Energy transfer between CdSe/ZnS core/shell quantum dots and fluorescent proteins

Fluorescent proteins from the green fluorescent protein (GFP) family interact strongly with CdSe/ZnS quantum dots. Photoluminescence of GFP5 is suppressed by red-emitting CdSe/ZnS quantum dots with high efficiency in a pH-dependent manner. The elevated degree of quenching, around 90%, makes it difficult to analyze the remaining signal, and it is not clear yet whether FRET is the reason behind the quenching. When the donor is a green-emitting CdSe/ZnS quantum dot and the acceptor is the HcRed1 protein, it is possible to detect quenching of the donor and sensitized emission from the acceptor. It was verified that the sensitized emission has the low anisotropy characteristic of FRET. The present characterization identifies donor-acceptor pairs formed by fluorescent proteins and CdSe/ZnS quantum dots that are suitable for the exploration of cellular events. These donor-acceptor pairs take advantage of the exceptional photochemical properties of quantum dots allied with the unique ability of fluorescent proteins to act as gene-based fluorescent probes.     

A nonfluorescent, broad-range quencher dye for Förster resonance energy transfer assays

We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Förster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 substrates were prepared by conjugating the quencher, IRDye^® QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum ∼540 nm), Cy3 (∼570 nm), Cy5 (∼670 nm), IRDye 680 (∼700 nm), IRDye 700DX (∼690 nm), or IRDye 800CW (∼790 nm). The Förster distance R₀ values are calculated as 41 to 65 Å for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change on complete cleavage by recombinant caspase-3 and ranged from 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes.     

Fluorescence stopped-flow studies of single turnover kinetics of E.coli RecBCD helicase-catalyzed DNA unwinding

We have developed and optimized a stopped-flow fluorescence assay for use in studying DNA unwinding catalyzed by Escherichia coli RecBCD helicase. This assay monitors changes in fluorescence resonance energy transfer (FRET) between a pair of fluorescent probes (Cy3 donor and Cy5 acceptor) placed on opposite sides of a nick in duplex DNA. As such, this is an "all-or-none" DNA unwinding assay. Single turnover DNA unwinding experiments were performed using a series of eight fluorescent DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. The time-courses obtained by monitoring Cy3 fluorescence display a distinct lag phase that increases with increasing duplex DNA length, reflecting the transient formation of partially unwound DNA intermediates. These Cy3 FRET time-courses are identical with those obtained using a chemical quenched-flow kinetic assay developed previously. The signal from the Cy5 fluorescence probe shows additional effects that appear to specifically monitor the RecD helicase subunit. The continuous nature of this fluorescence assay enabled us to acquire more precise time-courses for many more duplex DNA lengths in a significantly reduced amount of time, compared to quenched-flow methods. Global analysis of the Cy3 and Cy5 FRET time-courses, using an n-step sequential DNA unwinding model, indicates that RecBCD unwinds duplex DNA with an average unwinding rate constant of kU = 200(+/-40) steps s(-1) (mkU = 680(+/-12)bp s(-1)) and an average kinetic step size, m = 3.4 (+/-0.6) bp step(-1) (5 mM ATP, 10 mM MgCl(2), 30 mM NaCl, pH 7.0, 5% (v/v) glycerol, 25.0 degrees C), in excellent agreement with the kinetic parameters determined using quenched-flow techniques. Under these same conditions, the RecBC enzyme unwinds DNA with a very similar rate. These methods will facilitate detailed studies of the mechanisms of DNA unwinding and translocation of the RecBCD and RecBC helicases.     

量子点荧光技术标记肿瘤细胞中HSP的应用研究

目的探讨量子点荧光技术对人肾癌细胞株(ACHN)中不同HSP进行标记的可行性应用。方法利用量子点的荧光特性,免疫细胞化学方法检测体外培养的ACHN细胞中量子点特异性标记的HSP70、HSP90、HSPgp96的表达情况。结果共聚焦荧光显微镜下可见ACHN细胞中HSP70、HSP90、HSPgp96均有明显表达,呈现均匀分布的橙红色强荧光,且量子点在持续激发30分钟后无荧光淬灭发生。结论量子点荧光标记技术能够对不同HSP进行标记,且与传统的标记方法相比具有显著优点,可作为一种新型的检测技术应用于科研及临床标记检测中。     

