Development of an Enzyme-Linked ImmunoSorbent Assay (ELISA) with Enhanced Sensitivity in a Nanofluidic System

Abstract

Experimental studies were performed to evaluate the kinetics and equilibrium binding constants of biomolecules in nanofluidic channels. Binding events in the nanochannel were detected using electrical and fluorescence methods. We concluded that antibody-antigen binding constants in nanochannels were similar to experiments performed in microtiter plates at low antigen concentrations; however the bound fraction in nanochannels at high antigen concentration decreased due to steric hindrance. Binding kinetics in nanochannels was limited by convective transport of analytes, instead of diffusion or reaction. We also found that enzymatic reactions in nanochannels were very effective due to short diffusion length and high surface area to volume ratio. A bead based ELISA was developed to exploit the rapid binding reactions in the bulk and efficient enzymatic conversion in the nanochannels. Additionally, electrokinetic concentrators were integrated with multiplexed bead based ELISA to further improve the detection sensitivity of a sandwich immunoassay.