Biosensing and imaging based on bioluminescence resonance energy transfer. Curr Opin Biotechnol 20:37

Department of Radiology & Bio-X Program, Stanford University School of Medicine, Stanford, CA 94305-5484, USA.
Current Opinion in Biotechnology (Impact Factor: 7.12). 03/2009; 20(1):37-44. DOI: 10.1016/j.copbio.2009.01.001
Source: PubMed


Bioluminescence resonance energy transfer (BRET) operates with biochemical energy generated by bioluminescent proteins to excite fluorophores and offers additional advantages over fluorescence energy transfer (FRET) for in vivo imaging and biosensing. While fluorescent proteins are frequently used as BRET acceptors, both small molecule dyes and nanoparticles can also serve as acceptor fluorophores. Semiconductor fluorescent nanocrystals or quantum dots (QDs) are particularly well suited for use as BRET acceptors due to their high quantum yields, large Stokes shifts and long wavelength emission. This review examines the potential of QDs for BRET-based bioassays and imaging, and highlights examples of QD-BRET for biosensing and imaging applications. Future development of new BRET acceptors should further expand the multiplexing capability of BRET and improve its applicability and sensitivity for in vivo imaging applications.

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    • "Absence of these effects results in a low background and very low limits of detection for BRET assays [6]. Due to these characteristics, BRET has been used for a variety of applications including RNA detection [7], investigating protein-protein interactions [8]–[10], drug screening [5], [9], [11], imaging [8], [12], [13], and general biosensing [14], [15]. So far, however, uses of BRET have largely been restricted to fundamental research, often using sophisticated imaging equipment. "
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    ABSTRACT: Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now. We used a thrombin bioprobe based on a form of BRET (BRET(H)), which uses the BRET(1) substrate, native coelenterazine, with the typical BRET(2) donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H) ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay. The BRET(H) thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET(1)-based version but it is substantially brighter than the BRET(2) version. The normalised BRET(H) ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays. These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H) provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.
    PLoS ONE 02/2014; 9(2):e88399. DOI:10.1371/journal.pone.0088399 · 3.23 Impact Factor
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    • "We chose the budding yeast, Saccharomyces cerevisiae, because of its easiness to handle and its low cost of use (reviewed in [16]). By fusing the donor and acceptor moieties to the interacting proteins, BRET can be used to monitor protein interactions in vitro , in living cells and in vivo [17] [18] [19] [20]. "

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    • "Fusion of the tag with proteins of better stability and longer half-life could prolong fluorescent signal retention with target cells. Another potential application is to use HaloTag fusion proteins, in conjunction with bioluminescence, for in vivo BRET imaging [13]. With the current success and wide adaptation of the HaloTag technology for in vitro applications, our results suggest that the technology also has potential to become a versatile platform for in vivo imaging. "
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    ABSTRACT: Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.
    Current Chemical Genomics 09/2012; 6:48-54. DOI:10.2174/1875397301206010048
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