Detection of influenza A virus based on fluorescence resonance energy transfer from quantum dots to carbon nanotubes

In this paper, a simple and sensitive approach for H5N1 DNA detection was described based on the fluorescence resonance energy transfer (FRET) from quantum dots (QDs) to carbon nanotubes (CNTs) in a QDs-ssDNA/oxCNTs system, in which the QDs (CdTe) modified with ssDNA were used as donors. In the initial stage, with the strong interaction between ssDNA and oxCNTs, QDs fluorescence was effectively quenched. Upon the recognition of the target, the effective competitive bindings of it to QDs-ssDNA occurred, which decreased the interactions between the QDs-ssDNA and oxCNTs, leading to the recovery of the QDs fluorescence. The recovered fluorescence of QDs was linearly proportional to the concentration of the target in the range of 0.01–20 μM with a detection limit of 9.39 nM. Moreover, even a single-base mismatched target with the same concentration of target DNA can only recover a limited low fluorescence of QDs, illustrating the good anti-interference performance of this QDs-ssDNA/oxCNTs system. This FRET platform in the QDs-ssDNA/oxCNTs system was facilitated to the simple, sensitive and quantitative detection of virus nucleic acids and could have a wide range of applications in molecular diagnosis.     

Quantum dot FRET biosensors that respond to pH, to proteolytic or nucleolytic cleavage, to DNA synthesis, or to a multiplexing combination

Fluorescent acceptors have been immobilized on nanoparticulate quantum dots (QDs), which serve in turn as their FRET donors. The broad excitation and narrow emission bands of QDs mark them as having excellent potential as donors for FRET and, in principle, differently colored QDs could be excited simultaneously. The present work describes the preparation and operation of FRET-based QD bioprobes individually able to detect the actions of protease, deoxyribonuclease, DNA polymerase, or changes in pH. In addition, two such QD-mounted biosensors were excited at a single wavelength, and shown to operate simultaneously and independently of each other in the same sample solution, allowing multiplex detection of the action of a protease, trypsin, in the presence of deoxyribonuclease.     

Valence‐Engineering of Quantum Dots Using Programmable DNA Scaffolds

Precise control over the valency of quantum dots (QDs) is critical and fundamental for quantitative imaging in living cells. However, prior approaches on valence control of QDs remain restricted to single types of valences. A DNA‐programmed general strategy is presented for valence engineering of QDs with high modularity and high yield. By employing a series of programmable DNA scaffolds, QDs were generated with tunable valences in a single step with near‐quantitative yield (>95 %). The use of these valence‐engineered QDs was further demonstrated to develop 12 types of topologically organized QDs‐QDs and QDs‐AuNPs and 4 types of fluorescent resonance energy transfer (FRET) nanostructures. Quantitative analysis of the FRET nanostructures and live‐cell imaging reveal the high potential of these nanoprobes in bioimaging and nanophotonic applications.     

量子点:FRET的新发展

荧光共振能量转移(FRET)技术作为一种高效的光学“分子尺”,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。但是许多有机染料吸收光谱较窄而发射光谱较宽,并且光漂白现象比较严重,使得FRET的应用受到了限制,因此迫切需要寻找新的能量供-受体对。由于量子点(QDs)相对于有机染料有很多优点,可以较好地应用于FRET,可能成为FRET领域发展的一个有意义的新方向,近来已引起了人们的关注。本文就FRET的原理以及量子点应用于FRET的最新进展情况做了评述。     

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Recent developments in analytical applications of quantum dots

This review discusses the application of quantum dots (QDs) to chemical and biological detection, for which they have excellent features, particularly size-dependent optical properties.We can summarize the main areas discussed in this review as follows:(1) QDs associated with enzyme-linked immunosorbent assay (ELISA), chip detection and capillary electrophoresis (CE) enhance the sensitivity and the speed of detection of residues;(2) QDs are applied with other techniques, including polymerase chain reaction (PCR), fluorescence resonance-energy transfer (FRET) analysis, fluorescence in-situ hybridization (FISH) and western blot analysis; and,(3) QDs combined with the above techniques can successfully detect DNA and protein.We also cover perspectives and challenges in analytical applications of QDs.     

