluorescence quenching. Next, PBS was washed three times to remove residues. Finally, the fluorescence on the sensor surface was observed using a confocal laser scanning microscope (CLSM), and the fluorescence intensity was quantified using ImageJ. The process was repeated three times to ensure reproducibility.

[0084] 2.5 Electrochemical biosensor detection procedure

[0085] In the presence of 5.0mmol / L [Fe(CN) 6 ] 3- / 4- EIS was performed in 0.1 mol / L PBS (pH 7.4) with 0.1 mol / L KCl by applying different frequencies of 10 Hz to 1,000,000 Hz and 50 mV amplitude at 0 V constant potential. CV was performed in the same solution with a scanning potential range of -0.2 to 0.6 V. All experiments used 3 mL of electrolyte solution (pH 7.4). The optimal concentration of substrate ATCl, 5.0 mmol L -1Due to the redox reaction between the substrate and AChE, a clear oxidation peak at a potential of 650 mV was measured using the DPV method. Based on the reaction equation of ATCl as shown in Formula (2-1), ATCl is initially hydrolyzed by AChE into thiocholine (TCh) and acetic acid (HA). Subsequently, according to Formula (2-2), TCh can be further oxidized to dithiocholine on the electrode surface.

[0086]

[0087] 2TCh(rei)→TCh(ox)+2H + +2e - (2-2)

[0088] 2.6 Condition Optimization of ROCMCBs

[0089] 1) RBCMs concentration

[0090] The DPV signal responses of AChE and substrate ATC1 on RBCMs were investigated at RBCMs concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL and 3 mg / mL (n=3), and the peak currents were compared to obtain the optimal RBCMs concentration.

[0091] 2) Huperzine A incubation time

[0092] The ΔDPV signal response of RORCMBs was investigated when the incubation time of Huperzine A was 5min, 10min, 15min, 20min, 25min and 30min (n=3). 0 -I 1 , where I 0 is the peak current of ROCMCBs before incubation with Huperzine A, I 1 is the peak current on the same sensor after incubation with Huperzine A.

[0093] 3) Temperature

[0094] The peak currents of RORCMBs at 4, 25, 37, and 50 °C were investigated to obtain the optimal temperature.

[0095] 4) pH

[0096] The peak currents of RORCMBs in electrolytes with pH values ​​of 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 were investigated to obtain the optimal pH.

[0097] 2.7 Analytical performance of ROCMCBs

[0098] 1) Using the drug Huperzine A as a model compound, the relationship between the inhibition rate and the concentration of anti-AD drugs was evaluated. The concentration of Huperzine A was 4.1×10 -6 , 8.2×10 -7 4.1×10 -7 , 8.2×10 -8 4.1×10 -8 4.1×10 -9 4.1×10 -10 4.1×10 -11 4.1×10 -12 mol / L.

[0099] 2) The typical current-time response curve of ROCMCBs was obtained by continuously adding substrate (ATCl) to the stirred cell, i.e., preparing a 1.0 mol / L ATCl stock solution and adding ATCl once every 30 seconds in 1.0 mmol / L PBS.

[0100] 2.8 Methodological Review of ROCMCBs

[0101] 1) Selectivity

[0102] In order to evaluate the specific recognition of ROCMCBs against anti-AChE drugs, galantamine, oxytetracycline, furacillin and indomethacin were used as control groups. The experimental results of the above control groups were compared with those of huperzine A. Each group was repeated three times.

[0103] 2) Repeatability

[0104] The same method was used to prepare ROCMCBs and CMCBs groups, each containing 5 ROCMCBs and 5 CMCBs. The peak current of the two groups of sensors to ATCl was measured by DPV method and the RSD value was calculated. The experiments were repeated three times for both groups.

[0105] 3) Stability

[0106] The inhibitory responses of RORCMBs and CMCBs to 4.1 μmol / L huperzine A were measured daily. The 7-day DPV results were compared, and the experiments were repeated three times for both groups.

