Electrochemical detection method for mucinophilic akkermansia muciniphila amuc_1100 protein based on aptamer

Abstract

The present application relates to the electrochemical detection method of Amuc_1100 protein of mucinophilic Akkermansia based on aptamer. The method uses the nucleic acid aptamer capable of specifically binding to Amuc_1100 protein obtained by screening through SELEX technology, detects through DPV, the detection limit is 0.05 ng / mL, the linear range is 0.1-100 ng / mL. The content of Amuc_1100 protein is greater than or equal to 3 ng / mL. The present application completely bypasses the antibody route, and has the advantages of low cost and high stability.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an electrochemical detection method for the Amuc_1100 protein of Akkermansia bacillus based on aptamers. Background Technology

[0002] Amuc_1100 is one of the most abundant functional proteins expressed on the outer membrane of Akkermansia muciniphila (AKK), playing a role in metabolic and immunomodulatory functions through the Toll-like receptor 2 (TLR2) signaling pathway. Related studies (such as Otman N. et al., Plos ONE, 2017) have confirmed that Amuc_1100 protein is one of the most crucial indicators for evaluating the functional activity of AKK products.

[0003] Currently, the detection of Amuc_1100 protein mainly relies on two methods: one is enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies, which is cumbersome and time-consuming (usually 2-3 hours), and the difference between antibody batches will directly affect the stability of the detection results; the other is liquid chromatography-tandem mass spectrometry (LC-MS / MS), which has expensive equipment, high operating and maintenance costs, and extremely strict requirements for operators and detection environment, and cannot meet the needs of on-site rapid detection.

[0004] In recent years, electrochemical aptamer sensor technology has developed rapidly in the field of biological detection. This technology utilizes nucleic acid aptamers as recognition elements, combined with electrochemical detection methods, and possesses significant advantages such as high specificity, high sensitivity, label-free operation, fast detection speed, and portable equipment. However, there are currently no reports on combining Amuc_1100 protein-specific aptamers with electrochemical methods for rapid on-site detection of this protein.

[0005] To address the shortcomings of the existing technologies, this invention provides an electrochemical detection method for Amuc_1100 protein from AKK bacteria based on aptamers. This method enables rapid, accurate, and quantitative detection of Amuc_1100 protein using an electrochemical aptamer sensor, thereby meeting the practical needs of on-site quality control and rapid screening of end products. Summary of the Invention

[0006] ① Problem to be solved: The existing Amuc_1100 protein detection method is complex to operate and costly, and is not suitable for rapid on-site detection.

[0007] ② Technical solution: Amuc_1100 protein-specific nucleic acid aptamers obtained by screening using SELEX technology are detected by electrochemical methods.

[0008] ③ Beneficial effects: Completely bypasses the antibody route, resulting in low cost, high stability, and fast detection speed (approximately 20 minutes), making it suitable for rapid on-site testing. Detailed Implementation

[0009] Example 1: Screening of Amuc_1100 protein-specific aptamers and electrode preparation

[0010] (1) Aptamer screening: SELEX technology was used to screen Amuc_1100 protein-specific aptamers. After 12 rounds of screening, an enriched library was obtained, and high-throughput sequencing was used to determine the candidate aptamer sequences. Affinity (KD ≤ 10 nM) was determined by SPR.

[0011] (2) Electrode preparation: Glassy carbon electrode (GCE, 3 mm in diameter) was polished to a mirror finish with 1.0, 0.3, and 0.05 μm alumina slurries, and ultrasonically cleaned three times with deionized water (5 minutes each time). The electrode surface was activated by cyclic voltammetry scans at 50 mV / s in the range of -0.2 V to +1.0 V for 15 cycles in 0.5 M H2SO4 solution. Thiolized aptamer (5'-SH-aptamer sequence-3', 1 μM) was added to the treated electrode surface, incubated overnight at 4 °C, washed with PBS, and blocked with 1 mM 6-mercaptohexanol for 30 minutes.

[0012] Example 2: Sample Testing and Result Determination

[0013] (1) Sample pretreatment: Take 100 mg of the AKK bacteria sample to be tested, add 1 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% SDS, 1× protease inhibitor), incubate on ice for 30 minutes, sonicate (200W, 2s on and 2s off, 3 minutes in total), centrifuge at 12,000g for 20 minutes and take the supernatant.

[0014] (2) Electrochemical detection: The prepared electrochemical sensor was inserted into a PBS solution containing the sample to be tested and incubated at room temperature for 30 minutes to allow the Amuc_1100 protein to bind to the aptamer on the electrode surface. Differential pulse voltammetry (DPV) was used as the detection solution, with a scan range of -0.2V to +0.6V, a pulse amplitude of 50 mV, a pulse width of 50 ms, and a scan rate of 4 mV / s. The reduction peak current value (approximately +0.18V) was recorded.

[0015] (3) Calculation of results: The content of Amuc_1100 protein in the sample was calculated based on the standard curve (linear range 0.1-100 ng / mL).

[0016] (4) Result determination:

[0017] Judgment Result Standard Conclusion

[0018] A qualified Amuc_1100 protein content ≥ 3 ng / mL indicates normal expression and the product is qualified.

[0019] Unqualified (Low Expression): Amuc_1100 protein content < 3 ng / mL and ≥ 0.1 ng / mL is considered low, and the product is unqualified.

[0020] The Amuc_1100 protein content was severely substandard (< 0.1 ng / mL, below the detection limit), indicating almost no protein expression.

Claims

1. A method for detecting the protein of Akkermansia muciniphila Amuc_1100, characterized in that, The detection was performed using an electrochemical method with a nucleic acid aptamer that specifically binds to the Amuc_1100 protein.

2. The detection method according to claim 1, characterized in that, The nucleic acid aptamers were obtained through SELEX screening.

3. The detection method according to claim 1, characterized in that, The electrochemical method is differential pulse voltammetry (DPV) or electrochemical impedance spectroscopy (EIS).

4. The detection method according to claim 1, characterized in that, The detection limit of the method is 0.05 ng / mL, and the linear range is 0.1-100 ng / mL.

5. The detection method according to claim 1, characterized in that, The nucleic acid aptamer is labeled with an electrochemically active group or a thiol group at its 5' or 3' end and is fixed to the surface of the gold electrode by self-assembly via gold-sulfur bonds.

6. The detection method according to claim 3, characterized in that, The detection solution for the differential pulse voltammetry (DPV) is a PBS solution containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 (1:1) and 0.1 M KCl. The scan range is -0.2 V to +0.6 V, and the pulse amplitude is 50 mV.

7. The detection method according to claim 1, characterized in that, The sample is considered qualified when the calculated Amuc_1100 protein content is ≥3 ng / mL; the sample is considered unqualified when the calculated Amuc_1100 protein content is <3 ng / mL.