A fluoro-microbead guiding chip for simple and quantifiable immunoassay of cardiac troponin I (cTnI)

Abstract

We have developed a fluoro-microbead guiding chip (FMGC) to perform an optical immunoassay of cardiac troponin I (cTnI). The plasma marker protein cTnI is the currently preferred marker to use for a definitive diagnosis and prognosis of myocardial infarction. The FMGC has four immunoreaction regions on a silicon oxide substrate, with five gold patterns imprinted on each region for multiple simultaneous assays. The FMGC assay clearly distinguished immunospecific binding from nonspecific binding by comparing optical signals from inside and outside of the patterns. To detect cTnI, a sandwich immunoassay was performed using antibody-tagged fluoro-microbeads. The cTnI-specific capture antibody was conjugated to the FMGC surface by reaction with 3-3'-dithiobis-propionic acid N-hydroxysuccinimide ester to create a self-assembling antigen-sensing monolayer (DTSP SAM) on the chip. A sample containing cTnI was applied to the antigen-sensing monolayer and allowed to react. To generate a binding signal, a cTnI detection antibody-linked fluoro-microbead preparation was added. The cTnI concentration in a sample was determined by counting the number of biospecifically bound fluoro-microbeads on the corresponding five patterns on the FMGC. The optical signal showed a linear correlation with cTnI concentrations in plasma samples containing from 3.4 pM to 3.4 nM (0.1-100 ng/ml) cTnI. The sensitivity of cTnI detection could be increased by reducing the non-specific binding of the beads to the antigen-sensing surfaces of the chip. Optical detection and quantification of binding by fluorescence microscopy gave results that correlated well with results from a commercial ELISA for cTnI in human plasma. Based on these findings, we propose that the FMGC-based immunoassay system may be adapted to detect and quantify a variety of clinically important targets in human samples.