[0107] 2.9 Screening and validation experiments of potential inhibitors of ROCMCBs

[0108] Previous studies have found eight active ingredients in traditional Chinese medicine that may have an inhibitory effect on AChE: berberine, baicalin, gardenia glycoside, gastrodin, rhynchophylline, ligusticum chuanxiong lactone A, tanshinone IIA, and echinacoside. The DPV method was used to test the current inhibition of AChE by drugs at different concentrations to obtain the inhibition rate of the drug on AChE activity and calculate its IC50 value.

[0109] 2.10 Molecular docking experiment

[0110] To investigate the interaction between TCM and AChE, molecular docking was performed using SYBYL-X 2.0. The AChE structure used for docking was from the Protein Data Bank (PDB, PDB ID, 1VOT). During protein preparation, cocrystal ligands and water molecules were removed, and H atoms were added. The side chains were fixed, and the force field was AMBER7 FF99. A sketch of the chemical structure of the drug molecule was drawn and incorporated into the Tripos force field, and the Gasteiger-Huckel charge type was specified for energy minimization. Surflex-Dock was used for molecular docking studies. The Surflex-Dock score (total score) represents the binding affinity of the drug. Finally, the binding interaction between the drug ligand and the AChE protein was visualized using PyMOL software.

[0111] 3 Statistical analysis

[0112] The experimental data were expressed as the mean value of three repeated experiments plus or minus the standard deviation (Mean±SD). All experimental data were processed, statistically analyzed and graphically processed using Origin2024b, GraphPad Prism 9.5 and Image J software. The differences between the two groups were compared using the t test, and more than two groups were analyzed using One-Way ANOVA. In the statistical results, P<0.05 was considered statistically significant. (*P<0.05, **P<0.01, and ***P<0.001).

[0113] 4 Results and discussion

[0114] 4.1 Preparation and characterization of ROCMCBs

[0115] 1) Acetylcholinesterase (AChE) is present on RBCMs

[0116] To prove that AChE is anchored on RBCMs, immunofluorescence experiments of AChE and RBCMs were performed by CLSM. Figure 2 , RBCMs were labeled with red fluorescence, while AChE was labeled with green fluorescence. And the red and green signals had obvious co-localization signals, which confirmed that AChE was anchored on RBCMs. RORCMBs prepared with RBCMs can be used for subsequent screening of potential components in traditional Chinese medicine that inhibit AChE activity.

[0117] 2) Antifouling performance of RORCMBs

[0118] Figure 3 The CLSM images in Figure 4 show that a large amount of BSA-FITC accumulated on the surface of bare GCEs and SABG / GCEs, demonstrating that the nonspecific adsorption of proteins was very significant. In contrast, very little fluorescence was observed on ROCMCBs after modification of RBCMs on the SABG surface and subsequent treatment with BSA-FITC. Quantitative analysis showed that the fluorescence intensity on ROCMCBs was significantly different from that on bare GCEs and SABG / GCEs. In addition, the fluorescence intensity of the control group (BSA-FITC-labeled CMCBs) was similar to that of ROCMCBs, highlighting that the RBCMs coating has effective protein repellency properties.

[0119] 3) Water contact angle

[0120] The antibacterial efficacy of RBCM coatings depends mainly on three factors: steric hindrance of glycoproteins, abundant amphiphilic head groups in the phospholipid bilayer, and excellent hydrophilicity at the interface. Fundamentally, building an antifouling surface requires creating a hydrophilic interface. To demonstrate the strong hydrophilicity of the RBCM-coated sensor surface, we measured the water contact angles of sensors modified with various materials. Figure 4 As shown, the initial angle of bare GCEs was 64.6°. After modification with SABG, the contact angle dropped to 46.8°. In addition, after coating with RBCMs, the water contact angles of ROCMCBs and CMCBs dropped to 22.1° and 28.6°, respectively. These findings indicate that ROCMCBs lay a solid foundation for the development of antifouling sensing interfaces. At the same time, wettability is also a key parameter for improving the biocompatibility of materials. It plays an important role in determining how effectively a material interacts with biological components such as cells and tissues, so RBCMs-modified interfaces can improve the biocompatibility of electrodes and enhance the performance of sensors.

