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Time-gated Förster resonance energy transfer (TG-FRET) between Tb complexes and luminescent semiconductor quantum dots (QDs) provides highly advantageous photophysical properties for multiplexed biosensing. Multiplexed Tb-to-QD FRET immunoassays possess a large potential for in vitro diagnostics, but their performance is often insufficient for their application under clinical conditions. Here, we developed a homogeneous TG-FRET immunoassay for the quantification of carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and prostatespecific antigen (PSA) from a single serum sample by multiplexed Tb-to-QD FRET. Tb–IgG antibody donor conjugates were combined with compact QD-F(ab’)2 antibody acceptor conjugates with three different QDs emitting at 605, 650, and 705 nm. Upon antibody–antigen–antibody Sandwich complex formation, the QD acceptors were sensitized via FRET from Tb, and the FRET ratios of QD and Tb TG luminescence intensities increased specifically with increasing antigen concentrations.
Although limits of detection (LoDs: 3.6 ng/mL CEA, 3.5 ng/mL NSE, and 0.3 ng/mL PSA) for the triplexed assay were slightly higher compared to the single-antigen assays, they were still in a clinically relevant concentration range and could be quantified in 50 μL serum samples on a B·R·A·H·M·S KRYPTOR Compact PLUS clinical immunoassay plate reader. The simultaneous quantification of CEA, NSE, and PSA at different concentrations from the same serum sample demonstrated actual multiplexing Tb-to-QD FRET immunoassays and the potential of this technology for translation into clinical diagnostics.
In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Förster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies. Methods: We coupled the fluorophores DY-682 (donor; absorption [abs]/emission [em], 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system with multispectral analysis features. Results: When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization of the DY-682-IgGDY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice. Conclusion: Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.
DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye−dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA Origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pHresponsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences.
DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye–dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pH-responsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences.
In Part II of this Review series on lanthanide-doped photon-upconversion nanoparticles (UCNPs), we present and critically discuss the Performance and suitability of UCNPs as background-free luminescent Reporters in bioimaging and bioanalytical applications. The preparation of a biocompatible nanoparticle surface is an integral step for all life - science-related applications. UCNPs have found their way into a large number of diagnostic platforms, homogeneous and heterogeneous assay formats, and sensor applications. Many bioanalytical detection schemes involve Förster resonance energy transfert (FRET), which is still debated for UCNPs and Needs to be much improved. The Need for dedicated and standardized instruments as well as recent studies on the Dissolution and potential toxicity of UCNPs are addressed. Finally we outline future Trends and challenges in the field of upconversion.
The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short-sequence nucleic acids next-generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single-step, single-temperature, and amplification-free assay provides a unique combination of performance parameters compared to state-of-the-art miRNA detection technologies. Precise multiplexed quantification of miRNA-20a, -20b, and -21 at concentrations between 0.05 and 0.5 nm in a single 150 mL sample and detection limits between 0.2 and 0.9 nm in 7.5 mL serum samples demonstrate the feasibility of both highthroughput and point-of-care clinical diagnostics.
Upconversion nanoparticles (UCNPs) are attractive candidates for energy transfer-based analytical applications. In contrast to classical donor−acceptor pairs, these particles contain many emitting lanthanide ions together with numerous acceptor dye molecules at different distances to each other, strongly depending on the particle diameter.
UCNPs with precisely controlled sizes between 10 and 43 nm were prepared and functionalized with rose bengal and sulforhodamine B by a ligand-exchange procedure. Timeresolved studies of the upconversion luminescence of the UCNP donor revealed a considerable shortening of the donor lifetime as a clear hint for Förster resonance energy transfer (FRET). FRET was most pronounced for 21 nm-sized UCNPs, yielding a FRET efficiency of 60%. At larger surface-to-volume ratios, the FRET efficiency decreased by an increasing competition of nonradiative surface deactivation. Such dye-UCNP architectures can also provide an elegant way to shift the UCNP emission color, since the fluorescence intensity of the organic dyes excited by FRET was comparable to that of the upconversion emission of smaller particles.
DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single-molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G-quadruplex structures in the presence of monovalent cations (e.g. Na+ and K+) is currently used for the detection of K+ ions, however, with insufficient selectivity towards Na+. By means of FRET between two suitable dyes attached to the 3'- and 5'-ends of telomeric DNA we demonstrate that the formation of G-quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.
Artificial light harvesting complexes find applications in artificial photosynthesis, photovoltaics and light harvesting chemical sensors. They are used to enhance the absorption of light of a reaction center which is often represented by a single acceptor. Here, we present different light harvesting systems on DNA origami structures and analyze systematically the light harvesting efficiency. By changing the number and arrangement of different fluorophores (FAM as donor, Cy3 as transmitter and Cy5 as acceptor molecules) the light harvesting efficiency is optimized to create a broadband absorption and to improve the antenna effect 1 (including two energy transfer steps) from 0.02 to 1.58, and the antenna effect 2 (including a single energy transfer step) from 0.04 to 8.7, i.e. the fluorescence emission of the acceptor is significantly higher when the light-harvesting antenna is excited at lower wavelength compared to direct excitation of the acceptor. The channeling of photo energy to the acceptor proceeds by Förster Resonance Energy Transfer (FRET) and we carefully analyze also the FRET efficiency of the different light harvesting systems. Accordingly, the antenna effect can be tuned by modifying the stoichiometry of donor, transmitter and acceptor dyes, whereas the FRET efficiency is mainly governed by the spectroscopic properties of dyes and their distances.
The folding of single-stranded telomeric DNA into guanine (G) quadruplexes is a conformational change that plays a major role in sensing and drug targeting. The telomeric DNA can be placed on DNA origami nanostructures to make the folding process extremely selective for K+ ions even in the presence of high Na+ concentrations. Here, we demonstrate that the K+-selective G-quadruplex formation is reversible when using a cryptand to remove K+ from the G-quadruplex. We present a full characterization of the reversible switching between single-stranded telomeric DNA and G-quadruplex structures using Förster resonance energy transfer (FRET) between the dyes fluorescein (FAM) and cyanine3 (Cy3). When attached to the DNA origami platform, the G-quadruplex switch can be incorporated into more complex photonic networks, which is demonstrated for a three-color and a four-color FRET cascade from FAM over Cy3 and Cy5 to IRDye700 with G-quadruplex-Cy3 acting as a switchable transmitter.