Author ORCID Identifier
Document Type
Thesis
Date of Award
Spring 4-26-2024
Keywords
Thiamine, thiaminase, lateral flow assay, thiamine deficiency, enzymatic digestion assay, thiochrome, resource-deficient setting, thiamine-binding protein, fish tissue, food safety
Degree Name
Chemistry: Materials (BA, BS)
Department
CHEMISTRY
First Advisor
Dr. L. Nathan Tumey
Subject Heading(s)
Thiamine Deficiency--diagnosis ; Enzyme Assays--methods
Abstract
Thiamine (vitamin B1) is vital for various metabolic functions within the human body, such as the TCA cycle and pentose-phosphate pathway. In vertebrates, it cannot be produced by the body itself, and therefore must be taken in through diet. Thiamine deficiency can lead to symptoms such as weakness, fatigue, and loss of appetite, and certain neurological disorders. HPLC is a traditional method utilized for thiamine analysis, but the maintenance and expertise required to properly operate an HPLC instrument makes it unsuitable for resource-deficient settings. Therefore, two assay formats are considered for the detection of thiamine: an enzymatic plate digestion assay format and a lateral flow assay format.
The enzymatic plate digestion assay format relies on the relationship between thiamine and the enzyme that inhibits its activity: thiaminase. By subjecting biological sample solutions in parallel to incubation in the presence and absence of thiaminase I (an enzyme that degrades thiamine) we can compare the signal from the background matrix relative to that including thiamine. Thiamine (non-fluorescent) is then oxidized to thiochrome (fluorescent), and the fluorescence of solutions can be determined relative to an external standard curve. The thiaminase enzyme is utilized to create control matrices depleted of thiamine. Developing a diluent for thiamine standards that is similar to the matrix of the biological samples in the assay is key for achieving accurate results.
The lateral flow assay format is directly suited for an at-home user to detect thiamine at the point-of-care. A thiamine derivative is immobilized on the membrane strip, and dye-encapsulating liposomes that are functionalized with a periplasmic thiamine-binding protein are pre-mixed with samples to provide a visible signal indicative of thiamine content when added to the strip. The utilization of this format is intended for future applications in human biological samples.
Recommended Citation
Matesic, Sarah, "Establishing Methods for the Detection of Thiamine in Resource-Deficient Settings" (2024). Undergraduate Honors Theses. 34.
https://orb.binghamton.edu/undergrad_honors_theses/34