Preparation and Performance of Fluorescein Isothiocyanate⁃Labeled Fluorescent Starch and Polyvinyl Alcohol for Warp Sizing

In order to solve the problems of low accuracy and poor variety adaptability of the current measurement method of the permeability and coating property of sizing paste in textile industry, taking starch and polyvinyl alcohol(PVA) as the representatives of the most commonly used textile sizes, various concentrations of fluorescent molecules fluorescein isothiocyanate(FITC) were used to label starch and PVA to prepare fluorescein(F)-starch and F-PVA fluorescent sizes with different degrees of labeling(DLs) for the first time, respectively. Then the starch and PVA derivatives were employed to size pure cotton warp yarns. After preparing the sections of the sized yarns, the permeability and coating percentage of starch and PVA paste to the yarns were calculated by using a fluorescence microscope and the Photoshop software, respectively. The results demonstrate that F-starch and F-PVA with appropriate DL of 0.791% and 0.161%, respectively, exhibit good fluorescence property and similar sizing performance to the sizing performance of unlabeled starch and PVA. It is considered that fluorescence labeling of sizing agents with fluorescein units can provide an innovative way for the accurate determination of the permeability and coating property of sizing paste to warp yarns.

Nonlinear Intestinal Absorption of Fluorescein Isothiocyanate Dextran 4, 000 Caused by Absorptive and Secretory Transporting System

The mechanism of the nonlinear concentration dependence of the intestinal absorption of fluorescein isothiocyanate dextran 4,000 (FD-4) was studied using in situ rat intestinal loops and the in vitro Ussing-type chamber method. The intestinal absorption rate constant of FD-4, as evaluated by the intestinal loop method, increased significantly in a nonlinear fashion as the FD-4 concentration increased up to 0.2 mM and tended to decrease at concentrations higher than 0.2 mM. The mucosal-to-serosal permeation of FD-4 across rat ileal sheets, as evaluated by the in vitro Ussing-type chamber method, also increased in a nonlinear fashion in the low concentration range (0.01 - 0.02 mM), before decreasing as the concentration increased further, whereas serosal-to-mucosal permeation decreased in a concentration-dependent manner. In addition, mucosal-to-serosal flux and serosal-to-mucosal flux were increased and reduced in the presence of the metabolic inhibitor 2, 4-dinitrophenol, respectively. These results suggest that FD-4 is predominantly secreted into the intestinal lumen by an efflux transport system.

Tissue distribution of Lycium barbarum polysaccharides in rat tissue by fluorescein isothiocyanate labeling

To date, in vivo investigations of polysaccharide’s pharmacokinetics are significantly restricted by the difficulty in their detection. This study was conducted to establish the quantitative determination of Lycium barbarum polysaccharides(LBPs) based on fluorescein isothiocyanate(FITC) pre-labeling and to investigate their tissue distribution in rat. We obtained the calibration curves linear over the range of 0.0–25 μg/m L in rat tissue samples with correlation coefficients greater than 0.99. The inter-day and intra-day precisions(RSD, %) were within 15%, and the relative recovery ranged 95.2%–102.4%, with RSD range 1.48%–9.58%, indicating that this experiment was suitable for the determination of LBPs. The fluorescence intensity was measured after 24 h storage at room temperature, 3 times of freeze-cycle and cryopreservation at –20 ℃ for 15 day, these results indicated that the stability of the samples was good. LBP-FITC was mainly absorbed by the small intestine and stomach, and mainly excreted in the urine through the kidney;this distinct difference in the tissue distribution of LBPs could be attributed to the size of these LBPs in relation to the pore sizes of the vascular beds in the kidney and liver. Results showed in this study enable us to comprehensively understand the biological effects of LBPs following its oral ingestion.

抗青蒿酯钠伯氏疟原虫膜表面Con A结合位点结合量的研究

目的：研究疟原虫抗青蒿酯钠的产生机理。方法：以异硫氰酸荧光素－伴刀豆球蛋白Ａ（Ｆ－ＣｏｎＡ）为探针，采用荧光显微分光光度技术，对伯氏疟原虫滋养体膜表面的ＣｏｎＡ结合位点结合量进行了测定。结果：抗青蒿酯钠滋养体膜表面的ＣｏｎＡ结合位点结合量明显低于其正常株。结论：抗青蒿酯钠原虫膜表面的糖类已发生了改变。而膜的流动性变化可能是ＣｏｎＡ结合位点结合量改变的原因之一。

Pharmacokinetics and Biodegradation Performance of a Hydroxypropyl Chitosan Derivative

