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The establishment of rapid targeted identification and analysis of antibiotic resistance genes (ARGs) is very important. In this study, an electrochemical sensor, which can detect ARGs was obtained by modifying the sulfhydryl single-stranded DNA probe onto the thin-film gold electrode through self-assembly. The sensor can perform a hybridization reaction with a target sequence to obtain an electrochemical impedance spectroscopy signal. The results showed that when the concentration of the probe used to modify thin-film gold electrodes during preparation was 1 μM, the hybridization time was 1 h, and the hybridization temperature was 35 °C, the self-assembled sensor showed good detection performance for the ARGs encoding β-lactam hydrolase. The measurement ARG concentration linear range is 6.3–900.0 ng/mL, and the R 2 is 0.9992. The sensor shows good specific recognition ability for single-base, double-base, and three-base mismatch DNA. In addition, after 30 days of storage at 4 °C, the accurate identification and analysis of ARGs can still be maintained.

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