Agarase-based bacterial biosensor for detection of cadmium in environmental samples

Cadmium (Cd) contamination, which requires sensitive and cost-effective detection methods for environmental monitoring. In this study, the Cd-responsive promoter P cadR from P. aeruginosa strains RGS16 and RGS23 was functionally characterized and compared with Escherichia coli strains carrying pUCP-P cadR - gfp mut2 . The Cd sensing ability of this construct was standardized and analyzed by fluorescence microscopic imaging, high-throughput cell imaging, flow cytometric, fluorometric, and colorimetric analysis. Promoter activity was first evaluated using a GFP-based reporter, revealing significantly higher GFP expression in P. aeruginosa strains and a linear response to CdCl 2 concentrations between 56.2 and 562 ppm (0.5-5 mM). To extend biosensor applicability, a 938 bp β-agarase ( dagA ) reporter gene was fused downstream of the P cadR promoter, generating pUCP-P cadR - dagA 39 and pUCP-P cadR - dagA 40 constructs. The engineered P. aeruginosa BC15 strain exhibited a broad linear detection range from 10 ppb to 500 ppm CdCl 2 , with a high correlation coefficient (R 2 = 0.9851). Furthermore, immobilized Pa-CadBacS -GSR39 and GSR40 biosensors showed strong linear responses in both ppb and ppm ranges (R 2 = 0.94 - 0.963). Cd concentrations measured using the biosensor system correlated with ICP-MS analysis (R 2 = 0.967), confirming its accuracy. These results demonstrate that the P cadR -based whole-cell biosensor is a low-cost, sensitive, and reliable platform for quantitative Cd detection across environmentally relevant concentrations. • Designed a fluorescent based optical biosensor for quantification of Cd was constructed and optimized. • A simple, cost-effective colorimetry whole cell biosensor for specific detection of Cd was constructed. • Developed a low cost immobilized colorimetry whole cell bacterial biosensor for Cd using agarase as a novel reporter gene. • The response was greatly enhanced with increasing concentration of Cd and the limit of detection was 0.1ppb to 500 ppm. • The Bland Altman correlation analysis of ICP-MS and biosensor showed 95% limit of agreement and mean difference is -0.00022.