Erythrocyte membrane positive coating electrochemical biosensor and its preparation method and application

Abstract

The present invention discloses a red blood cell membrane positively coated electrochemical biosensor (ROCMCB), its preparation method, and application, belonging to the field of biosensor technology. ROCMCBs are based on antibodies modified on the surface of the electrochemical biosensor by specifically binding the extracellular binding region of the CD47 receptor on the cell membrane. Through immunoaffinity interactions between the antibodies, positive assembly of red blood cell membranes (RBCMs) on the surface of glassy carbon electrodes (GCEs) is achieved. The positive orientation enables the cell membrane to bind more firmly to the electrochemical biosensor and maximizes the exposure of the conformation and catalytic activity of the peripheral membrane-anchored protein AChE, enabling accurate measurement of the biological activity of acetylcholinesterase (AChE), thereby serving as a platform for anti-AD drug screening.

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 an erythrocyte membrane positively coated electrochemical biosensor (ROCMCBs) and a preparation method and application thereof. Background Art

[0002] The effective ingredients of traditional Chinese medicine (TCM) are structurally diverse, with enormous chemical diversity and multi-target characteristics. Screening for effective ingredients in complex TCMs is a bridge between the efficacy and mechanism of action of TCMs. Existing methods for screening potential active ingredients in TCMs mainly include molecular docking technology, high-throughput screening technology, cell membrane technology, etc. They utilize 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 TCM, the field of TCM still urgently needs an ideal strategy.

[0003] Designing cell membrane-based natural product screening methods, leveraging the cell membrane's drug-specific targeting properties, has become a research hotspot. In recent decades, numerous methods for screening and isolating target compounds have been proposed and refined. However, long analysis times, poor peak shape, and short lifetimes have limited the widespread application of these methods. Therefore, improving analytical methods that suffer from long analysis times and poor quality is crucial for developing more effective analytical methods for traditional Chinese medicines.

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

[0005] Acetylcholinesterase (AChE) is a serine protease with both aminopeptidase and carboxypeptidase activities. It is primarily present in the postsynaptic membrane of motor nerve end plates and in brain synapses. Its primary function is to hydrolyze the neurotransmitter acetylcholine, thereby terminating neurotransmission. Acetylcholinesterase inhibitors (AChE-I) are commonly used to improve cognitive function in patients with mild to moderate Alzheimer's disease (AD). These inhibitors increase ACh levels in neuronal synapses. To date, AChE-I remains the gold standard treatment for AD. Currently, the main inhibitors used clinically include donepezil (aricept), rivastine (exelon), galantamine (razadyne), and huperzine A. Huperzine A, a plant alkaloid isolated from Huperzia serrata, is a reversible and selective inhibitor of acetylcholinesterase with neuroprotective properties, and 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 precision and speed of physical transduction, biosensors provide a promising alternative tool for the quantification of target analytes in complex biological samples. Electrochemical biosensors not only have the characteristics of miniaturization and high selectivity of biosensors, but also embody the advantages of rapid and high sensitivity of electrochemical analysis. They have positive significance in drug design, drug testing, molecular screening and other aspects. AChE-based biosensors use the substrate thioacetylcholine to produce the product TCh under AChE catalysis. The concentration of TCh is proportional to the measured oxidation current. 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 universal detection principle for AChE inhibitor drugs.

[0007] Traditional Chinese medicine (TCM) has multiple components, pathways, and targets. In recent years, the design of cell membrane-based natural product screening methods, leveraging the biocompatibility and drug-specific targeting properties of cell membranes, has become a research hotspot. Cell membrane-coated electrochemical biosensors combine the miniaturization and high selectivity of biosensors with the rapidity and high sensitivity of electrochemical analysis, offering promising applications in drug design, drug testing, and molecular screening. However, practical applications of cell membrane-coated electrochemical biosensors still face several challenges. For example, the random orientation used in traditional methods can cause the cell membrane to detach from the biosensor surface, preventing drugs from effectively binding to specific sites. Furthermore, only correctly oriented cell membranes can maintain the natural conformation and function of membrane proteins or receptors, achieving optimal activity during drug screening. Therefore, the development of more robust and correctly oriented cell membrane-coated electrochemical biosensors is a key area of ​​current research. Summary of the Invention