Förster Resonance Energy Transfer Investigations Using Quantum‐Dot Fluorophores

F?rster resonance energy transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited donor fluorophore to a proximal ground-state acceptor fluorophore, is a well-characterized photophysical tool. It is very sensitive to nanometer-scale changes in donor-acceptor separation distance and their relative dipole orientations. It has found a wide range of applications in analytical chemistry, protein conformation studies, and biological assays. Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores with unique optical and spectroscopic properties that could enhance FRET as an analytical tool, due to broad excitation spectra and tunable narrow and symmetric photoemission. Recently, there have been several FRET investigations using luminescent QDs that focused on addressing basic fundamental questions, as well as developing targeted applications with potential use in biology, including sensor design and protein conformation studies. Herein, we provide a critical review of those developments. We discuss some of the basic aspects of FRET applied to QDs as both donors and acceptors, and highlight some of the advantages offered (and limitations encountered) by QDs as energy donors and acceptors compared to conventional dyes. We also review the recent developments made in using QD bioreceptor conjugates to design FRET-based assays.     

Compact,Polyvalent Mannose Quantum Dots as Sensitive,Ratiometric FRET Probes for Multivalent Protein–Ligand Interactions

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.     

Compact,Polyvalent Mannose Quantum Dots as Sensitive,Ratiometric FRET Probes for Multivalent Protein–Ligand Interactions

A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.     

A Brief Overview of Some Physical Studies on the Relaxation Dynamics and Förster Resonance Energy Transfer of Semiconductor Quantum Dots

This article highlights some physical studies on the relaxation dynamics and Förster resonance energy transfer (FRET) of semiconductor quantum dots (QDs) and the way these phenomena change with size, shape, and composition of the QDs. The understanding of the excited‐state dynamics of photoexcited QDs is essential for technological applications such as efficient solar energy conversion, light‐emitting diodes, and photovoltaic cells. Here, our emphasis is directed at describing the influence of size, shape, and composition of the QDs on their different relaxation processes, that is, radiative relaxation rate, nonradiative relaxation rate, and number of trap states. A stochastic model of carrier relaxation dynamics in semiconductor QDs was proposed to correlate with the experimental results. Many recent studies reveal that the energy transfer between the QDs and a dye is a FRET process, as established from 1/d⁶ distance dependence. QD‐based energy‐transfer processes have been used in applications such as luminescence tagging, imaging, sensors, and light harvesting. Thus, the understanding of the interaction between the excited state of the QD and the dye molecule and quantitative estimation of the number of dye molecules attached to the surface of the QD by using a kinetic model is important. Here, we highlight the influence of size, shape, and composition of QDs on the kinetics of energy transfer. Interesting findings reveal that QD‐based energy‐transfer processes offer exciting opportunities for future applications. Finally, a tentative outlook on future developments in this research field is given.     

In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications.     

Impact of FRET between Molecular Aggregates and Quantum Dots

Energy transfer has been employed in third‐generation solar cells for the conversion of light into electrical energy. Long‐range nonradiative energy transfer from semiconductor quantum dots (QDs) to fluorophores has been demonstrated by using CdS QDs and thiophene?BODIPY (boron dipyrromethene, abbreviated as TG2). TG2 shows a broad photoluminescence (PL) spectrum, which varies with concentration. At very low concentrations, monomeric units are present; then, upon increasing the concentration, these monomers form a mixed (J‐/H‐)aggregated state. Energy transfer between the CdS QDs and TG2 was confirmed by separately investigating the interactions between CdS and the monomer of TG2 and between CdS and the aggregated states of TG2. Size‐dependent PL quenching confirmed that nonradiative Förster resonance energy transfer (FRET) from photoexcited CdS QDs to the J‐aggregate state of TG2 was the major energy‐relaxation channel, which occurred on the timescale of hundreds of fs. These results have broad applications in the field of light harvesting based on the assembly of molecular aggregates.     

