Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer

We introduce a sensitive, rapid, label-free and general fluorescent method for the determination of tartrazine by competitive binding to reduced graphene oxide (rGO) against fluorescein, and the fluorescence recovery upon fluorescein desorption from rGO provides a quantitative readout for tartrazine, giving a detection limit of 0.53 ng mL(-1).     

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.     

Continuous-flow protease assay based on fluorescence resonance energy transfer

A homogeneous continuous-flow assay using fluorescence resonance energy transfer (FRET) for detection was developed to measure the hydrolysis of HIV Protease Substrate 1 (to which two choromophores, EDANS and DABCYL are covalently attached) by a protease (e.g. Subtilisin Carlsberg) and the influence of inhibitors. In the continuous-flow assay, an inhibitor solution and an enzyme solution were first eluted into the system and allowed to react with each other in a reaction coil. Subsequently, the substrate solution was added to an enzyme-inhibitor mixture in a second reaction coil and incubated for 1 min. Finally, the fluorescence intensity was monitored.The system was also utilized to measure the inhibition of the protease by two weak acidity inhibitors which are 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylenediaminetetraacetic acid (EDTA). Using the obtained optimum conditions for AEBSF, a detection limit of 0.3 mmol/l was achieved and the relative standard deviation was below 3.7% in the 2.5-7.5 mmol/l range. For EDTA, which required a 20 times higher substrate concentration than AEBSF, a detection limit of 0.2 mmol/l was obtained and the relative standard deviation was below 9.6% in the 0.5-7.5 mmol/l range.The optimization of pH, substrate concentration, enzyme concentration, reaction time and temperature are described. Organic modifier effects were also investigated. Methanol, acetonitrile and DMSO could be tolerated up to 30%.     

Shape-specific detection based on fluorescence resonance energy transfer using a flexible water-soluble conjugated polymer

We present the detection of the shape-specific conformation of DNA based on the fluorescence resonance energy transfer (FRET) by using a novel flexible water-soluble cationic conjugated polymer (CCP). The flexible backbone of CCP has more conformational freedom with the potential to be responsive to analyte shape by electrostatic interaction between flexible CCP and negatively charged DNA. The analyte shape dependent recognition is accomplished by structural changes that compressed or extended the flexible CCP. The morphology-dependent spectral properties of the novel flexible polymer related to the analyte shapes are investigated in detail, where two types of chromophores, referred to as "isolated" segment and "packed" segment aggregates, within the flexible polymer are identified by means of ensemble and single molecule measurements upon binding with different geometric DNA. The change in fluorescence intensity upon binding with shape-specific DNA without obvious color shifts makes this novel flexible polymer a suitable CCP donor for FRET measurements. The results provide insights for understanding the spectral properties of flexible water-soluble CCP and CCP/DNA interaction related to the geometry of target analyte.     

一种萘酰亚胺类荧光共振能量转移型光酸探针

合成了一种未见文献报道的键合螺噁嗪单元的1,8-萘二甲酰亚胺类化合物3,通过核磁共振谱和高分辨质谱确证了其结构.化合物在某些有机溶剂中和粉末状态下都能发射较强的荧光,研究了化合物3在二甲基亚砜中聚集诱导荧光增强(AIE)性质.在滤纸上以及用薄层层析硅胶(TLC)都能检测到螺噁嗪单元的光致变色现象;螺噁嗪单元酸致开环产物...     

Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin-quencher is bound to Eu-streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher-biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2-2 nmol L⁻¹. In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay.     

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(...     

Development of a new fluorescent probe for transition metal cations based upon fluorescence resonance energy transfer

A novel fluorescent probe for metal cations, which has a large Stokes shift, was synthesized from the reaction of N-(3-carboxy-2-naphthyl)-ethylenediamine-N,N′,N′-triacetic acid (CNEDTA) with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED). The large Stokes shift is due to the FRET phenomenon between a donor (CNEDTA) and an acceptor (DBD-ED) fluorophore. When the fluorescent probe, DBD-ED-CNEDTA, was excited at 240, 340 and 440 nm, an emission maximum was observed only at 560 nm. However, the fluorescence (FL) at 480 nm, based upon the CNEDTA moiety, was not detected with excitation at 340 nm. The FL intensity of DBD-ED-CNEDTA was dependent upon the acidity of the medium and highest at pH 4.1. DBD-ED-CNEDTA reacted with metal cations, i.e., Zn, Cd, Al, Y, and La, in aqueous medium to form chelates. The spectral change of FL excitation and emission was small before and after the addition of the metal ions. However, the FL intensity was dependent upon the concentrations of the metal ions. In the case of Zn²⁺, the molar ratio bound with DBD-ED-CNEDTA was calculated as 1:1. The FL intensities after chelate formation of Zn/DBD-ED-CNEDTA (1:1) were enhanced by 3.8-fold (excitation at 340 nm, emission at 560 nm), 4.2-fold (excitation at 440 nm, emission at 560 nm), and 5.9-fold (excitation at 240 nm, emission at 560 nm), respectively. The FL probe was applied to the determination of Zn in a food supplement.     

量子点荧光共振能量转移检测赭曲霉毒素A

基于量子点与荧光猝灭基团之间构成的荧光共振能量转移体系,以量子点标记赭曲霉毒素A适配体与荧光猝灭基团标记的补体杂交构成荧光传感探针,当有赭曲霉毒素A存在时,由于其适配体与赭曲霉毒素A的高度亲和作用,使传感探针上结合的荧光猝灭剂减少,荧光增强,从而建立了一种检测赭曲霉毒素A的荧光分析方法.该方法简单、快速、特异性强,在适...     

A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.     

Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer

Intramolecular distances in proteins and other biomolecules can be studied in living cells by means of fluorescence resonance energy transfer (FRET) in steady-state or pulsed-excitation experiments. The major uncertainty originates from the unknown orientation between the optical dipole moments of the fluorescent markers, especially when the molecule undergoes thermal fluctuations in physiological conditions. We introduce a statistical method based on the von Mises-Fisher distribution for the interpretation of fluorescence decay dynamics in donor-acceptor FRET pairs that allows us to retrieve both the orientation and the extent of directional fluctuations of the involved dipole moments. We verify the method by applying it to donor-acceptor pairs controllably attached to DNA helices and find that common assumptions such as complete rotational freedom or fully hindered rotation of the dipoles fail a physical interpretation of the fluorescence decay dynamics. This methodology is applicable in single-molecule and ensemble measurements of FRET to derive more accurate distance estimates from optical experiments, without the need for more complex and expensive NMR studies.     

Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer

We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and F?rster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.     

A DNA hybridization detection based on fluorescence resonance energy transfer between dye-doped core-shell silica nanoparticles and gold nanoparticles

A novel and efficient method to evaluate the DNA hybridization based on a fluorescence resonance energy transfer (FRET) system, with fluorescein isothiocyanate (FITC)-doped fluorescent silica nanoparticles (SiNPs) as donor and gold nanoparticles (AuNPs) as acceptor, has been reported. The strategy for specific DNA sequence detecting is based on DNA hybridization event, which is detected via excitation of SiNPs-oligonucleotide conjugates and energy transfer to AuNPs-oligonucleotide conjugates. The proximity required for FRET arises when the SiNPs-oligonucleotide conjugates hybridize with partly complementary AuNPs-oligonucleotide conjugates, resulting in the fluorescence quenching of donors, SiNPs-oligonucleotide conjugates, and the formation of a weakly fluorescent complex, SiNPs-dsDNA-AuNPs. Upon the addition of the target DNA sequence to SiNPs-dsDNA-AuNPs complex, the fluorescence restores (turn-on). Based on the restored fluorescence, a homogeneous assay for the target DNA is proposed. Our results have shown that the linear range for target DNA detection is 0-35.0 nM with a detection limit (3σ) of 3.0 picomole. Compared with FITC-dsDNA-AuNPs probe system, the sensitivity of the proposed probe system for target DNA detection is increased by a factor of 3.4-fold.     

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.     

Quantum dot-based multiplexed fluorescence resonance energy transfer

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.     

Amylase substrate based on fluorescence energy transfer

A fluorigenic substrate for measuring α-amylase (E.C. 3.2.1.1) activity was prepared by double labeling soluble starch with 5-(4,6-dichlorotrizain-2-yl)aminofluorescein and Procion Red MX8B. Because the absorption spectrum of Procion Red MX8B overlaps the fluorescein emission spectrum, Procion Red efficiently quenches fluorescein emission when it is closer than the critical radius for fluorescence energy transfer. When amylase catalyzes cleavage of a starch molecule between a fluorescein and a Procion Red MX8B, the distance between the two labels increases and the degree of quenching decreases. The rate at which the fluorescence intensity increases is proportional to amylase activity. To maximize the sensitivity it is critical to maximize the amount of Procion Red MX 8B coupled to the starch and to use a high-precision spectrofluorimeter which can measure a small rate of increase in fluorescence above a large constant background.     

Fiber-optic biosensors based on fluorescence energy transfer

A new optical homogeneous biochemical method for the assay of glucose has been developed, based on fluorescence energy transfer between a glucose analog, dextran labeled with fluorescein isothiocyanate (FITC-dextran), and a glucose-receptor protein, Rhodamine-labeled Concanavalin A (Rh-ConA). When FITC-dextran binds to Rh-ConA in solution, and is light-activated, the FITC label transfers its absorbed energy to the Rhodamine label, which then emits light according to its own characteristic fluorescence spectrum. When glucose is added to this solution, the FITC fluorescence intensity increases as FITC-dextran is released from the Rh-ConA and is replaced by glucose. Thus it is possible to determine glucose concentrations directly from the level of FITC fluorescence.     

A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy

An ultrasensitive fluorescence resonance energy transfer (FRET) bioassay was developed to detect staphylococcal enterotoxin B (SEB), a low molecular exotoxin, using an aptamer-affinity method coupled with upconversion nanoparticles (UCNPs)-sensing, and the fluorescence intensity was prominently enhanced using an exonuclease-catalyzed target recycling strategy. To construct this aptasensor, both fluorescence donor probes (complementary DNA₁–UCNPs) and fluorescence quencher probes (complementary DNA₂–Black Hole Quencher₃ (BHQ₃)) were hybridized to an SEB aptamer, and double-strand oligonucleotides were fabricated, which quenched the fluorescence of the UCNPs via FRET. The formation of an aptamer–SEB complex in the presence of the SEB analyte resulted in not only the dissociation of aptamer from the double-strand DNA but also both the disruption of the FRET system and the restoration of the UCNPs fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I, an exonuclease specific to single-stranded DNA, for analyte recycling by selectively digesting a particular DNA (SEB aptamer). Based on this exonuclease-catalyzed target recycling strategy, an amplified fluorescence intensity could be produced using different SEB concentrations. Using optimized experimental conditions produced an ultrasensitive aptasensor for the detection of SEB, with a wide linear range of 0.001–1 ng mL⁻¹ and a lower detection limit (LOD) of 0.3 pg mL⁻¹ SEB (at 3σ). The fabricated aptasensor was used to measure SEB in a real milk samples and validated using the ELISA method. Furthermore, a novel aptasensor FRET assay was established for the first time using 30 mol% Mn²⁺ ions doped NaYF₄:Yb/Er (20/2 mol%) UCNPs as the donor probes, which suggests that UCNPs are superior fluorescence labeling materials for food safety analysis.