Electrochemical evaluation of avidin-biotin interaction using N-iodoacetyl-N-biotinylhexylenediamine

The avidin-biotin assay was investigated by an electrochemical procedure based on the chemical reaction between cysteine containing a thiol group and N-iodoacetyl-N-biotinylhexylenediamine (IB), In the presence of avidin and biotin this reaction, whereby the thiol group combines with IB, is controlled because IB has a biotin part; that is, avidin and biotin are detected indirectly by measurement of iodide ion or cysteine. To achieve a high sensitivity of detection, Hg(II) was introduced as a marker that interacts with cysteine and the oxidation peak from Hg(0) to HE(II) was measured. The sensitivity of detection of avidin was at the level of 10(-9) M. The relative standard deviation at 1 x 10(-8) M avidin was 4.8% (n = 5). On the other hand, a response curve to detect biotin was obtained by the competitive reaction between IB and biotin for the limited binding sites of avidin. The change in peak current enables the detection of biotin at the level of 10(-9) M. This method has the advantage that it is not necessary to separate free IB from bound IB.