Improved QD-BRET conjugates for detection and imaging

Molecular Imaging Program at Stanford (MIPS), Department of Radiology, School of Medicine, Stanford University, Stanford, CA 94305, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 09/2008; 372(3):388-94. DOI: 10.1016/j.bbrc.2008.04.159
Source: PubMed


Self-illuminating quantum dots, also known as QD-BRET conjugates, are a new class of quantum dot bioconjugates which do not need external light for excitation. Instead, light emission relies on the bioluminescence resonance energy transfer from the attached Renilla luciferase enzyme, which emits light upon the oxidation of its substrate. QD-BRET combines the advantages of the QDs (such as superior brightness and photostability, tunable emission, multiplexing) as well as the high sensitivity of bioluminescence imaging, thus holding the promise for improved deep tissue in vivo imaging. Although studies have demonstrated the superior sensitivity and deep tissue imaging potential, the stability of the QD-BRET conjugates in biological environment needs to be improved for long-term imaging studies such as in vivo cell tracking. In this study, we seek to improve the stability of QD-BRET probes through polymeric encapsulation with a polyacrylamide gel. Results show that encapsulation caused some activity loss, but significantly improved both the in vitro serum stability and in vivo stability when subcutaneously injected into the animal. Stable QD-BRET probes should further facilitate their applications for both in vitro testing as well as in vivo cell tracking studies.

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    • "Bioluminescence resonance energy transfer (BRET) is similar to FRET (Fluorescence Resonance Energy Transfer), except that the energy comes from a chemical reaction catalyzed by the donor enzyme rather than from absorption of excitation photons. Compared to fl uorescence imaging, bioluminescence has tremendously high sensitivity for in vivo imaging purposes (Xing et al. 2008) (Figure 4). "
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    • "However, two challenges facing in vivo fluorescent imaging are that the strong background autofluorescence is difficult to avoid, and a part of the external exciting light is absorbed and scattered by the tissue,19 so that the fluorescence intensity emitted by the QDs in deep tissue is sometimes too low to be detected or just simply too weak to be distinguished from the background autofluorescence. To overcome this limitation, So et al,20,21 Xing et al,22 Ma et al,23 and Kim et al,24 developed self-illuminating QDs based on the principle of bioluminescence resonance energy transfer (BRET). The fluorescence emitted by these QDs can be illuminated by the bioluminescence produced by the reaction between the enzymes and substrate around the QD surface. "
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    ABSTRACT: Quantum dots (QDs) show promise as novel nanomaterials for sentinel lymph node (SLN) mapping through their use in noninvasive in vivo fluorescence imaging, and they have provided remarkable results. However, in vivo fluorescence imaging has limitations mainly reflected in the strong autofluorescence and low deepness of tissue penetration associated with this technique. Here, we report on the use of self-illuminating 3-mercaptopropionic acid-capped CdTe/CdS QDs for mouse axillary SLN mapping by bioluminescence resonance energy transfer, which was found to overcome these limitations [corrected]. We used CdTe/CdS QDs synthesized in aqueous solution to conjugate a mutant of the bioluminescent protein, Renilla reniformis luciferase. The nanobioconjugates obtained had an average hydrodynamic diameter of 19 nm, and their luminescence catalyzed by the substrate (coelenterazine) could penetrate into at least 20 mm of hairless pigskin, which could be observed using an in vivo imaging system equipped with a 700 nm emission filter. Conversely, the fluorescence of the nanobioconjugates penetrated no more than 10 mm of pigskin and was observed with a strong background. When 80 μL of the nanobioconjugates (containing about 0.5 μmol/L of QDs) and 5 μL of coelenterazine (1 μg/μL) were intradermally injected into a mouse paw, the axillary SLN could be imaged in real time without external excitation, and little background interference was detected. Furthermore, the decayed luminescence of QD-Luc8 in SLNs could be recovered after being intradermally reinjected with the coelenterazine. Our data showed that using self-illuminating QDs, as opposed to fluorescence QDs, has greatly enhanced sensitivity in SLN mapping, and that the SLN could be identified synchronously by the luminescence and fluorescence of the self-illuminating QDs.
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    ABSTRACT: Quantum dots (QDs) are novel nanocrystal fluorophores with extremely high fluorescence efficiency and minimal photobleaching. They also possess a constant excitation wavelength together with sharp and symmetrical tunable emission spectra. These unique optical properties make them near-perfect fluorescent markers and there has recently been rapid development of their use for bioimaging. QDs can be conjugated to a wide range of biological targets, including proteins, antibodies, and nucleic acid probes, rendering them of particular interest to pathology researchers. They have been used in multiplex immunohistochemistry and in situ hybridization, which when combined with multispectral imaging, has enabled quantitative measurement of gene expression in situ. QDs have also been used for live in vivo animal imaging and are now being applied to an ever-increasing range of biological problems. These are detailed in this review, which also acts to outline the important advances that have been made in their range of applications. The relative novelty of QDs can present problems in their practical use and guidelines for their application are given.
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