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Abstract
Fluorescence resonance energy transfer (FRET) has broad applications in the study of the interactions of biological macromolecules, immunoassay, nucleic acid detection and so on. However many organic dyes conventionally used in FRET have functional limitations such as low resistance to photobleaching, narrow excitation bands coupled with broad emission bands, all of which have limited the development and the application of FRET. Therefore new pairs of energy donor and acceptor are needed. Because of the advantages over organic dyes, quantum dots (QDs) have expanded the range of FRET and have been considered as new and significant development trend of FRET nowadays. The theory of FRET and the applications of QDs in FRET are reviewed in this article.
... 荧光共振能量转移(Föster resonance energy transfer, FRET)是一种发生在供体和受体之间的光物理进程, 当 供体的荧光发射光谱与受体的吸收光谱很好重叠, 并且 二者之间的距离在 1～10 nm 范围时, 供体荧光大幅度 降低, 受体荧光显著增强 [24] . 根据这种原理, 可以对受 体荧光信号进行放大, 从而提高检测的灵敏度. ...
Simple and sensitive detection of proteins is crucial in biological analysis and medical diagnosis. Conjugated polymers (CPs) with π-conjugated backbones were recognized as having excellent light-harvesting capability and high fluorescent quantum yield. They have been widely used as an energy donor to amplify fluorescence signal via high efficient Föster resonance energy transfer (FRET). In particular, conjugated polymer brush with high charge density provides more possibilities due to stronger electrostatic interactions with negatively charged biomolecules. Here, we developed a highly sensitive protein biosensor for thrombin detection based on a conjugated polymer brush (PFNI) and a fluorescein-labeled aptamer (FAM-apt15). PFNI is a water-soluble cationic polyfluorene derivate with extremely high charge density (78 positive charges per repeat unit). PFNI can attract negatively charged aptamer through strong electrostatic interactions. In this case, the energy donor (PFNI) and acceptor (FAM) are in a close proximity, which results in an efficient FRET process and a high FRET signal. However, when the FAM-apt15 combines with the target protein, a rigid and big-sized G-quadruplex/thrombin complex formed. Due to the steric hindrance from the densely brush of PFNI, the distance between the two fluorophores increased significantly, leading to an inefficient FRET process and a low FRET signal. The strategy exhibits excellent specificity and the limit of detection (LOD) for thrombin in buffer was estimated to be 0.05 nmol/L. It also works well in diluted serum and a LOD of 0.2 nmol/L can be obtained. Compared to the biosensors based on traditional linear conjugated polymers, the sensitivity was improved by one order of magnitude. In addition, our strategy also shows the merits of simple, label- free, and low-cost because labeled DNA is much more expensive than unlabeled one. Based on the specific binding of aptamer and protein, this novel method can be extended to a highly sensitive detection of more proteins. © 2016 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
... 11,12 Several parameters need to be satisfied for efficient FRET to occur. 13,14 The resonance condition can be easily achieved by an appropriate selection of photoactive components, 13,15 and it is limited to a distance of a few nanometers. 16 Appropriate intermolecular distances between interacting molecules as well as suitable molecular orientation are further factors influencing FRET efficiency. ...
This work describes the multistep Förster resonance energy transfer (FRET) among laser dyes adsorbed onto the nanoparticles of a layered silicate, saponite (Sap). Six cationic laser dyes with appropriate spectral properties to fit the resonance conditions were selected. Fluorescence spectroscopy proved cascade FRET in the experiments based on hybrid colloidal systems. The emission from preferentially excited energy donor molecules was reduced in favor of the emission from energy acceptors. The colloidal systems were used for the preparation of thin solid films with embedded dye cations. The films were characterized by X-ray diffraction, absorption spectroscopy, and linearly polarized absorption spectroscopy. Results from steady-state fluorescence, fluorescence anisotropy measurements, and time-resolved fluorescence spectroscopy confirmed the occurrence of a cascade FRET in the films. The effect of the concentration of embedded dye molecules on FRET efficiency was proved. The multicomponent films based on Sap and laser dyes are affordable materials useful for the manipulation of light energy at a molecular level, thus mimicking a cascade process occurring in photosynthesis systems in green plants.
... [44,45] Their combination may possess energy transfer, and thereby the resulting co-assembled nanomaterials could find some applications in detection systems with different stimulators. [46,47] In this work, Py-CHO and TPE-CHO are grafted onto PAH via a Schiff base reaction to obtain PAH-g-Py-g-TPE with different ratios of Py-CHO and TPE-CHO (Scheme 1). The PAH-g-Py-g-TPE forms spherical micelles in solution at pH 6.5, which are then incubated in acidic solution to explore possible co-assembly behaviors of Py-CHO and TPE-CHO. ...