Creating self-illuminating quantum dot conjugates

Semiconductor quantum dots are inorganic fluorescent nanocrystals that, because of their unique optical properties compared with those of organic fluorophores, have become popular as fluorescent imaging probes. Although external light excitation is typically required for imaging with quantum dots, a new type of quantum dot conjugate has been reported that can luminesce with no need for external excitation. These self-illuminating quantum dot conjugates can be prepared by coupling of commercially available carboxylate-presenting quantum dots to the light-emitting protein Renilla luciferase. When the conjugates are exposed to the luciferase's substrate coelenterazine, the energy released by substrate catabolism is transferred to the quantum dots through bioluminescence resonance energy transfer, leading to quantum dot light emission. This protocol describes step-by-step procedures for the preparation and characterization of these self-illuminating quantum dot conjugates. The preparation process is relatively simple and can be done in less than 2 hours. The availability of self-illuminating quantum dot conjugates will provide many new possibilities for in vivo imaging and detection, such as monitoring of in vivo cell trafficking, multiplex bioluminescence imaging and new quantum dot-based biosensors.     

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy

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.     

Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase,an enzyme essential for DNA replication,repair, and recombination

DNA ligase is an enzyme essential for DNA replication, repair, and recombination in all organisms. Bacterial DNA ligases catalyze a NAD(+)-dependent DNA ligation reaction, i.e., the formation of a phosphodiester bond between adjacent 3'-OH and 5'-phosphate termini of dsDNA. Due to their essential nature, unique cofactor requirement, and widespread existence in nature, bacterial DNA ligases appear to be valuable targets for identifying novel antibacterial agents. To explore bacterial DNA ligases as antibacterial targets and further characterize them, we developed a simple, robust, homogeneous time-resolved fluorescence resonance energy transfer assay (TR-FRET) for measuring Streptococcus pneumoniae DNA ligase activity. This assay involves the use of one dsDNA molecule labeled with biotin and another dsDNA molecule labeled with Cy5, an acceptor fluorophore. During ligation reactions, the donor fluorophore europium (Eu(3+)) labeled with streptavidin was added to the assay mixtures, which bound to the biotin label on the ligated products. This in turn resulted in the FRET from Eu(3+) to Cy5 due to their close proximity. The formation of ligation products was measured by monitoring the emission at 665nm. This assay was validated by the experiments showing that the DNA ligase activity required NAD(+) and MgCl(2), and was inhibited by NMN and AMP, products of the ligase reaction. Using this assay, we determined the K(m) values of the enzyme for dsDNA substrates and NAD(+), and the IC(50) values of NMN and AMP, examined the effects of MgCl(2) and PEG(8000) on the enzyme activity, optimized the concentrations of Eu(3+) in the assay, and validated its utilities for high-throughput screening and biochemical characterizations of this class of enzymes.     

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.     

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd²⁺ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.     

Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

Fluorescent probe for detection of Cu2+ using core‐shell CdTe/ZnS quantum dots

Core‐shell CdTe/ZnS quantum dots capped with 3‐mercaptopropionic acid (MPA) were successfully synthesized in aqueous medium by hydrothermal synthesis. These quantum dots have advantages compared to traditional quantum dots with limited biological applications, high toxicity and tendency to aggregate. The concentration of Cu²⁺ has a significant impact on the fluorescence intensity of quantum dots (QDs), therefore, a rapid sensitive and selective fluorescence probe has been proposed for the detection of Cu²⁺ in aqueous solution. Under optimal conditions, the fluorescence intensity of CdTe/ZnS QDs was linearly proportional to the concentration of Cu²⁺ in the range from 2.5 × 10^–9 M to 17.5 × 10^–7 M with the limit of 1.5 × 10^–9 M and relative standard deviation of 0.23%. The quenching mechanism is static quenching with recoveries of 97.30–102.75%. Copyright © 2015 John Wiley & Sons, Ltd.     

OFF-to-ON type fluorescent probe for the detection of 8-oxo-dG in DNA by the Adap-masked ODN probe

We have recently reported that Adap (adenosine-1,3-diazaphenoxazine) is an artificial nucleoside analogue for the specific recognition by multiple hydrogen bonding and that its fluorescence is selectively quenched with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA. We now report the development of a new OFF-to-ON type FRET probe, in which one strand contains Adap and another contains natural nucleotides for the formation of a less stable double strand. Each strand was labeled with Cy3 or BHQ2 at the 5'-end or 3'-end, respectively. It was expected in this system that fluorescence of the duplex probe is first quenched by FRET, but the target DNA strand containing 8-oxo-dG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair exhibited a complete discrimination between 8-oxo-dG and dG in DNA by fluorescence enhancement.     

