Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations

The use of F?rster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym "FRET" returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation.     

基于锰掺杂量子点的噁喹酸荧光共振能量转移检测研究

基于噁喹酸对锰掺杂硫化锌量子点的荧光猝灭作用,建立了一种噁喹酸荧光共振能量转移检测方法.噁喹酸对量子点的荧光猝灭是由于生成了新的复合物而造成的静态猝灭,二者相互作用过程中焓变ΔH ＜ 0,熵变ΔS ＜ 0,分子间作用力为氢键或范德华力.在0～65 μg/L线性范围内,噁喹酸质量浓度与量子点荧光抑制率呈现良好的线性关系(...     

A homogeneous assay for highly sensitive detection of CaMV35S promoter in transgenic soybean by förster resonance energy transfer between nitrogen-doped graphene quantum dots and Ag nanoparticles

In this work, a novel homogeneous assay for DNA quantitative analysis based on förster resonance energy transfer (FRET) was developed for cauliflwer mosaic virus 35s (CaMV35S) promoter of transgenic soybean detection. The homogenous FRET of fluorescence signal was fabricated by DNA hybridization with probe modified nitrogen-doped graphene quantum dots (NGQDs) and silver nanoparticles (AgNPs), which acted the donor-acceptor pairs for the first time. The highly efficient FRET and unique properties of the NGQDs made the proposed FRET system as a functionalized detection platform for labelling of DNA. Upon the recognition of specific target DNA (tDNA), the FRET between NGQDs and AgNPs was triggered to produce fluorescence quenching, which could be used for tDNA detection. The fabricated homogeneous FRET assay displayed a wide linear range of 0.1–500.0 nM and a low limit of detection 0.03 nM for the detection of CaMV35S (S/N = 3). This proposed biosensor revealed high specificity to detect tDNA, with acceptable intra-assay precision and excellent stability. This method was successfully applied to identify the real sample of 0.5% containing transgenic soybean, which achieved the most of national law regulations. This assay was further validated by polymerase chain reaction as the genetically modified organisms, suggesting that the proposed FRET system is a feasible tool for the further daily genetically modified organism detection.     

Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing

Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody–antigen interaction in the presence of casein kinase II (CK2) and adenosine 5′-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis.     

金纳米微粒表面能量转移及半胱氨酸的高灵敏度高选择性分析法

根据荧光染料在金纳米粒子表面的能量转移,本文建立了一种具有高灵敏和高选择性半胱氨酸分析方法.研究表明,通过静电作用吸附在柠檬酸根包被的金纳米粒子表面的阳离子荧光染料如罗丹明B分子在受光激发时,发生从荧光染料到金属纳米微粒的能量转移,导致荧光染料的荧光猝灭.但当体系中存在半胱氨酸时,由于半胱氨酸与金纳米粒子之间具有更强的共价作用,罗丹明B分子远离金纳米粒子表面,降低了能量转移效率,使得罗丹明B的荧光得到恢复.恢复的荧光强度与0.025～4.5μmol/L半胱氨酸呈很好的线性关系,检测限为8.0nmol/L(3σ),而其他十九种基本氨基酸的响应非常微弱.     

Nonblinking plasmonic quantum dot assemblies for multiplex biological detection

Nonblinking nanosystems are prepared by layer-by-layer polyelectrolyte deposition, which precisely controls the stoichiometry and the distance between quantum dots (QDs) and gold nanoparticles (GNPs). Conjugation of biorecognition molecules to these nanobarcodes enables cell targeting and entry with prolonged retention and minimal toxicity.     

Electrophoretic Deposition of a Reduced Graphene–Au Nanoparticle Composite Film as Counter Electrode for CdS Quantum Dot‐Sensitized Solar Cells

A reduced graphene (RG)‐Au nanoparticle composite film is successfully fabricated by electrophoretic deposition and used as counter electrode for quantum dot‐sensitized solar cells. The RG‐Au composite is prepared by one‐step microwave‐assisted reduction of chloroaurate in alkaline solution with graphite oxide dispersion. Under one sun illumination (AM 1.5 G, 100 mW cm^?2), the cell with a RG‐Au counter electrode shows an energy conversion efficiency of 1.36 %, which is higher than those of cells employing conventional Pt or Au counter electrodes, due to the superior combination of highly catalytic Au nanoparticles and the conductive graphene network structure.     

Cyclodextrin-promoted energy transfer for broadly applicable small-molecule detection

Reported herein is the development of non-covalent, proximity-induced energy transfer from small-molecule toxicants to organic fluorophores bound in the cavity of γ-cyclodextrin. This energy transfer occurs with exceptional efficiency for a broad range of toxicants in complex biological media, and is largely independent of the spectral overlap between the donor and acceptor. This generally applicable phenomenon has significant potential in the development of new turn-on detection schemes.     

Stable Sugar‐Chain‐Immobilized Fluorescent Nanoparticles for Probing Lectin and Cells

Sugar chains are important molecules in cellular recognition and signaling, and quantum dots (QDs) are a very powerful tool for in vitro and in vivo imaging. Herein, we report the preparation of stable sugar‐chain‐immobilized fluorescent nanoparticles (SFNPs) and their application to the analysis of sugar‐chain–protein interactions and cellular imaging. SFNPs were easily prepared by mixing CdTe/CdS core/shell QDs with our previously developed sugar‐chain–ligand conjugates. The obtained SFNPs were very stable and could be stored for several months. In the binding analysis, β‐galactose‐ and α‐glucose‐immobilized SFNPs were specifically interacted with Ricinus communis agglutinin I and concanavalin A, respectively, and made into aggregates. The binding interaction was detected visually, fluorescently, or both. In the experiment for cellular imaging, it was found that SFNPs were predominantly taken up by human hepatocyto carcinoma cells (HepG2), suggesting the possible usage of our designed SFNPs for various biochemical analyses of sugar chains.     