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

[0009] To achieve the above objectives, the present invention designs and prepares a red blood cell membrane forward-coated electrochemical biosensor. The electrochemical biosensor is based on the extracellular binding region of the CD47 receptor on the cell membrane specifically bound to an antibody modified on the surface of the electrochemical biosensor. The forward assembly of red blood cell membranes (RBCMs) on the surface of glassy carbon electrodes (GCEs) is achieved through immunoaffinity interactions between the antibodies. The forward orientation enables the cell membrane to bind more firmly to the electrochemical biosensor and maximizes the exposure of the conformation and catalytic activity of the peripheral membrane-anchored protein AChE, thereby accurately measuring the biological activity of AChE, thereby serving as a platform for anti-AD drug screening.

[0010] Specifically, the technical solution for achieving the purpose of the present invention is as follows: a method for preparing a red blood cell membrane forward-coated electrochemical biosensor, the method comprising the following steps:

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

[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 thoroughly 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 the mixture was centrifuged 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 then rinsed with PBS 1 to 5 times to remove unbound cell membranes, thereby obtaining ROCMCBs, a red blood cell membrane-coated electrochemical biosensor.

[0015] Further preferably, in the method for preparing the red blood cell membrane forward-coated electrochemical biosensor as described above, the specific method of step (1) GCEs pretreatment is: polishing the exposed GCEs with 1.0, 0.3 and 0.05 μm alumina powder 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 onto the surface of GCEs in step (2) is 5 to 10 μL.

[0018] Further preferably, in the method for preparing the erythrocyte 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 at a ratio 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, the whole blood is centrifuged at low speed at 4°C to separate red blood cells and plasma, and then the precipitate is collected and washed three times with pH7.4 PBS pre-cooled 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.

[0020] In addition, the present invention also provides an erythrocyte membrane forward-coated electrochemical biosensor obtained according to the above-mentioned preparation method. Furthermore, the purpose of designing the erythrocyte membrane forward-coated electrochemical biosensor in the present invention is to explore its ability to screen for potential active ingredients against Alzheimer's disease (AD). Therefore, the present invention also provides the use of the above-mentioned erythrocyte membrane forward-coated electrochemical biosensor in screening for potential active ingredients against Alzheimer's disease. Further preferably, the active ingredient is an effective fraction or monomer of a 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 was anchored on the surface of RBCMs, which can 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 membrane coating and better membrane coating stability.

[0023] 2) Under 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 approximately 0.41 pmol / L. Interfering drug experiments demonstrated that ROCMCBs possessed good selective recognition capabilities. The relative standard deviation (RSD) of the DPV test results for different ROCMCBs (n=5) was 3.39%, indicating that the prepared ROCMCBs had acceptable reproducibility. After 7 days of storage, the average inhibition rate of RORCMBs remained at approximately 72% of the initial value, demonstrating that the ROCMCBs possessed good storage stability.

[0024] 3) Six potential anti-AD active ingredients were screened out, including berberine, baicalin, geniposide, gastrodin, ligusticum 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, respectively. The molecular docking results were consistent with the DPV results.

[0025] 4) The successful fabrication of a cell membrane-based electrochemical biosensor for forward coating has established a highly efficient and rapid drug screening platform. The application of this cell membrane-based electrochemical biosensor for forward coating has broadened the scope and methodologies for drug lead discovery. This innovative approach 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 active ingredients of 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 antibodies; (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] Figure 9 : Schematic diagram of two series of immunofluorescence assays.

[0035] Figure 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] Figure 11 : Scanning electron microscopy images of (a) bare GCEs, (b) SABG / GCEs, (c) CMCBs, and (d) RORCMCBs. Scale bar: 2 μm.

