Multiple and simultaneous detection for cytokines based on the nanohole array by electrochemical sandwich immunoassay

Monitoring of cytokine storm markers is critical for determining the disease progression and treatment efficacy. In this study, we developed a novel fourplex electrochemical (EC) biosensor using nanohole array (NHA) structures containing several nanoholes on a single electrode for detecting cytokines with a low limit. A framework was developed for fabricating and optimizing multiplexed EC sandwich immunoassays using NHA electrodes because an NHA can enhance the EC detection of molecular binding by controlling the binding sites of captured biomolecules. For detecting multiple cytokines, the captured antibodies were immobilized on the surface-modified NHA and sandwich immunoassays with amperometric signal amplifications using poly-streptavidin horseradish peroxidase conjugates and 3,3′,5,5′-tetramethylbenzidine. Under the optimal conditions, the EC NHA immunosensors exhibited high sensitivity and specificity with a limit of detection of 1 pg/mL and a detection range of 1–300 pg/mL. The results revealed that the developed NHA sensor exhibits reliable EC characteristics and could be used to analyze multiple cytokines using small sample volume and subsequently developed for other applications. © 2023 The Authors