Study on the fluorescence resonance energy transfer between CdTe QDs and butyl-rhodamine B in the presence of CTMAB and its application on the detection of Hg(II)

The thioglycolic acid-functionalized CdTe quantum dots (QDs) were synthesized in aqueous solution using safe and low-cost inorganic salts as precursors. Fluorescence resonance energy transfer (FRET) system was constructed between CdTe QDs (donor) and butyl-rhodamine B (BRB) (acceptor) in the presence of cetyltrimethylammonium bromide (CTMAB). CTMAB micelles formed in water reduced the distance between the donor and the acceptor significantly and thus improved the FRET efficiency, which resulted in an obvious fluorescence enhancement of the acceptor. Several factors which impacted the fluorescence spectra of the FRET system were studied. The energy transfer efficiency (E) and the distance (r) between CdTe and BRB were obtained. The feasibility of the prepared FRET system as fluorescence probe for detecting Hg(II) in aqueous solution was demonstrated. At pH 6.60, a linear relationship could be established between the quenched fluorescence intensity of BRB and the concentration of Hg(II) in the range of 0.0625-2.5mumolL(-1). The limit of detection was 20.3nmolL(-1). The developed method was proved to be sensitive and repeatable to detect Hg(II) in a wide range in aqueous solutions.     

A double regio- and stereoselective glycosylation strategy for the synthesis of N-glycans

A building block approach for biantennary N-linked oligosaccharides from glycoproteins (N-glycans) has been developed. Starting from a core trisaccharide (beta-mannosyl chitobiose) containing a benzylidene-protected beta-mannoside, the attachment of the disaccharide building blocks for the antennae can be performed in a double regio- and stereoselective manner. A short synthesis of a GlcNPhtbeta1,2Man donor was developed. The benzylidene acetal moiety, as a minimal protection of the beta-mannoside, allows selective alpha-glycosylation at OH-3 of the 2,3-diol with GlcNbeta1,2Man trichloroacetimidate donors. Subsequent debenzylidenation leads to a 4,6-diol, which can be selectively extended at OH-6. Overreaction at OH-4 was generally low when phthalimido-protected donors were used. This general strategy represents a modular synthesis of N-glycans and their glycoconjugates.     

Resonance energy transfer methods of RNA detection

Quantitation of RNA is important in diagnostics, environmental science, and basic biomedical research. RNA is considered a signature for pathogen identification, and its expression profile is linked with disease pathogenesis, allowing for biomarker identification. RNA-based diagnostics is an emerging field of research. This expansion of interest in studying RNA has generated demand for its accurate and sensitive detection. Several methods have therefore been developed to detect RNA. Resonance energy transfer methods of RNA detection are highly promising in terms of simplicity and high sensitivity. In this review, we have focused on the latest developments in resonance energy transfer methods of RNA detection that utilize various probe designs. The probe designs discussed here are molecular beacons, quenched autoligation probes, and linear oligonucleotide probes. Resonance energy transfer methods based on both fluorescence and bioluminescence detection are discussed.     

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.     

In‐capillary probing of quantum dots and fluorescent protein self‐assembly and displacement using Förster resonance energy transfer

Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real‐time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His₆‐mCherry/quantum dot molar ratio. The kinetics of the self‐assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of‐ and in‐capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in‐capillary probing system.     

Comparative efficiency of energy transfer from CdSe-ZnS quantum dots or nanorods to organic dye molecules

Fluorescence resonance energy transfer (FRET) in conjugates of CdSe-ZnS semiconductor nanocrystals of different shapes (FRET donors) and an Alexa Fluor organic dye (FRET acceptors) is examined. The dye molecules are chemically conjugated with quantum dots (QDs) or nanorods (NRs) in dimethyl sulfoxide colloidal solutions, and FRET efficiency in the purified conjugates is measured. The FRET from NR to a single dye molecule is less efficient than that of the QD-dye conjugates and this effect is explained in terms of distance-limited energy-transfer rate in the case of a point-like acceptor and extended donor dipoles. However, the larger surface area of NRs allows for many more dye acceptors to be bound, and the total FRET efficiency in NR-dye conjugates approaches those of QD-dye conjugates.     

Fluorescence resonance energy transfer in doubly-quantum dot labeled IgG system

The mouse immunoglobulin G (mouse IgG) as a kind of bio-molecule was labeled with two different luminescent colloidal semiconductor quantum dots (QDs), green-emitting CdTe quantum dots and red-emitting CdTe quantum dots in this work. As a result of the fluorescence resonance energy transfer (FRET) between the two different sizes nanoparticles with mouse IgG as the binding bridge, a significant enhancement of the emission of the red-emitting CdTe quantum dots and the corresponding quenching of the emission of green-emitting CdTe quantum dots were observed. The relationship between the concentration of the mouse immunoglobulin G and the fluorescence intensity ratio (I_a/I_d) of acceptors and donors was studied also. Under optimal conditions, the calibration graph is linear over the range of 0.1–20.0 mg/L mouse IgG.     

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min.