荧光标记在药物分析领域的应用进展

荧光染料具有高灵敏度、高稳定性和高选择性等特点。荧光标记作为一种非放射性标记技术,已成为现代药物分析检测中不可或缺的方法之一。而新型荧光标记材料量子点的出现,克服了传统荧光染料的某些缺点,更有利于荧光标记技术在药物分析领域的推广应用。综述药物的荧光标记及其在药物分析领域的应用进展。     

化学标记技术及其在药物和临床检测中的应用

概述了多种化学标记技术（如荧光标记、化学发光标记、稀土离子标记、酶标记、生物素标记、地高辛标记、胶体金标记、同位素标记、自旋标记等）的原理及其在药物分析和临床检测中的应用。     

荧光标记多糖及其体内药动学应用进展

多糖研究和应用越来越普及,多糖荧光标记技术也在不断发展。由于多糖自身分子结构的特点,目前尚无标准的多糖体内吸收代谢的检测方法,使多糖在生物医药领域的应用受到限制。体外荧光标记技术已广泛用于多糖体内药动学研究。本文总结了目前用于多糖标记的各种荧光素,及其在多糖口服、静脉注射和腹腔注射给药的体内药动学研究中的应用。并进一步结合荧光标记多糖在体内药动学研究中的优势,阐述多糖口服吸收机制,增加多糖口服吸收的策略,影响多糖药动学的因素,以及多糖体内降解机制等,旨在为多糖体内药动学研究提供参考。     

近红外标记技术在生物医药领域的应用

文章主要介绍了与IGG类似的几类近红外菁染料：ICG衍生物、Cy系列染料及IRDye系列，总结了它们的结构特征、光谱性质和修饰部位，着重介绍了它们与生物大分子结合形成的一些荧光标记复合物，并且就其与抗体、多肽、药物骨架等结合的复合物在生物医药领域的应用进行了阐述。     

放射性同位素示踪技术在药物研发过程中的应用

药物的吸收、分布、代谢和排泄过程决定了药物分子在体内的过程和命运。作为常规生物样本检测技术的有效补充,放射性同位素标记示踪技术如今也被广泛应用于药物发现和开发过程中的各个阶段。特别是其高灵敏性、适用范围广的特点使其具有不可比拟的优势。通过对近年来放射性标记技术在药物评价领域中的应用进行了归纳和梳理,为药物研发相关领域研究人员提供可借鉴的参考。     

小动物体内可见光成像技术及其在药物研发领域的应用

小动物体内可见光成像是对小动物体内的细胞运动，基因表达及蛋白行为等生物学现象进行直接观察的一种新方法。利用一套非常灵敏的光学成像系统，我们可以观察荧光素酶基因或者荧光蛋白以及其他荧光染料标记的细胞，基因，蛋白或其他大分子在活体动物体内的表达，生长，运动和相互作用。因为其极高的灵敏度，安全性及使用方便等特点，小动物体内可见光成像技术在基础医学和药物研发等领域得到了广泛应用。短短几年内，利用这个技术已经发表了六百多篇文章，在Nature和Science上就发表了近五十篇文献。最近的一篇文献中，利用这个技术，研究人员在小动物体内观察到了荧光素酶基因标记的一个细胞，展现了这个技术在动物体内生物学现象研究领域的极大潜力。基于基础医学研究的成功，这个技术也广泛的应用于药物研发领域，尤其是抗肿瘤药物的早期疗效和抗生素的研发等领域。仅在最近几年，就有十一个肿瘤药物和抗生素的研发在上报给美国FDA的实验数据中采用了小动物体内成像技术。这个技术主要应用于药物研发的以下几个方面：     

羧基荧光素琥珀酰亚胺酯标记肿瘤细胞的条件优化

目的:探讨化学染料羧基荧光素二乙酸盐琥珀酰亚胺酯(CFDA-SE)标记肿瘤细胞的可行性和最适条件.方法:用不同标记时间、不同浓度、不同培养浓度的CFDA-SE标记肿瘤细胞TCA(舌癌).结果:20min内荧光强度与标记时间成正比;荧光强度的持续时间与标记时间成正比,但标记时间超过20min后,荧光持续时间无明显变化;提高标记时染料浓度,可适当延长荧光持续时间;将试剂盒浓度1/4染料加入培养基中,荧光持续效果最好.结论:CFSE标记肿瘤细胞的影响因素较多,不可用于肿瘤细胞长时间培养的实验;但可用于短时肿瘤细胞标记,确定最佳标记时间为10 min,标记浓度为试剂盒所示浓度,共培养时将试剂盒浓度1/4的染料加入培养基中荧光持续效果最好,且实验操作简单,结果易观察.     

