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

• Synthetic scheme for spatially-controlled polymeric, nano-scale tracer was devised.
• Commercial fluorophores employed in the nano-tracer showed minimal self-quenching.
• The fluorescent nano-tracer was used in 2D-chromatographic immunosensor for hs-cTnI.
• The immunosensor revealed a detection limit of 0.002–0.007 ng/mL hs-cTnI.

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 µm 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 (R2)>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.