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Detecting metabolites\n                      <jats:italic toggle=\"yes\">in situ<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Objective<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Using\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      strain BL21(DE3) transformed with the pPro24-GFP plasmid, which contains the green fluorescent protein (\n                      <jats:italic toggle=\"yes\">GFP<\/jats:italic>\n                      ) 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 \u03bcL).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The fluorescence-based biosensor functioned in both Luria\u2013Bertani and Dulbecco\u2019s modified Eagle media, producing fluorescence intensities of 3863 \u00b1 957 arbitrary units. Validation using a portable Qubit\n                      <jats:sup>\u00ae<\/jats:sup>\n                      3.0 fluorometer yielded a receiver operating characteristic curve with a Cohen\u2019s 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  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