[0121] 4) Fourier transform infrared spectroscopy (FT-IR)

[0122] In order to prove that ROCMCB was successfully constructed step by step, the FT-IR spectra of SABG (curve a), RBCM / CIPA (curve b), and RBCM / CIPA / SABG (curve c) were tested, as shown in Figure 2. Figure 5 As shown, 1600-1700cm -1 and 1510-1580cm -1 The peaks in the range of 2800-3000 cm-1 represent the amide I and amide II bands, respectively. On the other hand, curves b and c fully retain the 2800-3000 cm-1 -1 and 1080-1085cm -1 The peaks representing lipids (C-Hs) and phospholipids (CO-Cs) in the range are shown in Table 1. The characteristic spectral properties in RBCMs are effectively preserved.

[0123] 5) Energy dispersive X-ray spectroscopy (EDS) elemental images

[0124] EDS spectrum image Figure 6 It is worth noting that only four elements, C, O, N, and P, are evenly distributed on the surface of RBCMs, while SABG also contains S. The even distribution of C, O, N, S, and P in ROCMCBs indicates that the modification of RBCMs on SABG is successful.

[0125] 6) Atomic force microscopy (AFM) images

[0126] Figure 7 Atomic force microscopy images of ROCMCBs and SABG / GCEs are shown. It was observed that the average thickness of the SABG layer was about 2nm, and the thickness of this layer increased by about 10nm after the addition of the RBCMs layer. Measuring the roughness of the coating showed that the RBCMs were successfully encapsulated on the sensor surface.

[0127] 7) X-ray Photoelectron Spectroscopy (XPS)

[0128] XPS analysis was used to characterize the chemical compositions of surface materials such as SABG, CIPA / RBCMs and ROCMCBs. Figure 8 As shown, ROCMCBs retained the characteristic peaks of SABG and CIPA / RBCMs, indicating that SABG successfully bound to CIPA, and next, RBCMs could bind to ROCMCBs.

[0129] 4.2 Film orientation experiments of ROCMCBs and CMCBs

[0130] Cluster of differentiation 47 (CD47) is a transmembrane receptor consisting of a heavily glycosylated N-terminal IgV-like extracellular domain (ECD) and a small C-terminal domain (CTD). Therefore, the CD47 receptor is a suitable marker to demarcate the orientation of RBCMs on ROCMCBs. Fig. 9 Shown is a schematic diagram of two series of immunofluorescence assays designed to demonstrate the directed assembly of RBCMs onto ROCMCBs. Fig.10 The CLSM image of A shows that after the ROCMCBs were coated with RBCMs and SABG, only the extracellular part of the CD47 receptor was exposed on the sensor surface. Therefore, there was no binding between these receptors and CIPA. Subsequent results confirmed that these receptors could not further bind to the secondary antibody to produce blue fluorescence. On the contrary, Fig.10B shows that the control CMCBs retained both the intracellular and extracellular parts of the CD47 receptor on their surface, thus effectively binding to CIPA and CEPA, resulting in detectable blue fluorescence after staining with secondary antibodies. The difference in fluorescence intensity clearly indicates that the positive directional coating of RBCMs on ROCMCBs was successfully achieved.

[0131] 4.3 Experimental study on the superiority of ROCMCBs cell membrane positive coating

[0132] 1) SEM

[0133] Fig.11 The SEM analysis shown shows that the uncoated electrode surface has micro scratches, while the SABG / GCEs surface exhibits a complete and uniform SABG coating. Compared with CMCBs, the surface of ROCMCBs achieves a flatter coverage of RBCMs. These observations emphasize the directional and stable deposition of RBCMs in ROCMCBs.

