Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Chen, Meijun; Wu, Yixuan; Lin, Guanfeng; Hou, Jingyuan; Li, Ming; Liu, Tiancai

doi:10.1016/j.aca.2012.06.042

Cited by 63 publications

(36 citation statements)

References 38 publications

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter <13 nm, PL quantum yield up to 66% at 705 nm, and colloidal stability of several months in various buffers. They were applied as FRET acceptors in homogeneous, time-gated immunoassays using Tb-antibodies as FRET donors, both coupled by an immunological sandwich complex between the two antibodies and a PSA (prostate specific antigen) biomarker. The advantages of the compact surface coating for FRET could be demonstrated by an 6.2 and 2.5 fold improvement of the limit of detection (LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL(-1) obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL(-1)) and demonstrate their direct applicability in clinical diagnostics.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…Luminescent terbium chelates (LTC) are also conjugated to an anti-AFP antibody, and in the presence of AFP, the target is sandwiched, and brings the QDs and LTC in close proximity to each other, initiating FRET. Adapted with permission from ref 198. Copyright 2012 Elsevier.…”

Section: Figurementioning

confidence: 99%

Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence

et al. 2015

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“…Dilution is a very common method to reduce the interference of the serum autofluorescence [30][31][32][33][34]. The effects of the concentration of BSA in reaction buffer are shown in Fig.…”

Section: Influences Of Serum Autofluorescencementioning

confidence: 99%

Homogeneous immunoassay for the cancer marker alpha-fetoprotein using single wavelength excitation fluorescence cross-correlation spectroscopy and CdSe/ZnS quantum dots and fluorescent dyes as labels

et al. 2015

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The article describes sensitive and selective homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP) in human serum by using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). Both competitive and sandwich immunoassay modes were applied, and AFP served as a model analyte. Fluorescent CdSe/ZnS quantum dots (with a 655 nm emission peak) and the fluorophore Alexa Fluor 488 (520 nm emission) were chosen to label the antibodies in the sandwich mode, and the antibody and the antigen in the competitive mode. Under optimized conditions, the sandwich assay has a linear dynamic range that covers the 20 pM to 5.0 nM concentration range. The competitive assay, in turn, extends from 180 pM to 15.0 nM. The respective detection limits are 20 pM and 180 pM. The method was successfully applied to directly determine AFP in (spiked) clinical samples, and results were in good agreement with data obtained via ELISAs.

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Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Cited by 63 publications

References 38 publications

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

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

Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence

Homogeneous immunoassay for the cancer marker alpha-fetoprotein using single wavelength excitation fluorescence cross-correlation spectroscopy and CdSe/ZnS quantum dots and fluorescent dyes as labels

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