时间分辨荧光免疫定量分析与肿瘤临床应用进展

时间分辨荧光免疫分析（Timeresolved Fluoroimmunoassay，TRFIA）是一种以镧系元素螫合物为标记物，测量其发射荧光的超灵敏的无放射性污染的标记分析技术。是近年来发展起来的一种新的非同位素标记免疫分析技术，作为一种新型的非放射性免疫标记技术，其应用已不再局限于临床诊断，已渗透入生物学研究的各个领域。TRFIA技术为免疫学诊断开辟了一条崭新的途径。本文就TR—FRA技术及其在肿瘤学应用进展做一简要综述。     

一种阻断乙肝病毒衣壳组装的抗病毒药物的体外荧光筛选方法

病毒组装过程通常不作为开发抗病毒药物的靶标，部分由于缺乏易于操作的体外筛选方法。本研究基于染料标记的衣壳蛋白荧光猝灭原理，建立了体外检测乙型肝炎病毒（HBV）衣壳组装的方法，用于筛选可能的抑制剂。此方法适用于高通量筛选，能快速筛选出抑制病毒组装过程的小分子化合物，它们能阻止、不适当地加快和／或误导衣壳形成从而产生异常颗粒的小分子。     

绿色荧光蛋白在药物发现研究中的应用进展

目的药物的设计与筛选是药物研究的重要环节,绿色荧光蛋白(green fluorescent protein,GFP)在药物发现研究中有着重要的意义和价值。方法通过综述22篇中、英文文献,在化学药物基因药物等方面介绍了绿色荧光蛋白及其在药物发现研究中的应用。结果绿色荧光蛋白最早发现于美国西北海岸的水母中,在紫外照射下可以产生明亮的绿色荧光。它具有很多理想性的特征,如对酸、碱、氧化还原剂等许多化学试剂有极强的稳定性,因此常被于活体细胞或组织的跟踪、标记中,被喻为"活的"分子探针。通过监测绿色荧光蛋白可以对体内基因表达、细胞内蛋白质原位定位,观测肿瘤发生、生长、转移等过程,提供重要生物学靶标有效信息。结论绿色荧光蛋白在药物设计和筛选等领域展示了广阔前景,它与药物设计、药物筛选的结合将为新药研究和开发注入新的活力。     

Visualisation of real-time oral biodistribution of fluorescent labeled self-microemulsifying drug delivery system of olmesartan medoxomil using optical imaging method

In the present study, the biodistribution of self-microemulsifying drug delivery system of hydrophobic olmesartan medoxomil (OM-SMEDDS) was determined by labeling with a fluorescent dye VivoTag®680 XL and Xenolight® DiR. Labeled OM-SMEDDS and control dye solution administered orally to mice; real-time dynamic biodistributions over 7 h were determined by 2D-fluorescent imaging to verify their anatomic location. Fluorescent Emissions by Vivotag 680® XL and Xenolight® DiR labeled OM-SMEDDS emitted 2 to 24 times stronger emission than control dye administered group. To further confirm the results, organs were removed and examined using the same technique at the end of 7 h. VivoTag®680XL and Xenolight® DiR emitted 4 and 1.7 times stronger emission respectively than control dye administered mice in ex-vivo organ imaging studies. This study showed that OM-SMEDDS can be succesfully labeled with fluorescent dye and tracked with optical imaging method for the visualisation of biodistribution of drugs and is also useful for enhanced bioavailability.     

An assay for measurement of protein adsorption to glass vials

Protein adsorption to primary packaging is one of the problems faced by biopharmaceutical drug companies. An assay was developed to quantify loss of proteins to glass vial surfaces. The assay involves the labeling of protein with a fluorescent dye, incubation of the labeled protein with the vial surface, elution of the adsorbed protein using a stripping buffer, and determination of fluorescence of the adsorbed protein using a fluorometer. The assay is simple to set up, accurate, sensitive, and flexible. The assay can be modified for indirect measurement of protein adsorption and offers an attractive alternative for researchers to quantify protein adsorption to glass vials and syringes.     

