Automated Electrical Quantification of Vitamin B1 in a Bodily Fluid using an Engineered Nanopore Sensor

• Published in: Angewandte Chemie Vol. 133; no. 42; pp. 23031 - 23037
• Main Authors: Lucas, Florian Leonardus Rudolfus, Piso, Tjemme Rinze Cornelis, Heide, Nieck Jordy, Galenkamp, Nicole Stéphanie, Hermans, Jos, Wloka, Carsten, Maglia, Giovanni
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 11-10-2021
• Subjects: Automation; bayesian; Biological properties; Biological samples; Biosensors; Body fluids; Chemistry; Data analysis; Metabolites; nanopores; nanotechnology; Thiamine; Vitamin B
• ISSN: 0044-8249; 1521-3757
• DOI: 10.1002/ange.202107807

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.
Quantification of small molecules in biological samples is challenging. Using the concentration‐dependent current fluctuations elicited by a thiamine binding protein inside a ClyA nanopore, we show the automated quantification of thiamine in urine in less than a minute. By swapping the internal biosensor, this principle can be applied for the development of multiple next‐generation home‐diagnostic small‐molecule sensors.