The poly(allylamine hydrochloride)-g-pyrene-tetraphenylethylene (PAH-g-Py-g-TPE) copolymers with different ratios of Py and TPE are synthesized by grafting 1-pyrenecarboxaldehyde (Py-CHO) and tetraphenylethylenecarboxaldehyde (TPE-CHO) to PAH via a Schiff base reaction in methanol. The PAH-g-Py-g-TPE forms spherical micelles in water regardless of the ratios of Py and TPE, which can transform into different nanostructures after being incubated in pH 0 and pH 2 solutions, respectively. These nanomaterials including nanoparticles (NPs), nanorods (NRs), nanotubes (NTs) and nanoribbons (NBs) are composed of Py-CHO and TPE-CHO with different ratios, and emit fluorescence with colors different from the pure Py NRs and NTs, and TPE NPs.
As a newly developed fluorescent probe, DNA functionalized quantum dots (QDs) with unique optical properties play a critical role in the bioanalytical applications. The present review provides an overview on the conjugation technologies of DNA to QDs, including electrostatic attraction, covalent attachment, specific effect and direct synthesis. In addition, we summarize recent advances on DNA functionalized QDs in the bioapplications, such as targeting nucleic acids, ions, small molecules and the protein detection, cell imaging and disease diagnosis. Finally, we envision aspects concerning current challenges on the DNA functionalized fluorescent QDs.
CdTe semiconductor nanoparticle are synthesized in aqueous solution by using thioglycolic acid (HSCH2COOH) as the stabilizer. Based on it, the inorganic quantum dots ( QDs ) CdTe-conjugated molecular beacons (MBs) are developed. The products are characterized by using UV-vis absorption spectra, fluorescence spectra and Gel electrophoresis techniques and so on. Compared with the traditional probe, the CdTe conjugated MBs is proved to be fluorescent longevity.
A novel DNA biosensor system on silica microspheres as solid carriers which based on the fluorescence resonance energy transfer (FRET) was presented in this work when CdTe quantum dots (QDs) were as energy donors and Au nanoparticles (AuNPs) were as energy accepters. Compared with the fluorescent intensity of CdTe QDs, the fluorescent intensity of DNA biosensors decreased extremely, which indicated that the FRET occurred between CdTe QDs and AuNPs. The biosensor system would have a certain degree recovery of fluorescence when the complementary single stranded DNA was introduced into this system. The DNA detection results indicated that this novel fluorescent DNA probe system could recognize the existence of complementary target DNA or not.
DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5′-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 ° at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5′-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.
The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide such possibilities, combining the high spatial resolution of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules. Methods to visualize interaction can be based on FRET (fluorescence detected resonance energy transfer), for example in fluorescence lifetime imaging microscopy (FLIM). Another method is based on fluorescence correlation spectroscopy (FCS) by which the diffusion rate of single molecules can be determined, giving insight into whether a protein is part of a larger complex or not. Here, both FRET- and FCS-based approaches to study protein-protein interactions in vivo are reviewed.
In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell. This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique. This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site. The change of the FRET ratio upon cleavage was larger than 4-fold. Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation. Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8. Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells.
The potential of luminescent semiconductor quantum dots (QDs) to enable development of hybrid inorganic-bioreceptor sensing materials has remained largely unrealized. We report the design, formation and testing of QD-protein assemblies that function as chemical sensors. In these assemblies, multiple copies of Escherichia coli maltose-binding protein (MBP) coordinate to each QD by a C-terminal oligohistidine segment and function as sugar receptors. Sensors are self-assembled in solution in a controllable manner. In one configuration, a beta-cyclodextrin-QSY9 dark quencher conjugate bound in the MBP saccharide binding site results in fluorescence resonance energy-transfer (FRET) quenching of QD photoluminescence. Added maltose displaces the beta-cyclodextrin-QSY9, and QD photoluminescence increases in a systematic manner. A second maltose sensor assembly consists of QDs coupled with Cy3-labelled MBP bound to beta-cyclodextrin-Cy3.5. In this case, the QD donor drives sensor function through a two-step FRET mechanism that overcomes inherent QD donor-acceptor distance limitations. Quantum dot-biomolecule assemblies constructed using these methods may facilitate development of new hybrid sensing materials.
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 a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein–nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.
