Surface Modified Single Molecules Free-Diffusion Evidenced by Fluorescence Correlation Spectroscopy

We report on the free diffusion of single molecule near an interface studied using fluorescence correlation spectroscopy. In particular, we show that the chemical nature of the substrate may modify the free diffusion of a widely used molecule (rhodamine 6G), thus inducing unexpected effects in fluorescence correlation spectroscopy measurements. Our results show a strong influence, up to a few micrometer from the interface, of the surface polarity. This effect is assessed through the relative weight of the two dimensions diffusion process observed close to the surface. Our results are discussed in terms of competition between surface-solvent, solvent-molecule and molecule-surface specific interactions.     

活细胞核浆粘滞系数的荧光相关谱研究

细胞核浆粘滞系数直接反映一定生理状态下核内微环境的特性,是判断病变细胞和研究核内分子功能机制的重要依据。为了实时、灵敏和无损地测定单个活细胞的核浆粘滞系数,提出了一种基于荧光相关谱(fluorescence correlation spectroscopy, FCS)技术的新型检测方法。研究中利用FCS技术测定绿荧光蛋白分子(EGFP)在细胞核内的扩散系数,并且根据Stokes-Einstein关系式计算出核浆粘滞系数,同时实现了对特定生理条件下核浆粘滞系数的跟踪测量。结果表明：在pH 7.4以及37 ℃的条件下,人肺腺癌细胞(ASTC-a-1)和人宫颈癌细胞(HeLa)的核浆粘滞系数分别是(2.55±0.61)cP和(2.04±0.49)cP, 与传统方法的测量结果一致,并且发现核浆粘滞系数大于细胞质的粘滞系数。以上研究表明FCS技术可精确无损地检测单个活细胞内达飞升量级的微观环境,是探索细胞内生物分子动态行为的有力工具。     

Statistical Analysis of Diffusion Coefficient Determination by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. The parameter most frequently addressed by FCS is the diffusion of molecules in solution. Due to the highly non-linear connection between the diffusion coefficient and a measured autocorrelation function, it is extremely difficult to analyse the accuracy of the diffusion-coefficient determination in a FCS experiment. Here, we present a simplified analysis based on some general maximum-likelihood considerations, and numerical result are given for the dependence of the accuracy of the diffusion-coefficient determination on sample concentration, brightness, and measurement time. Optimal concentration values for performing FCS are found.     

基于二维相关荧光谱土壤中PAHs检测方法研究

传统荧光光谱技术已被用于土壤中多环芳烃(PAHs)的检测,但由于土壤体系的复杂性、PAHs污染物的多样化和微量化,传统的荧光光谱技术无法有效提取土壤中PAHs的特征信息。为了解决上述问题,提出并建立一种基于二维相关荧光谱土壤中多环芳烃的检测方法。以土壤中典型的多环芳烃蒽和菲为研究对象,配置38个蒽菲混合标准土壤样品(蒽和菲的浓度范围均为0.000 5～0.01 g·g-1),在激发波长265～340 nm,发射波长350～500 nm范围内采集了所有样品的三维荧光谱。以激发波长为外扰,对外扰变化的动态一维荧光谱进行相关计算,得到每一样品的同步二维相关荧光谱。研究了浓度均为0.005 g·g-1蒽菲混合土壤样品的三维荧光谱和同步二维相关荧光谱特性,在同步谱主对角线398,419,444和484 nm处存在自相关峰,其中,398和484 nm荧光峰来自土壤中的菲,419和444 nm荧光峰来自土壤中的蒽;在主对角线外侧,蒽和菲两组荧光峰之间存在负的交叉峰,进一步验证了其来源不同;同时,在(408,434) nm和(434,467) nm处出现交叉峰,其中408和434 nm荧光峰来自土壤中的菲,467 nm荧光峰来自土壤中的蒽。指出与三维荧光谱表征的信息相比,二维相关荧光谱不仅能提取更多的特征信息(408和467 nm的特征峰在三维荧光谱中未被表征),而且还能提供荧光峰之间的相互关系,对其来源进行有效解析。在上述研究二维相关荧光谱特性的基础上,基于同步相关谱矩阵(38×151×151)建立了定量分析土壤中蒽和菲污染物浓度的多维偏最小二乘(N-PLS)模型,对蒽的校正和预测相关系数分别为0.986和0.985,校正均方根误差(RMSEC)和预测均方根误差(RMSEP)分别为4.33×10-4和5.55×10-4 g·g-1;对菲的校正和预测相关系数分别为0.981和0.984,RMSEC和RMSEP分别为5.20×10-4和4.80×10-4 g·g-1。为了比较,基于三维荧光光谱矩阵(38×16×151)建立了定量了分析土壤中蒽和菲的N-PLS模型,对蒽的校正和预测相关系数分别为0.981和0.972,RMSEC和RMSEP分别为5.09×10-4和6.74×10-4 g·g-1;对菲的校正和预测相关系数分别为0.957和0.956,RMSEC和RMSEP分别为7.36×10-4和7.77×10-4 g·g-1。指出,对于土壤中的蒽和菲检测,基于二维相关荧光谱的N-PLS模型的相关系数r,RMSEC和RMSEP都要优于基于三维荧光谱的N-PLS模型。研究结果表明：所提出和建立的方法-二维相关荧光谱直接检测土壤中PAHs污染物不仅可行,而且能提供更好的分析结果。该研究为激光诱导荧光结合相关谱技术现场直接检测土壤中多环芳烃污染物提供了理论和实验基础,具有较好的应用前景。     

