A Label-Free Detection Method for P9 Protein of Akkermansia muciniphila Based on Electrochemical Sensors

Abstract

This invention relates to a label-free detection method for P9 protein of Akkermansia myxophilus based on an electrochemical sensor. The method employs an electrochemical biosensor with a surface-modified recognition element, detecting changes in the electrochemical signal caused by P9 protein binding via EIS or DPV, achieving label-free quantitative detection. This invention features rapid detection (approximately 20 minutes), simple operation, a detection limit of 0.03 ng / mL, and a linear range of 0.1–100 ng / mL. A measured P9 protein concentration ≥3 ng / mL is considered acceptable. It is suitable for rapid quality control in Akkermansia myxophilus production sites.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a label-free detection method for Akkermansia p9 protein based on an electrochemical sensor. Background Technology

[0002] Existing methods for P9 protein detection are mainly based on ELISA or mass spectrometry, which are relatively complex to operate and require sophisticated equipment. Electrochemical biosensors offer advantages such as fast detection speed, high sensitivity, and portable equipment, making them suitable for rapid on-site detection. Currently, there is no label-free detection method for P9 protein based on electrochemical sensors. Summary of the Invention

[0003] ① Problem to be solved: To provide a label-free rapid detection method for P9 protein based on an electrochemical sensor, in order to solve the technical problems of existing detection methods being complex to operate, expensive to use, and unsuitable for on-site detection.

[0004] ② Technical solution: This invention provides a label-free detection method for AKK bacteria P9 protein based on an electrochemical sensor. The electrode surface is modified with a recognition element (antibody, aptamer or molecularly imprinted polymer) that can specifically recognize the P9 protein. The electrochemical signal change caused by P9 protein binding is detected by EIS or DPV to achieve label-free quantitative detection.

[0005] ③Beneficial effects: Label-free detection, simple operation; fast detection speed, detection time is about 20 minutes; portable equipment, suitable for on-site quality control in production; high sensitivity, detection limit is 0.03 ng / mL. Detailed Implementation

[0006] Example 1: Fabrication of an electrochemical sensor (1) The glassy carbon electrode (GCE, diameter 3mm) was polished to a mirror finish on chamois leather with 1.0, 0.3 and 0.05 μm alumina paste in sequence, and then ultrasonically cleaned with deionized water 3 times for 5 minutes each time.

[0007] (2) The cleaned electrode was placed in 0.5 M H2SO4 solution and cyclically scanned for 15 cycles in the range of -0.2 V to +1.0 V at a scan rate of 50 mV / s to activate the electrode surface.

[0008] (3) Add 5 μL of P9 protein-specific antibody solution (10 μg / mL, dissolved in PBS) to the electrode surface and incubate overnight at 4°C to fix the antibody on the electrode surface.

[0009] (4) After washing the electrode with PBS, add 50 μL of 1% BSA solution and block at room temperature for 1 hour to block the non-specific binding sites on the electrode surface.

[0010] Example 2: Label-free electrochemical detection (1) Insert the prepared electrochemical sensor into PBS solutions containing different concentrations of P9 protein (0.01-1000 ng / mL) and incubate at room temperature for 30 minutes to allow the P9 protein to bind to the antibody on the electrode surface.

[0011] (2) Using PBS containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 (1:1) and 0.1 M KCl as the detection solution, differential pulse voltammetry (DPV) was used for scanning with the following parameters: scan range -0.2V to +0.6V, pulse amplitude 50 mV, pulse width 50 ms, and scan speed 4 mV / s.

[0012] (3) Record the reduction peak current values ​​(approximately +0.18V) corresponding to different concentrations of P9 protein. As the concentration of P9 protein increases, the reduction peak current value gradually decreases. Plot a standard curve with the logarithm of P9 protein concentration on the x-axis and the peak current change value on the y-axis. The linear range is 0.1-100 ng / mL, the detection limit is 0.03 ng / mL, and the detection time is approximately 20 minutes.

[0013] Example 3: Sample Testing and Result Determination 3.1 Sample preparation Take the AKK bacteria sample (lyophilized powder, fermentation broth, or finished product) and process it as follows: (1) Lyophilized powder sample: Accurately weigh 100 mg of lyophilized powder and add 1 mL of lysis buffer (containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton) Mix X-100, 0.5% SDS, and 1x protease inhibitor (mixture), vortex to mix, incubate on ice for 30 minutes, sonicate (200W power, 2s on, 2s off, 3 minutes in total), centrifuge at 12,000g at 4°C for 20 minutes, and use the supernatant as the test solution.

[0014] (2) Fermentation broth sample: Take 1 mL of fermentation broth, centrifuge at 12,000 g for 20 minutes at 4°C, and take the supernatant as the test solution.

[0015] (3) End products (capsule contents, solid beverages, etc.): Take a sample equivalent to 100 mg of the original sample, add 1 mL of lysis buffer, process it according to the method for lyophilized powder samples, and take the supernatant as the test solution.

[0016] 3.2 Testing Operation (1) Insert the prepared electrochemical sensor into the sample solution to be tested and incubate at room temperature for 30 minutes.

[0017] (2) Using PBS containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 (1:1) and 0.1 M KCl as the detection solution, DPV was used for scanning.

[0018] (3) Record the value of the reduction peak current.

[0019] 3.3 Result Calculation and Judgment (1) Plot a standard curve with the logarithm of P9 protein concentration on the x-axis and the peak current change on the y-axis. R² should be ≥0.995.

[0020] (2) Substitute the sample peak current value into the standard curve to calculate the measured concentration (ng / mL) of P9 protein in the sample.

[0021] 3.4 Standards for Passing and Failure Validity determination: The standard curve R² should be ≥0.995, otherwise the batch of experiments is invalid.

[0022] Sample judgment: Qualified product (normal P9 protein expression): The peak current value of the sample is within the linear range of the standard curve, and the calculated actual concentration of P9 protein is ≥ 3 ng / mL.

[0023] Non-compliant sample (low P9 protein expression): The peak current value of the sample is within the linear range of the standard curve, but the calculated actual concentration of P9 protein is < 3 ng / mL.

[0024] Seriously unqualified (P9 protein not detected): The sample peak current value is lower than the detection limit of the standard curve (<0.03 ng / mL).

[0025] Completely unqualified: If the test sample is found to have both P9 protein and Amuc_1100 protein or Amuc_1409 protein in a "seriously unqualified" state, it will be judged as completely unqualified and will not pass.

Claims

1. A label-free detection method for P9 protein of Akkermansia muciniphila, characterized in that, The P9 protein is detected using an electrochemical biosensor, wherein the electrode surface of the electrochemical biosensor is modified with a recognition element capable of specifically recognizing the P9 protein.

2. The detection method according to claim 1, characterized in that, The recognition element is a P9 protein-specific antibody, a nucleic acid aptamer, or a molecularly imprinted polymer.

3. The detection method according to claim 1, characterized in that, The detection method is based on electrochemical impedance spectroscopy (EIS) or differential pulse voltammetry (DPV) to detect changes in electrochemical signals caused by the binding of P9 protein to recognition elements.

4. The detection method according to claim 1, characterized in that, The electrode surface modification method includes: immobilizing antibodies or aptamers on the electrode surface through self-assembly or covalent coupling.

5. The detection method according to claim 1, characterized in that, The linear range of the method is 0.1-100 ng / mL, the detection limit is 0.03 ng / mL, and the detection time is 20 minutes.

6. The application of the detection method according to any one of claims 1 to 5 in the rapid detection or on-site detection of P9 protein in AKK bacteria.