Quantum Dots and FRET-Nanobeads for Probing Genes, Proteins, and Drug Targets in Single Cells

2003 ◽  
Author(s):  
Amit Agrawal ◽  
Xiaohu Gao ◽  
Nitin Nitin ◽  
Gang Bao ◽  
Shuming Nie
Keyword(s):  
Quantum Dots ◽  
Drug Targets ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Length Scale ◽  
Imaging And Spectroscopy

Quantum dots are tiny light-emitting particles on the length scale of 2–10 nm, and FRET-nanobeads for fluorophore-embedded nanoparticles on the length scale of 40–200 nm based on the phenomenon of fluorescence resonance energy transfer (FRET). These materials are emerging as a new class of biological labels with properties and applications that are not available with traditional organic dyes and fluorescent proteins. In this ASME contribution, we report new developments in using semiconductor quantum dots for quantitative imaging and spectroscopy of single cancer cells. We also show results from intracellular staining of actin filaments using FRET-nanobeads. These results raise new possibilities in disease diagnostics, drug and biochemical discovery, cancer imaging, molecular profiling, and disease staging.

Semiconductor Quantum Dots for Multicolor Fluorescence Imaging and Spectroscopy of Single Cancer Cells

2003 ◽  
Vol 773 ◽  
Author(s):  
Xiaohu Gao ◽  
Shuming Nie ◽  
Wallace H. Coulter
Keyword(s):  
Quantum Dots ◽  
Cancer Cells ◽  
Fluorescence Imaging ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Semiconductor Quantum Dots ◽  
Single Cancer ◽  
Imaging And Spectroscopy

AbstractLuminescent quantum dots (QDs) are emerging as a new class of biological labels with unique properties and applications that are not available from traditional organic dyes and fluorescent proteins. Here we report new developments in using semiconductor quantum dots for quantitative imaging and spectroscopy of single cancer cells. We show that both live and fixed cells can be labeled with multicolor QDs, and that single cells can be analyzed by fluorescence imaging and wavelength-resolved spectroscopy. These results raise new possibilities in cancer imaging, molecular profiling, and disease staging.

THE ROLES OF PHOTOLUMINESCENT QUANTUM DOTS IN GENERATION OR DETECTION OF REACTIVE OXYGEN SPECIES: CULPRITS OR DETECTIVES?

COSMOS ◽  
2010 ◽  
Vol 06 (02) ◽  
pp. 149-158
Author(s):  
SUHUA WANG ◽  
DEJIAN HUANG
Keyword(s):  
Reactive Oxygen Species ◽  
Quantum Dots ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Reactive Oxygen ◽  
Resonance Energy ◽  
Oxygen Species ◽  
Positive Side ◽  
Biological Importance ◽  
Organic Sensitizers

In this review, we systematically analyzed the complicated interrelationship between photoluminescent quantum dots (QDs) and reactive oxygen species of biological importance. QDs, when photoexcited, generate reactive oxygen species (ROS), which are partially blamed for the cytotoxicity of QDs. On the positive side, the ability of generating ROS by QDs are exploited in photodynamic therapy using QDs alone or in combination with QD-surface bound organic sensitizers via resonance energy transfer from QDs to the organic dyes. Lastly, depending on the chemical composition and the functionalization of the QDs, ROS are known to quench or switch-on the QD photoluminescence. The selectivity and sensitivity toward specific ROS can be achieved through judicious chemical modification of QD surface coating layers by taking into account the reactivity difference among different ROS. The flexible QD surface functionalization opens up the unprecedented possibility of designer-made nanoprobes for sensing and quantifying ROS of biological importance.

scholarly journals Regulation of β cell glucokinase by S-nitrosylation and association with nitric oxide synthase

2003 ◽  
Vol 161 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Mark A. Rizzo ◽  
David W. Piston
Keyword(s):  
Nitric Oxide ◽  
Fluorescent Proteins ◽  
Signal Sequence ◽  
Secretory Granules ◽  
Quantitative Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
No Production ◽  
Β Cells ◽  
Pancreatic Β Cells

Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic β cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured β cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic β cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

scholarly journals Bioluminescence-Based Energy Transfer Using Semiconductor Quantum Dots as Acceptors

Sensors ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 2909 ◽  
Author(s):  
Anirban Samanta ◽  
Igor L. Medintz
Keyword(s):  
Quantum Dots ◽  
Energy Transfer ◽  
Organic Dyes ◽  
Stokes Shift ◽  
Resonance Energy Transfer ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Semiconductor Quantum Dots ◽  
Renilla Luciferase ◽  
Bioluminescent Protein

