Immunoassay using disulfide-terminated aryldiazonium salt compounds as linkers

Fluorescence and Electrochemical Sandwich Immunoassay Using DSAD Linker Electrodeposition

This technology is a sandwich immunoassay that electrodeposits DSAD salt compounds onto an electrode and converts disulfide groups into thiosulfinate or thiosulfonate groups via electrochemical oxidation to covalently bond primary antibodies. It offers two methods: detection via TRITC fluorescence on an ITO microelectrode array, and an ELISA-based approach using cyclic voltammetry to measure HRP catalytic reactions on ITO electrodes modified with PEI and ERGO.

Conventional immunosensors struggle to selectively immobilize antibodies at specific electrode locations, limiting the ability to analyze multiple regions on a single chip. Furthermore, graphene oxide-based electrochemical sensors often suffer from reduced catalytic activity during surface functionalization, preventing optimal detection sensitivity.

This technology enables high-sensitivity detection by electrochemically activating only specific microelectrodes to immobilize antibodies at desired locations, while maintaining catalytic activity on the ERGO surface to quantify benzoquinone generated from hydrogen peroxide and hydroquinone reactions. Applicable to multi-target diagnostic chips, point-of-care electrochemical immunosensors, and fluorescence-based protein analysis equipment, it allows for diagnostic processes that can distinguish and measure multiple antigens simultaneously from small samples.

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Key Features:

• Electrodepositing the DSAD salt compound of Formula 2 onto a microelectrode array and electrochemically oxidizing only specific electrodes to activate them with thiosulfinate or thiosulfonate groups
• Spatially selectively immobilizing primary antibodies with thiol groups only on the activated electrode portions, followed by sequential binding of target antigens and TRITC-labeled secondary antibodies to detect fluorescence
• Pretreating piranha-cleaned ITO electrodes with PEI, forming graphene oxide, and electrochemically reducing it to prepare an ERGO/PEI/ITO surface
• Forming an HRP-labeled antibody complex on the DSAD-functionalized ERGO electrode and detecting benzoquinone generated from hydrogen peroxide and hydroquinone reactions via cyclic voltammetry

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This invention was developed with support from the Ministry of Education, Science and Technology for the development of electrochemical biosensors using bacteriophages.

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