High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition

摘要

Although antibodies and aptamers are commonly used bioaffinity recognition elements, they are not available for many important analytes. As an alternative, we demonstrate use of a periplasmic binding protein (PBP) to provide high affinity recognition for thiamine (vitamin BI), an analyte of great importance to human and environmental health for which, like so many other small molecules, no suitable biorecognition element is available. We demonstrate that with an appropriate competitive strategy, a highly sensitive (limit of detection of 0.5 nM) and specific bioassay for thiamine and its phosphorylated derivatives can be designed. The high throughput method relies upon the thiamine periplasmic binding protein (TBP) from Escherichia coli for thiamine biorecognition and dye-encapsulating liposomes for signal enhancement. A thiamine monosuccinate-PEG-biotin derivative was synthesized to serve as an immobilized competitor that overcame constraints imposed by the deep binding cleft and structural recognition requirements of PBPs. The assay was applied to ambient environmental samples with high reproducibility. These findings demonstrate that PBPs can serve as highly specific and sensitive affinity recognition elements in bioanalytical assay formats, thereby opening up the field of affinity sensors to a new range of analytes.