Study of Organized Media Using Time-Resolved Fluorescence Spectroscopy

Many photophysical processes which occur on an ultrafast time scale in ordinary liquids become significantly retarded in organized assemblies, by two to three orders of magnitude. Recently many groups have applied ultrafast laser spectroscopy and theoretical methods to elucidate this dramatic phenomenon. Although the implications of this phenomenon in biology and chemistry are not yet fully understood, it has been demonstrated that ultrafast time-resolved fluorescence spectroscopy is a very powerful tool to study the microscopic properties of the organized assemblies and that water or other liquids confined inside these assemblies are fundamentally different from the corresponding liquid in bulk.     

Progress in Boronic Acid-Based Fluorescent Glucose Sensors

Effective management of diabetes relies on the frequent monitoring of blood glucose concentrations. The current approach of blood sampling and glucose concentration determination in vitro) presents many problems. Therefore, there is a drive for the development of non-invasive and continuous monitoring of glucose concentrations. The use of implanted glucose fluorescent sensors represents a promising approach. Due to its strong interaction with diol moieties, the boronic acid group often plays a critical role in the design of such glucose sensors. This paper reviews the progress in this area during the last ten years.     

Lanthanide Complex-Based Fluorescence Label for Time-Resolved Fluorescence Bioassay

Different from organic fluorescence dyes, fluorescent lanthanide complexes have the fluorescence properties of long fluorescence lifetime, large Stokes shift and sharp emission profile, which makes them favorable be used as the fluorescent labeling reagents for microsecond time-resolved fluorescence bioassay. Lanthanide complex-based fluorescence labels have been successfully used for highly sensitive time-resolved fluorescence immunoassay, DNA hybridization assay, cell activity assay, and bioimaging microscopy assay. Since the technique allows easy distinction of the specific fluorescence signal of the long-lived label from short-lived background noises associated with biological samples, scattering lights (Tyndall, Rayleigh and Raman scatterings) and the optical components (cuvettes, filters and lenses), the sensitivity of fluorescence bioassay has been remarkably improved. This paper summarized the recent developments of lanthanide complex-based fluorescence labels and their applications in time-resolved fluorescence bioassays mainly based on the authors’ researches and relative publications.     

Boronic Acid Based Modular Fluorescent Sensors for Glucose

Modular photoinduced electron transfer (PET) sensors bearing two phenylboronic acid groups, one or two fluorophores: pyrene(a), phenanthrene(b), anthracene(c), 1-naphthalene(d), 2-naphthalene(e) and alkylene linkers, from trimethylene(3) to octamethylene(8), have been evaluated. Systems with a single pyrene fluorophore 34a, 35a and 36a bind the strongest with D-glucose (36a also binds well with D-melibiose). Whilst 37a and 38a bind the strongest with D-galactose. Changing the fluorophore, also, influences the binding, 36a, 36b and 36c are D-glucose selective, whilst 36d and 36e are D-galactose selective. Systems with two fluorophores 36a-a and 36a-b show an overall decrease in binding efficiency. Energy transfer in 36a-b results in enhanced sensitivity and selectivity towards D-glucose.     

用时间分辨光谱研究红细胞悬液的荧光发射

实验获得了激光照射红细胞悬液的荧光光谱,并分别监测不同荧光峰值波长处强度随时间的衰变过程,测试了其相应的荧光寿命。结果表明,在波长为407nm的激光照射下,红细胞悬液向外发射中心波长分别位于596,628,692nm的荧光光谱,各荧光峰对应衰变过程的平均荧光寿命分别为1.97,13.31,14.58ns。利用荧光强度和吸收率的加和性表示了混合物的总吸收率和总荧光发射强度,通过理论计算获得了红细胞悬液中锌卟啉、原卟啉和其他游离物参与荧光发射的相对含量和相对强度在不同荧光峰位的变化关系,进一步解释了不同峰位处荧光发射强度和平均荧光寿命的变化原因。     

