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 D3 (25(OH)D3) 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)D3 into 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 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 D3 isomers were produced in addition to 1,25(OH)2D3. 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)D3 in buffer within the physiological range (5-200 ng/ml).