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。     

Study on the Binding Behavior of Lysozyme with Cephalosporin Analogues by Fluorescence Spectroscopy

It was first found that the intrinsic fluorescence of lysozyme at 340 nm can be quenched by cephalosporin analogues through the static quenching and non-radiative energy transferring procedure. In the acetate buffer solution with pH 7.0 and 298 K, the quenching fluorescence intensity was in a good linearity over the concentration of drugs in the range of 1–100 μmol L⁻¹, 0.1–100 μmol L⁻¹, 0.5–100 μmol L⁻¹ and 0.05–100 μmol L⁻¹ for cefradine, cefuroxime, cefotaxime and ceftriaxone, respectively. The quenching ability or the binding ability of the studied drugs followed the pattern: ceftriaxone > cefotaxime > cefuroxime > cefradine, which was close to the order of their antibacterial ability. The binding parameters including the association constant and the number of binding potential point were calculated at different temperatures (288, 298 and 308 K), and thermodynamic parameters ΔH°, ΔS° and ΔG° were given. The binding mode of lysozyme with cephalosporins showed that the hydrophobic effect might play a major role. The binding distance between cephalosporin and tryptophan residue in lysozyme was obtained. The results provided the quantitative information for the binding of cephalosporin to lysozyme, and it was suggested that the drugs probably bound to the active site near Trp62 in lysozyme.     

Helium-Implantation-Induced Damage in NHS Steel Investigated by Slow-Positron Annihilation Spectroscopy

Evolutions of defects and helium contained defects produced by atomic displacement and helium deposition with helium implantation at different temperatures in novel high silicon （NHS） steel are investigated by a slow positron beam. Differences of the defect information among samples implanted by helium to a fluence of 1×1017 ions/cm2 at room temperature, 300°C, 450°C and 750°C are discussed. It is found that the mobility of vacancies and vacancy clusters, a recombination of vacancy-type defects and the formation of the He-V complex lead to the occurrence of these differences. At high temperature irradiations, a change of the diffusion mechanism of He atoms/He bubbles might be one of the reasons for the change of the S-parameter.     

Sugar Binding Effects on the Enzymatic Reaction and Conformation Near the Active Site of Pokeweed Antiviral Protein Revealed by Fluorescence Spectroscopy

In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.     

基于表面增强拉曼光谱技术的金表面耐腐蚀性分析

金属表面耐腐蚀性能与其微观结构密切相关。以金为分析对象,采用不同粒径氧化铝抛光粉抛光处理金片表面,经扫描电镜和原子力显微镜观察,表明金片表面具备与实际工件类似的微观结构,在此基础上,研究金片表面微观划痕结构对表面增强拉曼散射(SERS)以及耐腐蚀性能的影响。以罗丹明B为探针分子,对不同特征尺寸的微观划痕进行SERS分析,获得了罗丹明B的SERS信号强度随划痕平均特征尺寸的变化规律。通过改变拉曼激发光偏振方向与划痕方向的夹角大小,发现SERS信号主要归因于激发光与划痕相互作用产生的横向表面等离激元。划痕平均特征尺寸在50 nm附近时,横向表面等离激元最强,之后随着划痕平均特征尺寸增加,横向表面等离激元逐渐变弱。另一方面,对具有不同特征尺寸微观划痕的金片进行电化学极化测试,获得了各样品的自腐蚀电位,发现金片的自腐蚀电位随着划痕平均特征尺寸增大而降低。结果表明,当划痕平均特征尺寸大于50 nm,金表面的SERS信号与自腐蚀电位间存在良好的线性对应关系,这为SERS技术用于工件耐蚀性分析提供了依据。通过在工件表面滴涂探针分子并使用便携式拉曼光谱仪测量有望可以对工件耐蚀性进行现场快速预判。     