The influence of solvent refractive index on the photoluminescence decay of thick-shell gradient-alloyed colloidal quantum dots investigated in a wide range of delay times

Colloidal semiconductor quantum dots have many potential optical applications, including quantum dot light-emitting diodes, single-photon sources, or biological luminescent markers. The optical properties of colloidal quantum dots can be affected by their dielectric environment. This study investigated the photoluminescence (PL) decay of thick-shell gradient-alloyed colloidal semiconductor quantum dots as a function of solvent refractive index. These measurements were conducted in a wide range of delay times to account for both the initial spontaneous decay of excitons and the delayed emission of excitons that has the form of a power law. It is shown that whereas the initial spontaneous PL decay is very sensitive to the refractive index of the solvent, the power-law delayed emission of excitons is not. Our results seem to exclude the possibility of carrier self-trapping in the considered solvents and suggest the existence of trap states inside the quantum dots. Finally, our data show that the average exciton lifetime significantly decreases as a function of the solvent refractive index. The change in exciton lifetime is qualitatively modeled and discussed.     

Plasmonically Induced Energy Flow in Monodisperse Quantum Dot Solids

We studied the excitation-power-dependent red shift and broadening of the emission spectra of monodisperse CdSe/ZnS quantum dot solids when they are in close proximity of gold metallic nanoparticles. Our results suggest that these features are the signs of plasmonic enhancement of the interdot energy transfer in such solids leading to (a) efficient funneling of excitons to the locations where quantum dots with large CdSe cores are and (b) near complete depletion of excitons in regions where the quantum dots with incomplete shells or/and small core sizes are located. We studied the impacts of the heat generated by the metallic nanoparticles and discussed the effects of excitation-power-dependent photoionization rates. The uneven spatial distribution of excitons in monodisperse quantum dot solids in the presence of metallic nanoparticles suggests that plasmonic fields can drive significant spatial migration of energy in such structures.     

Spectroscopic investigation of alloyed quantum dot‐based FRET to cresyl violet dye

Quantum dots (QDs), bright luminescent semiconductor nanoparticles, have found numerous applications ranging from optoelectronics to bioimaging. Here, we present a systematic investigation of fluorescence resonance energy transfer (FRET) from hydrophilic ternary alloyed quantum dots (CdSeS/ZnS) to cresyl violet dye with a view to explore the effect of composition of QD donors on FRET efficiency. Fluorescence emission of QD is controlled by varying the composition of QD without altering the particle size. The results show that quantum yield of the QDs increases with increase in the emission wavelength. The FRET parameters such as spectral overlap J(λ), Förster distance R₀, intermolecular distance (r) , rate of energy transfer k_T (r), and transfer efficiency (E) are determined by employing both steady‐state and time‐resolved fluorescence spectroscopy. Additionally, dynamic quenching is noticed to occur in the present FRET system. Stern–Volmer (K_D) and bimolecular quenching constants (k_q) are determined from the Stern–Volmer plot. It is observed that the transfer efficiency follows a linear dependence on the spectral overlap and the quantum yield of the donor as predicted by the Förster theory upon changing the composition of the QD. Copyright © 2015 John Wiley & Sons, Ltd.     

FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes.

The fate of fluorescently labeled pre-nsL-TP (Cy3-pre-nsL-TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning microscopy. The protein exhibited a distinct punctate fluorescence pattern and colocalized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the putative peroxisomal targeting sequence (AKL) resulted in a diffuse cytosolic fluorescence. These results indicate that microinjected Cy3-pre-nsL-TP is targeted to peroxisomes. The association of nsL-TP with peroxisomal enzymes was investigated in cells by measuring fluorescence resonance energy transfer (FRET) between the microinjected Cy3-pre-nsL-TP and Cy5-labeled antibodies against the peroxisomal enzymes acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, bifunctional enzyme, PMP70 and catalase. The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate the FRET efficiency on a pixel-by-pixel basis, revealed a specific association of nsL-TP with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and bifunctional enzyme in the peroxisomes. These observations were corroborated by subjecting a peroxisomal matrix protein fraction to affinity chromatography on Sepharose-immobilized pre-nsL-TP. Acyl-CoA oxidase was retained. These studies provide strong evidence for a role of nsL-TP in the regulation of peroxisomal fatty acid beta-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes.     

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.