Comparison Between Fluorescence Correlation Spectroscopy and Time-Resolved Fluorescence Anisotropy as Illustrated with a Fluorescent Dextran Conjugate

The motional properties of rhodamine green alone and conjugated to 10-kDa dextran have been studied by fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA). With FCS the translational diffusion times of the fluorescent particles can be determined, which are directly proportional to the shear viscosity as shown in aqueous solutions of different sucrose concentrations. With TRFA the rotational correlation times of the fluorescent particles can be determined. TRFA experiments in the case of fluorescent dextran reveal a distinct restricted internal motion of the fluorescent probe independent of the slower overall rotation of the polysaccharide. The fast depolarization is most likely due to internal motion and not to energy transfer between different rhodamine green molecules in the same dextran, since a higher viscosity of the solvent increases the correlation time for internal motion proportionally. FCS and TRFA yield complementary information in the sense that the correlation time for overall dextran rotation can be accurately determined from the translational diffusion coefficient.     

Photobleaching, Mobility, and Compartmentalisation: Inferences in Fluorescence Correlation Spectroscopy

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where sigma(B) is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when P(B) < 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like sigma(B)P/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.     

Fluorescence Correlation Spectroscopy (FCS): A Promising Tool for Biological Research

Abstract

Fluorescence correlation spectroscopy (FCS) is an important biophysical technique. FCS is currently being used in many areas of biology to solve several scientific problems. Its properties such as detection at the single molecular level, higher sensitivity, and use of lower sample volume make FCS a promising molecular diagnostic tool. The promising applications of FCS extend from DNA kinetics/dynamics studies to the comprehensive understanding of receptor–ligand interactions. In this article, we review various promising biological applications of FCS.     

荧光相关光谱测定溶液中胭脂红的含量

使用FLS920P型荧光光谱仪测量了20个合成色素胭脂红溶液样本的荧光发射谱,实验表明：胭脂红的最佳激发波长为300 nm,在此波长激发光下,荧光峰值波长为440 nm。同时测量相同条件下超纯水的光谱数据作为参考光谱,进行与胭脂红溶液光谱数据的相关计算,构建以浓度为外扰的荧光相关光谱。采用sym8小波函数4尺度降噪,将降噪后的同步相关光谱数据、自相关光谱数据应用偏最小二乘回归(PLSR)算法进行预测,建立溶液中胭脂红含量的定量模型,结果表明：采用同步相关光谱建模的预测相关系数为99.863%,预测均方根误差为0.414 μg·mL-1;而采用自相关光谱建模的预测相关系数为99.940%,预测均方根误差为0.303 μg·mL-1。对比可知,自相关光谱数据有效地避免了信息冗余,预测结果更为可靠。该方法无需样本处理,操作简单,为食品安全检测提供了一种新的思路。     

基于金纳米粒子比色法检测全氟辛烷磺酸

全氟辛烷磺酸(perfluorooctanesulfonate,PFOS)具有遗传毒性、生物蓄积性和持久性,且难以降解,因此对其进行分析研究具有十分重要的意义。实验发现,PFOS能使巯基乙胺包被的正电金纳米粒子发生聚集,引起体系吸收信号及颜色改变,据此建立了检测PFOS的紫外-可见分光光度法及比色法。线性方程为A=-0.346+0.049c,相关系数为0.992 4,线性范围0.8～8.0 μmol·L-1,检出限为80 nmol·L-1。研究表明：金纳米粒子在524 nm有特征吸收峰,在650 nm处有较宽吸收峰,PFOS的加入会使金纳米粒子524 nm吸收峰降低,650 nm吸收峰增强,随着PFOS浓度增大,体系颜色由酒红色向红紫色变化。表征了体系的扫描电镜显微成像(SEM)及紫外吸收光谱,考察了金纳米粒子的聚集情况,实验缓冲体系选用pH 5.0的HAc-NaAc缓冲溶液。本方法具有简单、快速等特点,可通过肉眼观察颜色变化来实现对环境污染物PFOS的检测。本方法用于实际水样中PFOS的检测,RSD≤4.4％。     