温度对矿井老空水激光诱导荧光光谱的影响

煤矿井下突水水源的快速判别对煤矿安全生产意义非常重大,激光荧光技术用于煤矿突水水源的识别打破了传统水化学方法耗时长的不足。矿井老空水是最常见也是危害最大的水源类型,而温度是影响物质特性的重要因素之一,研究激光诱导荧光检测老空水的温度特性有助于快速准确识别矿井突水水源,该研究具有重要的学术意义和实用价值。采用405 nm蓝紫光半导体激光器作为光源,设定激光器功率为120 mW,产生的激光经UV/Vis石英光纤由荧光探头照射待测水样,待测水样受激光激发产生荧光由荧光探头采集,通过石英光纤传输至光谱仪。以2017年3月在淮南市张集煤矿采集的老空水作为研究对象,首先过滤掉水样中的悬浮颗粒,随后将其放置在烧杯中,使用冰块使样品温度降低至5.0 ℃,随后放入恒温水浴锅中,使用铁架台固定荧光探头使其位于液面下1 cm处。在荧光光谱采集过程中,样品始终放置在恒温水浴锅中,通过水浴锅控制样品在10.0～60.0 ℃温度范围内获取荧光光谱,并讨论了温度变化对老空水激光诱导荧光光谱谱图、波峰位置及峰值、温度系数、谱图面积的影响。研究结果表明：随着温度升高,加速了分子运动,增加了分子间碰撞的概率,使得非辐射跃迁增加,老空水的荧光效率下降,荧光强度减弱,荧光光谱整体呈衰减变化主要集中在400～700 nm波段;老空水荧光光谱的两个波峰所对应的波长保持不变,并未随着温度变化发生漂移,两个波峰处(472和493 nm)荧光强度减弱最明显,同时荧光强度减弱与温度升高存在较好的线性关系,荧光强度和温度在472 nm处拟合相关系数r2为0.91,在493 nm处的拟合相关系数r2为0.963 36;472 nm处的温度系数在20.0 ℃时达到最小值0.34%,493 nm处的温度系数在20 ℃时达到最小值0.81%,两处的温度系数均在20.0 ℃时达到最低值即荧光光谱在20.0 ℃附近最稳定;温度升高,老空水在荧光光谱在400～700 nm波段与温度轴包围的面积逐渐减小,400～700 nm波段谱图所对应的面积与温度的拟合相关系数r2为0.975 39即面积的减小与温度的升高有良好的线性关系。通过研究矿井老空水的温度特性,矿井老空水的激光诱导荧光光谱在20 ℃最稳定,在该温度条件下采用激光诱导荧光技术进行矿井水源的识别效果最佳,同时利用老空水波峰以及面积与温度的线性关系进行温度补偿可以进一步提升利用LIF技术进行矿井突水水源识别的灵敏度和精度,该研究对实现矿井老空水的快速、准确判别具有重要意义。     

应用荧光相关光谱测定L-色氨酸的浓度

应用FLS920P型荧光光谱仪对L-色氨酸溶液进行三维荧光光谱检测,从中发现：L-色氨酸的特征荧光峰位于270/350 nm。设定发射波长为350 nm,测量激发谱。由测量结果发现在250~260 nm区间,谱线斜率较大、线性度好。因此选取250,255和260 nm三个激发波长,在每个激发波长下分别测量相应的荧光发射谱。基于三条不同的荧光发射谱,构建以激发波长为外扰变量的自相关光谱;而以浓度为外扰变量的自相关光谱,是以超纯水在不同激发波长的平均谱作为参考光谱,通过参考光谱与样本平均谱的相关计算得到。在此基础上,将相关光谱数据分别与偏最小二乘回归(PLSR)和径向基神经网络(RBFNN)相结合,建立溶液中L-色氨酸含量的预测模型,研究结果表明：采用浓度为外扰变量构造的荧光相关光谱信噪比较高,建模的预测效果要好;而在外扰变量相同时,基于径向基神经网络建立的预测模型比基于偏最小二乘回归建立的预测模型对溶液中L-色氨酸浓度的预测结果更为准确。其中,以浓度为外扰变量时的径向基神经网络预测模型准确度最高,该模型的预测相关系数为99.91%,预测均方根误差为0.033 μg·mL-1。研究结果表明,使用该方法能够对溶液中的物质含量进行准确测定,可为食品安全监管提供帮助。     

荧光光谱法研究紫草素与DNA的相互作用

在pH 7.4的Tris-HCl介质中,以盐酸小檗碱(BR)为荧光探针,研究了紫草素(SHI)与鲱鱼精DNA的相互作用。结果表明,SHI与BR竞争结合DNA上的位点;SHI对DNA-BR体系的荧光有猝灭作用,猝灭机制为静态猝灭和动态猝灭共存,SHI与DNA之间为嵌插和静电两种作用方式;热变性实验进一步证明嵌插结合是SHI与DNA的主要结合模式。最后由Scatchard方程求得二者的结合常数为3.48×104 L·mol-1。     