• maltose-binding protein
• three-dimensional structure
• nanotechnology
• nanocrystal
The scanning near-field optical microscope (SNOM) breaks the resolution limit set up by the diffraction of conventional optical microscope, hence it has wide application prospect in many fields. The shape and size of the scanning probe tip are two of the key factors affecting the resolution of SNOM. A novel hot drawing method for making probe was put forward which combined the hot drawing method with the chemical etching method in an experimental setup to improve the successful rate of probe fabrication. The results show that the optimal size of the probe tips obtained with the two methods can achieve 96 nm and 76 nm respectively.
Semiconductor quantum dots (QDs) are inorganic nanocrystals consisted of a cadmium selenide (CdSe) core which was wrapped in an outer shell of zinc sulfide (ZnS). In comparison with common organic dyes, this class of fluorescent labels is 20 times as bright, 100 times as stable against photobleaching. The emission wavelength of QDs was controlled by the size of the core and each single-color of QDs has narrow symmetrical emission peak. These advantages of the optical properties make them broad applications in medical diagnosis, high-speed screening drugs and high-throughput analysis of genes and proteins. Based on the stability and biocompatibility of the QDs, it is possible to make movies of long-term interaction of biological molecules in a living cell by tagging each biomolecule with different color of QDs.
Water-soluble CdSe quantum dots (QDs) capped by bovine serum albumin (BSA) is described as selective silver (I) ion probe based on their fluorescence (FL) quenched by silver ion at pH 5.7. The detection limit is 3×10-7 mol L-1 for silver ion. The interference was also investigated.
Luminescent CdSe-ZnS quantum dots (QDs) were modified with bovine serum albumin (BSA) and used as selective copper ion probe. The fluorescence of the water-soluble QDs can be quenched only by Cu2+ and Fe3+ in physiological buffer solution. Approximate concentrations of other physiologically important cations, such as Zn2+, Na+ and K+ etc. have no effect on the fluorescence. Adding F− to form the colorless complex FeF63− can eliminate the interference of Fe3+. The detection limit of Cu2+ ions was 10nM. The results can be explained in terms of strong binding of Cu2+ onto the surface of CdSe resulting in a chemical displacement of Cd2+ ions and the formation of CuSe on the surface of the QDs.
It is well known that the quantum confinement effects are closely related to the existence of different behavior for the same material composition. Due to the reduced size scale of the nanoparticles, the most part of their forming atoms are at the particle surface, which needs to be as stable as possible, to avoid phenomena such as dissolution and photodegradation. This way, methodologies for semiconductor nanoparticles obtention shall take into account the size, shape and energy of the final product. However, the relationship between these parameters is not yet clearly understood for nanometric systems, specially for those ones which are smaller than 20nm. In this work, we present and discuss experimental and theoretical data obtained for nanoparticles of the semiconductor cadmium sulfide (CdS), in order to contribute for the understanding of the correlation between energetical and structural properties of nanometric systems in the quantum confinement regime.
We demonstrate that the nanoscale assemblies of water-solubilized, oppositely charged CdSe/ZnS core/shell quantum dots and of quantum dots and nanogold particles make it possible to develop FRET-based sensors with a donor quenching efficiency close to 100%. The cw and time-resolved photoluminescence analysis of nanoassemblies with different donor-to-acceptor ratios demonstrates a prevalence of 1:1 complexes. Experimental data perfectly fit the mathematical simulation based on long-range FRET and a static model of the assembling of the oppositely charged nanoparticles. These assemblies are stable in biological fluids, suggesting new applications.
Complementary bioconjugates based on antibody−antigen interactions were synthesized from luminescent CdTe nanoparticles (NPs). Antigen (bovine serum albumin) was conjugated to red-emitting CdTe NPs, while green-emitting NPs were attached to the corresponding anti-BSA antibody (IgG). The NP bioconjugates were characterized by native and SDS−PAGE electrophoresis, gel-permeation HPLC, and circular dichroism. Antigen−antibody binding affinity was evaluated by enzyme-linked immunosorbent assay (ELISA). The formation of BSA−IgG immunocomplex resulted in the Förster resonance energy transfer (FRET) between the two different NPs: the luminescence of green-emitting NPs was quenched whereas the emission of the red-emitting NPs was enhanced. The luminescence recovered when the immunocomplex was exposed to an unlabeled antigen. The immunocomplexes can be considered as a prototype of NP superstructures based on biospecific ligands, while the competitive FRET inhibition can be used in an immunoassay protocol.
