Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET.

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 prostate-specific antigen (PSA) from a single serum sample by multiplexed Tb-to-QD FRET.

Sensitivity Enhancement of Förster Resonance Energy Transfer Immunoassays by Multiple Antibody Conjugation on Quantum Dots.

Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (AB) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many AB per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the AB on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate.

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.

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.

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.

Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials.

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells.

Simplistic attachment and multispectral imaging with semiconductor nanocrystals.

Advances in spectral deconvolution technologies are rapidly enabling researchers to replace or enhance traditional epifluorescence microscopes with instruments capable of detecting numerous markers simultaneously in a multiplexed fashion. While significantly expediting sample throughput and elucidating sample information, this technology is limited by the spectral width of common fluorescence reporters. Semiconductor nanocrystals (NC's) are very bright, narrow band fluorescence emitters with great potential for multiplexed fluorescence detection, however the availability of NC's with facile attachment chemistries to targeting molecules has been a severe limitation to the advancement of NC technology in applications such as immunocytochemistry and immunohistochemistry.

The controlled display of biomolecules on nanoparticles: a challenge suited to bioorthogonal chemistry.

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture.

Rapid screening of genetic biomarkers of infectious agents using quantum dot barcodes.

The development of a rapid and sensitive infectious disease diagnostic platform would enable one to select proper treatment and to contain the spread of the disease. Here we examined the feasibility of using quantum dot (QD) barcodes to detect genetic biomarkers of the bloodborne pathogens HIV, malaria, hepatitis B and C, and syphilis. The genetic fragments from these pathogens were detected in less than 10 min at a sample volume of 200 μL and with a detection limit in the femtomol range.

Tracking spatial disorder in an optical ruler by time-resolved NSET.

For biomolecular applications, potential interactions between newly developed dye molecules and the biomolecule of interest can dramatically influence the accuracy of optical ruler techniques. By utilizing nanometal surface energy transfer (NSET), an optical technique is developed that allows the nature of interactions between dyes and a biomolecule, namely DNA, to be directly assessed. To demonstrate the method, interactions between well-known molecular dyes based on carboxyfluorescein (FAM, noninteracting) and Cy5 (known intercalator) with DNA is probed.