A fluorescent immunosensor for high-sensitivity cardiac troponin I using a spatially-controlled polymeric, nano-scale tracer to prevent quenching.
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.
Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 136-701, Korea.
Sung-Min
SM
Seo
Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 136-701, Korea.
Seung-Wan
SW
Kim
Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 136-701, Korea.
Ji-Na
JN
Park
Digital Optics, 204, Sicox Tower, 513-14, Sangdaewon-dong, Seongnam 462-120, Korea.
Jung-Hwan
JH
Cho
Digital Optics, 204, Sicox Tower, 513-14, Sangdaewon-dong, Seongnam 462-120, Korea.
Hee-Soo
HS
Kim
Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 136-701, Korea; Department of Biotechnology, Korea University, 2511 Sejong-ro, Sejong 339-700, Korea. Electronic address: shpaek@korea.ac.kr.
Se-Hwan
SH
Paek
D000906:Antibodies / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D011108:Polymers / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry*; D019210:Troponin I / Q000097:blood*
D000906:Antibodies; D005456:Fluorescent Dyes; D011108:Polymers; D019210:Troponin I; D019809:Streptavidin
Cross-flow chromatography; Distal fixation of fluorophore in the conjugate; High-sensitivity cardiac troponin I; Nano-scale fluorescent tracer; Rapid immunosensor; Self-quenching of fluorescence; Stepwise protein polymerization