Dopamine-mediated signal amplification integrated with CCK-8 detection for highly sensitive colorimetric immunoassay

The sensitive detection of proteins with a simple colorimetric readout is of great significance for the early diagnosis of diseases and the monitoring of therapeutic efficacy. Herein, we report a novel colorimetric immunoassay that integrates the dopamine-mediated signal amplification with Cell Counting Kit-8 (CCK-8) detection. Through the construction of a sandwich immunoassay system, the horseradish peroxidase conjugated to the detection antibody rapidly catalyzes the deposition and in situ self-polymerization of dopamine on the immune-complex, forming a versatile nanoscale polydopamine (PDA). The resulting PDA serves as a universal scaffold for high-density binding of lactic dehydrogenase (LDH). Upon the addition of L-lactic acid and nicotinamide adenine dinucleotide (NAD⁺), the bound LDH efficiently catalyzes the conversion of L-lactic acid to pyruvate, and simultaneously reduces NAD⁺ to NADH, which subsequently drives the colorimetric reaction of CCK-8 system, producing a strong absorbance signal at 450 nm. This process greatly enhances the immunoassay sensitivity by converting immune recognition events into a CCK-8 colorimetric readout and integrating multi-step cascade signal amplification. This method exhibits a broad dynamic range spanning five orders of magnitude for the model analytes, carcinoma embryonic antigen and alpha-fetoprotein, with detection limit as low as 5.4 pg/mL and 4.1 pg/mL respectively. Moreover, the distinct color gradient enables reliable semi-quantitative assessment with the naked eye. This colorimetric immunoassay provides a general, straightforward, and potent method in clinical diagnostics and prognosis of diseases.