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Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum
Published on: December 13, 2017
A fluorescent immunosensor for high-sensitivity cardiac troponin I using a spatially-controlled polymeric, nano-scale
Sung-Min Seo1, Seung-Wan Kim1, Ji-Na Park1
1Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 136-701, Korea.
Insights
This study developed a novel fluorescently-labeled polymeric antibody for highly sensitive cardiac troponin I detection. The new immunosensor achieved a limit of detection as low as 0.002 ng/mL, outperforming existing methods.
Area of Science:
- Biomedical Engineering
- Analytical Chemistry
- Immunosensor Technology
Background:
- High-sensitivity cardiac troponin I (hs-cTnI) detection is crucial for diagnosing acute myocardial infarction.
- Fluorescent quenching of detection antibodies can limit assay sensitivity.
- Novel signal amplification strategies are needed for improved hs-cTnI detection.
Purpose of the Study:
- To develop a novel fluorescently-labeled polymeric detection antibody for enhanced hs-cTnI detection.
- To minimize fluorescent quenching in polymeric tracers for improved signal amplification.
- To evaluate the performance of the developed immunosensor for hs-cTnI detection in human serum.
Main Methods:
- Synthesized a polymeric detection antibody by stepwise polymerization of fluorophore-coupled streptavidin (FL-SA) with biotinylated detection antibody (b-Ab).
- Controlled fluorophore spatial distribution using di-biotinylated oligonucleotides and filtered for uniform polymer size (~400nm).
- Applied the polymeric tracers (Dylight 650, Alexa 647) to a two-dimensional chromatography-based immunosensor for hs-cTnI detection.
Main Results:
- Achieved limits of detection of 0.002 ng/mL (Dylight 650) and 0.007 ng/mL (Alexa 647) for hs-cTnI.
- Standard curves showed strong linearity (R(2)>0.97) after log-logit transformation.
- Demonstrated comparable performance to commercial systems (Pathfast, i-STAT) with high correlation (R(2)>0.91) for low concentration ranges.
Conclusions:
- The developed fluorescently-labeled polymeric antibody enables highly sensitive hs-cTnI detection.
- The novel polymerization strategy effectively minimizes fluorescent quenching, enhancing signal amplification.
- This immunosensor technology offers a promising alternative for rapid and accurate cardiac troponin detection.
Abstract:
For detection of high-sensitivity cardiac troponin I (hs-cTnI<0.01ng/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 10nm 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 0.45µm filter to obtain a polymer of uniform size (i.e., ~400nm 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 0.002ng/mL for Dylight 650 and 0.007ng/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 (R(2)>0.91) for the concentration range <100pg/mL.