A Study of Boronic Acid Based Fluorescent Glucose Sensors

Boronic acid based anthracene dyes were designed, synthesized, and immobilized to solid phase, creating a continuous glucose sensor. Glucose sensitivities of dyes can decrease drastically after immobilization, therefore how to immobilize a dye to solid phase without changing the dye property is a key issue in developing the sensor. The glucose sensitivity of the simplest 1st generation sensor, which is based on an immobilized mono-phenylboronate/single-arm type, came short of the sensitivity requirement for practical use, because of the very moderate fluorescence intensity change over the physiological glucose range. However, the 2nd generation, an immobilized bis-phenylboronate/double-arm type sensor, which contained two boronate groups in the dye moiety in expectation of a large intensity change, brought about considerable improvement on its glucose sensitivity. We tried to introduce functional groups onto an anthracene ring in order to improve the dies' fluorescence properties. Acetyl or carboxyl substitution on anthracene contributed to shift the fluorescence wavelength into the more visible range (red-shift) and a divergence of wavelength between an excitation peak and an emission peak. This improvement is advantageous to the design of an optical detection system. Furthermore, single arm immobilization to this carboxyl group, thus linking directly to the fluorophore led to a 3rd generation sensor, an immobilized bis-phenylboronate/single-arm type, that was twice as sensitive as that of the 2nd generation sensor, presumably due to increased mobility of the dye moiety. The results of our study advance closer toward a clinically useful continuous fluorescent glucose sensor.     

Live Imaging of Glucose Homeostasis in Nuclei of COS-7 Cells

Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.     

Nonenzymatic Direct Assay of Hydrogen Peroxide at Neutral pH Using the Eu<Subscript>3</Subscript>Tc Fluorescent Probe

A detailed study is presented on the use of an easily accessible probe (the europium–tetracycline 3:1 complex; referred to as Eu₃Tc) for determination of hydrogen peroxide (HP). Eu₃Tc undergoes a 15-fold increase in luminescence intensity on exposure to an excess of HP. Data are given on the time dependence of the reaction, on the pH dependence of the absorption and emission spectra of both the probe and its complex with HP, and on the effect of stoichiometry between Eu³⁺ and Tc on selectivity and signal change. HP can be quantified in aqueous solution of pH 6.9 over a 2–400 μM concentration range with a limit of detection of 960 nM. The assay is validated using standard additions, and mean recoveries are found to be between 97.0 and 101.8%. Species that interfere in concentrations below 1 mM include phosphate, copper(II), fluoride and citrate. The addition of detergents causes the response curves towards HP to shift to higher HP concentrations. The method is critically assessed with respect to other common optical methods for determination of HP.     

用于飞秒时间分辨分子反应动力学研究的千赫兹离子/电子速度成像仪

A new velocity map imaging spectrometer is constructed for molecular reaction dynamics studies using time-resolved photoelectron/ion spectroscopy method.By combining a kHz pulsed valve and an ICCD camera,this velocity map imaging spectrometer can be run at a repetition rate of 1 kHz,totally compatible with the fs Ti:Sapphire laser system,facilitating time-resolved studies in gas phase which are usually time-consuming.Time-resolved velocity map imaging study of NH₃ photodissociation at 200 nm was performed and the time-resolved total kinetic energy release spectrum of H+NH₂ products provides rich information about the dissociation dynamics of NH₃.These results show that this new apparatus is a powerful tool for investigating the molecular reaction dynamics using time-resolved methods.     

Fluorescence and Time-Resolved Delayed Luminescence of Porphyrins in Organic Solvents and Polymer Matrices

Porphyrin dyes fulfill an essential function in photosynthesis and are important in photodynamic therapy and in a range of electronic devices. Their spectroscopic characteristics may play a crucial role in these processes. The spectral properties of two porphyrin dyes: tetraphenylporphyrin and tetraphenysulfporphyrin in organic solvents (acetone, chloroform, methyl alcohol, and dimethyl-sulfoxide) and in polyvinyl alcohol and poly(methylmethacrylate) films have been investigated. Absorption, fluorescence, and microsecond time-resolved delayed luminescence spectra have been measured at room temperature. The existence of different aggregated dye forms in the ground and excited states has been demonstrated. The manifold of dye species depends on the solvent/polymer. In the case of the polymers, it also depends on the solvent used to coat the polymer film. Delayed luminescence spectra and decay times of the two porphyrins in the different solutions and in polymeric matrices suggest that different mechanisms of deexcitation of the singlet excited states may be responsible for their generation in these and other porphyrin dyes.     