荧光光谱法研究黄豆黄素与牛血清白蛋白的相互作用

用荧光光谱法在pH 7.4的PBS中研究了黄豆黄素(Glycitein,GL)与牛血清白蛋白(Bovine serum albumin,BSA)分子之间的相互作用方式及机理.结果表明GL可静态猝灭BSA的内源荧光.310K和315K的结合位点数与表观结合常数分别为1.30,8.09×104L·mol-1和1.73,5....     

肌红蛋白与生物碱相互作用的光谱性质研究

用分子荧光光谱法和紫外可见分光光度法研究了肌红蛋白（myoglobin,Mb）与咖啡因、茶碱相互作用的光谱性质变化。据Stern-Volmer图得到猝灭常数,由紫外-可见吸收光谱判断均为静态猝灭,通过热力学常数得出其相互作用力为静电作用,依据Foerster偶极-偶极无辐射能量转移理论计算出Mb与生物碱间的距离,并用同步荧光光谱技术考察了生物碱对Mb构象的影响。     

荧光光谱法研究PNA-DNA的杂交和解链

本文建立了一种以阳离子型荧光染料藏红 T(ST)作为荧光试剂的荧光猝灭新方法 ,ST首次被用于PNA- DNA杂交和解链的检测。实验结果发现当 PNA- DNA杂交形成双链时 ,可以在一定程度上猝灭 ST的荧光 ,而当双螺旋解链时 ,对 ST荧光的猝灭程度减小 ,逐渐接近于 ST本身的荧光 ,根据荧光强度的变化研究了 PNA- DNA的杂交和解链过程。通过 UV光谱实验证实了这种荧光猝灭由它们之间的静电作用以及 ST插入到双螺旋内部引起     

荧光光谱法研究咖啡因与肌红蛋白的相互作用

采用荧光光谱法在生理pH 7.4条件下研究了药物咖啡因与肌红蛋白分子间的相互作用,表明这种相互作用能使肌红蛋白的内源荧光猝灭。通过猝灭常数,结合常数和结合位点数的计算,证明此猝灭为静态猝灭机制。咖啡因和肌红蛋白形成1∶1稳定配合物,形成常数(18 ℃)K_A=1.82×104 L·mol-1;根据热力学参数确定了它们之间的主要作用力为疏水力和静电力。利用同步荧光光谱法研究了咖啡因对肌红蛋白构象的影响。咖啡因能使肌红蛋白的构象发生改变,导致蛋白质分子中色氨酸和酪氨酸残基所处微环境由原来的疏水环境不同程度地向亲水环境转变。     

Studies on Molecular Structure of Ethanol-Water Clusters by Fluorescence Spectroscopy

It was found that ethanol and water molecules mixed together can form new clusters. Based on the steady state spectral characteristic and the mole ratio method, three possible bonding constants of ethanol-water clusters were deduced. To confirm the structure of these clusters, the fluorescence lifetimes of different emission bands were investigated, and the average lifetime of the entire decay process was found to vary as the volume percent of ethanol changed. Furthermore, the possible bonding constant n of molecular clusters was determined to be 2 based on the time-resolved spectra, and the cluster was concluded to be a chain structure formed by one ethanol molecule and two water molecules with hydrogen bonds.     

盐酸吡格列酮与BSA反应机理的荧光猝灭法、同步荧光法比较研究

利用荧光猝灭法和同步荧光法进行了盐酸吡格列酮(PGH)与牛血清白蛋白(BSA)之间反应机理的比较研究.结果表明:两种方法在研究PGH-BSA体系的猝灭类型、结合常数、结合位置、作用力类型、协同性及能量转移距离等方面,得到了相同的结果,表明同步荧光光谱法可以用于药物与蛋白反应机理的研究.     

