Electrochemical Enzyme‐Linked Immunoassay for the Determination of Estriol Using Methyl Red as Substrate

A new electrochemical substrate for horseradish peroxidase, methyl red, is reported. In this reaction system, horseradish peroxidase can catalyze the redox reaction of methyl red and H2O2. Methyl red exhibits a sensitive voltammetric peak at−0.51 V vs. Ag/AgCl reference electrode, the decrease of the peak current of methyl red is in proportion to the concentration of horseradish peroxidase (HRP). The linear range for determination of horseradish peroxidase is 5.0×10−8∼5.0×10−7 g mL−1 and the detection limit is 1.8×10−8 g mL−1. The relative standard deviation is 3.3% when 2.0×10−7 g mL−1 HRP was sequentially determined 11 times. A voltammetric enzyme‐linked immunoassay method for the determination of estriol was developed, based on this electrochemical system. The linear range for determination of estriol is 1.0∼1000.0 ng mL−1, and the detection limit is 0.33 ng mL−1. The relative standard deviation for 11 parallel determinations with 200 ng mL−1 estriol is 4.8%. Some pregnancy serum samples were analyzed with satisfactory results.