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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biosensor Development

Background:

• Antibodies and aptamers are limited in detecting many small molecules, hindering bioassay development.
• Thiamine (vitamin B1) is crucial for health, but lacks suitable biorecognition elements for sensitive detection.
• Periplasmic binding proteins (PBPs) present an alternative for high-affinity molecular recognition.

Purpose of the Study:

• To develop a sensitive and specific bioassay for thiamine and its derivatives using a PBP.
• To demonstrate the utility of PBPs as biorecognition elements for small molecule detection.
• To expand the range of analytes addressable by affinity sensors.

Main Methods:

• Utilized the thiamine-binding PBP (TBP) from Escherichia coli for thiamine recognition.
• Employed dye-encapsulating liposomes for signal enhancement in a competitive assay format.
• Synthesized a thiamine derivative for use as an immobilized competitor, overcoming PBP binding constraints.

Main Results:

• Developed a high-throughput bioassay with a limit of detection of 0.5 nM for thiamine.
• Achieved high specificity for thiamine and its phosphorylated derivatives.
• Demonstrated reproducible thiamine detection in ambient environmental samples.

Conclusions:

• Periplasmic binding proteins (PBPs) are effective high-affinity recognition elements for bioanalytical assays.
• This PBP-based assay enables sensitive and specific detection of thiamine and related compounds.
• The findings broaden the applicability of affinity sensors to a wider range of small molecule analytes.