Bacterial Fluorescence-Based Biosensor for Detecting Propionate in Culture Media for Diagnostic Applications

Background Engineering bacteria for disease diagnosis and treatment is an emerging area of medical research that involves assessing short-lived molecules in complex, dynamic environments. Detecting metabolites in situ offers significant opportunities for identifying responsive cellular populations. Among suitable targets for this technology are short-chain fatty acids, such as propionate, that play crucial roles in human health and disease. Objective To develop a bacterial fluorescence-based biosensor capable of detecting propionate levels at the millimolar scale in low-volume samples across different culture media and optimize its application protocol. Methods Using Escherichia coli strain BL21(DE3) transformed with the pPro24-GFP plasmid, which contains the green fluorescent protein ( GFP ) gene under the control of a propionate-inducible promoter, we achieved reliable propionate detection with a threshold of 10 mM in a small volume (750 μL). Results The fluorescence-based biosensor functioned in both Luria–Bertani and Dulbecco’s modified Eagle media, producing fluorescence intensities of 3863 ± 957 arbitrary units. Validation using a portable Qubit ® 3.0 fluorometer yielded a receiver operating characteristic curve with a Cohen’s kappa of 0.716, indicating substantial agreement. Proof-of-concept experiments using flow cytometry and mock fecal samples (healthy feces + 100 mM propionate) successfully detected the elevated propionate levels. Conclusion This cost-effective, easy-to-use protocol provides a proof-of-concept for propionate detection in biological samples, with potential future applications in clinical diagnostics, particularly for gastrointestinal disorders in which propionate serves as a biomarker, where it could be used to monitor disease progression and therapeutic interventions.