Disposable Paper-Based Electrochemical Biosensor Employing g-C₃N₄/Carbon Dots and Toll-like Receptor for Ultrasensitive Detection of Gram-Negative Bacteria

Abstract

Waterborne bacterial contamination remains a pressing global health concern, demanding point-of-care (POC) devices for rapid and efficient on-site detection. High costs, long processing times and reliance on sophisticated equipment limit conventional methods. Thus, this study proposes the fabrication of a low-cost, disposable paper-based electrochemical biosensor for the effective and selective detection of gram-negative bacterial samples. The developed biosensor was modified with g-C3N4/amine-functionalised carbon dots composite to boost signal transduction and offer stable immobilisation of TLR-4/MD-2 bioreceptor, which detects explicitly the lipopolysaccharide layer of gram-negative bacteria samples. The developed paper-based biosensor revealed an excellent analytical performance with remarkable specificity and achieved a low theoretical limit of detection of 0.66 CFU/mL and 0.88 CFU/mL for E. coli and P. aeruginosa, respectively, across a wide dynamic range of 1.5 to 1.5 × 10⁵ CFU/mL. Furthermore, the biosensor demonstrated good stability, reproducibility and ability to attain a satisfactory low LOD in the spiked tap and pond water samples. Moreover, the simple disposability of the paper electrodes lowers the cross-contamination issues and ensures the safety of the environment. Collectively, this work introduces a sustainable, low-cost, and portable biosensing platform that effectively integrates nanomaterial for enhanced transduction with receptor-based specificity, offering significant potential for early diagnosis of waterborne bacterial contamination and advancing public health protection through POC application.