茶多酚锰-壳聚糖微球的制备、控释和诱导肝癌细胞凋亡的研究

选用壳聚糖为微米粒包被材料, 制备茶多酚锰(Tea Polyphenol Manganese, TPMn)-壳聚糖微球. 用荧光显微技术研究了TPMn-壳聚糖微球的荧光特性, 用扫描和透射电子显微镜证实TPMn-壳聚糖微球尺寸和分布规律. RP-HPLC定量分析TPMn-壳聚糖微球包封率为68%, 符合微米级微粒控释药物包封率的要求. 动力学研究结果表明, 茶多酚(TP)-壳聚糖和TPMn-壳聚糖的微球均有控释TP的能力, 控释时间高达40 h以上, 但前者释放速率稍快于后者. TPMn和TPMn-壳聚糖微球均能诱导肝癌细胞凋亡, 但TPMn-壳聚糖微球诱导肿瘤细胞的凋亡速率稍高于TPM. 实验结果证实, 以TPMn-壳聚糖微球方式控释TPMn有利于提高诱导肿瘤细胞凋亡速率. TPMn-壳聚糖微球具有研发成注射型抗肿瘤新药的可能性.     

量子点荧光微球免疫层析试纸条定量检测恶性疟原虫

以羧基化Cd Te/Zn Se量子点荧光微球为荧光标记物,采用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)法偶联抗恶性疟原虫富组氨酸蛋白(Pf)单克隆抗体制备荧光探针;以羊抗恶性疟原虫组氨酸多克隆抗体和驴抗鼠二抗分别喷涂硝酸纤维膜,形成试纸条检测线和质控线,建立了免疫层析试纸条定量检测血清中恶性疟原虫的方法。所使用的羧基化量子点荧光微球的荧光强度为单个量子点的2800倍。实验结果表明,该荧光试纸条定量检测血清中恶性疟原虫线性范围为5.8~8010 Parasite/μL,最低灵敏度达到5.8 Parasite/μL,单个样品检测时间只需15 min。加标回收实验显示,试纸条批内回收率为93.0%~111.8%,批间回收率为98.3%~115.1%,且批内、批间的相对标准偏差均小于5%。     

荧光标记聚苯乙烯微球的激光捕获及其原位稳态和时间分辨荧光光谱研究

本工作设计研制了一套激光捕获与原位稳态及时间分辨荧光光谱检测系统,该系统将激光捕获技术与稳态及时间分辨荧光光谱检测技术相结合,实现了对亚微米级微粒的原位稳态及时间分辨发光性质研究.利用研制的仪器研究了荧光标记聚苯乙烯微球的激光捕获过程、以及捕获微粒的原位稳态及时间分辨荧光光谱.本工作为单个纳米粒子发光性质的研究开辟了一条新的途径.     

微型传感器:荧光纳米微球的制备及表征

以丙烯酰胺为基体,N,N-甲叉双丙烯酰胺为交联剂,荧光分子Fura-2作为探针,构造荧光纳米微球。考察了乳化剂用量、引发剂用量、交联剂单体浓度对纳米微球直径和油水比对乳液稳定性的影响。含Fura-2的荧光纳米微球对钙离子非常敏感,可作为钙离子传感器。     

一种聚苯乙烯荧光微球的制备及发光机理研究

以聚苯乙烯(PS)微球为基质,与乙酰氯通过Friedel-Crafts反应得到具有荧光性质的乙酰化聚苯乙烯微球(PS-AC).红外光谱(FTIR)分析表明苯环上已成功偶联了乙酰基.我们推测PS-AC微球的荧光现象是由聚苯乙烯分子链中的苯环与羰基双键通过π-π共轭形成的刚性平面结构引起的.为了验证该发光机理,以乙基苯和乙酰氯为原料通过Friedel-Crafts酰基化反应合成了一种与荧光微球发光基团具有相似结构的小分子化学物-对乙基苯乙酮.荧光发射光谱分析表明,对乙基苯乙酮与荧光微球在350~400 nm范围内激发光源的激发作用下都能在400~600 nm范围内产生稳定的荧光发射峰.而紫外-可见吸收光谱表明,与PS微球、乙基苯和乙酰氯相比,PS-AC微球与对乙基苯乙酮在300~400 nm范围内出现了新的吸收带,属于新形成的共轭体系的吸收.这种新的共轭体系可以将微球吸收的紫外光或紫光转变成可见的荧光.以上实验结果表明,本研究制备的荧光微球的发光现象是由苯环π键与羰基双键的共轭作用产生的.     