Specific binding of biotinilated bovine serum albumin (bBSA) and tetramethylrhodamine-labeled streptavidin (SAv−TMR) was observed by conjugating bBSA to CdSe−ZnS core−shell quantum dots (QDs) and observing enhanced TMR fluorescence caused by fluorescence resonance energy transfer (FRET) from the QD donors to the TMR acceptors. Because of the broad absorption spectrum of the QDs, efficient donor excitation could occur at a wavelength that was well resolved from the absorption spectrum of the acceptor, thereby minimizing direct acceptor excitation. Appreciable overlap of the donor emission and acceptor absorption spectra was achieved by size-tuning the QD emission spectrum into resonance with the acceptor absorption spectrum, and cross-talk between the donor and acceptor emission was minimized because of the narrow, symmetrically shaped QD emission spectrum. Evidence for an additional, nonspecific QD−TMR energy transfer mechanism that caused quenching of the QD emission without a corresponding TMR fluorescence enhancement was also observed.
Conjugates of bovine serum albumin and CdTe nanoparticles capped with l-cysteine have been synthesized via a one-pot glutaric dialdehyde cross-linking procedure. Diads (1:1) with some amount of 2:1 albumin−nanoparticle assemblies preferably form in this reaction, as evidenced by gel electrophoresis. Circular dichroism spectroscopy demonstrates that the tertiary structure of the protein remains largely intact after the conjugation. Attachment of protein moieties result in a significant increase of CdTe emission, which is attributed to the resonance energy transfer from the tryptophan moieties of albumin to CdTe nanoparticles acting as receptors for the protein antennae.
We report results on the observation of energy transfer between CdTe nanocrystals and AlexaFluor dye molecules. The nanocrystals were attached to the surface of polystyrene latex beads of 450 nm in diameter by means of the layer-by-layer technique. Energy transfer was observed as signal enhancement in confocal imaging of a single bead covered with an AlexaFluor film. This was verified by detecting quenching of the donor emission and enhancement of the acceptor emission in spectral measurements. A fluorescence-resonance energy transfer scanning near-field optical microscopy (FRET-SNOM) tip was made from a single bead coated with CdTe attached to a near-field tip. We report first results on FRET-SNOM imaging with this tip.
Fluorescence resonance energy transfer (FRET) is a technique to detect the structural changes in biomolecules. We extended this technique to the single-molecule level in aqueous solution, by combining it with total internal reflection fluorescence microscopy. Both multiple color images and fluorescence spectra at the single-molecular level were obtained to determine FRET from Cy3 to Cy5 attached to α-tropomyosin (αTm), a coiled-coil of homodimer. The FRET properties observed between single fluorophores were consistent with the premise of FRET. On excitation at the donor, the fluorescence of the donor decreased and the fluorescence of the acceptor increased. Photobleaching of one of the fluorophore affected the fluorescence of the other as predicted by the mechanism of FRET. Photobleaching occurred in a single step, confirming that the donor and acceptor fluorophores were single. Large FRET efficiency (78%) was obtained in agreement with a coiled-coil structure and the FRET decreased when the protein was denatured into two polypeptide chains. Thus, we demonstrated that it is possible to detect the assembly–disassembly of individual protein molecules as well as conformational changes occurring within a single protein molecule.
Enhanced blue fluorescent protein (EBFP) and enhanced green fluorescent protein (EGFP) mutants of GFP in close proximity to one another can act as a fluorescence resonance energy transfer (FRET) pair. Unstructured amino acid linkers of varying length were inserted between EBFP and EGFP, revealing that linkers even as long as 50 amino acids can be accommodated and still allow FRET to occur. This led to the development of a novel biosensor for Rac/Cdc42 binding to their effector proteins based on the insertion of amino acids 75–118 of p21-activated kinase (PAK) between the GFP mutants. We demonstrate that this protein construct allows significant FRET between EBFP and EGFP and retains the ability to bind to Rac in its GTP-bound form with a binding affinity similar to the uncomplexed PAK fragment, and furthermore, on binding to Rac or Cdc42 a marked change in FRET takes place. This forms the basis for a simple, sensitive, and rapid method to measure binding of Rac/Cdc42 to their effector proteins. Since the signal is dependent upon the interaction with active GTP-bound forms it acts as a biosensor for the activation of Rac/Cdc42. It has the potential for use in live cells and for identifying localization of Rac/Cdc42 within subcellular compartments.
We have developed semiconductor quantum dots (QDs)-conjugated molecular beacons (MBs). The organic fluorophores are replaced with inorganic fluorescent ZnS-capped CdSe quantum dots covered with mercaptoacetic acid monolayer. We confirmed the overlap between emission spectrum of the QD after the surface modification and the absorption spectrum of 4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL) as an organic quencher molecule. About 90% overlap between QDs and DABCYLs’ spectra promotes fluorescent resonance energy transfer (FRET). FRET efficiency between QD-DABCYL and 6-FAM (organic fluorophore)-DABCYL are compared by using 3-D molecular modeling. The farthest and nearest possible separation between the fluorophore and the quencher are predicted using 3-D molecular model considering the rotational freedom of the bonds defined by the type of chemical linker between MB-fluorophore and MB-DABCYL. Finally, QD-modified MBs are tested with their complementary target deoxyribonucleic acid (DNA) sequence and for non-specific sequences. The use of QDs instead of organic fluorophores may offer multiplexed imaging during DNA or RNA analysis due to their broad UV absorption spectrum.
