Luo Qiangqiang, Zhang Faying, Zhang Mengjie, Hu Shantong, Li Xin, Pan Li, Lu Zhenghui, Wu Pan, Zhang Guimin
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, Hubei, 430062, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, Hubei, 430062, China; College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Anal Chim Acta. 2025 Oct 8;1370:344404. doi: 10.1016/j.aca.2025.344404. Epub 2025 Jul 8.
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 HbpR-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.
We developed a BL21(DE3)/HbpR-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 HbpR 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 μM). Structural docking revealed that HbpR 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 μM) and fluorescence intensity.
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.
多氯联苯（PCBs）是广泛分布于生态系统中的持久性致癌物。它们结构复杂，氯原子数量和位置多样，这给使用酶联免疫吸附测定（ELISA）或仪器分析等传统方法进行检测带来了挑战，这些传统方法仅限于检测单一的同系物。全细胞生物传感器（WCBs）通过实现多同系物检测提供了一种有前景的替代方法，但其灵敏度仍受限于效率低下的PCB传感蛋白。为了克服这一限制，我们构建了一种基于大肠杆菌的WCB，该传感器将bphAB介导的PCB降解与基于HbpR的传感相结合，利用代谢转化来提高检测能力。这种双回路设计旨在实现广谱的PCB识别，同时通过底盘优化和酶预处理提高灵敏度。
我们开发了一种BL21(DE3)/HbpR-bphAB WCB，它通过两步机制检测多种PCB同系物：BphAB酶首先将PCBs转化为羟基化衍生物（OH-PCBs）,然后由HbpR转录因子识别。该生物传感器实现了已报道的2-CBP（2-氯联苯）最低检测限，并在低微摩尔浓度范围（0.06-1 μM）检测到3-CBP、4-CBP、2,3-二氯联苯和2,2'-二氯联苯等。结构对接显示，HbpR与OH-PCBs的结合亲和力（结合能：-6.39 kcal/mol）高于天然PCBs（-6.14 kcal/mol），这验证了bphAB在提高灵敏度方面的关键作用。为了实现现场应用，我们将WCB固定在转谷氨酰胺酶交联的水凝胶中，并将其与基于智能手机的检测平台配对使用。剂量反应曲线显示2-CBP浓度（1-100 μM）与荧光强度之间呈线性对数关系。
本研究通过将代谢预处理与灵敏的转录激活相结合，推进了PCB监测，克服了单靶点检测的局限性。与智能手机兼容的水凝胶平台实现了实时、现场检测。我们将分解代谢途径与优化的传感蛋白相结合的策略，不仅提升了环境监测能力，还为开发代谢途径传感蛋白组合生物传感器提供了创新策略。