Hydroxypropyl chitosan(HP-chitosan) has been shown to have promising applications in a wide range of areas due to its biocompatibility, biodegradability and various biological activities, especially in the biomedical and pharmaceutical fields. However, it is not yet known about its pharmacokinetics and biodegradation performance, which are crucial for its clinical applications. In order to lay a foundation for its further applications and exploitations, here we carried out fluorescence intensity and GPC analyses to determine the pharmacokinetics mode of fluorescein isothiocyanate-labeled HP-chitosan(FITC-HP-chitosan) and its biodegradability. The results showed that after intraperitoneal administration at a dose of 10 mg per rat, FITC-HP-chitosan could be absorbed rapidly and distributed to liver, kidney and spleen through blood. It was indicated that FITC-HP-chitosan could be utilized effectively, and 88.47% of the FITC-HP-chitosan could be excreted by urine within 11 days with a molecular weight less than 10 k Da. Moreover, our data indicated that there was an obvious degradation process occurred in liver(< 10 k Da at 24 h). In summary, HP-chitosan has excellent bioavailability and biodegradability, suggesting the potential applications of hydroxypropyl-modified chitosan as materials in drug delivery, tissue engineering and biomedical area.

Pharmacokinetics and Biodegradation of Chitosan in Rats

Chitosan, an excellent biomedical material, has received a widespread in vivo application. In contrast, its metabolism and distribution once being implanted were less documented. In this study, the pharmacokinetics and biodegradation of fluorescein isothiocyanate(FITC) labeled and muscle implantation administrated chitosan in rats were investigated with fluorescence spectrophotometry, histological assay and gel chromatography. After implantation, chitosan was degraded gradually during its distribution to diverse organs. Among the tested organs, liver and kidney were found to be the first two highest in chitosan content, which was followed by heart, brain and spleen. Urinary excretion was believed to be the major pathway of chitosan elimination, yet 80% of chitosan administered to rats was not trackable in their urine. This indicated that the majority of chitosan was degraded in tissues. In average, the molecular weight of the degradation products of chitosan in diverse organs and urine was found to be <65 k Da. This further confirmed the in vivo degradation of chitosan. Our findings provided new evidences for the intensive and safe application of chitosan as a biomedical material.

Preparation, Characterization and Pharmacokinetics of Fluorescence Labeled Propylene Glycol Alginate Sodium Sulfate

A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate(PSS), in rat plasma. Fluorescein isothiocyanate(FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS(F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney(MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

Adsorption and Desorption Behaviors of Bovine Serum Albumin on Gelatin/Chitosan Sponge

Gelatin (Gel) and chitosan (CTS) have several biomedical applications because of their biodegradability and biocompatibility. Crosslinking of Gel and Gel/CTS systems was evaluated using N-acetyl-D-glucosamine (GlcNAc) formed into sponges by lyophilization. The prepared sponges were used to study the adsorption and desorption of fluorescein isothiocyanate (FITC) labeled bovine serum albumin (BSA) as a model instead of a growth factor. The effect of FITC-BSA concentration and temperature on the adsorption behavior of Gel/CTS sponges was investigated. The Langmuir adsorption isotherm model was used on the basis of the assumption that monolayer adsorption occurs on the surface;the results fit with the experiment data. The adsorption constants were 5.77 and 9.68 mL/mg for Gel and Gel/CTS sponges, respectively. The adsorption thermodynamic constants were found;adsorption onto sponges was an exothermic reaction. In particular, Gibbs free energy (ΔG) exhibited negative values in the range of 283 - 343 K for both Gel and Gel/CTS sponges, demonstrating the spontaneous nature of adsorption reaction. In addition, desorption behavior was evaluated for different concentrations and pH values of the FITC-BSA solution. The high adsorbed amounts of FITC-BSA on sponge resulted in high desorbed amounts in sponge, up to 55% from 3.5 mg/mL adsorbed concentration (around 1.5 mg from 3 mg adsorb amount). Desorption decreased following the buffer solution pH decrease, from 7.4 to 4 and 2 in Gel and Gel/CTS sponges, respectively. Based on the results of this preliminary study, these composite sponges could have significant application in biomedical materials.

Effect of Gardenia extract ZG on the adsorption quantity of herpes simplex virus type 1 (HSV-1)

To observe the effect of Gardenia extract ZG on the adsorption quantity of herpes simplex virus type 1 （HSV-1） so as to explore the mechanism of its antiviral activity, fluorescein isothiocyanate （FITC） was used as the fluorescent probe to label viruses and heparin sodium was used as control. Meanwhile, the effect of Gardenia extract ZG on the adsorption quantity on the surface of Hep-2 cells was determined by flow cytometry. It was demonstrated that adsorption of HSV-1 on the surface of Hep-2 cells exhibited the character of saturation and specificity and heparin sodium could prevent attachment of viruses on these ceils. These results are in accord with those reported previously. It was also proved that the manner of drug-use prior to adsorption or simultaneous use of drug and adsorption was better than adsorption prior to drug-use, and the inhibition rates of the former and latter manner were 84.76% and 82.92% respectively. Three manners of drug-use with Gardenia extract ZG were all effective to reduce the adsorption quantity of viruses, especially the manner of simultaneous use of drug and adsorption with an adsorption inhibition rate of 68.46%. From the above observation, it is apparent that the mechanism of anti-viral activity of Gardenia extract ZG may be via several steps involved in the HSV-1 adsorption.

