Development of a simple and robust absorbance-based carbon dot platform for rapid escherichia coli DNA detection

Abstract Reliable DNA-based detection of Escherichia coli is essential for monitoring bacterial contamination in food, water, and environmental samples. In this work, we developed a label-free and amplification-free UV-Vis absorbance biosensor using carbon dots (Cdots) as optical transducers for the detection of E. coli DNA. Cdots synthesized from chitosan, citric acid, and thiourea were functionalized with NH 2 -terminated DNA probes through carbodiimide-mediated covalent coupling, enabling stable attachment of amine-modified DNA probes to carboxyl-containing Cdots. Among the tested probe configurations, NH 2 -BL21 produced the strongest optical response, with a maximum absorbance change of 31.6%, highlighting the importance of both probe sequence and terminal functionalization in enhancing sensor performance. The optimized biosensor exhibited excellent linearity over the 200–2000 fM range, with an R 2 of 0.9992, a limit of detection of 147.53 fM, and a limit of quantification of 491.76 fM. In addition, the sensor exhibited sequence-dependent recognition and preferential selectivity toward the tested E. coli DNA target, while its response remained statistically unchanged over four weeks of storage, demonstrating short-term stability under the tested conditions. Mechanistic analysis suggests that the absorbance response reflects multiple hybridization-induced interfacial and optical effects, including increased nucleic-acid absorption near 258 nm and possible contributions from changes in surface charge, the local dielectric environment, particle organization, and light scattering. This label-free and amplification-free absorbance strategy provides a potentially accessible platform for E. coli DNA detection. However, matrix-specific calibration and further validation are required before practical environmental or food-safety deployment.