钙黄绿素-藏红T与DNA作用机理的光谱研究

应用能量转移方法和光谱技术，研究了钙黄绿素-藏红T荧光能量转移体系与ctDNA的作用机理以及抑制作用的影响因素．在pH8．0和低离子强度条件下，ctDNA与Caleein-ST发生了静电电荷作用，降低了钙黄绿素-藏红T荧光体系的能量转移效率，对荧光体系的能量转移产生了明显的抑制作用．     

钙黄绿素-刚果红荧光光度法测定片剂及尿液中莫西沙星含量

在pH6.0的Britton-Robinson(B-R)缓冲溶液中,刚果红与钙黄绿素能够发生有效的能量转移,使钙黄绿素荧光猝灭.莫西沙星的加入,又使得钙黄绿素重现荧光,据此建立了测定莫西沙星的新方法.将该方法用于片剂和尿液中莫西沙星的测定,结果满意.实验表明,该方法简单、快速、灵敏、准确.     

基于钙黄绿素－臧红T荧光共振能量转移检测六偏磷酸钠

在pH8．5的Tris－HCl缓冲溶液中,钙黄绿素作为能量供体（D）可以与藏红T受体（A）发生有效的荧光共振能量转移（FRET）,但加入六偏磷酸钠（SHMP）后,因其与受体发生静电作用破坏了该能量转移体系,使得荧光供体钙黄绿素荧光强度的增加（△FD）与受体藏红T荧光强度的降低（△FA）的比值（△FD／△R）-9SHMP浓度（csHMP）呈良好的线性关系．基于此,建立了一种检测六偏磷酸盐的新方法．在优化条件下,该方法的检测范围为3．0×10^-6-1．0×10^-5mol／L,对6．0×10拍mol／L的六偏磷酸盐连续平行测定11次,其相对标准偏差（RSD）为3．1％．该方法具有选择性好、操作简单和检测速度快等优点,已成功应用于饮料中六偏磷酸钠的分析检测．     

荧光共振能量转移测定杀虫单

1引言 研究了钙黄绿素（Calcein）与酚藏花红（PF）的荧光共振能量转移现象，并且将其作为探针用来测定杀虫单，扩大了荧光共振能量转移的应用领域。该方法准确、简便，灵敏度高，检出限低，同时能消除仪器噪音影响，结果令人满意。     

钙黄绿素-钯（Ⅱ）荧光光度法测定土壤中甲拌磷残留

基于钙黄绿素在pH 6.4的NaoH-KH2PO4缓冲溶液中有荧光,钯(Ⅱ)与钙黄绿素反应使其荧光猝灭,甲拌磷与钙黄绿素-钯(Ⅱ)反应置换出钙黄绿素,重显荧光,分别以493 nm和514 nm为激发波长和发射波长,建立了一种测定甲拌磷的新方法.方法的线性范围为2.0×10-5～2.0×10-7mol·L-1,相关系数为0.9988,检出限为5.3×10-9mol·L-1.用该法对土壤样品进行分析,并与色谱法对照,结果比较满意.     

金纳米粒子-钙黄绿素荧光共振能量转移体系测定青霉胺

利用金纳米粒子-钙黄绿素荧光共振能量转移体系,建立了一种简单、灵敏、快速测定药物青霉胺的方法。初步探讨了方法机理,并对体系pH、反应时间、Au NPs和钙黄绿素的浓度等实验条件进行了优化。优化实验条件下,方法的线性范围为1. 87×10~(-7)~1. 68×10~(-5)mol·L~(-1),检出限(3S/N)为6. 87×10~(-8)mol·L~(-1)。该法用于青霉胺药品中青霉胺的测定,结果满意。     

碳点-钙黄绿素荧光共振能量转移体系在山奈素检测中的应用研究

研究了碳点(CDs)-钙黄绿素(CA)能量转移体系,并利用该体系建立了测定山奈素(KF)的新方法。在λex=320 nm下,pH 8.0的磷酸氢二钠-柠檬酸缓冲液中,CDs与CA能发生有效的能量转移,使得CA的荧光增强,而KF的加入则可有效猝灭CA的荧光,且在一定范围内CA的荧光猝灭值与KF的加入量呈良好的线性关系,基于此建立了测定KF的新方法。在最佳实验条件下,方法的线性范围为1.0×10-8~3.4×10-6mol/L,检出限为3.33×10-9mol/L。方法应用于复方金银花颗粒中KF含量的测定,回收率为98.4%~103.5%,RSD(n=6)不大于2.7%。     

荧光猝灭法测定中草药中痕量钴

在pH 7.4的磷酸盐缓冲溶液和十六烷基三甲基溴化铵(CTMAB)介质中,钙黄绿素与罗丹明B之间发生能量转移而导致后者的荧光增强(λem581nm)。当在此体系中加入痕量Co2+后,于发射波长581nm处产生荧光猝灭现象。据此提出了荧光猝灭法测定中草药中痕量钴含量。Co2+的质量浓度在4.0×10-4～1.35×10-2g.L-1范围内与反应液荧光强度的降低程度呈线性关系,方法的检出限(3S/N)为1.8×10-7g.L-1。方法用于中草药样品分析,回收率在97.6%～102%之间,测定值的相对标准偏差(n=6)在0.98%～1.5%之间。     

钙黄绿素-铜(Ⅱ)荧光体系测定痕量硫离子

在pH 8.0的KH2PO4-NaOH缓冲液中,Cu(Ⅱ)与钙黄绿素配位引起荧光猝灭。由于硫离子与Cu(Ⅱ)的亲和力很强,可从钙黄绿素-Cu(Ⅱ)的络合物中夺取Cu(Ⅱ)而使钙黄绿素游离出来,从而使体系的荧光得以恢复,并且荧光恢复的程度与加入硫离子的量在一定范围内呈线性。据此建立了一种测定硫离子的新方法,该方法的线性范...     