无皂种子乳液聚合制备含稀土磁性荧光微球及其表征

以FeCl3·6H2O和FeCl2·4H2O为原料,通过化学共沉淀方法制备Fe3O4磁性纳米粒子,用油酸和十一烯酸钠对纳米粒子进行双重改性,得到固含量为4%的稳定水基磁流体.在该磁流体存在下,以苯乙烯和甲基丙烯酸缩水甘油酯为单体进行无皂乳液聚合,制备出磁性种子微球;在种子磁性微球存在下,以对苯乙烯磺酸钠,稀土配合物等为功能单体,通过无皂种子乳液聚合法制备磁性荧光微球,该微球表现出优异的磁学性能以及荧光性能.用傅立叶红外光谱仪、透射电子显微镜、X射线衍射仪、振动样品磁强计、荧光分光光度计对磁性荧光微球结构形貌以及磁性荧光性能表征.测试结果表明,所制备的种子微球以及磁性荧光微球呈良好的单分散性,Z均粒径分别为147 nm和228 nm,热重分析表明磁性荧光微球中Fe3O4的含量为4.7%,与之对应的饱和磁化强度为0.396 emu/g,在595 nm和619 nm处观测到Eu3+的特征发射光谱.     

量子点@SiO_2微球的水相合成及其在流式分析技术中的应用

通过表面配体交换将巯基硅烷包覆在量子点(QDs)表面,并与过量的硅烷共水解制备出包覆QDs的荧光硅球。该方法通过化学键合避免了QDs的泄露以及QDs荧光性质的改变,而且微球表面富集大量的巯基,为后续的反应提供活性位点。研究了反应时间、温度和搅拌速度对荧光微球形成的影响,优化条件得到了尺寸均一、单分散性的荧光微球。将荧光微球用于流式细胞分析检测,微球的数量和荧光强度能够满足流式分析检测的需要,适合制备成编码微球。     

基于NaYF_4∶Yb~(3+),Er~(3+)上转换发光纳米晶的复合微球组装及其光动力活性

采用微乳液法,以NaYF4∶Yb3+,Er3+纳米晶为发光基元,肽菁锌(Zn Pc)光敏分子与十八碳烯-马来酸酐共聚物(PMAO)为功能分子,一步组装获得了NaYF4-Zn Pc-PMAO复合微球,此微球同时具备成像与光动力活性功能,NaYF4可作为低生物背景的荧光成像剂,同时其上转换发光可以敏化Zn Pc用于光动力活性研究,PMAO分子经过简单的水解反应即可实现表面羧基功能化。TEM,Zeta电位与PL测试证实了微球的结构与性能。利用荧光共聚焦成像技术实现了对Hela细胞的发光成像;进一步通过单线态氧监测及980 nm光照下的MTT法细胞活性测试表明微球具有光动力活性功能。     

磁性微球与牛血清白蛋白的相互作用

以纳米级四氧化三铁为磁性载体,以苯乙烯为单体,用微悬浮聚合法制备了聚苯乙烯磁性微球;以牛血清白蛋白(BSA)为模型蛋白,用荧光光谱仪和紫外-可见吸收光谱仪研究了磁性微球与BSA的相互作用.结果表明,磁性微球与BSA结合反应的猝灭机理为静态猝灭.     

淀粉基荧光微球的制备及其布洛芬包载和释药性能

以淀粉为原料,用乙酸酐酰化后,将异硫氰酸荧光素（FITC）接枝到淀粉醋酸酯大分子链上,以自组装的方法制备出包载布洛芬的淀粉基荧光微球,同时考察了淀粉基荧光纳米微球对布洛芬的控制释放性能。 荧光淀粉酯的用量、布洛芬的加入量及丙酮与水的体积比可影响载药微球的包封率和药物释放速度。 研究结果表明,当荧光淀粉酯用量为200 mg、布洛芬的量为60 mg、水和丙酮体积分别为50和20 mL时所合成的载药微球包封率最高,为69.5%,其药物体外释放也最快,48 h可释放62.7%。 用扫描电子显微镜和激光共聚焦显微镜(CLSM)对包药微球的体外释放过程进行了表征。     

单波长荧光交叉相关光谱单分子检测系统

基于激光共焦构型建立了一套单波长荧光交叉相关光谱检测系统, 并对检测系统进行了优化, 阐述了荧光交叉(相关)光谱基本理论. 以荧光量子点和有机染料为标记探针, 以人免疫球蛋白与羊抗人免疫球蛋白的结合反应为研究对象, 利用该系统成功地实现了在单分子水平上对免疫结合产物的分子数、浓度、特征扩散时间和动力学半径等参数的表征.     

Fluorescence cross-correlation spectroscopy using single wavelength laser

In this paper, we first introduced the basic principle of fluorescence cross-correlation spectroscopy (FCCS) and then established an FCCS setup using a single wavelength laser. We systematically optimized the setup, and the detection volume reached about 0.7 fL. The homebuilt setup was successfully applied for the study of the binding reaction of human immunoglobulin G with goat antihuman immunoglobulin G. Using quantum dots (745 nm emission wavelength) and Rhodamine B (580 nm emission wavelength) as labeling probes and 532 nm laser beam as an excitation source, the cross-talk effect was almost completely suppressed. The molecule numbers in a highly focused volume, the concentration, and the diffusion time and hydrodynamic radii of the reaction products can be determined by FCCS system. __________ Translated from Chemical Journal of Chinese Universities, 2008, 29(5) (in Chinese)     

Label-free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label-free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium-doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time-correlated single photon counting detection. This allowed recording of photon timing with sub-nanosecond precision. Thereby fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3-phenoxy-1,2-propanediol and tryptophan were electrophoretically separated using a fused-silica microchip. Average lifetimes were independently determined for each compound via bi-exponential tail fitting. Time-correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal-to-noise-ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.     