链霉亲和素-异硫氰酸荧光素偶联核壳型Fe_3O_4/Au纳米晶的合成及性质

采用改进的Polyol合成法,以PEO-PPO-PEO为表面活性剂制备了链霉亲和素-异硫氰酸荧光素偶联的Fe3O4/Au纳米粒子;利用透射电镜和X射线衍射仪分析证实了Fe3O4/Au的核壳型纳米结构,确定了其粒径和分布;采用紫外-可见吸收光谱仪和荧光光谱仪测定了所制备的纳米粒子的光学活性和荧光特性,并采用振动样品磁强计(VSM)测量了其磁化率.结果表明,所制备的Fe3O4/Au纳米粒子具有光学活性和荧光特性,以及优异的磁性.

小鼠抗星形胶质细胞上调基因1(AEG-1)单克隆抗体的制备及应用

目的制备并纯化具有高特异性的抗星形胶质细胞上调基因1(AEG-1)单克隆抗体(mAb),并进行异硫氰酸荧光素(FITC)标记,鉴定标记的mAb与肿瘤细胞的结合能力。方法小鼠腹水诱生法制备并纯化抗AEG-1 mAb,Western blot法和免疫组织化学染色检测mAb的纯度及免疫活性。流式细胞术检测FITC标记mAb的标记效率,流式细胞术及免疫细胞化学染色检测FITC标记mAb与肿瘤细胞的结合能力。结果纯化得到了具有较高纯度的抗AEG-1 mAb,免疫活性良好;流式细胞术检测结果显示,FITC标记的mAb标记的肿瘤细胞表面荧光强度明显增加,标记率可达到90%以上;FITC标记的mAb可特异性识别并结合AEG-1阳性表达的肿瘤细胞,还能与小鼠体内注射的肿瘤细胞结合,在BALB/c小鼠血液中可检测出荧光染色阳性的肿瘤细胞。结论成功制备出小鼠抗AEG-1 mAb并进行了FITC标记,标记的mAb在体内外均可与肿瘤细胞特异性结合。

蛋白质的非定点荧光标记——推荐一个化学生物学实验

本论文介绍了一项化学生物学实验,旨在利用异硫氰酸荧光素的异硫氰基与蛋白质氨基共价结合的原理,实现对体外蛋白(以牛血清白蛋白为例)和体内蛋白(以大肠杆菌总蛋白为例)的非定点荧光标记。实验采用凝胶过滤法作为后续纯化手段,并运用紫外-可见分光光度计、流式细胞术以及荧光显微镜对标记结果进行全面表征。该实验涵盖了多种基础和前沿实验技能,与科研工作中常见的抗体荧光标记技术密切相关,难度适中。通过参与这一实验,学生不仅能够深入理解化学生物学基础知识,提高综合实践能力,还能够拓宽科研视野并培养科研思维。

异硫氰酸荧光素-酚藏花红双发光磷光探针测定DNA

以Pb2+为离子微扰剂时,酚藏花红(PF)与异硫氰酸荧光素(FITC)均能在滤纸上分别发射强而稳定的室温磷光(RTP)信号;当两者混合时,发现PF和FITC的RTP信号均显著增强;而1.12 ag DNA spot-1均使PF和FITC的RTP信号剧烈增强,在634与659 nm处PF和FITC的ΔIp与DNA含量成线性关系,据此建立了FITC-PF双发光磷光探针测定蛋白质的新方法.该方法的检出限(LD)分别为14zg DNA spot–1(PF)和18zg DNA spot–1(FITC),灵敏度高,并成功用于花蜜样品中DNA含量的测定.同时探讨了FITC-PF双发光磷光探针测定痕量DNA的反应机理.

应用FRAP技术评价蛋白在聚丙烯酰胺-丙烯酸凝胶中的扩散行为

药物分子从药物载体中的释放行为与载体的结构有密切关系.本实验中采用丙烯酰胺和丙烯酸等材料,运用水相沉淀的方法,制备了4种不同单体配比的聚丙烯酰胺-丙烯酸(P(Am-co-Ac))共聚物水凝胶.运用红外分析方法对P(Am-co-Ac)组成进行表征.使用荧光漂白恢复法(FRAP,fluorescence recovery after photobleaching)观察荧光素FITC标记的牛血清白蛋白(BSA)在聚丙烯酰胺-丙烯酸中的扩散行为,并以激光共聚焦显微镜进行实时成像.实验表明,FITC-BSA在不同单体配比的共聚物中的扩散系数是不同的.通过调节聚合物中单体的配比能够达到控制蛋白释放速率的作用,从而为调控蛋白和多肽类药物的缓控释放提供了可能性.

