HaloTag Protein-Mediated Site-Specific Conjugation of Bioluminescent Proteins to Quantum Dots (original) (raw)
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Bioconjugation of quantum dots: review & impact on future application
TrAC Trends in Analytical Chemistry, 2016
Nowadays luminescent semiconductor quantum dots (QDs) are widely applied in different areas due to their unique optical properties. QDs can be used as photoluminescent labels with excellent possibilities for high-throughput detection and diagnostics. For most of such applications QDs must be coupled to biomolecules, which often represents a fundamental challenge. Although QDs have a lot of advantages over organic dyes, most of the techniques that have been developed for QD functionalization and bioconjugation, are more complicated than the corresponding techniques for organic fluorescent dyes. Here, the importance of choosing a suitable bioconjugation strategy in different applications, such as imaging and assays is described. The main goal of this review is to give a structured and detailed overview and comparison of the most widely used conjugation strategies in function of the active groups (carboxyl, amine, thiol, epoxy, hydroxyl and aldehyde groups) present on QD surface.
Quantum dot bioconjugates for imaging, labelling and sensing
Nature Materials, 2005
One of the fastest moving and most exciting interfaces of nanotechnology is the use of quantum dots (QDs) in biology. The unique optical properties of QDs make them appealing as in vivo and in vitro fl uorophores in a variety of biological investigations, in which traditional fl uorescent labels based on organic molecules fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water soluble and target them to specifi c biomolecules has led to promising applications in cellular labelling, deep-tissue imaging, assay labelling and as effi cient fl uorescence resonance energy transfer donors. Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop fl exible bioconjugation techniques. In this review, we look at current methods for preparing QD bioconjugates as well as presenting an overview of applications. The potential of QDs in biology has just begun to be realized and new avenues will arise as our ability to manipulate these materials improves.
Bio-Mediated Synthesis of Quantum Dots for Fluorescent Biosensing and Bio-Imaging Applications
Materials research foundations, 2021
Quantum dots (QDs) have received great attention for development of novel fluorescent nanoprobe with tunable colors towards the near-infrared (NIR) region because of their unique optical and electronic properties such as luminescence characteristics, wide range, continuous absorption spectra and narrow emission spectra with high light stability. Quantum dots are promising materials for biosensing and single molecular bio-imaging application due to their excellent photophysical properties such as strong brightness and resistance to photobleaching. However, the use of quantum dots in biomedical applications is limited due to their toxicity. Recently, the development of novel and safe alternative method, the bio-mediated greener approach is one of the best aspects for synthesis of quantum dots. In this Chapter, bio-mediated synthesis of quantum dots by living organisms and biomimetic systems were highlighted. Quantum dots based fluorescent probes utilizing resonance energy transfer (RET), especially Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and chemiluminescence resonance energy transfer (CRET) to probe biological phenomena were discussed. In addition, quantum dot nanocomposites are promising ultrasensitive bioimaging probe for in vivo multicolor, multimodal, multiplex and NIR deep tissue imaging. Finally, this chapter provides a conclusion with future perspectives of this field.
Proceedings of the National Academy of Sciences, 2004
The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core–shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has ...
Resonance energy transfer between luminescent quantum dots and diverse fluorescent protein acceptors
2009
We characterized the resonance energy-transfer interactions for conjugates consisting of QD donors selfassembled with three distinct fluorescent protein acceptors, two monomeric fluorescent proteins, the dsRed derivative mCherry or yellow fluorescent protein, and the multichromophore b-phycoerythrin light-harvesting complex. Using steady-state and time-resolved fluorescence, we showed that nonradiative transfer of excitation energy in these conjugates can be described within the Förster dipole-dipole formalism, with transfer efficiencies that vary with the degree of spectral overlap, the donor-acceptor separation distance, and the number of acceptors per QD. Comparison between the quenching data and simulation of the conjugate structures indicated that while energy transfer to monomeric proteins was identical to what was measured for QD-dye pairs, interactions with b-phycoerythrin were more complex. For the latter, the overall transfer efficiency results from the cumulative contribution of individual channels between the central QD and the chromophores distributed throughout the protein structure. Due to the biocompatible nature of fluorescent proteins, these QD assemblies may have great potential for use in intracellular imaging and sensing.
Targeting quantum dots to surface proteins in living cells with biotin ligase
Proceedings of the National Academy of Sciences, 2005
Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.
Bioconjugated quantum dots for in vivo molecular and cellular imaging☆
Advanced Drug Delivery Reviews, 2008
Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology.
Quantum Dot Molecules Assembled with Genetically Engineered Proteins
Nano Letters, 2003
We report the assembly of quantum dots using an approach that attempts to capitalize on the self-assembling properties of naturally occurring proteins. Colloidal quantum dots made of cadmium selenide core and a zinc sulfide shell, (CdSe)ZnS, were incubated with a genetically modified, bacterial cellulosomal protein, cohesin, and the resultant mixture subjected to fractionation using high-pressure size exclusion chromatography (HPSEC). We note that the HPSEC profile is distinctly bimodal. The peak corresponding to particles of larger effective hydrodynamic radius (R e ) contains a plethora of sizable protein-coated, quantum dot assemblies with a predominance of structures that are trefoil-shaped. The fraction corresponding to particles of smaller R e contains individual, protein-coated quantum dots that are chemically different from their larger counterparts. This overall procedure is shown to successfully assemble novel quantum dot bioconjugates that exhibit light harvesting properties as well as providing an effective method of purifying quantum dot samples.
Fluorescence Resonance Energy Transfer Between Quantum Dot Donors and Dye-Labeled Protein Acceptors
Journal of The American Chemical Society, 2004
We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 Å and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dyelabeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding Fö rster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.
Colloidal quantum dots for fluorescent labels of proteins
IOP Conference Series: Materials Science and Engineering, 2016
The work is devoted to the synthesis of colloidal quantum dots (QDs) and their bioconjugates with proteins. Various QDs were obtained as well with synthesis method in an organic solvent followed by hydrophilization and functionalization or synthesis in aqueous phase provides obtaining hydrophilic QDs directly. Particular attention is paid to the synthesis of QDs as fluorescent tags in the near infrared where minimum absorption occurs and the fluorescence of biological tissue and synthetic materials used in analytical systems. A method for the QDs synthesis of type fluorescent core/shell CdTeSe/CdS/CdZnS-PolyT with mixed telluride, selenide cadmium core with a high quantum yield and high resistance to photoaging. It is shown that these quantum dots may be effectively used in the immunoassay.