Periplasmic binding protein-based magnetic isolation and detection of thiamine in complex biological matrices

Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine periplasmic binding protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5 nM ranging up to 240 nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2 nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0 nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.