Bioluminescence resonance energy transfer (BRET) is the non-radiative transfer of energy from a bioluminescent protein donor to a fluorophore acceptor. It shares all the formalism of Förster resonance energy transfer (FRET) but differs in one key aspect: that the excited donor here is produced by biochemical means and not by an external illumination. Often the choice of BRET source is the bioluminescent protein Renilla luciferase, which catalyzes the oxidation of a substrate, typically coelenterazine, producing an oxidized product in its electronic excited state that, in turn, couples with a proximal fluorophore resulting in a fluorescence emission from the acceptor. The acceptors pertinent to this discussion are semiconductor quantum dots (QDs), which offer some unrivalled photophysical properties. Amongst other advantages, the QD’s large Stokes shift is particularly advantageous as it allows easy and accurate deconstruction of acceptor signal, which is difficult to attain using organic dyes or fluorescent proteins. QD-BRET systems are gaining popularity in non-invasive bioimaging and as probes for biosensing as they don’t require external optical illumination, which dramatically improves the signal-to-noise ratio by avoiding background auto-fluorescence. Despite the additional advantages such systems offer, there are challenges lying ahead that need to be addressed before they are utilized for translational types of research.

scholarly journals Spectral-Time Multiplexing in FRET Complexes of AgInS2/ZnS Quantum Dot and Organic Dyes

Nanomaterials ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1569 ◽  
Author(s):  
Vera Kuznetsova ◽  
Anton Tkach ◽  
Sergei Cherevkov ◽  
Anastasiia Sokolova ◽  
Yulia Gromova ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Visible Range ◽  
Additional Parameter ◽  
Time Resolved ◽  
Cd Toxicity ◽  
Multiplex Analysis ◽  
Luminescent Markers

Nowadays, multiplex analysis is very popular, since it allows to detect a large number of biomarkers simultaneously. Traditional multiplex analysis is usually based on changes of photoluminescence (PL) intensity and/or PL band spectral positions in the presence of analytes. Using PL lifetime as an additional parameter might increase the efficiency of multiplex methods. Quantum dots (QDs) can be used as luminescent markers for multiplex analysis. Ternary in-based QDs are a great alternative to the traditional Cd-based one. Ternary QDs possess all advantages of traditional QDs, including tunable photoluminescence in visible range. At the same time ternary QDs do not have Cd-toxicity, and moreover they possess long spectral dependent lifetimes. This allows the use of ternary QDs as a donor for time-resolved multiplex sensing based on Förster resonance energy transfer (FRET). In the present work, we implemented FRET from AgInS2/ZnS ternary QDs to cyanine dyes absorbing in different spectral regions of QD luminescence with different lifetimes. As the result, FRET-induced luminescence of dyes differed not only in wavelengths but also in lifetimes of luminescence, which can be used for time-resolved multiplex analysis in biology and medicine.

scholarly journals Optical Sensors Based on II-VI Quantum Dots

Nanomaterials ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 192 ◽  
Author(s):  
Anna Lesiak ◽  
Kamila Drzozga ◽  
Joanna Cabaj ◽  
Mateusz Bański ◽  
Karol Malecha ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Optical Sensors ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Emission Spectra ◽  
Chemical Mechanisms ◽  
Physico Chemical ◽  
High Emission ◽  
Analyte Detection

Fundamentals of quantum dots (QDs) sensing phenomena show the predominance of these fluorophores over standard organic dyes, mainly because of their unique optical properties such as sharp and tunable emission spectra, high emission quantum yield and broad absorption. Moreover, they also indicate no photo bleaching and can be also grown as no blinking emitters. Due to these properties, QDs may be used e.g., for multiplex testing of the analyte by simultaneously detecting multiple or very weak signals. Physico-chemical mechanisms used for analyte detection, like analyte stimulated QDs aggregation, nonradiative Förster resonance energy transfer (FRET) exhibit a number of QDs, which can be applied in sensors. Quantum dots-based sensors find use in the detection of ions, organic compounds (e.g., proteins, sugars, volatile substances) as well as bacteria and viruses.

scholarly journals A FRET based biosensor for measuring Gα13 activation in single cells

2017 ◽  
Author(s):  
Marieke Mastop ◽  
Nathalie R. Reinhard ◽  
Cristiane R. Zuconelli ◽  
Fenna Terwey ◽  
Theodorus W. J. Gadella ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Coupling Efficiency ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protease Activated Receptors ◽  
Heterotrimeric G Protein ◽  
Gpcr Activation ◽  
P115 Rhogef

AbstractFörster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit. Three variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gβ1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were treated with thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor could be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe Gα13 activation.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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