Determination of Protolytic Constants by Trilinear Fluorescence Spectroscopy

Protolytic equilibria often have profound effects on chemical activity, since protolytic species usually behave quite differently. It is therefore important to characterize the protolytic properties of important chemicals. Here we present a new approach to study protolytic equilibria of fluorescent species that is extremely accurate and relies on minimum assumptions. We show that by measuring 2-dimensional excitation/emission scans of samples at different pH. the 3-dimensional experimental data set, I(lambda(ex), lambda(em), C(pH)), can be unambiguously decomposed into the spectral responses of the protolytic species present as well as their concentration. The approach is demonstrated on the protolytic equilibrium of fluorescein. Although the fluorescein monoanion cannot be obtained in pure form, the spectra and concentrations of both fluorescein species, as well as the protolytic constant, are determined with excellent accuracy. The proposed method is general and can be applied not only for studies of protolytic equilibria, but on any chemical equilibria and chemical reactions involving fluorescent species.     

Fluorescence Correlation Spectroscopy of the Binding of Nucleotide Excision Repair Protein XPC-hHr23B with DNA Substrates

The interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 90-mer single-strand DNA was investigated in solution by fluorescence correlation spectroscopy. Autocorrelation functions obtained in the presence of an excess of protein show larger diffusion times (tau (d)) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (theta), as a way to describe the percentage of protein bound to DNA, was directly estimated from FCS data. A significantly stronger binding capability for the cholesterol modified substrate (78% DNA bound) than for other double-stranded DNA substrates was observed, while the lowest affinity was found for the single-stranded DNA (27%). This is in accordance with a damage recognition role of the XPC protein. The similar affinity of XPC for undamaged and 'bubble' DNA substrates (58% and 55%, respectively) indicates that XPC does not specifically bind to this type of DNA substrate comprising a large (30-nt) central unpaired region.     

Control of Immobilization and Hybridization on DNA Chips by Fluorescence Spectroscopy

A new methodology for the analysis of DNA polymorphisms has been developed using specific oligonucleotide strand arrays bound to a solid silicon support recovered by a thin layer of silica. Arrays of directly synthesized oligonucleotides covalently fixed on Si/SiO₂ wafers have been designed at the macroscopic scale. Using suitable nucleotide-labeled units, the fluorescence emission technique has been used as an experimental control of the molecular network bound to the support and as a method for analyzing the hybridizing abilities of the corresponding oligonucleotide array. Fluorescein has allowed us to control the molecular density of the DNA strand resulting from a complete synthetic growing process. A specific protocol using both complementary and noncomplementary units labeled with two probes, Cy3 and Cy5, was used to distinguish clearly nucleotide units fixed on the array either as hybridized sequences or by the unavoidable adsorption process. The present performance of this fluorescence detection procedure will now be used with a scanning fluorescence device to perform the analysis at the microscopic scale.     

荧光光谱法研究虎杖苷与牛血清白蛋白的相互作用

在不同温度下,采用荧光光谱,紫外-可见吸收光谱研究了虎杖苷与牛血清白蛋白(BSA)的相互作用。虎杖苷对BSA的荧光有较强的猝灭作用。根据292K和311K时虎杖苷对BSA的荧光猝灭作用,利用Stern-Volmer方程及双倒数方程处理实验数据,表明虎杖苷对BSA的荧光猝灭作用属于动态猝灭过程,根据Foerster非辐射能量转移理论计算出了虎杖苷与BSA间的结合距离r=3.61nm,结合常数(Kb)分别为5.189×105L·mol-1(292K),1.382×105L·mol-1(311K)及对应温度下的热力学参数。实验结果表明二者主要靠疏水作用力结合,采用同步荧光光谱探讨了虎杖苷对BSA构象的影响。     

Electrically induced microflows probed by Fluorescence Correlation Spectroscopy

We report on the experimental characterisation of electrically induced flows at the micrometer scale through Fluorescence Correlation Spectroscopy (FCS) measurements. We stress the potential of FCS as a useful characterisation technique in microfluidics devices for transport properties cartography. The experimental results obtained in a model situation are in agreement with previous calculations (F. Nadal, F. Argoul, P. Kestener, B. Pouligny, C. Ybert, A. Ajdari, Eur. Phys. J. E 9, 387 (2002)) predicting the structure and electric-field dependency of the induced flow. Additionally, the present study evidences a complex behaviour of the probe nanobeads under electric field whose precise understanding might prove relevant for situations where nano-objects interact with an external electric field.