Engineered nanopore biosensor for thiamine quantification: Experimental design.

Biosensing using protein nanopores has become a powerful tool for the analysis of single molecules, enabling for example portable devices for long-read DNA sequencing. By inserting a single pore into a lipid bilayer and applying an electric field, one can create a single-molecule sensor using the ionic current flowing through the pore. Thiamine can be quantified with such a technique using the Cytolysin A pore and a thiamine-binding protein TbpA, which imparts its affinity to the nanopore. When thiamine passes through the pore, it gets captured by TbpA, causing a conformational change that alters the flow of ions through the pore and thus causes a change in current. The result is a biosensor pair with high selectivity that allows quantification of thiamine even in complex solutions. Here, we provide instructions for the implementation of this measurement technique, which includes the production of the required recombinant proteins as well as construction and operation of the electrochemical measuring setup.