不同聚合度壳寡糖单体在小鼠体内的吸收分布

荧光探针标记操作简单且荧光的检测灵敏度比紫外-可见光谱等传统方法高3~4个数量级,故本实验借助荧光探针测定并分析不同聚合度壳寡糖单体在小鼠体内的吸收分布。制备异硫氰酸荧光素标记的壳寡糖单体并灌胃小鼠,借助小动物活体光学成像系统和荧光定量检测,分析聚合度2~5的壳寡糖单体在小鼠体内吸收分布的差异性。结果显示,荧光标记的壳寡糖单体灌胃小鼠1 h后,在小鼠体内的荧光信号达到最大分布,壳寡糖能吸收入血,并跨过血脑屏障分布到脑组织中,主要分布在肾和肝脏中,其次分布在心和脾,少量分布在肺和脑;不同聚合度壳寡糖单体在肾和肝的分布量与聚合度呈正相关,而在血清、心、脾、肺和脑中的峰值分布量为壳二糖最多,壳四糖其次,壳五糖最少。明确不同聚合度壳寡糖单体的体内吸收分布的差异性,对于壳寡糖功能活性的构效关系研究具有一定的指导意义,能为壳寡糖的功能活性机制研究以及加快壳寡糖在食品功能性食品的开发和应用提供参考。

传统与光谱流式细胞仪对自发荧光样本检测的比较

目的比较传统与光谱流式细胞仪检测自发荧光样本的性能。方法以annexin V(AV)/碘化丙啶(PI)染色检测细胞凋亡为模型,制备带有自发荧光的单细胞样本,分别用传统与光谱流式细胞仪采集同一样本荧光信号。结果自发荧光干扰异硫氰酸荧光素(FITC)通道荧光信号,传统流式细胞仪应避免使用FITC通道的荧光素;而光谱流式细胞仪可以清楚分辨FTIC通道的荧光素和自发荧光。同时优化了样本制备方法和annexin V/PI染色方案。结论光谱流式细胞仪对于自发荧光样本的检测在一定条件下具有相对优良的性能。

羧基化聚苯乙烯荧光微球的合成及其在织物防伪中的应用

为解决防伪技术中荧光织物制备方法复杂和成本高的问题,采用分散聚合法制备表面带有负电荷的羧基化聚苯乙烯微球(CPS),再以阳离子表面活性剂十八烷基三甲基溴化铵为改性剂,利用静电自组装法吸附荧光染料异硫氰酸荧光素(FITC),制备羧基化聚苯乙烯荧光微球(CPS-FITC),最后将其负载于织物上制备荧光织物。结果表明:所制备的荧光微球和荧光织物在紫外灯(365 nm)下具有明亮的黄绿色荧光,且放置不同时间(1~9 d)、在不同的pH值(3~11)下,经多次摩擦和水洗后均能保持良好的荧光稳定性。该制备方法操作简便、成本低且荧光强度高,可用于不同织物的防伪检测。

A NEW FLUORESCEIN-LABELED DERIVATIVE OF ESTRADIOL

<正> Estradiol method for estrogen receptors by ~3H-estradiol has been used almost routinely, but application of fluorescein-labeled derivative of estradiol are only at outset. Dandliker has synthesized such a conjugate by condensing 1,3,5, [10]-estratriene-3, 17β-diol-6-iminooxyacetic acid with fluorescein amine. He found that this conjugate binds E, receptor or antibody, thus changing its fluorescent polarization. However,

荧光标记的叶酸修饰壳聚糖纳米载体研制

制备荧光标记的叶酸偶联壳聚糖纳米粒,为抗肿瘤药物给药系统提供载体材料。通过叶酸活性酯与壳聚糖上的氨基反应,使叶酸与壳聚糖偶联。将异硫氰基荧光素与叶酸偶联壳聚糖进行化学嫁接,以离子交联法制成具有荧光的叶酸偶联壳聚糖纳米粒,并与肝癌HepG2细胞进行体外细胞实验。实验结果表明:叶酸活性酯用量和反应温度及试剂滴加速度是影响偶联比的主要因素;在叶酸活性酯与壳聚糖用量质量比为1:1,反应温度为30℃,滴加速度为2mL/min,反应时间为12h的条件下可得到偶联稳定的叶酸偶联壳聚糖;所制得的纳米粒粒径为290nm,形态规则,细胞荧光效果明显;此方法能用于制备荧光标记的叶酸修饰壳聚糖纳米粒载体。