Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.

Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide-mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

Fluorescence imaging of cells and subcellular compartments is an essential tool to investigate biological processes and to evaluate the development and progression of diseases. In particular, protein-protein interactions can be monitored by Förster resonance energy transfer (FRET) between two proximal fluorophores that are attached to specific recognition biomolecules such as antibodies. We investigated the membrane expression of E- and N-cadherins in three different cell lines used as model systems to study epithelial to mesenchymal transition (EMT) and a possible detection of circulating tumour cells (CTCs). EMT is a key process in cancer metastasis, during which epithelial markers (such as E-cadherin) are down-regulated in the primary tumour whereas mesenchymal markers (such as N-cadherin) are up-regulated, leading to enhanced cell motility, intravasation, and appearance of CTCs. Various FRET donor-acceptor pairs and protein recognition strategies were utilized, in which Lumi4-Tb terbium complexes (Tb) and different organic dyes were conjugated to several distinct E- and N-cadherin-specific antibodies. Pulsed excitation of Tb at low repetition rates (100 Hz) and time-gated (TG) imaging of both the Tb-donor and the dye-acceptor photoluminescence (PL) allowed efficient detection of the EMT markers as well as FRET in the case of sufficient donor-acceptor proximity. Efficient FRET was observed only between two E-cadherin-specific antibodies and further experiments indicated that these antibodies recognized the same E-cadherin molecule, suggesting a limited accessibility of cadherins when they are clustered at adherens junctions. The investigated Tb-to-dye FRET systems provided reduced photobleaching compared to the AlexaFluor 488-568 donor-acceptor pair. Our results demonstrate the applicability and advantages of Tb-based TG FRET for efficient and stable imaging of antibody-antibody interactions on different cell lines. They also reveal the limitations of interpreting colocalization on cell membranes in the case of lacking FRET signals.

Lanthanides and quantum dots as Förster resonance energy transfer agents for diagnostics and cellular imaging.

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.

Nanobodies and nanocrystals: highly sensitive quantum dot-based homogeneous FRET immunoassay for serum-based EGFR detection.

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.

Quantum-dot-basedFörster resonance energy transfer immunoassay for sensitive clinical diagnostics of low-volume serum samples.

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.

Activated phosphonated trifunctional chelates for highly sensitive lanthanide-based FRET immunoassays applied to total prostate specific antigen detection.

The first example of an activated phosphonated trifunctional chelate (TFC) is presented, which combines a non-macrocyclic coordination site for lanthanide coordination based on two aminobis-methylphosphonate coordinating arms, a central bispyrazolylpyridyl antenna and an N-hydroxysuccinimide ester in para position of the central pyridine as an activated function for the labeling of biomaterial. The synthesis of the TFC is presented together with photo-physical studies of the related Tb and Eu complexes. Excited state lifetime measurements in H2O and D2O confirmed an excellent shielding of the cation from water molecules with a hydration number of zero. The Tb complex provides a high photoluminescence (PL) quantum yield of 24% in aqueous solutions (0.01 M Tris-HCl, pH 7.4) and a very long luminescence lifetime of 2.6 ms. The activated ligand was conjugated to different biological compounds such as streptavidin, and a monoclonal antibody against total prostate specific antigen (TPSA). In combination with AlexaFluor647 (AF647) and crosslinked allophycocyanin (XL665) antibody (ABs) conjugates, homogeneous time-resolved Fluorescence Resonance Energy Transfer (FRET) immunoassays of TPSA were performed in serum samples. The Tb donor-dye acceptor FRET pairs provided large Förster distances of 5.3 nm (AF647) and 7.1 nm (XL665). A detailed time-resolved FRET analysis of Tb donor and dye acceptor PL decays revealed average donor-acceptor distances of 4.2 nm (AF647) and 6.3 nm (XL665) within the sandwich immunocomplex and FRET efficiencies of 0.79 and 0.68, respectively. Very low detection limits of 1.4 ng mL(-1) (43 pM) and 2.4 ng mL(-1) (74 pM) TPSA were determined using a KRYPTOR fluorescence immunoanalyzer. These results demonstrate the applicability of our novel Tb-bioconjugates for highly sensitive clinical diagnostics.