An ultrasensitive electrochemical nucleic acid biosensor with a triple amplification strategy for bacterial detection in water.

Rapid and sensitive determination of total bacterial count in water is essential for safeguarding water safety. In this work, an ultrasensitive electrochemical nucleic acid biosensor was developed for the detection of total bacteria in water. A conserved fragment of bacterial 16S rRNA gene (16S rDNA) was screened as the universal target, and the corresponding hairpin capture probe was designed and immobilized on magnetic particles for specific recognition and enrichment. By combining electrochemical detection with a triple signal amplification strategy-including hybridization chain reaction, G-quadruplex-assisted signal enhancement, and artificial nuclease-mediated signal activation-the sensor achieved highly sensitive quantification of aquatic bacteria. It displayed a linear response to bacterial 16S rDNA over a concentration range from 0.1 fM to 100 pM, with an ultralow detection limit of 1.0 aM. For total bacterial determination in real water, it achieved a practical detection limit of 1 CFU mL-1, showing strong concordance with the gold-standard plate count method. It also exhibited good reproducibility and stability, satisfying the demand for total bacterial count monitoring in drinking water and environmental water bodies, and thus providing an efficient and reliable platform for water safety assurance.