Citation: DING Jia, LIANG Wen-Xu, ZHOU Yun-Lei, YIN Huan-Shun, LI Yi-Jing, AI Shi-Yun. An Electrochemical Immunosensor for Detection of Ten-eleven Translocation 1 Protein[J]. Chinese Journal of Analytical Chemistry, ;2022, 50(2): 217-224. doi: 10.19756/j.issn.0253-3820.210791 shu

An Electrochemical Immunosensor for Detection of Ten-eleven Translocation 1 Protein

College of Chemistry and Material Science, Food Safety Analysis and Test Engineering Technology Research Center of Shandong Province, Shandong Agricultural University, Taian 271018, China

Corresponding author: ZHOU Yun-Lei, LI Yi-Jing,
Received Date: 13 October 2021
Revised Date: 24 November 2021

Fund Project: Supported by the National Natural Science Foundation of China (Nos.21775090, 41807484, 41977345, 42077363)

An electrochemical method for sensitive and specific detection of ten-eleven translocation 1 (TET1) protein was developed based on TET1 protein catalyzing the oxidation of 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and the immunological recognition of 5hmC by anti-5hmC antibody (Ab). The bare gold electrode modified with gold nanoparticles (AuNPs/Au) was employed as the substrate electrode, while the double-stranded DNA (dsDNA, formed from the hybridization reaction between probe DNA and its complementary DNA that contains 5mC base)was self-assembled onto the AuNPs/Au electrode through the formation of Au—S bond between AuNPs and —SH at 3'-end of probe DNA. In the presence of α-ketoglutaric acid and Fe²⁺, TET1 protein catalyzed the oxidation of 5mC to produce 5hmC, which triggered the immune-recognition reaction, leading to the immobilization of anti-5hmC antibody. Based on the increasing steric hindrance effect, the electron transmission rate on the electrode surface decreased, and the electrochemical resistance increased, resulting in a decreased electrochemical reduction signal in detection buffer solution. On the basis of the relationship between the decreased electrochemical signal and the concentration of TET1 protein, the detection of TET1 protein was realized. Under the optimal conditions, the developed method presented a wide linear range from 0.5 to 10 μg/mL, with a low detection limit of 0.17 μg/mL (3σ). Moreover, the effect of Pb²⁺ on the activity of TET1 protein was investigated. The results showed that Pb²⁺ could inhibit the activity of TET1 protein with IC₅₀ value of 41.72 nmol/L. In addition, this method showed many advantages such as simple operation, inexpensive instrument, high detection sensitivity and selectivity. This work not only provided a new method for detection of TET1 protein, but also presented alternative biomarker and evaluation method for the investigation of ecotoxicological effects of environmental pollutants.
- DNA hydroxymethylation,
- Ten-eleven translocation 1 protein,
- Electrochemical immunosensor,
- Anti-5-hydroxymethylcytosine antibody
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