Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities,and purification of the dye

Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.     

On the mode of liposome-cell interactions. Biotin-conjugated lipids as ultrastructural probes

An efficient method for labeling and visualizing phospholipids at the ultrastructural level is described. Biotin was coupled to the amines of appropriate phospholipids via the N-hydroxysuccinimide ester. The biotinylated lipid could be specifically labeled by ferritin-avidin conjugates and detected by transmission electron microscopy. The lipid derivatives were analyzed and evaluated in terms of their resemblance to the original lipid. Although differing in some aspects from the parent lipid molecules, the biotinyl derivatives still retain the basic characteristics of lipids vis-a-vis their orientation and position in the membrane bilayer. The latter property renders the biotinylated lipid qualitatively suitable for tracing the fate of the lipid component(s) of liposomes during their interaction with biological membranes of various cell types. Using this system, we propose that the extent and pattern of the liposome-cell interaction depends, at least in part, on the cell type employed. This observation may be due to intrinsic variations in cell surface structure and properties relative to those of the liposome.     

A nanogram-level colloidal gold single reagent quantitative protein assay

We have developed a nanogram-level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein–gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10–20 μl) containing 1 to 5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (2–16 h), rinsed with water, and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than do conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples and compared with the Pierce Micro BCA Protein Assay Kit. In addition, the Golddots assay is evaluated with several known proteins with different physical properties.     

Immunocytochemistry in plants with colloidal gold conjugates

Summary The chitin-binding lectin wheat germ agglutinin (WGA) is found at the periphery of wheat embryos, and a similar lectin is present at the root tips of older plants (Mishkind et al. 1982). Although a ferritin-conjugated secondary antibody is adequate for localizing WGA in embryos, native electron-opaque particles make the electron microscope identification of added label equivocal in other wheat tissues. As reported here, however, unambiguous ultrastructural localization of WGA-like lectin in adult wheat roots can be obtained with rabbit anti-WGA followed by colloidal gold-labeled goat anti-rabbit (GAR) IgG. Colloidal gold (CG) was prepared by the reduction of gold chloride with citrate, ascorbate or phosphorous. GAR IgG, prepared from serum by antigen affinity chromatograhy, was adsorbed to the gold particles to produce a stabilized suspension of GAR-CG. Localization was performed on 8–12 M frozen sections of tissue fixed in 4% paraformaldehyde, 0.3% glutaraldehyde, and 0.75% acrolein in phosphate-buffered saline containing 1M sucrose. Localization with GAR-CG was first compared to that ascertained in embryos using other probes and was then extended to the roots of adult plants. An advantage of the GARCG method is that it permits the visualization of antigen at both the light and electron microscope levels in the same section. At the light level, the anti-WGA-GAR-CG complex appears as a red stain that is localized in specific tissues of embryos and in the caps and outer layers of adult roots. Sections in which lectin was detected at the light microscope level were embedded in plastic and sectioned for subcellular examination. Electron dense gold particles indicative of WGA are found at the periphery of protein bodies in wheat embryos and in vacuoles of the roots of adult plants. Sections incubated with control IgG lack reaction product.     

A protein assay based on colloidal gold conjugates with trypsin

The standard sol particle immunoassay (SPIA) is based on a biospecific aggregation of gold nanoparticle conjugates, followed by conventional spectrophotometry. Here we propose a novel SPIA format that uses microtitration immunological plates and an enzyme-linked immunosorbent assay reader. The novel and standard assays are exemplified by determination of immunoglobulin G by using 15-nm colloidal gold-protein A conjugates. We also describe a novel sol particle-trypsin assay using conjugates of gold nanoparticles with trypsin. The method is based on measuring spectral extinction changes caused by the addition of protein to a conjugate solution. The changes in the extinction spectra are presumed to be related to aggregation of gold nanoparticles caused by polyvalent binding of protein molecules to the trypsin molecules of the conjugates.     

Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments

Summary The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (<25 nm) gold particles into 40–50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (>25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but not 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.     

Spectroimmunochemistry using colloidal gold bioconjugates

Using surface-enhanced infrared absorption (SEIRA) spectroscopy of dry films of colloidal gold (CG) bioconjugates with protein A, it is shown that certain characteristic bands of the protein (e.g., amide I, amide II and some other vibration modes) are essentially affected by the metal surface. Thus, the method may be used for controlling the quality of such bioconjugates. Moreover, it is demonstrated that the biospecific reaction of protein A attached to CG particles with human immunoglobulin G (IgG) results in further essential changes in SEIRA spectra, providing a means for an easy and rapid IR spectroscopic detection of biospecific immunochemical interactions (i.e., spectroimmunochemistry). The results obtained can form a basis for developing test systems for detecting various biospecific interactions.     