Nicking endonuclease and target recycles signal amplification assisted quantum dots for fluorescence detection of DNA

An ultrasensitive fluorescence detection method for DNA based on nicking endonuclease (NEase) and target recycles assisted with CdTe quantum dots (QDs) is reported. In the detection system, when the target DNA is present, it hybridizes with a linker strand to from a duplex, in which the NEase recognizes specific nucleotide sequences and cleaves the linker strand. After nicking, the fragments of the linker strand spontaneously dissociate from the target DNA and another linker strand hybridizes to the target to trigger another strand-scission cycle. On the other hand, when the target was absent, no duplex is formed and no fragment of linker strand is produced. Then CdTe QDs and magnetic beads (MBs), which were all modified with DNA sequences complementary to that of the linker strands are added to the solution to detect the presence of a target DNA. The signal was generated through the difference in F?rster resonance energy transfer (FRET) between the MB and CdTe QDs. This method indicates that one target DNA leads to cleavage of hundreds of linker DNA, increasing detection sensitivity by nearly three orders of magnitude. This method should be applicable whenever there is a requirement to detect a specific DNA sequence and can also be used for multicomponent detection.     

Gold nanoparticle-based homogeneous fluorescent aptasensor for multiplex detection

We developed a homogeneous fluorescence assay for multiplex detection based on the target induced conformational change of DNA aptamers. DNA aptamers were immobilized on quantum dots (QDs), and QDs conjugated ssDNA was adsorbed on the surface of gold nanoparticles (AuNPs) by electrostatic interaction between uncoiled ssDNA and the AuNPs. Subsequently the fluorescence of QDs was effectively quenched by the AuNPs due to fluorescence resonance energy transfer (FRET) of QDs to AuNPs. In the presence of targets, the QDs conjugated aptamers were detached from AuNPs by target induced conformational change of aptamers, consequently the fluorescence of the QDs was recovered proportional to the target concentration. In this study, three different QD/aptamer conjugates were used for multiplex detection of mercury ions, adenosine and potassium ions. In a control experiment, all of the three targets were simultaneously detected with high selectivity.     

How Quantum Dots Aggregation Enhances Förster Resonant Energy Transfer

As luminescent quantum dots (QDs) are known to aggregate themselves through their chemical activation by carbodiimide chemistry and their functionalization with biotin molecules, we investigate both effects on the fluorescence properties of CdTe QDs and their impact on Förster Resonant Energy Transfer (FRET) occurring with fluorescent streptavidin molecules (FA). First, the QDs fluorescence spectrum undergoes significant changes during the activation step which are explained thanks to an original analytical model based on QDs intra-aggregate screening and inter-QDs FRET. We also highlight the strong influence of biotin in solution on FRET efficiency, and define the experimental conditions maximizing the FRET. Finally, a free-QD-based system and an aggregated-QD-based system are studied in order to compare their detection threshold. The results show a minimum concentration limit of 80 nM in FA for the former while it is equal to 5 nM for the latter, favouring monitored aggregation in the design of QDs-based biosensors.     

