Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbium-to-Quantum Dot Förster Resonance Energy Transfer.

Although antibodies remain a primary recognition element in all forms of biosensing, functional limitations arising from their size, stability, and structure have motivated the development and production of many different artificial scaffold proteins for biological recognition. However, implementing such artificial binders into functional high-performance biosensors remains a challenging task. Here, we present the design and application of Förster resonance energy transfer (FRET) nanoprobes comprising small artificial proteins (αRep bidomains) labeled with a Tb complex (Tb) donor on the C-terminus and a semiconductor quantum dot (QD) acceptor on the N-terminus. Specific binding of one or two protein targets to the αReps induced a conformational change that could be detected by time-resolved Tb-to-QD FRET. These single-probe FRET switches were used in a separation-free solution-phase assay to quantify different protein targets at sub-nanomolar concentrations and to measure the conformational changes with sub-nanometer resolution. Probing ligand-receptor binding under physiological conditions at very low concentrations in solution is a special feature of FRET that can be efficiently combined with other structural characterization methods to develop, understand, and optimize artificial biosensors. Our results suggest that the αRep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live-cell imaging of ligand-receptor interactions.

Advanced microRNA-based cancer diagnostics using amplified time-gated FRET.

MicroRNAs (miRNAs) play an important role in cellular functions and in the development and progression of cancer. Precise quantification of endogenous miRNAs from different clinical patient and control samples combined with a one-to-one comparison to standard technologies is a challenging but necessary endeavor that is largely neglected by many emerging fluorescence technologies. Here, we present a simple, precise, sensitive, and specific ratiometric assay for absolute quantification of miRNAs. Isothermally amplified time-gated Förster resonance energy transfer (TG-FRET) between Tb donors and dye acceptors resulted in miRNA assays with single-nucleotide variant specificity and detection limits down to 4.2 ± 0.5 attomoles. Quantification of miR-21 from human tissues and plasma samples revealed the relevance for breast and ovarian cancer diagnostics. Analysis of miR-132 and miR-146a from acute monocytic leukemia cells (THP-1) demonstrated the broad applicability to different miRNAs and other types of clinical samples. Direct comparison to the gold standard RT-qPCR showed advantages of amplified TG-FRET concerning precision and specificity when quantifying low concentrations of miRNAs as required for diagnostic applications. Our results demonstrate that a careful implementation of rolling circle amplification and TG-FRET into one straightforward nucleic acid detection method can significantly advance the possibilities of miRNA-based cancer diagnostics and research.

Self-Assembled Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery.

Nanoparticles combining enhanced cellular drug delivery with efficient fluorescence detection are important tools for the development of theranostic agents. Here, we demonstrate this concept by a simple, fast, and robust protocol of cationic polymer-mediated gold nanocluster (Au NCs) self-assembly into nanoparticles (NPs) of ca. 120 nm diameter. An extensive characterization of the monodisperse and positively charged NPs revealed pH-dependent swelling properties, strong fluorescence enhancement, and excellent colloidal and photostability in water, buffer, and culture medium. The versatility of the preparation is demonstrated by using different Au NC surface ligands and cationic polymers. Steady-state and time-resolved fluorescence measurements give insight into the aggregation-induced emission phenomenon (AIE) by tuning the Au NC interactions in the self-assembled nanoparticles using the pH-dependent swelling. In vitro studies in human monocytic cells indicate strongly enhanced uptake of the NPs compared to free Au NCs in endocytic compartments. The NPs keep their assembly structure with quite low cytotoxicity up to 500 µg Au/mL. Enhanced drug delivery is demonstrated by loading peptides or antibodies in the NPs using a one-pot synthesis. Fluorescence microscopy and flow cytometry confirmed intracellular colocalization of the biomolecules and the NP carriers with a respective 1.7-fold and 6.5-fold enhanced cellular uptake of peptides and antibodies compared to the free biomolecules.

Thin-coated water soluble CdTeS alloyed quantum dots as energy donors for highly efficient FRET.

The synthesis of highly luminescent water soluble CdTe(x)S(y) quantum dots (QDs) is described and their elemental composition and optical properties are fully characterized. Glutathione (GSH)-capped nanocrystals were obtained from an aqueous solution of CdCl2, Na2TeO3 and GSH in the presence of NaBH4 upon heating at 100 °C. Spherical CdTe(x)S(y) alloyed nanoparticles with diameters ranging from 2 to 4 nm were formed, and characterized by X-ray powder diffraction and Transmission Electron Microscopy. Their elemental composition was determined from Inductively Coupled Plasma Atomic Emission Spectroscopy and CHN elemental analysis experiments. A model for the determination of their molecular formulas, molecular weights and extinction coefficients is proposed. Surface GSH molecules were involved in amide bond formation with fluorescent Nile-Red molecules, to be used as energy acceptor in Förster resonance energy transfer (FRET) experiments. FRET was observed from the CdTe(x)S(y) core (λ(ex) = 430 nm) to the Nile-Red dye (λ(em) = 648 nm) with an almost quantitative FRET efficiency (η(FRET) = 98%). A detailed analysis of the FRET is presented, revealing a core-dye distance of 24 Å, in very good agreement with the estimated radius of the core (13 Å) as measured by TEM. The QDs present excellent photophysical properties (QY up to 29%), easy synthesis and can be isolated as solids and redispersed in water without loss of their photoluminescence efficiency.

A novel mixed valent Cu(II)-Cu(I) 2D framework made of a hydrazone and µ-SCN bridged metallacyclic loops cross-linked by µ3-SCN chains.

A mixed valent copper complex [Cu(II)Cu(I)(L)(µ-SCN)(µ(3)-SCN)](n) (LH = N'-((pyridin-2-yl)methylene)acetohydrazide) has been synthesized and characterized. It is a unique example of a 2D mixed valent Cu(II)-Cu(I) interlinked molecular assembly with a very unusual bridging property of the hydrazone ligand. An extraordinary in situ partial Cu(II)→ Cu(I) reduction is observed in this system at room temperature.