[0037] Figure 12 EIS images of different incubation methods, including bare GCE (a); RBCMs droplets incubated with GCE for 2 hours, then rinsed with PBS solution and dried under nitrogen (b); RBCMs droplets incubated with GCE overnight at 4°C in humid conditions to prevent 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 rinsed with PBS solution and dried under nitrogen (f).

[0038] Figure 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 the inhibition rate of DPV and current signals in 1 mol / L Huperzine A (b, d) (***P<0.001).

[0039] Figure 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] Figure 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] Figure 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] Figure 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] Figure 18 : 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] Figure 19 : It curve of ROCMCBs under an applied potential of 650 mV and continuous addition of ATCl under stirring. The inset is the calibration diagram for the determination of ATCl.

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

[0046] Figure 21 :(A) berberine, baicalin; (B) geniposide, gastrodin; (C) rhynchophylline, ligusticum lactone A; (D) tanshinone IIA, echinacoside concentration and RORCMBs inhibition rate (n=3).

[0047] Figure 22: DPV curves of the effects of different concentrations of (A) berberine; (B) baicalin; (C) geniposide; (D) gastrodin; (E) ligusticum lactone A; and (F) rhynchophylline on RORCMBs.

[0048] Figure 23 Docking diagram of (A) berberine, (B) baicalin, (C) geniposide, (D) gastrodin, (E) ligusticum lactone A, (F) rhynchophylline, (G) tanshinone IIA, and (H) echinacoside at the AChE active site. (PDB ID: 1VOT). DETAILED DESCRIPTION

[0049] In this embodiment, a red blood cell membrane positive coating electrochemical biosensor (ROCMCBs) was designed. Figure 1 As shown in the figure, the biological activity of AChE is accurately measured as a platform for anti-AD drug screening. The constructed ROCMCBs were used to determine the inhibition rate of 8 active ingredients of traditional Chinese medicine (berberine, baicalin, geniposide, gastrodin, rhynchophylline, ligusticum lactone A, tanshinone IIA and echinacoside) on AChE activity on the cell membrane, and the IC 50 The molecular docking of 8 compounds with AChE was performed using 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 is drawn and injected into a centrifuge tube filled with anticoagulant. The whole blood is centrifuged at 1000g (low speed) for 10 minutes at 4°C to separate red blood cells and plasma. The precipitate is then collected and washed three times with 1×PBS (pH 7.4) pre-cooled at 4°C to obtain pure red blood cells. The washed red blood cells are 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 are fully swollen and hemolyzed. The suspension is then centrifuged at 12000r (high speed) and 4°C for 20 minutes, and the supernatant is removed. RBCMs can be obtained through the above operations. Finally, the RBCMs precipitate is rinsed with 1×PBS and the above process is 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 powder. After each polishing step, the GCEs were ultrasonically treated for 3 minutes in dilute nitric acid, ethanol, and doubly distilled water, respectively, to remove any adsorbed materials. Finally, the GCEs were dried in a nitrogen atmosphere.

[0060] 2) Preparation of SABG / GCEs

[0061] Prepare SABG by diluting 10 μL of secondary antibody (gold-conjugated anti-rabbit IgG) with 0.5% BSA at a ratio of 20:1, then adding 5 μL of 25% glutaraldehyde. After thorough mixing, apply 8 μL of SABG to the GCE surface and allow to dry at room temperature. Glutaraldehyde helps the secondary antibody adhere firmly to the GCE surface.

[0062] 3) Preparation of CIPA / RBCMs

[0063] CIPA was diluted 50:1 with 1% BSA. 100 μL of the diluted CIPA was thoroughly mixed with 100 μL of extracted RBCMs and incubated at 37°C for 1 hour to allow CIPA to fully bind to the CD47 receptor on the surface of the RBCMs. 500 μL of 0.1% BSA (washing buffer) was added and the mixture was centrifuged at 12,000 rpm and 4°C for 15 minutes. The supernatant was discarded and the resulting CIPA / RBCMs were resuspended in 50 μL of PBS.