Detector based on time-resolved ion-induced voltage in laser multiphoton ionization and laser-induced fluorescence

The time-resolved ion-induced voltages obtained from multiphoton ionization (MPI) in gases were applied, for the first time, together with time-resolved laser-induced fluorescence (TR-LIF) for fast detection of organic compounds in ambient air. The target compounds were polycyclic aromatic hydrocarbons (PAHs) and nitroaromatics. The concepts of the proposed method are presented and examined. It is shown that the MPI signals allow for estimation of the molecular weight, while the fluorescence data supply additional quantitative and qualitative information. A 3D data presentation is suggested for obtaining selectivity. The proposed dimensions are the MPI-based mass estimation, the maximum LIF wavelength and the measured fluorescence lifetime. Since all required data are acquired simultaneously and since the setup can be continuously operated, this analytical concept is suggested for on-line applications. The common air components are not ionized at the selected wavelengths and only the organic pollutants are detected under these conditions. Moreover, it is shown that the resonant MPI mode provides a certain level of selectivity. Therefore, this setup may become the basis of an air sniffer.     

Spectroscopic evidence of monomeric aluminium phthalocyanine tetrasulphonate in aqueous solutions

Aqueous solutions of aluminium phthalocyanine tetrasulphonate (AlPcS(4)) were investigated by means of absorption and fluorescence spectroscopy. The absorption spectrum of AlPcS(4) is independent of concentration in a wide range (from 10(-8) to 10(-4) M). The fluorescence spectrum measured with a standard setup is strongly dependent on AlPcS(4) concentration, and the fluorescence maximum is gradually red-shifted with increasing concentration. Calculations that take into account reabsorption of fluorescence (inner-filter effect) fit the experimental observations at low concentrations (up to 10(-6) M). Disagreement between the calculations and spectra recorded at higher concentrations (above 10(-5) M) shows that the reabsorbed light may be reemitted as fluorescence. The influence of inner-filter effects on the spectral shape was demonstrated by the experiments where a fibre-optic front-face fluorescence setup was applied: Under such conditions the shape of the fluorescence spectra for a high concentration (10(-3) M) coincided with that of a low concentration (10(-8) M). In conclusion, the present spectroscopic results show that AlPcS(4) does not form aggregates and is a very stable compound in aqueous solutions.     

Fast quantitative determination of diuretic drugs in tablets and human urine by microchip electrophoresis with native fluorescence detection

Microchip electrophoresis (MCE) with native fluorescence detection has been applied for the fast quantitative analysis of pharmaceutical formulations. For this purpose, methods for fast separation and sensitive detection of the unlabeled diuretic drugs, amiloride, triamterene, bendroflumethiazide (BFMTZ), and bumetanide were developed. An epifluorescence setup was used enabling the coupling of different lasers into a commercial fluorescence microscope. The detection sensitivity of different excitation light sources was compared utilizing either a HeCd laser (lambda(exc) = 325 nm), a frequency quadrupled Nd:YAG laser (lambda(exc) = 266 nm), or a mercury lamp (lambda(exc) = 330-380 nm). At optimal conditions using the HeCd laser, the drugs were separated within 15 s with LODs less than 1 mug/mL for the four compounds. A linear relationship between concentration and peak area was obtained in the concentration range of 0.05-20 microg/mL with a mean correlation coefficient of around 0.996 for all analytes. The method was successfully applied to the analysis of the respective drugs in commercial formulations and in human urine without interference from other constituents. These data show that MCE has a great potential for reliable drug analysis.     

高分辨阿达玛变换显微荧光图像分析(Ⅱ)单细胞DNA荧光图像生成、处理及自动定量分析研究

本研究把荧光显微镜，阿达玛变换多通道成像技术和计算机图象处理技术结合起来，建立了一种单细胞DNA荧光图像自动定量分析系统，对系统的成像原理、信号编码和解码方法及单细胞定量分析方法进行了讨论，分析结果表明，本系统提供的单细胞定量分析数据稳定可靠。     

阿达玛变换显微荧光成像在单细胞定量分析中的应用研究

本文探讨了用阿达玛变换（HT）显微荧光成像系统获得基于256灰度级图像的单细胞荧光强度和解码定标值之间的关系,将归一化的HT图像用于定量分析,建立了适合不同样品的定量分析方法.在此条件下,定量分析数据有很好的准确度和精密度;将系统用于单个乳腺肿瘤细胞的DNA定量分析,对乳腺肿瘤的良恶性及癌变程度进行判断,分析结果与病理学诊断结论一致.     

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.