[0134] 2) EIS

[0135] In addition, EIS measurements verified the efficacy of the RBCMs forward oriented coating method to produce bioelectronic sensors. To this end, GCEs were comparatively evaluated using various electrode preparation techniques. Fig.12 As shown, it is noteworthy that a high charge transfer resistance up to 1200 ohms was achieved by the immunoaffinity-based RBCMs forward directed coating approach, thus proving to be the most efficient approach compared to methods such as direct droplet casting.

[0136] 3) DPV

[0137] To further demonstrate the superiority of the directional coating method for RBCMs, ROCMCBs and CMCBs were prepared simultaneously. Their electrochemical behaviors were studied by DPV in 5.0mmol / LATCl. It is worth noting that Fig.13 It was shown that under the same experimental conditions, the current response of ROCMCBs was significantly higher than that of CMCBs. This performance improvement was mainly attributed to the use of an immunoaffinity-based forward-oriented coating method for RBCMs, which prevented the RBCMs from detaching from the electrode surface and thus improved the stability of ROCMCBs. Typically, RBCMs are attached to gold electrodes through electrostatic adsorption or covalent interactions (such as gold-sulfur bonds). However, the surface of GCEs lacks these interaction capabilities and cannot maintain the RBCMs coating during application. If RBCMs are simply drop-coated on GCEs, perturbations from electrical stimulation may result in their shedding and low efficiency.

[0138] 4.4 Electrochemical behavior of RORCMBs

[0139] like Fig.14 As shown in the figure, the stepwise construction process of RORCMBs was compared by EIS and CV. These experiments were carried out in the presence of 0.1 mol / L KCl and 5 mmol / L K 3 [Fe(CN) 6 ] 3- / 4- It is carried out in an electrolyte composed of. Fig.14 A shows the peak current graph of bare GCEs, SABG / GCEs, and ROCMCBs. With the modification of SABG and CIPA / RBCMs, the peak current of the sensor gradually decreased and the peak reversibility became worse. Fig.14 B shows the typical Nyquist plots of the three sensors. The charge transfer resistance (Ret) can be obtained by calculating the semicircle diameter, thereby comparing the changes in interfacial impedance of different modified sensors during the preparation process. In curve a, bare GCEs showed a reversible redox peak current during the ferrocyanidation reaction, with a particularly low Ret, which was almost linear in the EIS analysis. In contrast, when SABG was immobilized on the surface of GCEs using glutaraldehyde, an increase in impedance coupling was observed in curve b due to the low conductivity of SABG. After coating with CIPA / RBCMs, the impedance further increased due to the immunoaffinity binding of the primary and secondary antibodies, and the semicircle in curve c was enhanced. The CV and EIS results corresponded, indicating that RORBCMs were gradually constructed successfully. Subsequently Fig.14 The DPV experiment of C recorded the reaction in PBS buffer solution (pH 7.4, containing 5.0mmol / L ATCl). The image shows a strong oxidation peak of AChE at 650mV (curve a). At the same time, AChE has no obvious current response in the electrolyte without substrate ATCl (curve b).

[0140] Next, the electrochemical behaviors of ROCMCBs in the presence and absence of 5.0 mmol / LATCl were evaluated. Fig.15 The CV values ​​of different biosensors are shown. The results show that in the absence of 5.0mmol / L ATCl, no redox peaks were observed for the three biosensors. However, in the presence of 5.0mmol / L ATCl, the CV responses of ROCMCBs showed an irreversible oxidation peak at 650mV, which was in sharp contrast to bare GCEs and SABG / GCEs. The observed oxidation peak can be attributed to the electrochemical oxidation of electroactive TCh, which was produced by the hydrolysis of ATCl catalyzed by the immobilized acetylcholinesterase. Subsequently, the effect of scan rate on ROCMCB was studied in 0.1mol / L PBS solution containing 5.0mmol / L ATCl, see Fig.16. The CV analysis results show that the oxidation of TCh is an irreversible electrode process, as only one oxidation peak is observed. In addition, as the scan rate changes from 10mV / s to 100mV / s, the oxidation peak current of TCh also increases, indicating that the oxidation of ATCl on ROCMCB is a typical diffusion-controlled process. The figure confirms that the TCh oxidation peak current (Ipa) is proportional to the square root of the scan rate (v 1 / 2 ), indicating that this is a diffusion-controlled oxidation process (R 2 =0.9934, n=3).