基于金纳米粒子比色法检测全氟辛烷磺酸

全氟辛烷磺酸(perfluorooctanesulfonate, PFOS)具有遗传毒性、生物蓄积性和持久性, 且难以降解, 因此对其进行分析研究具有十分重要的意义。实验发现, PFOS能使巯基乙胺包被的正电金纳米粒子发生聚集, 引起体系吸收信号及颜色改变, 据此建立了检测PFOS的紫外-可见分光光度法及比色法。线性方程为A=-0.346+0.049c, 相关系数为0.992 4, 线性范围0.8～8.0 μmol·L-1, 检出限为80 nmol·L-1。研究表明: 金纳米粒子在524 nm有特征吸收峰, 在650 nm处有较宽吸收峰, PFOS的加入会使金纳米粒子524 nm吸收峰降低, 650 nm吸收峰增强, 随着PFOS浓度增大, 体系颜色由酒红色向红紫色变化。表征了体系的扫描电镜显微成像(SEM)及紫外吸收光谱, 考察了金纳米粒子的聚集情况, 实验缓冲体系选用pH 5.0的HAc-NaAc缓冲溶液。本方法具有简单、快速等特点, 可通过肉眼观察颜色变化来实现对环境污染物PFOS的检测。本方法用于实际水样中PFOS的检测, RSD≤4.4％。     

The Application of Two-dimensional Fluorescence Correlation Spectroscopy on the Interaction Between Bovine Serum Albumin and Paeonolum in the Presence of Fe(III)

The effect of Fe³⁺ on the interaction between bovine serum albumin (BSA) and paeonolum (PAL), which was extracted from the traditional Chinese herb, Paeonia suffruticosa Andr, was investigated by UV and fluorescence spectroscopy. Two-dimensional correlation spectroscopy was applied to the analysis of fluorescence spectra. The results of spectroscopic measurements suggested that PAL had a strong ability to quench the intrinsic fluorescence of BSA through static quenching procedure in the presence of Fe(III). Thermodynamic parameter enthalpy changes (ΔH) and entropy changes (ΔS) were calculated. The binding parameters including binding constant (K), and the distance (r) between PAL and BSA were evaluated on the basis of the theory of Föster energy transfer. Owing to the spectral resolution enhancement in 2D correlation spectroscopy, the structure change of PAL–Fe³⁺ can be observed.     

New Perspectives of Fluorescence Correlation Spectroscopy

The principle of fluorescence correlation spectroscopy is outlined. The technique has been applied to a mutant of the well-known green fluorescent protein. A comparative study has been made with time-resolved fluorescence anisotropy. The latter experiment shows that the fluorophore is rigidly bound inside the protein matrix follows the rotation of the whole protein and does not show any fast restricted motion. It is evident from fluorescence correlation spectroscopy that some excited-state reaction plays a role, since the autocorrelation traces show a significant effect on the incident laser power. Other potential applications of fluorescence correlation spectroscopy are presented as taken from very recent publications.     

Quenched Emission of Fluorescence by Ligand Functionalized Gold Nanoparticles

A fluorescence-based detection scheme that uses ligand functionalized gold nanoparticles is proposed. The transduction scheme is based on the strong quenching of the fluorescence emission exerted by metallic surfaces on fluorophores positioned in their immediate vicinity (< 5 nm). Binding of fluorophore-labeled anti-biotin to biotinylated gold nanoparticles resulted in decreased fluorescence emission intensity. Subsequent competitive dissociation of labeled anti-biotin with D-biotin resulted in increased fluorescence emission intensity. These interactions occurred by means of specific molecular recognition because when the binding sites of anti-biotin were saturated with D-biotin prior to interaction with the gold nanoparticles; changes in the fluorescence emission intensity were not observed.     

