A Novel Homogeneous Time-Resolved Fluoroimmunoassay for Carcinoembryonic Antigen Based on Water-Soluble Quantum Dots

Abstract

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.

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

“…632 To investigate and prove this concept, Liu et al utilized in-house-synthesized Tb-complexes and QDs and demonstrated TG Tb-to-QD FRET homogeneous immunoassays for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in buffer. 629,630 Extending their applicability to clinical diagnostics and multiplexing, Hildebrandt et al…”

Compact quantum dot–antibody conjugates for FRET immunoassays with subnanomolar detection limits

“…FRET immunoassays have been developed with QD-donors [12][13][14][15][16][17][18][19] as well as QD-acceptors. 11,[20][21][22][23][24] The latter case is of particular interest, because these FRET assays used Tb-complexes as donors, whose extremely long excited-state lifetimes allow for time-gated detection and a concomitant suppression of directly excited QD PL and autofluorescence background. 3,8,9 Such Tb-to-QD energy transfer systems have been shown to follow FRET theory 25,26 and were successfully used in various homogeneous FRET biosensing applications, ranging from molecular rulers, 27,28 over nucleic acid detection and enzyme kinetics, 29,30 to molecular logic gates.…”

Multiplex immunofluorescence methods in neurodegenerative diseases

“…More recently, alternative methods for in situ detection were proposed, such as those replacing fluorescent-based reporters for quantum dots that are directly conjugated in primary antibodies. Quantum dots allow for a higher number of probes to be detected at the same tissue because of reduced spectral overlap (Byers and Hitchman, 2011;Chan et al, 2005;Chen et al, 2013;Dixon et al, 2015;Krenacs et al, 2010;Mansfield, 2017;Prost et al, 2016;Sweeney et al, 2008;Tholouli et al, 2008;Wu et al, 2003;Xu et al, 2013), however, quantum dots and other similar techniques require expensive, specialized equipment.…”