[0064] 4) Preparation of ROCMCBs

[0065] 8 μL of CIPA / RBCMs was dropped onto SABG / GCEs and allowed to stand at room temperature for 2 hours. The cells were then rinsed three times with PBS to remove any unbound cell membranes. Due to the immunoaffinity between the antibodies, the RBCMs were coated onto the sensor surface in a right-side-out orientation, effectively creating ROCMCBs.

[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 unbound cell membranes to obtain CMCBs.

[0068] 2.3 Characterization of ROCMCBs

[0069] 1) Acetylcholinesterase on the cell membrane

[0070] To demonstrate that acetylcholinesterase is anchored to 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). Samples were incubated with the antibodies for 2 hours and 40 minutes at room temperature, respectively. After treatment, the RBCMs were rinsed three times with PBS. The samples were then incubated in DiI staining reagent for 15 minutes and then rinsed three times with PBS to stain the RBCMs.

[0071] 2) Contact angle test

[0072] GCEs, SABG / GCEs, RORCMBs, and CMCBs were placed separately. Ultrapure water droplets were injected electrically, and the sample platform was raised to receive the droplet, allowing the solid sample to contact the droplet. The results were captured and analyzed.

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

[0074] Take 1 ml of the RBCMs prepared in 2.1, 1 ml of the SABG prepared in 2.2, and 1 ml of the RBCMs / CIPA / SABG solution, place them in a freeze dryer and dry them. Add 100 mg of potassium bromide (KBr) to the resulting freeze-dried powders, grind them, and place them under an infrared lamp to fully dry the powders. Then take an appropriate amount of powder and place it in a tableting mold for tableting (pressure 10 MPa, pressurization for 1 minute). Finally, place the sample in an FT-IR detector and observe the fluorescence intensity 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 the 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 double-sided tape on 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] Appropriate amounts of RBCMs, SABG, and SABG / CIPA / RBCMs powders were respectively evenly coated on the surface of the conductive adhesive. The residual powder was blown away with an ear bulb, and the sample was sprayed with gold to make it conductive. The surface morphology of the sample was observed and photographed under SEM, and the EDS energy spectrum was scanned to determine its elemental composition.

[0081] 2.4 Film orientation experiments of ROCMCBs and CMCBs

[0082] First, following the preparation method described in Section 2.2, two complete sets of RORCMBs and CMCBs were assembled using DiI-labeled RBCMs, each containing at least two identical sensors. Subsequently, CIPA and CEPA solutions were drop-coated on the surface of each sensor set. The sensors were incubated at room temperature for 2 hours and then rinsed three times with PBS. Finally, a secondary antibody labeled with the Abflo405 blue fluorophore was added to each sensor set. 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 any residual traces. 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, the RBCM was labeled red with the membrane dye DiI. Next, ROCMCBs and CMCBs were assembled separately according to the preparation method in Section 2.2, with at least two of each type. 8 μL of CIPA and CEPA solution were applied to the surface of each set of sensors, respectively, followed by incubation at room temperature for 2 hours and washing three times with PBS. Subsequently, 8 μL of Alexa Fluor 405-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, fluorescence on the sensor surface was observed using a confocal laser scanning microscope (CLSM), and the fluorescence intensity was quantified using ImageJ. This 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 AC impedance spectroscopy at different frequencies from 10 Hz to 1,000,000 Hz and 50 mV amplitude at a constant potential of 0 V. 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 Equation (2-1), ATCl is initially hydrolyzed by AChE to thiocholine (TCh) and acetic acid (HA). Subsequently, according to Equation (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 after incubation with Huperzine A for 5, 10, 15, 20, 25, and 30 minutes (n = 3). ΔDPV = I0 - I1, where I0 is the peak current of the RORCMBs before incubation with Huperzine A, and I1 is the peak current of 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 Huperzine A as a model compound, the relationship between inhibition rate and anti-AD drug concentration 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) By continuously adding substrate (ATCl) to the stirred cell, i.e., preparing a 1.0 mol / L ATCl stock solution in 1.0 mmol / L PBS, adding ATCl once every 30 seconds, the typical current-time response curves of ROCMCBs were obtained.

