A smartphone-based genetically recombinant whole-cell biosensor for highly sensitive monitoring of polychlorinated biphenyls (PCBs)

Background: Polychlorinated biphenyls (PCBs) are persistent carcinogens widely distributed in ecosystems. Their structural complexity-diverse chlorine numbers and positions-challenges detection using conventional methods like ELISA or instrumental analysis, which are limited to single congeners. Whole-cell biosensors (WCBs) present a promising alternative by enabling multi-congener detection, yet their sensitivity remains constrained by inefficient PCB-sensing proteins. To overcome this limitation, we engineer an Escherichia coli-based WCB that integrates bphAB-mediated PCB degradation with HbpRCBP6-based sensing, exploiting metabolic conversion to enhance detection. This dual-circuit design aims to achieve broad-spectrum PCB recognition while improving sensitivity through chassis optimization and enzymatic pre-processing.<br /> Results: We developed a BL21(DE3)/HbpRCBP6-bphAB WCB that detects multiple PCB congeners via a two-step mechanism: BphAB enzymes first convert PCBs to hydroxylated derivatives (OH-PCBs), which are then recognized by the HbpRCBP6 transcriptional factor. The biosensor achieved the lowest reported LOD for 2-CBP (2-chlorobiphenyl) and detected 3-CBP, 4-CBP, 2,3-diCBP, and 2,2 '-diCBP at low micromolar limits (0.06-1 mu M). Structural docking revealed that HbpRCBP6 binds OH-PCBs with higher affinity (binding energy:-6.39 kcal/mol) than native PCBs (-6.14 kcal/mol), validating the critical role of bphAB in enhancing sensitivity. To enable field applications, we immobilized the WCB in a transglutaminase-crosslinked hydrogel and paired it with a smartphone-based detection platform. Dose-response curves showed a linear logarithmic relationship between 2-CBP concentration (1-100 mu M) and fluorescence intensity. Significance: This study advances PCB monitoring by integrating metabolic pre-processing with sensitive transcriptional activation, overcoming the limitations of single-target assays. The smartphone-compatible hydrogel platform enables real-time, on-site detection. Our strategy, which couples catabolic pathways with optimized sensing proteins, not only advances environmental monitoring capabilities but also provide an innovative strategy for developing metabolic pathway-sensing proteins combined biosensors.