Active Analyte Import Improves the Dynamic Range and Sensitivity of a Vitamin B12 Biosensor

Cell-free systems provide a versatile platform for the development of low-cost, easy-to-use sensors for diverse analytes. However, sensor affinity dictates response sensitivity, and improving binding affinity can be challenging. Here, we describe efforts to address this problem while developing a biosensor for vitamin B-12, a critical micronutrient. We first use a B-12-responsive transcription factor to enable B-12-dependent output in a cell-free reaction, but the resulting sensor responds to B-12 far above clinically relevant concentrations. Surprisingly, when expressed in cells, the same sensor mediates a much more sensitive response to B-12. The sensitivity difference is partly due to regulated import that accumulates cytoplasmic B-12. Overexpression of importers further improves sensitivity, demonstrating an inherent advantage of whole-cell sensors. The resulting cells can respond to B-12 in serum, can be lyophilized, and are functional in a minimal-equipment environment, showing the potential utility of whole-cell sensors as sensitive, field-deployable diagnostics.