[0100] 2.8 Methodological Review of ROCMCBs

[0101] 1) Selectivity

[0102] In order to evaluate the specific recognition of ROCMCBs for anti-AChE drugs, galantamine, oxytetracycline, nitrofurazone 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] ROCMCBs and CMCBs were prepared using the same method, with each group containing five ROCMCBs and five CMCBs. The peak currents of the two sensor groups to ATC1 were measured using the DPV method, and the relative standard deviations (RSDs) were calculated. Three replicates were performed 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 results of the 7-day DPV 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 identified eight active ingredients from traditional Chinese medicine that may inhibit AChE: berberine, baicalin, geniposide, gastrodin, rhynchophylline, ligusticum lactone A, tanshinone IIA, and echinacoside. The DPV method was used to test the inhibitory effects of different concentrations of drugs on AChE current to obtain the inhibition rate of the drug on AChE activity and calculate its IC value. 50 value.

[0109] 2.10 Molecular docking experiment

[0110] In order to study the interaction between Chinese medicine 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 the protein preparation process, the cocrystallized 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 assigned 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] All data are presented as the mean ± SD of three replicates. Data processing, statistical analysis, and graphics were performed using Origin 2024b, GraphPad Prism 9.5, and Image J software. Differences between two groups were compared using the t-test, and more than two groups were analyzed using one-way ANOVA. 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) 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. The red and green signals clearly colocalized, confirming that AChE was anchored to the RBCMs. RORCMBs prepared using RBCMs can be used to subsequently screen for potential components in traditional Chinese medicine that inhibit AChE activity.

[0117] 2) Antifouling performance of RORCMBs

[0118] Figure 3The CLSM images in Figure 3 show that a large amount of BSA-FITC accumulates on the surfaces of bare GCEs and SABG / GCEs, demonstrating that nonspecific protein adsorption is highly pronounced. In contrast, minimal fluorescence is observed on ROCMCBs after modification of the SABG surface with RBCMs and subsequent treatment with BSA-FITC. Quantitative analysis reveals that the fluorescence intensity on ROCMCBs is significantly different from that on bare GCEs and SABG / GCEs. Furthermore, the fluorescence intensity of the control group (BSA-FITC-labeled CMCBs) is similar to that of the ROCMCBs, highlighting the effective protein-repelling properties of the RBCM coating.

[0119] 3) Water contact angle

[0120] The antibacterial efficacy of RBCM coatings is primarily due to 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. Therefore, 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 appearing 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 RBCMs were effectively preserved.

[0123] 5) Energy Dispersive X-ray Spectroscopy (EDS) Elemental Images

[0124] EDS spectrum image is shown in 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. The SABG layer was observed to have an average thickness of approximately 2 nm, and the addition of the RBCMs layer increased this thickness by approximately 10 nm. Measuring the coating's roughness indicated 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 composition 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 for demarcating the orientation of RBCMs on ROCMCBs. Figure 9 Schematic diagram showing two series of immunofluorescence assays designed to demonstrate the directed assembly of RBCMs onto ROCMCBs. Figure 10 The CLSM image in A shows that after the ROCMCBs were coated with RBCMs and SABG, only the extracellular portion 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. In contrast, Figure 10Figure B shows that the control CMCBs retained both the intracellular and extracellular portions of the CD47 receptor on their surface, allowing them to effectively bind to CIPA and CEPA, resulting in detectable blue fluorescence after staining with secondary antibodies. The difference in fluorescence intensity clearly demonstrates the successful positive-directed coating of RBCMs on ROCMCBs.

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

[0132] 1) SEM

[0133] Figure 11 The SEM analysis shown here reveals micro-scratches on the uncoated electrode surface, while the SABG / GCE surface exhibits a complete and uniform SABG coating. Compared to CMCBs, the surface of ROCMCBs achieves a flatter coverage of RBCMs. These observations highlight the directional and stable deposition of RBCMs in ROCMCBs.