Because of the coatings needed to solubilize and passivate quantum dots for biological applications, their use
in fluorescent resonance energy transfer (FRET) has been limited. However, hydrophobic particles without
polymer coatings may be embedded into lipid membranes, as demonstrated here with biomimetic vesicles.
FRET is seen to a lipid-soluble dye (DiD) and a water-soluble dye (Cy3.5) in which the vesicles are suspended.
The degree of energy transfer to each dye suggests that most of the QDs are located deep within the lipid, as
confirmed by electron microscopy of whole mounts and thin sections of vesicles. Energy transfer is also seen
to a voltage-sensitive, lipid-soluble dye (di-4-ANEPPS) only when the potassium ionophore valinomycin is
present in the membrane. The effect is dependent upon potassium ion concentration rather than absolute
membrane potential.
We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin.
Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.
Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation.
We describe the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe-ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays. The conjugation strategy employs an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies. In this method, the number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed-composition conjugates of the molecular adaptor and a second two-domain protein that allows the use of affinity chromatography. QD-antibody conjugates were successfully used in fluoroimmunoassays for detection of both a protein toxin (staphylococcal enterotoxin B) and a small molecule (2,4,6-trinitrotoluene).
Phosphatidylethanolamine and -choline derivatives equipped with fluorescent donor-acceptor pairs of dyes connected to the tips of the fatty acids were synthesised and shown to be suitable substrates for phospholipase A2.
CdSe-ZnS core-shell quantum dots (QDs) act as photochemical centers for lighting-up the dynamics of telomerization or DNA replication.
We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.
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 A 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 dye-labeled 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 Förster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.
Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.
Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein-protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric gamma-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.
CdSe quantum dots (QDs) have been prepared and modified with mercaptoacetic acid. They are water-soluble and biocompatible. To improve their fluorescence intensity and stability in water solution, bovine serum albumin (BSA) was absorbed onto their surface. Based on the quench of fluorescence signals of the functionalized CdSe QDs in the 543 nm wavelength and enhancement of them in the 570-700 nm wavelength range by Ag(I) ions at pH 5.0, a simple, rapid and specific method for Ag(I) determination was proposed. In comparison with single organic fluorophores, these nanoparticles are brighter, more stable against photobleaching, and do not suffer from blinking. Under the optimum conditions, the response is linearly proportional to the concentration of Ag(I) between 4.0 x 10(-7) and 1.5 x 10(-5) mol L(-1), and the limit of detection is 7.0 x 10(-8) mol L(-1). The mechanism of reaction is also discussed.
A novel functionally PEGylated quantum dot (QD) was prepared by a coprecipitation method in the presence of the biotin-PEG/polyamine block copolymer. When CdCl2 and Na2S were mixed in aqueous media in the presence of the biotin-PEG-b-poly(2-(N,N-dimethylamino)ethyl methacrylate) [biotin-PEG/PAMA], a CdS QD with a size of ca. 5 nm was prepared. The polyamine segment was anchored on the surface of the formed CdS nanoparticle, whereas the PEG segment was tethered on the surface to form a hydrophilic palisade, thus improving the dispersion stability in aqueous media even under a high salt concentration condition. An effective fluorescent resonance energy transfer (FRET) was observed by the specific interaction of the biotin-PEG/PAMA stabilized CdS QD with TexasRed-labeled streptavidin of the physiological ionic strength of 0.15 M. The extent of the energy transfer was in proportion to the concentration of the TexasRed-streptavidin. This FRET system using the PEGylated CdS QD coupled with fluorescent-labeled protein can be utilized as a highly sensitive bioanalytical system.
Fluorescent semiconductor nanocrystals, known as quantum dots (QDs), have several unique optical and chemical features. These features make them desirable fluorescent tags for cell and developmental biological applications that require long-term, multi-target and highly sensitive imaging. The improved synthesis of water-stable QDs, the development of approaches to label cells efficiently with QDs, and improvements in conjugating QDs to specific biomolecules have triggered the recent explosion in their use in biological imaging. Although there have been many successes in using QDs for biological applications, limitations remain that must be overcome before these powerful tools can be used routinely by biologists.