The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics.H ere we present an anopore-based biosensor and automated data analysis for quantification of thiamine in urine in less than am inute,w ithout the need for recalibration. Fort his we use the Cytolysin An anopore and equip it with an engineered periplasmic thiamine binding protein (TbpA). To allow fast measurements we tuned the affinity of TbpA for thiamine by redesigning the p-p stacking interactions between the thiazole group of thiamine and TbpA. This substitution resulted furthermore in am arked difference between unbound and bound state,a llowing the reliable discrimination of thiamine from its two phosphorylated forms by residual current only.Using an arrayofnanopores,this will allowt he quantification within seconds,p aving the way for next-generation single-molecule metabolite detection systems.

The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics. Here we present a nanopore‐based biosensor and automated data analysis for quantification of thiamine in urine in less than a minute, without the need for recalibration. For this we use the Cytolysin A nanopore and equip it with an engineered periplasmic thiamine binding protein (TbpA). To allow fast measurements we tuned the affinity of TbpA for thiamine by redesigning the π‐π stacking interactions between the thiazole group of thiamine and TbpA. This substitution resulted furthermore in a marked difference between unbound and bound state, allowing the reliable discrimination of thiamine from its two phosphorylated forms by residual current only. Using an array of nanopores, this will allow the quantification within seconds, paving the way for next‐generation single‐molecule metabolite detection systems.