[0134] 2) EIS

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

[0136] 3)DPV

[0137] To further demonstrate the superiority of the RBCMs forward oriented coating method, ROCMCBs and CMCBs were prepared simultaneously. Their electrochemical behaviors were studied by DPV in 5.0 mmol / LATCl. Figure 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-directed 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 RBCM coating during application. If the RBCMs are simply drop-coated on GCEs, the perturbation of electrical stimulation may cause them to fall off and have low efficiency.

[0138] 4.4 Electrochemical behavior of RORCMBs

[0139] like Figure 14 As shown, the stepwise construction process of RORCMBs was compared by EIS and CV. These experiments were carried out in a mol / L KCl and 5 mmol / L K3[Fe(CN)6] 3- / 4- It is carried out in an electrolyte composed of. Figure 14 A shows the peak current diagram 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. Figure 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, and the Ret was particularly low, 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 successfully constructed step by step. Subsequently Figure 14 The DPV experiment of C was recorded in PBS buffer solution (pH 7.4, containing 5.0 mmol / L ATC1). The image shows a strong oxidation peak of AChE at 650 mV (curve a). At the same time, AChE has no obvious current response in the electrolyte without the substrate ATC1 (curve b).

[0140] Next, the electrochemical behaviors of ROCMCBs in the presence and absence of 5.0 mmol / L ATCl were evaluated. Figure 15 The CV values ​​of different biosensors are shown. The results show that in the absence of 5.0 mmol / L ATCl, no redox peaks were observed for the three biosensors. However, in the presence of 5.0 mmol / L ATCl, the CV responses of ROCMCBs showed an irreversible oxidation peak at 650 mV, 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 is produced by the hydrolysis of ATCl catalyzed by immobilized acetylcholinesterase. Subsequently, the effect of scan rate on ROCMCB was studied in 0.1 mol / L PBS solution containing 5.0 mmol / L ATCl, see Figure 16The CV analysis results show that the oxidation of TCh is an irreversible electrode process, as only one oxidation peak is observed. In addition, the oxidation peak current of TCh increases with the change of scan rate from 10mV / s to 100mV / s, which indicates 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 AChE content on the RBCMs. The effect of RBCM concentration (ranging 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. Figure 17 As shown in Figures A and B, the current response increases with increasing RBCM concentration, reaching a maximum at approximately 2 mg / ml. However, with further increases in RBCM concentration, the current response decreases significantly. This phenomenon is likely due to the increased resistance to the electrochemical process caused by the thickening of the RBCM layer. Therefore, 2 mg / ml was selected as the optimal RBCM concentration.

[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 effect of different incubation times (5-30 minutes) on AChE activity was studied in 100 mol / L huperzine A. Where, I0 is the peak current of AChE before incubation of ROCMCBs with huperzine A, and I1 is the peak current of AChE on the same sensor after incubation with huperzine A. Figure 17 C and D show that the binding of AChE to the active target site reached saturation after 25 minutes. Therefore, 25 minutes was selected as the optimal incubation time.

[0146] 3) pH

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

[0148] 4) Temperature

[0149] The peak current of RORCMBs was tested at 4, 25, 37 and 50°C, and the highest peak current was recorded at 25°C. Figure 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. Figure 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] To date, there is no method for utilizing AChE enriched in RBCMs to replace pure AChE extracts for anti-AD drug screening. However, compared to previously reported AChE-based sensing platforms, this strategy demonstrated comparable or even superior sensitivity. These results demonstrate that the designed ROCMCBs are capable of detecting low-abundance AChE. Combined with the readily available and cost-effective advantages of RBCMs, this method holds great promise for ultrasensitive biomedical assays.