Attempted endocytosis of nano-environment produced by colloidal lithography by human fibroblasts

Control of the cells' nanoenvironment is likely to be important in the future of cell and tissue engineering. Microtopography has been shown to provide cues to cells that elicit a large range of cell responses, including control of adhesion, morphology, apoptosis and gene regulations. Now, researchers are focusing on nanotopography as techniques such as colloidal and electron beam lithography and polymer demixing have become available. In this study, human fibroblast response to nanocolumns (160-nm high, 100-nm diameter, 230-nm centre-centre spacing) produced by colloidal lithography are considered. Using electron microscopy and immunofluorescence to image the cytoskeleton, clathrin and dynamin, it was observed that the cells try to endocytose the nanocolumns. It also appeared that a small population of the cells changed to unusual morphologies with macrophage-like processes and highly disrupted cytoskeleton. These observations could have implications for nanomaterials science in areas such as cell transfection and drug delivery.     

Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (k_ass and k_diss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol⁻¹, respectively.     

The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity

The enhancement of a single strain DNA probe linked to the sensor surface is of crucial importance in DNA molecule recognition. By means of nanogold modification of the sensor surface in addition to the nanogold amplifier, DNA detection sensitivity higher than 10(-16)mol/L was obtained in a Quartz Crystal microbalance (QCM) system, much higher than the ordinary QCM sensor without surface modification by nanogold.     

Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (k_ass and k_diss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol⁻¹, respectively.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin–streptavidin interaction monitoring

A new electrochemical method to monitor biotin–streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin–streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at +0.08 V was recorded in NH₃ 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H₂SO₄ 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at −0.18 V for 2 min and anodic stripping voltammetry were carried out in NH₃ 1.0 M containing 2.0×10⁻⁵ M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25×10⁻¹⁵ to 2.24×10⁻¹² M and a limit of detection of 2.0×10⁻¹⁵ M were obtained.     

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I₅₀ and I₁₀ values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.     

基于胶体金的8-羟基-2′-脱氧鸟苷免疫层析试纸条北大核心CSCD

8-羟基-2′-脱氧鸟苷(8-hydroxy-2′-deoxyguanosine,8-OHdG)是评价DNA氧化损伤较灵敏和稳定的生物标志物。文中采用竞争法建立一种快速、灵敏检测8-OHdG的胶体金免疫层析试纸条。将样品垫(玻璃纤维素膜)、结合垫(玻璃纤维素膜)、硝酸纤维素膜和吸水垫依此粘贴在聚氯乙烯(polyvinyl chloride,PVC)底板上,构建试纸条。通过柠檬酸钠还原三水合四氯金酸制备胶体金(gold nanoparticles,AuNPs),8-OHdG配对的抗体(antibody,Ab)包被于AuNPs的外层(Ab coated AuNPs,Ab@AuNPs)作为探针。牛血清蛋白(bovine serum protein,BSA)与8-OHdG用碳二亚胺盐酸盐偶联制备人工抗原,作为检测线的包被抗原。羊抗鼠多抗(imunoglobulin G,IgG)作为质控线的包被抗体。对试纸条的硝酸纤维素膜、上样液的配方、金标抗体喷涂量等实验参数进行了优化。结果表明,硝酸纤维素膜(nitrocellulose film,NC)膜采用CN 95,上样液的最优配方为1%BSA+3%吐温-20+3%蔗糖+0.9%NaCl溶液,最适金标抗体喷涂量为4μL。利用试纸条在可见光下检测8-OHdG,根据检测线(test line,T线)和质控线(control line,C线)的显色强度对比,可初步判断尿液中8-OHdG的含量水平。并通过T线的灰度值计算尿液中的8-OHdG的浓度,检测限为2.55μg/L。该方法简单、快速且有较好的特异性,可检测人体尿液中的8-OHdG含量,以初步评价人体的健康状态。     