[0141] 4.5 Optimization Experiment

[0142] 1) Cell membrane concentration

[0143] An important aspect of biosensor preparation is the content of AChE on RBCMs. The effect of RBCMs concentration (from 0.5 mg / mL to 3 mg / mL) was studied in 0.1 mol / L PBS (pH 7.4) containing 5.0 mmol / L ATCl. Fig.17 As shown in A and B, the current response increases with the increase of RBCMs concentration and reaches a maximum value at about 2 mg / ml. However, with the further increase of RBCMs concentration, the current response decreases significantly. This phenomenon may be due to the increase in resistance of the electrochemical process caused by the thickening of the RBCMs layer. Therefore, 2 mg / ml was selected as the optimal concentration of RBCMs.

[0144] 2) Huperzine A incubation time

[0145] The effect of inhibition time on the biosensor response is one of the most important parameters. -6 The inhibitory effects of different incubation times (5-30 minutes) on AChE activity were studied in 1 mol / L Huperzine A. 0 is the peak current of ATCl before ROCMCBs were incubated with Huperzine A, I 1 is the peak current of ATC1 on the same sensor after incubation with Huperzine A. Fig.17 C and D show that the binding of AChE to the active target site reached saturation after 25 min. Therefore, 25 min was selected as the optimal incubation time.

[0146] 3) pH

[0147] like Fig.17 As shown in E and F, in the pH range of 5.5 to 8.0, the peak current was the largest at pH 7.4. Therefore, a solution pH of 7.4 was selected for subsequent experiments.

[0148] 4) Temperature

[0149] The peak currents of RORCMBs were tested at 4, 25, 37, and 50°C, and the highest peak current was recorded at 25°C. Fig.17 G and H. Therefore, a room temperature of 25°C was selected for subsequent experiments.

[0150] 4.6 Electrochemical response of RORCMBs to Huperzine A

[0151] Using the drug Huperzine A as a model compound, the relationship between the inhibition rate and the concentration of anti-AD drugs was evaluated. Fig.18 As shown in Figure 2, with the increase of Huperzine A concentration in ATCl solution, the oxidation current of AChE decreased. Under the optimized experimental conditions, at 4.2×10 -6 -4.2×10 -12 mol / L, the inhibitory effect of Huperzine A on AChE is proportional to its concentration. The linear equation representing this relationship is inhibition rate (%) = 10.32logC + 136.99 (%), and the correlation coefficient (R 2 ) was 0.9986. The limit of detection (LOD) was approximately 0.41 pmol / L.

[0152] As far as we know, there is no way to utilize the AChE enriched on RBCMs to replace pure AChE extracts for anti-AD drug screening. However, compared with previously reported AChE-related sensing platforms, this strategy showed comparable or even higher sensitivity. These results indicate that the designed ROCMCBs are capable of detecting low-abundance AChE. Combined with the advantages of RBCMs being easily accessible and cost-effective, this method has broad application prospects in ultrasensitive biomedical assays.

[0153] 4.7 Current-time response of ROCMCBs to ATCl

[0154] The typical current-time response curves of ROCMCBs were obtained by continuously adding substrate (ATCl) to the stirred cell. Fig.19 As shown, the addition of ATCl to ROCMCBs produces a fast and sensitive current response, indicating that the reaction product (ACh) diffuses rapidly. The correlation coefficient is 0.9799. In order to determine the enzyme activity of AChE when reacting with ATCl as a substrate, the Michaelis-Menten constant (Km) was calculated according to formula (4). Km is 1.995mmol / L. A small Km means that AChE immobilized on ROCMCBs can maintain its catalytic activity and show greater affinity for ATCl.