钙黄绿素蓝荧光猝灭测定茶叶中的锰

本文研究了Ｍｎ（Ⅱ）－钙黄绿素蓝的荧光体系的测定方法及条件，在ＰＨ９．５－１０．０的硼砂－氢氧化钾缓冲溶液中，钙黄绿素蓝的λｅｘ为３６２ｎｍ，λｅｍ为４４６ｎｍ，Ｍｎ（Ⅱ）与钙黄绿素蓝生成的１：１络合物猝灭钙黄绿素蓝的荧光。锰量的线性范围为０．０５－１．０μｇ／１０ｍＬ，方法灵敏度高，检测限为５ｎｇ／ｍＬ。该方法用于茶叶中锰的测定，结果满意。     

Method for determination of xanthene dyes in guava fruits and its application in a field dissipation study

Xanthene dyes, i.e., phloxine B and uranine or phloxine B alone, are phototoxic to tephritid fruit flies infesting guava fruits. An analytical method was developed for determination of residues of these dyes used in bait solutions for suppression of the tephritid fruit fly population in guava fruits. The procedure involved solvent extraction, anion-exchange cleanup, and determination by liquid chromatography or capillary zone electrophoresis. The dyes were extracted from 50 g guava fruit at 45 degrees degrees with 400 mL methanol-acetonitrile (1 + 1) and 5 g magnesium oxide added as an alkaline and clarifying agent. The guava extract was adjusted to pH 8.5 and subjected to an amino column cleanup. Average recoveries of xanthene dyes added to guava purees ranged from 77 to 99% for phloxine B and from 79 to 102% for uranine at spiking levels of 0.05-1.00 microg/g. The method was applied to the determination of phloxine B residues in guava fruits collected from a dye-sprayed orchard. After phloxine B was applied at a rate of 62.5 g/ha for 14 weekly sprayings, it was found on guava fruits at an average concentration of 111 +/- 18 ng/g 4 h after the llth spraying. The concentration of phloxine B was 426 +/- 94 ng/g in selected fruits with high deposits of the dye 4 h after spraying. Average concentrations of phloxine B 5 days after the 7th and 14th sprayings were 29 +/- 7 and 19 +/- 8 ng/g, respectively.     

Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins.

Detection of F?rster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater F?rster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

Two-dimensional fluorescence resonance energy transfer as a probe for protein folding: a theoretical study

We describe a two-dimensional (2D), four-color fluorescence resonance energy transfer (FRET) scheme, in which the conformational dynamics of a protein is followed by simultaneously observing the FRET signal from two different donor-acceptor pairs. For a general class of models that assume Markovian conformational dynamics, we relate the properties of the emission correlation functions to the rates of elementary kinetic steps in the model. We further use a toy folding model that treats proteins as chains with breakable cross-links to examine the relationship between the cooperativity of folding and FRET data and to establish what additional information about the folding dynamics can be gleaned from 2D, as opposed to one-dimensional FRET experiments. We finally discuss the potential advantages of the four-color FRET over the three-color FRET technique.     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors

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 F?rster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.     

Quantitative FRET (qFRET) Technology for the Determination of Protein–Protein Interaction Affinity in Solution

Protein–protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein–protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein–protein interaction affinity (K_D), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.     

Molecularly imprinted coated graphene oxide solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of phloxine B in coffee bean

A method was developed to sensitively determine phloxine B in coffee bean by molecularly imprinted polymers (MIPs) coated graphene oxide (GO) solid-phase extraction (GO-MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC–LIF). The GO-MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using GO as supporting material, phloxine B, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade GO-MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions. The GO-MIPs were characterized by scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FT-IR). The mean recoveries of phloxine B in coffee bean ranged from 89.5% to 91.4% and the intra-day and inter-day relative standard deviation (RSD) values all ranged from 3.6% to 4.7%. Good linearity was obtained over 0.001–2.0 μg mL⁻¹ (r = 0.9995) with the detection limit (S/N = 3) of 0.075 ng mL⁻¹. Under the selected conditions, enrichment factors of over 90-fold were obtained and extraction on the monolithic column effectively cleaned up the coffee bean matrix. The results demonstrated that the proposed GO-MISPE HPLC–LIF method can be applied to sensitively determine phloxine B in coffee bean.     

Calculation of transition dipole moment in fluorescent proteins--towards efficient energy transfer

F?rster Resonance Energy Transfer (FRET) between fluorescent proteins (FPs) is widely used to construct fluorescent sensor proteins, to study intracellular protein-protein interactions and to monitor conformational changes in multidomain proteins. Although FRET depends strongly on the orientation of the transition dipole moments (TDMs) of the donor and acceptor fluorophores, this orientation dependence is currently not taken into account in FRET sensor design. Similarly, studies that use FRET to derive structural constrains typically assume a κ(2) of 2/3 or use the TDM of green fluorescent protein, as this is the only FP for which the TDM has been determined experimentally. Here we used time-dependent density functional theory (TD-DFT) methods to calculate the TDM for a comprehensive list of commonly used fluorescent proteins. The method was validated against higher levels of calculation. Validation with model compounds and the experimentally determined TDM of GFP shows that the TDM is mostly determined by the structure of the π-conjugated fluorophore and is insensitive to non-conjugated side chains or the protein surrounding. Our calculations not only provide TDM for most of the currently used FPs, but also suggest an empirical rule that can be used to obtain the TDMs for newly developed fluorescent proteins in the future.