氨苄青霉素标记的胶体金颗粒的制备及活性研究

采用微波加热法制得15 nm的胶体金颗粒,并将小分子抗原氨苄青霉素分别以戊二醛和碳化二亚胺为偶联剂与牛血清蛋白偶联制成全抗原,再通过最佳标记量的确定分别将氨苄青霉素、戊二醛法全抗原、碳化二亚胺法全抗原同胶体金进行结合,红外检测和杯碟法的抑菌试验表明采用仅有戊二醛作为偶联剂的全抗原能够很好地与胶体金结合,并保持良好抗原活性。     

Thiazole orange: a useful probe for fluorescence sensing of G-quadruplex-ligand interactions

Fluorimetric titrations were performed to gain insight into parameters that govern the association of thiazole orange (TO) and G-quadruplex-DNA (G4-DNA). Use of loop-containing and loop-lacking quadruplexes evidenced the critical influence of the loops on the stoichiometry of the association and on the fluorescence exaltation of TO. We subsequently tried to benefit from this sensitivity to evaluate the influence of G4-DNA cationic environment on ligand binding via a recently reported G4-FID assay.     

狂犬病病毒抗体胶体金检测试纸的制备

通过胶体金免疫层析技术建立一种特异、便捷、快速的狂犬病病毒抗体检测方法,对犬等动物免疫狂犬病疫苗后的抗体水平监测提供参考。用醋酸锌沉淀法沉淀狂犬病病毒CVS11,Sepharose 4FF进行层析纯化。用柠檬酸三钠还原法制备的胶体金,标记纯化的狂犬病病毒,喷于试纸的结合释放垫 (金标垫),将SPA (葡萄球菌表面A蛋白) 和纯化的兔抗狂犬病病毒IgG分别喷于试纸的T (检测线) 处和C (对照线) 处,组装试纸条。用制备的试纸条对261份犬血清进行检测,与快速荧光灶抑制试验 (RFFIT) 检测的结果一致;对已知效价的犬狂犬病毒中和抗体 (VNA) 大于0.5 IU,结果为阳性;对狂犬病病毒中和抗体 (VNA) 小于0.5 IU/mL的血清,结果为阴性。制备的狂犬病病毒抗体胶体金检测试纸检测犬血清抗体,具有特异、便捷、快速的特点,能够检测出狂犬病病毒中和抗体大于0.5 IU的血清,适用于临床犬血清抗体水平监测,具有良好的应用前景。     

Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

诺如病毒常见流行株胶体金免疫层析快速检测方法

【背景】诺如病毒是全球引发急性胃肠炎的最主要病原之一,具有丰富的遗传多样性。【目的】建立一种简便快捷、适用于诺如病毒常见流行株的胶体金免疫层析检测方法。【方法】将抗诺如病毒衣壳蛋白的单克隆抗体1B10作为金标抗体、抗诺如病毒衣壳蛋白的单克隆抗体1D6作为检测线、羊抗鼠抗体作为质控线组装成胶体金试纸条。对试纸条的组装条件进行优化,确定最佳标记pH、金标抗体最佳浓缩比例及检测线(test line,T线)、质控线(control line,C线)最佳划线浓度等。对新方法进行性能评价,包括灵敏性试验、特异性试验、保存期试验及符合率实验等。【结果】所建立的诺如病毒胶体金试纸条检测方法最低检测浓度为5.9×105copies/μL。本方法与常见的腹泻病毒,如轮状病毒、星状病毒、腺病毒、肠道病毒均无交叉反应。批次间与批次内重复较好,保存期试验表明试纸条至少可以室温密封干燥保存一年时间。应用所建立的胶体金检测方法对24份临床粪便样本进行检测,检测结果与实时荧光RT-PCR方法的阳性符合率约为83%(15/18),常见流行株GII.2型、GII.4型、GII.17型均被成功检出。【结论】建立的胶体金试纸条检测方法具有较好的特异性与稳定性,可用于诺如病毒常见流行株检测及大规模流行病学调查。     

纳米金探针在基因检测中的应用

传统的核酸分析中常采用放射性元素、荧光色素以及酶标记等基因探针,这些探针都存在着一些不足之处。近年来,纳米金探针作为一种新型的基因探针,己引起了广泛的关注。该探针具有优良的光谱特征和光化学稳定性,对核酸的非特异吸附性小,与核酸等生物大分子结合后不改变生物分子的活性。将纳米金探针用于基因检测,具有操作简便、快速、安全、实验成本低等优点。本文就纳米金探针的发展过程、纳米金探针的制备、检测原理及其在基因分析中的应用等几个方面作了系统而全面地概述,同时介绍了纳米金探针的最新研究进展,并对其发展前景作了简要评述。