利用二维同步荧光相关光谱研究温度对胶原溶液中分子聚集行为的影响

采用恒波长同步荧光法和二维相关分析技术研究了不同浓度Ⅰ型胶原溶液中胶原分子聚集行为随温度升高(10~70 ℃)的变化规律。选取0.2,0.4,1.6 mg·mL-1的胶原溶液,在初始温度下各浓度溶液中胶原分子分别处于单分子状态、较低程度和较高程度的聚集态。研究表明：波长差为9 nm的同步荧光光谱中,激发波长282和292 nm处荧光峰分别归属于未参与形成氢键的Tyr(酪氨酸)残基和参与形成氢键的Tyr残基。对升温过程同步荧光数据进行二维相关分析,得两荧光值对温度的响应顺序,进而推测得到：当温度低于30 ℃时,0.2 mg·mL-1溶液中出现了胶原分子间形成Tyr残基参与的氢键的趋势。0.4和1.6 mg·mL-1的溶液中原有聚集体可能发生进一步聚集,形成疏水微区。当逐步接近胶原变性温度(36~38 ℃)时,推测0.4和1.6 mg·mL-1胶原溶液中的疏水微区和聚集体有被破坏的趋势,而0.2 mg·mL-1胶原溶液保持分子间形成氢键的趋势。超过胶原变性温度时,各浓度溶液中胶原分子三股螺旋结构发生松散。当超过45 ℃时,胶原分子三股螺旋结构松散的趋势更为明显。     

Fluorescence Correlation Spectroscopy (FCS) Analysis of Human Red Blood Cell System

Recent studies have revealed the importance of the lipid micro domain for signal transduction in cell membrane. To analyze the biophysical properties of the lipid micro domain at the single molecule level, we measured the diffusion of fluorescence probe in human red blood cell (RBC) membrane using fluorescence correlation spectroscopy (FCS). The value of diffusion constant of octadecyl rhodamine B chloride (R18), D = 4.7 × 10⁻⁹cm²/s, is close to that of phospholipid molecules in membrane. This indicates that the probed RBC with R18 could be a convenient model for analyzing membrane property under natural conditions. Using this model, we investigated how amyloid beta peptide (A-beta) interacts with plasma membrane. This paper demonstrates that A-beta was inserted into the phospholipid bilayer of the RBC membrane and predicts the existence of the micro domain, lipid raft, on this membrane by the heterologous insertion of A-beta.     

Multi-Photon Fluorescence Correlation Spectroscopy: a Quantification of Tryptophan Methylester Solutions by Visible Emission

A multi-photon excitation fluorescence correlation system has been developed. The emission from tryptophan methylester solution was observed by this system and analyzed by the intensity correlation function of the visible emission, which originates from the two-photon excitation of photo products generated through a five-photon process. The intensity and the product concentration were proportional to the concentration of tryptophan methylester at a lower concentration range and thus the generation process is a single molecular reaction. The correlation analysis determined the concentration of tryptophan methylester down to 5 μM. The photo product generation from tryptophan solution was enhanced by a potassium iodide addition. These results suggest a new quantification method of tryptophan derivatives.     

Nuclear Uptake of Gold Nanoparticles Deduced Using Dual‐Angle X‐Ray Fluorescence Mapping

Studies into the cell nucleus' incorporation of gold nanoparticles (AuNPs) are often limited by ambiguities arising from conventional imaging techniques. Indeed, it is suggested that to date there is no unambiguous imaging evidence for such uptake in whole cells, particularly at the single nanoparticle level. This shortcoming in understanding exists despite the nucleus being the most important subcellular compartment in eukaryotes and gold being the most commonly used metal nanoparticle in medical applications. Here, dual‐angle X‐ray flouresence is used to show individually resolved nanoparticles within the cell nucleus, finding them to be well separated and 79% of the intranuclear population to be monodispersed. These findings have important implications for nanomedicine, illustrated here through a specific exemplar of the predicted enhancement of radiation effects arising from the observed AuNPs, finding intranuclear dose enhancements spanning nearly five orders of magnitude.     

DNA Biosensor with High Sensitivity Amplified by Gold Nanoparticles

A biosensor based on quartz crystal microbalance (QCM) using 50 nm gold nanoparticles as the amplification probe for DNA detection was reported in this paper. It had been found that a DNA detection sensitivity of 10^–14 M could be obtained, which was higher than what has been ever reported using the same method. In the curve of weight increase (m) against the target DNA in solution, a plateau appeared as the concentration of analyte DNA increasing, implying the existence of monolayer of gold nanoparticles binding on the top of target DNA. The high sensitivity was explained not only by the weight of the larger particles, but also the larger area occupied by the larger particles which need less target DNA for their binding.     

荧光关联谱的数据处理改进方法

荧光关联谱方法凭借其独特的对溶质浓度及扩散系数的测量能力,在对复杂系统的分子物理化学性质测量方面,不断扩大和深化其应用领域。但是从经典上,传统的荧光关联谱数据处理方法容易在拟合自相关曲线的参数时引入一定的误差,而且图表上的直观信息不多。针对以上问题,在文章中作者尝试用带有微分的数据处理办法,来更直观地估测参数并判断荧光多组分问题。经典自相关曲线处理后的微分曲线,有特异的波谷位置、深度和半深宽度,由此可判断特征扩散时间和多荧光成分的组分数目。此改进方法为利用荧光涨落谱方法测量生物组织体内的复杂环境提供帮助。