A myriad of quantum dot (QD) biosensor examples have emerged from the literature over the past decade, but despite their photophysical advantages, QDs have yet to find acceptance as standard fluorescent reagents in clinical diagnostics. Lack of reproducible, stable, and robust immunoassays using easily prepared QD-antibody conjugates has historically plagued this field, preventing researchers from advancing the deeper issues concerning assay sensitivity and clinically relevant detection limits on low-volume serum samples. Here we demonstrate a ratiometric multiplexable FRET immunoassay using Tb donors and QD acceptors, which overcomes all the aforementioned limitations toward application in clinical diagnostics. We demonstrate the determination of prostate specific antigen (PSA) in 50 μL serum samples with subnanomolar (1.6 ng/mL) detection limits using time-gated detection and two different QD colors. This concentration is well below the clinical cutoff value of PSA, which demonstrates the possibility of direct integration into real-life in vitro diagnostics. The application of IgG, F(ab')2, and F(ab) antibodies makes our homogeneous immunoassay highly flexible and ready-to-use for the sensitive and specific homogeneous detection of many different biomarkers.
Luminescent lanthanide labels (LLLs) and semiconductor quantum dots (QDs) are two very special classes of (at least partially) inorganic fluorophores, which provide unique properties for Förster resonance energy transfer (FRET). FRET is an energy-transfer process between an excited donor fluorophore and a ground-state acceptor fluorophore in close proximity (approximately 1-20 nm), and therefore it is extremely well suited for biosensing applications in optical spectroscopy and microscopy. Within this cogent review, we will outline the main photophysical advantages of LLLs and QDs and their special properties for FRET. We will then focus on some recent applications from the FRET biosensing literature using LLLs as donors and QDs as donors and acceptors in combination with several other fluorophores. Recent examples of combining LLLs and QDs for spectral and temporal multiplexing from single-step to multistep FRET demonstrate the versatile and powerful biosensing capabilities of this unique FRET pair. As this review is published in the Forum on Imaging and Sensing, we will also present some new results of our groups concerning LLL-based time-gated cellular imaging with optically trifunctional antibodies and LLL-to-QD FRET-based homogeneous sandwich immunoassays for the detection of carcinoembryonic antigen.
Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain thick polyethylene glycol (PEG)-based coatings in order to retain the advantageous QD properties in biological media such as blood, serum or plasma. On the other hand, the application of QDs in Förster resonance energy transfer (FRET) immunoassays, one of the most sensitive and most common fluorescence-based techniques for non-competitive homogeneous biomarker diagnostics, is limited by such thick coatings due to the increased donor-acceptor distance. In particular, the combination with large IgG antibodies usually leads to distances well beyond the common FRET range of approximately 1 to 10 nm. Herein, time-gated detection of Tb-to-QD FRET for background suppression and an increased FRET range is combined with single domain antibodies (or nanobodies) for a reduced distance in order to realize highly sensitive QD-based FRET immunoassays. The "(nano)(2) " immunoassay (combination of nanocrystals and nanobodies) is performed on a commercial clinical fluorescence plate reader and provides sub-nanomolar (few ng/mL) detection limits of soluble epidermal growth factor receptor (EGFR) in 50 μL buffer or serum samples. Apart from the first demonstration of using nanobodies for FRET-based immunoassays, the extremely low and clinically relevant detection limits of EGFR demonstrate the direct applicability of the (nano)(2-) assay to fast and sensitive biomarker detection in clinical diagnostics.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.