Fluorometric Detection of Multiple Bacteria in Water Using Cell Imprinted Polymer Thin Films Integrated into a Microfluidic Channel

Abstract

Waterborne pathogens threaten public health, but standard detection methods—culture plating and molecular assays—are slow, laborious, and confined to centralized labs. Biosensors promise rapid, on-site monitoring, yet antibody-based designs demand cold-chain handling and are fragile. This thesis introduces a fluorescence microfluidic sensor built on whole-cell imprinted polymers (CIPs), offering a robust, reagent-free alternative for duplex detection in water. CIP thin films were formed in situ within microchannels by polymerizing around target bacterial cells, creating selective cavities. Multiple CIP regions on a single chip enable parallel capture of different species. After trapping cells, optimized FITC injection generates a fluorescence signal proportional to bacterial concentration. In singleplex tests with Salmonella, the sensor achieved a limit of detection (LOD) of 1.47 × 10³ CFU/mL, a limit of quantification (LOQ) of 5.28 × 10³ CFU/mL, and a linear response from 10³ to 10⁷ CFU/mL. Selectivity assays showed preferential Salmonella binding over non-targets. Duplex mode captured both Salmonella and E. coli concurrently at concentrations above 10⁵ CFU/mL. Although multiplexing reduced sensitivity, the CIP-based sensor requires minimal sample volume, no complex prep, and avoids cold storage, underscoring its field-ready potential. Future work will focus on polymer optimization, signal amplification, and validation with real environmental samples.