An urgent need for a concise and rapid detection method for Mycoplasma pneumoniae is required due to its severe impact on human’s health. To meet such a need, this study proposed and constructed an innovative point-of-care testing (POCT) platform that consists of a hydrogen ion-selective loop-mediated isothermal amplification (H+-LAMP) sensor and an electrochemical detection device. The H+-LAMP sensor successfully integrated the working and reference electrodes, and converted the H+ generated during the LAMP process into an electrochemical signal. High sensitivity and stability for Pathogen detection were also achieved by treating the working electrode with an electrodeposited polyaniline solid contact layer and by using an ion-selective membrane. As a result, the sensor shows a sensitivity of 68.26 mV/pH, a response time of less than 2s, and a potential drift of less than 5 mV within one hour, which well meets the urgent need.. The results also demonstrated that the detection limit for Mycoplasma pneumoniae was lowered to 1 copy/μL, the nucleic acid extraction and detection process could be completed in 30 minutes, and the impact of interfering ions on the sensor was negligible. Validation with 20 clinical samples yielded satisfactory results. More importantly, the storage lifespan of such an electrochemical sensor is over seven days, which is a great advantage for on-site pathogen detection. Therefore, the constructed hydrogen ion-selective sensor in this investigation is particularly suitable as a core component for an instant pathogen detection platform.

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