Homogentisic Acid-Based Whole-Cell Biosensor for Detection of Alkaptonuria Disease.

Clinicians require simple quantitative tools for the detection of homogentisic acid in alkaptonuria patients, a rare inherited disorder of amino acid metabolism. In this study, we report a whole-cell biosensor for homogentisic acid to detect alkaptonuria disease through the expression of green fluorescence protein. The assay system utilizes a promoter sequence (hmgA) isolated from the Pseudomonas aeruginosa genome. To increase the sensitivity, the sensor module harboring phmgA::GFP was further transformed into various transposon mutants debilitated in steps involved in the metabolism of phenylalanine and tyrosine via homogentisic acid as a central intermediate. The proposed biosensor was further checked for analytical features such as sensitivity, selectivity, linearity, and precision for the quantification of homogentisic acid in spiked urine samples. The limit of detection for the developed biosensor was calculated to be 3.9 μM, which is comparable to that of the various analytical techniques currently in use. The sensor construct showed no interference from all of the amino acids and its homolog molecules. The accuracy and precision of the proposed biosensor were validated using high-performance liquid chromatography (HPLC) with satisfactory results.