A Novel Homogeneous Time-Resolved Fluoroimmunoassay for Carcinoembryonic Antigen Based on Water-Soluble Quantum Dots
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Quantum dots are not widely used in clinical diagnosis. However, the homogeneous time-resolved fluorescence assay possesses many advantages over current methods for the detection of carcinoembryonic antigen (CEA), a primary marker for many cancers and diseases. Therefore, a novel luminescent terbium chelates- (LTCs) and quantum dots-based homogeneous time-resolved fluorescence assay was developed to detect CEA. Glutathione-capped quantum dots (QDs) were prepared from oil-soluble QDs with a 565 nm emission peak. Conjugates (QDs-6 F11) were prepared with QDs and anti-CEA monoclonal antibody. LTCs were prepared and conjugates (LTCs-S001) were prepared with another anti-CEA monoclonal antibody. The fluorescence lifetime of QDs was optimized for sequential analysis. The Förster distance (R0) was calculated as 61.9 Å based on the overlap of the spectra of QDs-6 F11 and LTCs-S001. Using a double-antibody sandwich approach, the above antibody conjugates were used as energy acceptor and donor, respectively. The signals from QDs were collected in time-resolved mode and analyzed for the detection of CEA. The results show that the QDs were suitable for time-resolved fluoroassays. The spatial distance of the donor-acceptor pair was calculated to be 61.9 Å. The signals from QDs were proportional to CEA concentration. The standard curve was LogY = 2.75566 + 0.94457 LogX (R = 0.998) using the fluorescence counts (Y) of QDs and the concentrations of CEA (X). The calculated sensitivity was 0.4 ng/mL. The results indicate that water-soluble QDs are suitable for the homogenous immunoassay. This work has expanded future applications of QDs in homogeneous clinical bioassays. Furthermore, a QDs-based homogeneous multiplex immunoassay will be investigated as a biomarker for infectious diseases in future research.
KeywordsWater-soluble quantum dots Homogeneous immunoassay FRET Terbium chelates Carcinoembryonic antigen
List of Abbreviations
- 6 F11
Anti-CEA monoclonal antibody
Bis(sulfosuccinimidyl) suberate sodium salt
Anhydrous dimethyl sulfoxide
Diethylene triamine pentacetic acid
Diethylenetriaminepentaacetic acid dianhydride
(N-(3-dimethyllaminopropyl) carbodiimide hydrochloride
Förster resonance energy transfer
Full-width at half maximum
Homogeneous time-resolved fluoroassays
Luminescent terbium chelates
Oil-soluble quantum dots
Phosphate buffered saline
Anti-CEA monoclonal antibody
N-hydroxysulfosuccinimide sodium salt
Time-resolved Förster resonance transfer
Water-soluble quantum dots
The work was supported by the National Natural Science Foundation of China (Grant No. 30901382, 81271931), the Natural Science Foundation of Guangdong Province (No. S2012010009547), the New Teacher for Doctoral Fund of Ministry of Education of China (Grant No. 20094433120008), Special Funds for College and University Talents by Guangdong Province (2009) and Scientific Research Foundation of Introducing Talents of Southern Medical University(2009).
The National Natural Science Foundation of China (Grant No. 30901382, 81271931), the New Teacher for Doctoral Fund of Ministry of Education of China (Grant No. 20094433120008), the Natural Science Foundation of Guangdong Province (No. S2012010009547), Special Funds for College and University Talents by Guangdong Province (2009), and the Scientific Research Foundation of Introducing Talents of Southern Medical University(2009).
The Ethical Committee of Science and Technology Department of Southern Medical University approved this study (REC number: 20121058B).
Mei-Jun Chen, Jing-Yuan Hou and Da Sun contributed to experimental work. Tian-Cai Liu and Ying-Song Wu researched literature and conceived the study. Mei-Jun Chen and Zhi-Qi Ren were involved in protocol development, gaining ethical approval, and data analysis. Mei-Jun Chen wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.
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