[0155] 4.8 Methodological Review of RORCMBs

[0156] The selective recognition ability of ROCMCBs is crucial for practical applications. To evaluate the selectivity of ROCMCBs, five drugs were used, namely, huperzine A, galantamine, oxytetracycline, nitrofurazone, and indomethacin. Fig. 20 As shown in A, only huperzine A and galantamine, which target AChE, have significant inhibition rates. However, ROCMCBs have lower inhibition rates against the other three drugs. The results confirmed the selectivity of ROCMCBs. Subsequently, ROCMCBs and CMCBs groups were prepared using the same method, each containing 5 ROCMCBs and 5 CMCBs. Both groups were subjected to three experiments. Fig. 20 As shown in Figure B, the relative standard deviation of ROCMCBs was 3.39%, while that of CMCBSs was 7.07%. The results showed that the prepared ROCMCBs had acceptable reproducibility. Then, to evaluate the storage stability of RORCMBs, the inhibition responses of RORCMBs and CMCBs to 4.1 μmol / L Huperzine A were measured every day. The sensors were stored at 4 °C when not in use. Fig. 20 As shown in Figure C, after 7 days of storage, the average inhibition rate of RORCMBs remained at about 72% of the initial value. But surprisingly, the average inhibition rate of CMCBs dropped to about 27% of the initial value. The results show that ROCMCBs have good storage stability.

[0157] 4.9 Screening and validation experiments of potential inhibitors of ROCMCBs

[0158] This work determined whether the proposed RORCMBs are suitable for rapid screening of potential anti-AChE active ingredients in drugs. The inhibitory effects of eight traditional Chinese medicine active ingredients at different concentrations on AChE current were observed using the DPV test, and their half-maximum inhibitory concentration (IC50) values ​​were calculated. Fig.21 and Fig. 22 The inhibitory effects of berberine, baicalin, gardenia glycoside, gastrodin, rhynchophylline, and ligusticum chuanxiong lactone A on AChE increased with the increase of drug concentration, and their IC50 values ​​were 4.7, 12.2, 8.3, 3.2, 86.6, and 66.3 μmol / L, respectively. Under the same conditions, tanshinone IIA and echinacoside had no obvious inhibitory effects on AChE, and no significant IC50 values ​​were found. The above results indicate that RORCMBs can be used to screen potential active ingredients of traditional Chinese medicine for inhibiting AChE activity.

[0159] 4.10 Molecular docking experiments of potential inhibitors with AChE

[0160] In order to study the interaction mode between the identified compounds and AChE, 8 traditional Chinese medicine compounds were docked with AChE using the Surflex-Dock mode of SYBYL-X 2.0. The screened compounds and the positive drug Huperzine A were docked with the active site of AChE (PDB ID: 1VOT). Fig.23 AF shows the simulated binding of berberine, baicalin, gardenia glycoside, gastrodin, rhynchophylline and ligusticum truncatum lactone A. The docking results show that they have similar activities to the binding pocket of huperzine A. In addition, Fig.23 GH shows the simulated binding of tanshinone IIA and echinacoside. These docking results showed that berberine, baicalin, gardenia glycoside, gastrodin, rhynchophylline and ligusticum bisporum A were identified as selective AChE inhibitors.

[0161] 5. Summary

[0162] In this chapter, a pioneering method for positive coating of erythrocytes on electrochemical biosensors using immunoaffinity was successfully prepared, which can screen active compounds in traditional Chinese medicine. The extracellular domain of CD47 receptor was used for the positive orientation of RBCM. The experimental results showed that the right-lateral oriented RBCMs coating based on immunoaffinity not only maintained the stability of the coating but also retained the activity of the peripheral membrane-anchored protein tetramer. Therefore, six potential anti-AD active ingredients including berberine, baicalin, jasmine, gastrodin, limonene lactone A and rhynchophylline were screened based on AChE activity using this method. The screening results were in good agreement with the molecular docking simulation results, indicating the effectiveness of this method in accurately monitoring the AChE activity inhibited by traditional Chinese medicine. In summary, the directional coating of erythrocytes using immunoaffinity is an effective technology for precise fixation in electrochemical screening of traditional Chinese medicine. This innovative method not only facilitates the exploration of cell interactions but also has great potential in accelerating the drug discovery process.

