An ultrasensitive colorimetric immunoassay based on glucose oxidase catalyzed cascade formation of blue–black iodine–starch complex

This work reports a novel colorimetric immunoassay for ultrasensitive quantitation of prostate specific antigen (PSA) based on glucose oxidase (GOx) catalyzed cascade formation of blue-black iodine–starch complex. In this design, a potassium iodide (KI)–starch system was integrated with a sandwich immunoassay, multivalent biotin–avidin interaction and high efficient GOx catalysis. In the presence of PSA, biotinylated anti–PSA and GOx–conjugated avidin was successively introduced via the specific immunobinding and the high affinity biotin–avidin interaction. GOx–catalyzed oxidation of glucose leads to cascade production of gluconic acid and hydrogen peroxide (H2O2), which subsequently oxidizes KI and results in a visual color change of the starch solution due to the formation of an intense colored iodine–starch complex. The color intensity is proportional to the analyte level, allowing a simple and rapid assay by direct readout with naked eyes. A quantitative immunoassay is easily accomplished by an UV–vis spectrophotometer. Under optimal condition, a colorimetric immunoassay with an ultralow limit of detection (LOD) at 0.46 pg mL−1 and a wide dynamic range from 1 pg mL−1 to 1 μg mL−1 was achieved with PSA as a model biomarker, demonstrating its great potential for early cancer diagnosis in clinical application.