Detection of 25-Hydroxyvitamin D3 with an Enzyme modified Electrode

Since testing for circulating vitamin D concentrations is relatively expensive and time consuming, rapid means of measurement are desired.As a step towards this goal, enzyme-modified electrodes responsive to 25-hydroxyvitamin D 3 (25(OH)D 3 ) have been developed.To make the enzyme, a synthetic gene encoding a human cytochrome P450 27B1 (CYP27B1) enzyme, which is a mitochondrial type heme-thiolate monooxygenase that converts 25(OH)D 3 into 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), was expressed and purified.The CYP27B1 enzyme was combined with NADPH-adrenodoxin reductase (ADR) and adrenodoxin (ADX) and activity was characterized using liquid chromatography mass spectrometry (LC-MS/MS).It was found that dihydroxyvitamin D 3 isomers were produced in addition to 1,25(OH) 2 D 3 .The enzyme was immobilized on glassy carbon electrodes using pH-adjusted Nafion ® along with cobalt sepulchrate trichloride (Co(sep) 3+ ) as a redox mediator and electrode performance was characterized using cyclic and square wave voltammetry.The results demonstrate detection of 25(OH)D 3 in buffer within the physiological range (5-200 ng/ml).