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A method for fast and highly sensitive detection of antibodies in serum would greatly facilitate the early diagnosis of disease and infection and dose optimization of therapeutic antibody. Bioluminescence detection with LUMABS (renamed mNeonG-LUMABS, where mNeonG is short for mNeonGreen) sensors based on bioluminescence resonance energy transfer (BRET) between blue-emitting luciferase Nluc and green fluorescent protein (FP) mNeonGreen has been demonstrated to enable fast detection of antibodies directly in serum with reasonable sensitivity. However, some mNeonG-LUMABS sensors exhibit low sensitivity, and thus, sensitivity improvement remains imperative. Here, we report a bright green FP, Clover4, obtained by structure-guided mutagenesis of green FP Clover. Despite similar brightness and fluorescence spectra of Clover and mNeonGreen, Clover4-LUMABS sensors exhibit a largely increased dynamic range (maximum 20-fold) and much lower limit of detection (LOD) (maximum 5.6-fold), most likely because Clover4 is positioned in a more parallel orientation to Nluc in LUMABS. Due to modular design, Clover4-LUMABS offers a general BRET system for fast and highly sensitive antibody detection in serum.

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.

MicroRNAs (miRNAs) have attracted extensive interest as promising biomarkers for the profiling of diseases. However, quantitative measurement of miRNAs presents a significant challenge in biochemical studies. In this work, we developed an innovative optofluidic platform to perform a rapid, simple, quantitative and high-specificity miRNA assay using the Förster resonance energy transfer (FRET) principle. A novel three-way junction FRET probe was proposed to enable rapid and enzyme-free miRNA detection. Using this platform, we performed one-step, amplification-free miRNA detection with simple device operation and achieved miRNA identification at a low concentration. The detection system could achieve high specificity for discrimination of three-base mismatches, and the sample volume was significantly reduced, favorable for low-level miRNA detection in material-limited samples. The establishment of a compact, low-cost, highly sensitive and selective miRNA analysis platform provides a valuable tool for point-of-care diagnosis. [ABSTRACT FROM AUTHOR]