Chemical Redox-Cycling for Improving the Sensitivity of Colorimetric Enzyme-Linked Immunosorbent Assay

Herein, a redox-cycling was proposed to amplify the signal of enzyme-linked immunosorbent assay (ELISA), which was performed in a polystyrene microplate based on a classic sandwich-type. After the sandwich immunoreactions were finished, the alkaline phosphatase captured on a microplate triggered the hydrolyzation of l-ascorbic acid 2-phosphate to generate ascorbic acid (AA), which then reduced colorless tris(bathophenanthroline) iron(III) (Fe(BPT)₃³⁺) encapsulated in the micelle of TX-100 to pink red tris(bathophenanthroline) iron(II) (Fe(BPT)₃²⁺). In the presence of tris(2-carboxyethyl)phosphine, the oxidation product, dehydroascorbic acid, was transformed to AA quickly which then reduced Fe(BPT)₃³⁺ again and again, resulting in the generation of abundant Fe(BPT)₃²⁺ that could be read out conveniently by a commercial microplate reader or the naked eye. Because the negative charged TCEP with large size could hardly pass through the micelle, the reduction of Fe(BPT)₃³⁺ by TCEP directly was negligible. Experiment results for assay of alpha-fetoprotein (a model antigen) showed the cycling greatly improved the detection limit to 5 pg/mL, 2 orders of magnitude lower than that of conventional ELISA. The cycling also exhibited the advantages of simplicity and high reproducibility, implying its great potential for practical applications in biological and clinical diagnosis.