A fluorescent immunosensor for high-sensitivity cardiac troponin I using a spatially-controlled polymeric, nano-scale tracer to prevent quenching

Abstract

For detection of high-sensitivity cardiac troponin I (hs-cTnI < 0.01 ng/mL), signal amplification was attained using a rapid immunosensor with a fluorescently-labeled, polymeric detection antibody. As fluorescent molecules tend to quench when they are less than 10 nm apart, a synthetic scheme for the labeled antibody was devised to control the molecular distance and so minimize the quenching effect in a single conjugate unit. To this end, we first performed novel polymerization of fluorophore-coupled streptavidin (FL-SA) with biotinylated detection antibody (b-Ab) in a stepwise manner by adding FL-SA to b-Ab five times sequentially. Relative spatial positions of the fluorophore molecules in the polymer were then distally fixed using di-biotinylated oligonucleotides and passed through a 0.45 gm filter to obtain a polymer of uniform size (i.e., 400 nm in diameter). We produced polymeric tracers using two different inexpensive fluorophores, Dylight 650 and Alexa 647, and applied it to the detection of hs-cTnI spiked in human serum using a two-dimensional chromatography-based immunosensor. The tracers showed a limit of detection of 0.002 ng/mL for Dylight 650 and 0.007 ng/mL for Alexa 647. The standard curves linearized via log-logit transformation exhibited regression lines with correlation coefficients (R-2) > 0.97. The total coefficient of variation for the overall standard curve was 3.4 +/- 3.3% for the Dylight fluorophore and 5.9 +/- 1.5% for the Alexa dye. Such performances were comparable to those of the reference systems employing sophisticated technologies, Pathfast (Mitsubishi, Japan) and i-STAT (Abbott, US), with a strong correlation (R2 > 0.91) for the concentration range < 100 pg/mL. (C) 2016 Elsevier B.V. All rights reserved.