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

[0154] By continuously adding substrate (ATCl) to the stirred cell, the typical current-time response curve of ROCMCBs was obtained. Figure 19 As shown, the addition of ATCl to ROCMCBs produces a rapid and sensitive current response, indicating that the reaction product (ACh) diffuses rapidly. The correlation coefficient is 0.9799. To determine the enzymatic activity of AChE when reacting with ATCl as a substrate, the Michaelis-Menten constant (Km) was calculated according to formula (4). Km is 1.995 mmol / L. A small Km means that AChE immobilized on ROCMCBs can maintain its catalytic activity and exhibit a 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. Figure 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, with each group containing 5 ROCMCBs and 5 CMCBs. Both groups were tested three times. Figure 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 response of RORCMBs and CMCBs to 4.1 μmol / L Huperzine A was measured every day. The sensors were stored at 4°C when not in use. Figure 20 As shown in Figure C, after 7 days of storage, the average inhibition rate of RORCMBs remained at around 72% of the initial value. However, surprisingly, the average inhibition rate of CMCBs dropped to approximately 27% of the initial value. These results indicate 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-maximal inhibitory concentration (IC50) values ​​were calculated. Figure 21 and Figure 22 The inhibitory effects of berberine, baicalin, geniposide, gastrodin, rhynchophylline, and ligusticum lactone A on AChE increased with increasing drug concentration, with IC50 values ​​of 4.7, 12.2, 8.3, 3.2, 86.6, and 66.3 μmol / L, respectively. Under the same conditions, tanshinone IIA and echinacoside showed no significant inhibitory effects on AChE, with no significant IC50 values. These results suggest that RORCMBs can be used to screen potential active ingredients in traditional Chinese medicine for inhibiting AChE activity.

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

[0160] To investigate the interaction patterns between the identified compounds and AChE, eight 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 AChE active site (PDB ID: 1VOT). Figure 23 AF shows the simulated binding of berberine, baicalin, geniposide, gastrodin, rhynchophylline and ligusticum lactone A. The docking results show that they have similar activities to the binding pocket of huperzine A. In addition, Figure 23 GH shows the simulated binding of tanshinone IIA and echinacoside. These docking results showed that berberine, baicalin, geniposide, gastrodin, rhynchophylline and ligusticum lactone A were identified as selective AChE inhibitors.

[0161] 5. Summary

[0162] This chapter successfully developed a pioneering method for positively coating erythrocytes (RBCs) onto electrochemical biosensors using immunoaffinity, enabling the screening of active compounds from traditional Chinese medicines (TCMs). The extracellular domain of the CD47 receptor was used for the positive orientation of the RBCMs. Experimental results demonstrated that the immunoaffinity-based coating of RBCs with the right lateral orientation not only maintained the stability of the coating but also preserved the activity of the peripheral membrane-anchored protein tetramer. Consequently, this method was used to screen six potential anti-AD compounds based on their AChE activity: berberine, baicalin, geniposide, gastrodin, ligustrin A, and rhynchophylline. The screening results were in good agreement with molecular docking simulations, demonstrating the effectiveness of this method in accurately monitoring the AChE activity inhibited by TCMs. In summary, immunoaffinity-based targeted coating of RBCs is an effective technique for precise immobilization in electrochemical TCM screening. This innovative approach not only facilitates the exploration of cellular interactions but also holds great potential for accelerating the drug discovery process.

Claims

1. A method for preparing an electrochemical biosensor coated with red blood cell membrane, characterized in that: The method comprises the following steps: (1) GCEs pretreatment: GCEs were taken and polished to a mirror finish; (2) Preparation of SABG / GCEs: dilute the gold-conjugated anti-rabbit IgG secondary antibody with 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 the mixture was centrifuged 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 then rinsed with PBS 1 to 5 times to remove unbound cell membranes, thereby obtaining ROCMCBs, a red blood cell membrane-coated electrochemical biosensor.

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 step (1) pretreatment of GCEs is as follows: the exposed GCEs are polished with 1.0, 0.3 and 0.05 μm alumina powder in sequence. 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 at a ratio of (25-100):

1.

6. The method for preparing the erythrocyte 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 low speed at 4°C to separate red blood cells and plasma, and then the precipitate is collected and washed three times with pH 7.4 PBS pre-cooled at 4°C to obtain pure red blood cells. The washed red blood cells are completely suspended in pH 7.4 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 for treating Alzheimer's disease.

9. The use according to claim 8, characterized in that The active ingredient is an effective part of traditional Chinese medicine or a monomer of traditional Chinese medicine.

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