Claims

1. A method for preparing a red blood cell membrane positive coating electrochemical biosensor, characterized in that: The method comprises the following steps: (1) GCEs pretreatment: GCEs are taken and polished to a mirror effect; (2) Preparation of SABG / GCEs: dilute the gold-conjugated anti-rabbit IgG secondary antibody with a BSA solution at a ratio of (10-40):1, then add glutaraldehyde, and mix thoroughly to obtain SABG; drop SABG on the surface of GCEs and dry at room temperature to obtain SABG / GCEs; (3) Preparation of CIPA / RBCMs: CIPA solution and RBCMs solution were fully mixed and allowed to stand at 36.5-37.5°C for 0.5-2 hours to allow CIPA to fully bind to the CD47 receptor on the surface of RBCMs. BSA solution was then added and placed in a centrifuge for centrifugation at 11,000-13,000 r / min and 4°C for 10-20 minutes. The supernatant was discarded and the CIPA / RBCMs were resuspended in PBS. (4) Preparation of ROCMCBs: The CIPA / RBCMs prepared in step (3) were dropped onto the SABG / GCEs prepared in step (2), and allowed to stand at room temperature for 1.5 to 2.5 hours. The mixture was rinsed with PBS for 1 to 5 times to remove the unbound cell membrane, thereby obtaining the red blood cell membrane positively coated electrochemical biosensor ROCMCBs.

2. The method for preparing the red blood cell membrane forward coated electrochemical biosensor according to claim 1, characterized in that: The specific method of pre-treating the GCEs in step (1) is as follows: the exposed GCEs are polished with 1.0, 0.3 and 0.05 μm alumina powders in sequence, and after each polishing, the GCEs are ultrasonically treated in dilute nitric acid, ethanol and double distilled water for 1 to 5 minutes to wash away the alumina powder adsorbed on the surface of the GCEs, and then the GCEs are dried in a nitrogen environment.

3. The method for preparing the red blood cell membrane forward coated electrochemical biosensor according to claim 1, characterized in that: The concentration of the BSA solution in step (2) is 0.3-0.8%.

4. The method for preparing the red blood cell membrane forward coated electrochemical biosensor according to claim 1, characterized in that: The amount of SABG dropped onto the surface of GCEs in step (2) is 5 to 10 μL.

5. The method for preparing the red blood cell membrane forward coated electrochemical biosensor according to claim 1, characterized in that: The CIPA solution described in step (3) is prepared by diluting CIPA with 0.8-1.5% BSA in a multiple of (25-100):

1.

6. The method for preparing the red blood cell membrane forward coated electrochemical biosensor according to claim 1, characterized in that: The RBCMs solution described in step (3) is prepared as follows: fresh blood is drawn and injected into a centrifuge tube containing an anticoagulant, the whole blood is centrifuged at 4°C to separate red blood cells and plasma by low-speed centrifugation, and then the precipitate is collected and washed three times with pH7.4 PBS precooled at 4°C to obtain pure red blood cells, the washed red blood cells are completely suspended in pH7.4 PBS and placed in a refrigerator at 4°C. Due to the osmotic pressure acting on the plasma membrane, the red blood cells are fully expanded and hemolyzed, and then the suspension is centrifuged at 11000-13000r and 4°C for 15-25 minutes, and the supernatant is removed to obtain RBCMs.

7. An erythrocyte membrane forward-coated electrochemical biosensor obtained according to the preparation method according to any one of claims 1 to 6.

8. Use of the erythrocyte membrane forward coating electrochemical biosensor according to claim 7 in screening potential active ingredients against Alzheimer's disease.

9. The use according to claim 8, characterized in that: The active ingredients are effective parts of traditional Chinese medicine or monomers of traditional Chinese medicine.

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