Specific biosensor of amino acid

Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. In maple syrup urine disease (MSUD), a metabolic defect due to decreased activity of the branched-chain alpha-ketoacid dehydrogenase complex (BCKD), which is the second enzymatic step in the degradation of branched-chain amino acids (BCAAs) leucine, valine and isoleucine. Affected patients will have elevated BCAAs and alloisoleucine that will result in encephalopathy if left untreated. Therefore, close monitoring of BCAAs levels in the blood are required. However, so far there are no real-time detection methods available for real time or continuous monitoring of BCAAs` levels in the blood, which highlights the importance of the development of more rapid analytical tools. Our biosensor relies on specific aminoacylation of tRNA and an enzymatic cascade to produce an electrochemical signal. Leucyl-tRNA-synthetase (LRS) is the recognition element and is connected to pyruvate oxidase (POX) which uses the aminoacylation products to produce H2O2 which can be measured using electrochemical measurement techniques. Two proteins were produced and purified, Leucyl-tRNA-synthetase and pyruvate oxidase. In addition, several experiments were conducted to test the activity and the ability of the enzymatic cascade to produce H2O2 which will be measured electrochemically and provide the concentration of the specific amino acid in the body.