Solvation and Solvent Relaxation in Swellable Copolymers as Studied by Time-Resolved Fluorescence Spectroscopy

The fluorescent probe dimethylaminonaphthylsulfonamide is covalently bound to the ends of the pol(ethylene glycol) chains of the swellable block copolymers poly(ethylene glycol)–polystyrene (PEG-PS) and poly(ethylene glycol)–poly(ethylene imine) (PEG-PEI) to investigate the molecular mobility inside the polymers, swollen by different liquids. Steady-state and time-resolved studies of the Stokes shift between absorption and fluorescence spectra reveal that the probe is solvated by both the polymer matrix and the liquid phase. The extent of solvation by the liquid and the mobility of the microenvironment of the probe depend on both the swelling volume of the polymer and the solubility of the probe in this liquid. Steady-state and time-resolved fluorescence depolarisation measurements show that the polymer matrix forms a very rigid solvent cage, which almost completely immobilizes the probe. Upon solvation of the probe by the liquid, the mobility of the probe increases. In PEG-PEI swollen by polar solvents, the mobilities of the probe itself and of its microenvironment, although not reaching the values observed in homogeneous solution, are significantly higher than in PEG-PS, due to the hydrophilic nature of the polymeric backbone in PEG-PEI.     

Preparation,Characterization and Application of Fluorescent Terbium Complex-Doped Zirconia Nanoparticles

Novel zirconia-based fluorescent terbium nanoparticles have been prepared as a fluorescent nanoprobe for time-resolved fluorescence bioassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion consisting of a strongly fluorescent Tb³⁺ complex, N,N,N¹, N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-phenylpyridine]tetrakis(acetate)-Tb³⁺(BPTA-Tb³⁺), Triton X-100, hexanol, and cyclohexane by controlling co-condensation of Zr(OCH₂CH₃)₄ and ZrOCl_2. The characterizations by transmission electron microscopy and fluorometric methods indicate that the nanoparticles are uniform in size, 33± 4 nm in diameter, and have a fluorescence quantum yield of 8.9% and a long fluorescence lifetime of 2.0 ms. The zirconia-based fluorescent terbium nanoparticles show high stability against basic dissolution in a high pH aqueous buffer compared to the silica-based nanoparticles. A surface modification and bioconjugation method for the fluorescent nanoparticles was developed, and the nanoparticle-conjugated streptavidin (SA) was used for time-resolved floroimmunoassy (TR-FIA) of human prostate specific antigen (PSA). The result shows that the zirconia-based fluorescent terbium nanoparticles are useful as a fluorescent nanoprobe for time-resolved fluorescence bioassay.     

聚电解质包覆的铕(Ⅲ)配合物@SiO2荧光纳米粒的合成与性质

以二苯甲酰甲烷(DBM)、邻菲罗琳(phen)和丙烯酸(AA)为配体,制备了铕的配合物Eu(Ⅲ)(DBM)2-(phen)(AA).利用St(o)ber法合成了SiO2纳米粒.通过超声辅助,将脂溶性的强荧光铕配合物吸附到SiO2纳米粒上,再包覆阳离子聚电解质聚二烯丙基二甲基氯化铵(PDAC)和阴离子聚电解质聚丙烯酸(P...     

Inter- and Intramolecular Dynamics of Pyrenyl Lipids in Bilayer Membranes from Time-Resolved Fluorescence Spectroscopy

The analysis of time-resolved fluorescence of monopyrenyl phospholipid monomer and excimer emission is reviewed using a model based on the diffusion equation for bimolecular reactions in two-dimensional planar membrane bilayers. This aspect is illustrated by the analysis of a real experiment. The method can also be extended to short-range diffusion, which can be measured for dipyrenyl phospholipids in membrane bilayers. The latter aspect is illustrated by the analysis of a simulated experiment to show the experimental requirements to recover the desired parameters.     