Detection of FRET efficiency in imaging systems by photo-bleaching acceptors

Fluorescence resonance energy transfer (FRET) is widely used to obtain the distance between a donor and an acceptor in biological research. However, the detection of FRET efficiencies with fluorescence microscopy imaging systems remains a great challenge due to the difficulties of transferring gray scales of the images into fluorescence intensities, and the absence of exact quantum yields of donors and acceptors. Herein, we presented a new method to detect the FRET efficiency in imaging systems by analyzing the photo-bleaching-induced changes in fluorescent intensities of quantum dots (QDs, donors) and Cy5 dyes (acceptors). Our method is different from the previous acceptor-photo-bleaching studies in imaging systems by theoretically analyzing the bleaching process, and bringing forward a new parameter which is universal for samples of the same kind. It is convenient for calculating FRET efficiencies. There is hardly any spectral crosstalk between 605QD and Cy5, thus the FRET result is more accurate than that of many other common FRET pairs. The lengths of single-stranded and double-stranded DNA fragments in solution were determined via the analysis of FRET efficiency values. This technique provides a reliable approach to study biomacromolecules in living cells through fluorescent imaging and in situ measurements.     

Quantum dots as simultaneous acceptors and donors in time-gated Förster resonance energy transfer relays: characterization and biosensing

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in F?rster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.     

Self-assembled quantum dot-sensitized multivalent DNA photonic wires

Combining the inherent scaffolding provided by DNA structure with spatial control over fluorophore positioning allows the creation of DNA-based photonic wires with the capacity to transfer excitation energy over distances greater than 150 ?. We demonstrate hybrid multifluorophore DNA-photonic wires that both self-assemble around semiconductor quantum dots (QDs) and exploit their unique photophysical properties. In this architecture, the QDs function as both central nanoscaffolds and ultraviolet energy harvesting donors that drive Fo?rster resonance energy transfer (FRET) cascades through the DNA wires with emissions that approach the near-infrared. To assemble the wires, DNA fragments labeled with a series of increasingly red-shifted acceptor-dyes were hybridized in a predetermined linear arrangement to a complementary DNA template that was chemoselectively modified with a hexahistidine-appended peptide. The peptide portion facilitated metal-affinity coordination of multiple hybridized DNA-dye structures to a central QD completing the final nanocrystal-DNA photonic wire structure. We assembled several such hybrid structures where labeled-acceptor dyes were excited by the QDs and arranged to interact with each other via consecutive FRET processes. The inherently facile reconfiguration properties of this design allowed testing of alternate formats including the addition of an intercalating dye located in the template DNA or placement of multiple identical dye acceptors that engaged in homoFRET. Lastly, a photonic structure linking the central QD with multiple copies of DNA hybridized with 4-sequentially arranged acceptor dyes and demonstrating 4-consecutive energy transfer steps was examined. Step-by-step monitoring of energy transfer with both steady-state and time-resolved spectroscopy allowed efficiencies to be tracked through the structures and suggested that acceptor dye quantum yields are the predominant limiting factor. Integrating such DNA-based photonic structures with QDs can help create a new generation of biophotonic wire assemblies with widespread potential in nanotechnology.     

High‐Performance Förster Resonance Energy Transfer (FRET)‐Based Dye‐Sensitized Solar Cells: Rational Design of Quantum Dots for Wide Solar‐Spectrum Utilization

High‐performance Förster resonance energy transfer (FRET)‐based dye‐sensitized solar cells (DSSCs) have been successfully fabricated through the optimized design of a CdSe/CdS quantum‐dot (QD) donor and a dye acceptor. This simple approach enables quantum dots and dyes to simultaneously utilize the wide solar spectrum, thereby resulting in high conversion efficiency over a wide wavelength range. In addition, major parameters that affect the FRET interaction between donor and acceptor have been investigated including the fluorescent emission spectrum of QD, and the content of deposited QDs into the TiO₂ matrix. By judicious control of these parameters, the FRET interaction can be readily optimized for high photovoltaic performance. In addition, the as‐synthesized water‐soluble quantum dots were highly dispersed in a nanoporous TiO₂ matrix, thereby resulting in excellent contact between donors and acceptors. Importantly, high‐performance FRET‐based DSSCs can be prepared without any infrared (IR) dye synthetic procedures. This novel strategy offers great potential for applications of dye‐sensitized solar cells.     

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.