Molecular conformation of ammonium 8-anilino-1-naphthalenesulfonate hemihydrate. A fluorescent probe for thyroxine binding to thyroxine binding globulin.

The crystal and molecular structure of the ammonium hemihydrate salt of the fluorescent dye, 8-anilino-1-naphthalenesulfonic acid (ANS), has been determined. There are two conformationally distinct molecules in the triclinic P1 lattice. The anilino nitrogen of one molecule has slightly distorted planar geometry, and the overall conformation of the molecule is similar to that observed for the potassium salt of the fluorescent dye 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS). The anilino nitrogen of the other molecule has slightly distorted tetrahedral geometry and the overall conformation of the molecule is similar to that observed for the thyroid hormones T3 and T4. The observation of two distinct conformational modifications of ANS in this crystal structure determination has shed light on the conformational flexibility of the ANS molecule itself and on the mode by which its acts as a competitive inhibitor in thyroid hormone transport proteins and as a signal for hydrophobic areas in macromolecular systems.     

A contribution to the evaluation of microcapsules by light microscopy.

Light microscopy has been used for the evaluation of the internal and external structure of dry microcapsules. The method involves surface and penetrative staining with various dyes after which the microcapsules were embedded in suitable optically translucent material. Using this method the core material, its shape and position within the microcapsules either in total or as subunits of the core are clearly distinguishable from the wall material. The surface characteristics of the microcapsules can be observed with either light or fluorescent microscopy after staining with a fluorescent dye. Furthermore, it is a relatively simple and inexpensive method by comparison with the scanning electron microscopy. The natural character of microcapsules, without any artificial structures, has been maintained. It could serve as a routine auxiliary method for complex evaluation or control of the microencapsulation process and its optimization.     

A method to monitor DNA transfer during transfection

This report describes a method for monitoring the transfer of DNA during transfection. This method involves random labeling of plasmid DNA with fluorescein-12-dUTP, flow cytometric detection and sorting of the fluorescent transfected cells, and laser confocal fluorescence microscopic determination of the intracellular location of the plasmid DNA. By this method, >95% of the sorted cells were labeled, indicating a >95% specificity of the sorting procedure. The sorted cells were viable, as indicated by their ability to exclude trypan blue dye (>98% cells excluded the dye) and to maintain cell growth. The results of the kinetics of the Lipofectin transfection technology show that the fraction of the cells that internalized plasmid DNA increased from 10% at 1 hr after initiation of the transfection procedures to 18% at 3 hr. This method does not require protein expression, does not require the use of selection pressure such as drug treatment to isolate the cells that internalized DNA, and can be used to study the early events of DNA transfection.     

Synthesis of support-bound peptides carrying color labels

In order to check the possibility of labeling sub-libraries, we synthesized a colored support by acylation of aminomethyl resin with a special Boc-amino acid (a red azo dye). Syntheses of model peptides proved that the colored resin is suitable for solid phase peptide synthesis. Color labeling was also made by coupling resin-bound peptides at their N-termini with a blue dye. Colored resins as well as labeling at the N-terminus offer a possibility to simplify the preparation of support-bound peptide sub-library kits and their use in sequence determination of bioactive peptides. © 1994 Wiley-Less, Inc.     

Site-specific fluorescent labeling of poly-histidine sequences using a metal-chelating cysteine

Coupling genetically encoded target sequences with specific and selective labeling strategies has made it possible to utilize fluorescence spectroscopy in complex mixtures to investigate the structure, function, and dynamics of proteins. Thus, there is a growing need for a repertoire of such labeling approaches to deploy based on a given application and to utilize in combination with one another by orthogonal reactivity. We have developed a simple approach to synthesize a fluorescent probe that binds to a poly-histidine sequence. The amino group of cysteine was converted into nitrilotriacetate to create a metal-chelating cysteine molecule, Cys-nitrilotriacetate. Two Cys-nitrilotriacetate molecules were then cross-linked using dibromobimane to generate a fluorophore capable of binding a His-tag on a protein, NTA(2)-BM. NTA(2)-BM is a potential fluorophore for selective tagging of proteins in vivo.