Effects of Internal Rotations on the Time-Resolved Fluorescence in a Bichromophoric Protein System Under the Energy Transfer Interaction

Effects of internal rotations of chromophores under the energy transfer interaction in proteins on the time-resolved fluorescence were examined by numerical calculations. Expressions used for the calculations are based on the approximations that the energy transfer takes place according to Foöurster's mechanism and the rotational motions of the energy donor and acceptor along the surfaces of cones are described by a set of rotational diffusion equations. The intensity decay of the donor depended a little on the rotational diffusion coefficient of the donor in some cases, while that of the acceptor did very little. Anisotropy of the donor decayed faster as the diffusion coefficient of the donor increased. Anisotropy decay of the acceptor markedly depended not only on the mutual configuration of the pair in the protein, but also on the diffusion coefficient of the donor. The dependence of the time-resolved fluorescence on the diffusion coefficient of the acceptor was not as great as that of the donor.     

Nano‐Ruby: A Promising Fluorescent Probe for Background‐Free Cellular Imaging

Bioprobes based on fluorescent ruby nanoparticles, which are suitable for ultrasensitive imaging, are reported. A stable aqueous/buffer colloid, permitting facile conjugation to proteins, is produced by femtosecond laser ablation of ruby and the nanoparticles (mean size 17 nm) are photostable, with long lifetime (1–4 ms) 694 nm emission. With time‐gating complete (>20 dB) suppression of cell autofluorescence and suppression of exogenous fluorophores is observed. Nanoparticles are imaged in as‐grown cells and those immunolabeled with quantum dots. Immunoassay binding to target biomolecules is also demonstrated.     

荧光分子层析中的全时间分辨图像重建法

在荧光分子层析(Fluorescence molecular tomography,FMT)中.全时间分辨(Time Resolved.TR)测量包含了最多的光子传输信息.基于有限元一有限差分扩散方程的正向模型和Newtown-Raphson的逆向模型,将全时间分辨方法用于时域荧光分子层析中.用模拟数据对算法在空间分辨率、定量性、重建尺寸和灰度的保真度以及噪声稳健性等方面进行了验证.结果表明,此方法能够实时重建荧光产率和荧光寿命图像.与以前发展的基于广义脉冲谱技术(Generalized pulse spectrum technique,GPST)的特征数据法进行图像重建相比较.整体上优于广义脉冲谱技术.     

BOD光化学传感敏感膜荧光响应的研究

利用自行构建的BOD光纤传感装置对海水中BOD含量进行了检测,考察了海洋耗氧菌种在四甲基硅氧烷(TMOS)、二甲基二甲氧基硅烷(DiMe-DMOS)和聚乙烯醇(PVA)包埋情况下,对BOD的荧光响应行为以及耗氧菌种的加入量对BOD浓度的响应情况。传感膜对5 mg·L-1 BOD溶液测定的相对标准偏差为1.2%(n＝6),响应时间为3.2 min,敏感膜使用寿命大于12个月。实际海水样品检测表明,利用BOD光纤传感检测的结果与国标BOD₅方法,存在较好的一致性。     

The Influence of Solvent Viscosity on the Fluorescence Decay and Time-Resolved Anisotropy of Green Fluorescent Protein

This report describes fluorescence decay and time-resolved anisotropy studies of green fluorescent protein (GFP) in various environments. The addition of glucose and fructose, NaCl, or polyethylene glycol changes the viscosity of the medium surrounding the GFP. Both the time-resolved anisotropy and the fluorescence decay of GFP are measured and it is shown that only the time-resolved anisotropy of GFP is affected by the viscosity, but not its fluorescence decay.     

Pitfalls and Their Remedies in Time-Resolved Fluorescence Spectroscopy and Microscopy

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.