K. David Wegner

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.

A Rapid, Amplification-Free, and Sensitive Diagnostic Assay for Single-Step Multiplexed Fluorescence Detection of MicroRNA.

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 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high-throughput and point-of-care clinical diagnostics.

Upconverting nanoparticle to quantum dot FRET for homogeneous double-nano biosensors

Both upconverting nanoparticles (UCNPs) and semiconductor quantum dots (QDs) have revolutionized optical biosensing because of their unique photophysical properties. However, their outstanding photostability, near-infrared (NIR) excitability, and colour tunability have never been combined for homogeneous mix-and-measure FRET (Forster resonance energy transfer) biosensors that do not require any washing or separation steps. Here we demonstrate that UCNP-to-QD FRET systems can be used for rapid homogeneous bioassays, which are essential tools for clinical diagnostics. One of the main drawbacks of UCNPs for FRET, namely their very low photoluminescence (PL) quantum yields, was efficiently overcome by using QD FRET acceptors with very strong spectral overlap with the UCNP donors. This resulted in unrivalled Forster distances for UCNP-based FRET pairs of up to 6 nm. We could quantify the prototypical analyte biotin (vitamin H) at low nanomolar concentrations and steady-state and time-resolved PL analysis showed that UCNP-to-QD FRET was caused by streptavidin-to-biotin binding. Immediate applicability in biosensing was demonstrated by biotin replacement assays over a large concentration range with IC50 values between 8 nM and 250 nM and detection limits down to 5 nM. The high photostability of the double-nanoparticle biosensor, the NIR excitation of UCNPs for minimal autofluorescence, and the spectral multiplexing capability of QDs offer a large potential for spectroscopy and imaging-based biosensing beyond in vitro diagnostics.

Influence of Luminescence Quantum Yield, Surface Coating, and Functionalization of Quantum Dots on the Sensitivity of Time-Resolved FRET Bioassays

In clinical diagnostics, homogeneous time-resolved (TR) FRET immunoassays are used for fast and highly sensitive detection of biomarkers in serum samples. The most common immunoassay format is based on europium chelate or cryptate donors and allophycocyanin acceptors. Replacing europium donors with terbium complexes and the acceptors with QDs offers large photophysical advantages for multiplexed diagnostics, because the Tb-complex can be used as FRET donor for QD acceptors of different colors. Water-soluble and biocompatible QDs are commercially available or can be synthesized in the laboratory using many available recipes from the literature. Apart from the semiconductor material composition, an important aspect of choosing the right QD for TR-FRET assays is the thickness of the QD coating, which will influence the photophysical properties and long-term stability as well as the donor-acceptor distance and FRET efficiency. Here we present a detailed time-resolved spectroscopic study of three different QDs with an emission maximum around 605 nm for their application as FRET acceptors (using a common Tb donor) in TR-bioassays: (i) Invitrogen/Life Technologies Qdot605, (ii) eBioscience eFluorNC605 and iii) ter-polymer stabilized CdSe/CdS/ZnS QDs synthesized in our laboratories. All FRET systems are very stable and possess large Förster distances (7.4-9.1 nm), high FRET efficiencies (0.63-0.80) and low detection limits (0.06-2.0 pM) within the FRET-bioassays. Shapes, sizes and the biotin/QD ratio of the biocompatible QDs could be determined directly in the solution phase bioassays at subnanomolar concentrations. Both commercial amphiphilic polymer/lipid encapsulated QDs and self-made ligand-exchanged QDs provide extremely low detection limits for highly sensitive TR-FRET bioassays.

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.

Chapter 2:Quantum Dots in the Analysis of Food Safety and Quality

The detection of chemical residues, toxins, pathogens and allergens contaminating food and water is of utmost importance to society. Although numerous strategies have been developed to detect, isolate and identify potential threats in food, there remains great demand for assays that enhance the speed, sensitivity and selectivity of detection in formats that are simple, portable and low cost. Quantum dots are brightly fluorescent semiconductor nanocrystals with many physical and optical properties that can help address the challenges associated with developing improved assays for food safety and quality. This chapter summarizes research toward the utilization of quantum dots in assays for the detection of analytes such as pathogens, pesticides, antibiotics and genetically modified organisms (GMOs). A short primer on the properties and bioconjugation of quantum dots is also included. Numerous studies have demonstrated the potential for quantum dots to enhance analytical figures of merit in food safety and quality assays; however, strategic research is needed to develop quantum dot-enabled assays that will have the greatest opportunity to impact food safety practices in industry and society.