| Summary: | Bioluminescent bacteria whole-cell biosensors (WCBs) have been widely used in a range of sensing applications in environmental monitoring and medical diagnostics. However, most of them use planktonic bacteria cells that require complicated signal measurement processes and therefore limit the portability of the biosensor device. In this study, a simple and low-cost immobilization method was examined. The bioluminescent bioreporter bacteria was absorbed on a filter membrane disk. Further optimization of the immobilization process was conducted by comparing different surface materials (polyester and parafilm) or by adding glucose and ampicillin. The filter membrane disks with immobilized bacteria cells were stored at −20 °C for three weeks without a compromise in the stability of its biosensing functionality for water toxicants monitoring. Also, the bacterial immobilized disks were integrated with smartphones-based signal detection. Then, they were exposed to water samples with ethanol, chloroform, and H<sub>2</sub>O<sub>2</sub>, as common toxicants. The sensitivity of the smartphone-based WCB for the detection of ethanol, chloroform, and H<sub>2</sub>O<sub>2</sub> was 1% (<i>v</i>/<i>v</i>), 0.02% (<i>v</i>/<i>v</i>), and 0.0006% (<i>v</i>/<i>v</i>), respectively. To conclude, this bacterial immobilization approach demonstrated higher sensitivity, portability, and improved storability than the planktonic counterpart. The developed smartphone-based WCB establishes a model for future applications in the detection of environmental water toxicants.
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