CE法测定CD45-FITC荧光抗体溶液中的游离FITC

建立了测定CD45-FITC荧光抗体样品溶液中游离FITC浓度的毛细管电泳(CE)方法。熔融石英毛细管45 cm(有效长度30 cm)×75μm i.d.;0.05 mol·L-1磷酸氢二钠-磷酸二氢钠缓冲液(含5g·L-1PEG4000,p H=7.14);分离电压+12 k V;进样压力0.5 psi(3.45 k Pa),进样时间3.0 s;分离温度25℃;UV-Vis检测器检测波长200 nm。该方法能有效测定CD45-FITC荧光抗体样品溶液中游离的FITC含量,线性回归方程相关系数r=0.9900,检测限LOQ=0.20μg·m L-1。样品加标回收率为105.49%,相对标准偏差小于5.36%。该方法快速、灵敏、重现性好,能用于CD45-FITC荧光抗体样品溶液中游离FITC的快速测定。     

直接竞争荧光免疫法测定中药材中的黄曲霉毒素B1

本文采用Mannich反应合成黄曲霉毒素B1(AFB1)人工抗原,免疫小鼠制备AFB1单克隆抗体。采用直接搅拌法将异硫氰酸荧光素(FITC)标记AFB1抗体,经Sephadex-50凝胶柱纯化,制得FITC-AFB1荧光标记抗体,分析其免疫学特性,从而建立了一种快速灵敏的直接竞争荧光免疫分析方法(FIA)以用于检测中药材中的AFB1含量。结果表明,AFB1-FITC标记抗体的结合比率为4.19。通过对检测体系多项影响因素的筛选优化,FIA检测方法的标准曲线方程为I=33.45 log C+25.55,R=0.9913,线性检测范围1~100 ng/m L,检测限0.69ng/m L,回收率90.4%~106.6%。该方法具有操作简单、快速灵敏、特异性高等特点,可用于中药材中AFB1的分析测定。     

基于石墨烯猝灭及核酸外切酶辅助循环放大适体传感器检测ATP

基于核酸外切酶Ⅲ(ExoⅢ)辅助分析物循环放大原理,结合石墨烯(G)的超高荧光猝灭能力,以三磷酸腺苷(ATP)作为模型分析物,构建了一种新型的荧光探针,实现了ATP的高灵敏检测。两条DNA链各包含一部分ATP适体序列,其中1条DNA链的3'端修饰有荧光染料异硫氰酸荧光素(FITC)。当模型分析物不存在时,两条DNA链通过非共价π-π堆积作用吸附在G表面,导致FITC的荧光猝灭。当目标物ATP存在时,两条DNA链与目标物ATP进行特异性结合,形成一双链夹心结构,随后加入ExoⅢ,由于ExoⅢ可对该双链夹心结构的3'凹陷末端进行逐步水解,释放出目标物ATP和游离的FITC。释放出的目标物ATP可继续进入下一循环,不断产生游离FITC,游离FITC将脱离G表面,从而导致体系的荧光恢复,并且恢复的荧光强度与ATP浓度在0.02~1μmol/L范围内存在良好的线性关系,检出限(S/N=3)为9 nmol/L。     

用荧光光谱跟踪钙-海藻酸水溶液的Sol-gel转变

为了克服传统方法在测定凝胶化点的同时, 作用力对物理交联点的破坏和对大分子链运动的干扰, 探索和建立不施加应力的凝胶化点测定方法, 采用荧光光谱跟踪了异硫氰酸荧光黄(FITC)标记海藻酸与钙离子在水溶液中螯合的物理凝胶化. 随着凝胶化的进行, 荧光相对强度和各向异性比在凝胶化时间曲线的80 min时出现了明显的转折点, 与Winter方法得到的凝胶化点(80 min)完全一致. 因此可以利用FITC标记的荧光发射相对强度及各向异性比来决定钙-海藻酸体系凝胶化点.     

新型有机荧光染料嵌合的核壳荧光纳米材料的研制

采用油包水的反相微乳液方法,首次以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功地制备了FITC的核壳荧光纳米颗粒,克服了采用传统方法制备核壳荧光纳米颗粒中存在的荧光染料泄露的问题.制备的这种核壳荧光纳米颗粒比细胞小很多,且具有生物亲和性,可为纳米生物传感器件提供新型材料.基于该核壳荧光纳米颗粒的标记方法也为生物医学提供了一种新型的非同位素分析方法.     

荧光光谱跟踪结冷胶水溶液的溶液-凝胶转变

将异硫氰酸荧光黄(FITC)标记在结冷胶分子链,用荧光光谱跟踪了结冷胶水溶液凝胶化过程中FITC荧光强度和各向异性比的变化.结果表明在结冷胶的凝胶化转变中,FITC的荧光相对强度和各向异性随温度降低而增大,在某一温度荧光相对强度和各向异性比对温度的曲线出现了明显的转折点,这个转折点的温度低于流变温度扫描曲线中G′=G″的温度.利用荧光的方法确定物理交联体系的关于重均聚合度和凝胶分数的相关临界指数γ和β.γ和β不符合Flory-Stockmayer和逾渗模型的预测.     

汞灯光源芯片毛细管电泳荧光电荷耦合器件检测系统

以宽谱带高压汞灯为光源的倒置荧光显微镜,配备电荷耦合器件(CCD)及微流控芯片,自组装芯片毛细管电泳荧光电荷耦合器件检测系统。选择不同激发波长(340~540 nm)荧光试剂,进行荧光检测,荧光素的检出限(S/N=3)为7.3×10-9mol/L,荧光素异硫氰酸酯(FITC)的检出限(S/N=3)为1.7×10-8mol/L,显示了该系统对荧光试剂选择范围宽、灵敏度高的特点。用该系统成功分离了FITC与荧光素、曙红与荧光素两种混合荧光试剂。实验表明,组装的芯片毛细管电泳CCD荧光检测系统是成功的。     

芯片毛细管电泳荧光检测器灵敏度的研究

本文采用倒置荧光显微镜,以汞灯为激发光源,自行设计组装了芯片毛细管电泳荧光检测系统。以荧光素异硫氰酸酯(FITC)为检测对象,对双通道门控光子计数器、CH151型光子计数探测器、电荷耦合器件(CCD)三种荧光检测器的灵敏度进行了比较研究。根据芯片毛细管电泳图谱分析,FITC在双通道门控光子计数器、CH151型光子计数探测器、CCD三种检测器中的检出限(S/N=3)分别为7.0×10-10mol/L,1.2×10-9mol/L,3.2×10-8mol/L。进一步采用CH151型光子计数探测器和CCD两种较常用检测器,对FITC和荧光素、曙红和荧光素两组荧光试剂的分离及检测进行了研究。结果表明,使用CH151型光子计数探测器作为检测器,灵敏度高,基线稳定,信噪比高。     

纳米探针芯片技术用于微量乙肝病毒DNA的检测

利用两组探针修饰的微粒:(1)表面标记有可与待测乙肝病毒(HBV) DNA另一端结合的纳米金探针1(信号探针)以及可与信号探针部分结合的纳米金探针2(检测探针);(2)表面标记有可与待测HBV DNA一端结合的磁珠探针(捕捉探针1).检测靶HBV DNA时,磁珠探针与信号探针在液相中可分别与HBV DNA靶序列一端结合最终形成三明治样结构.再以磁场将三明治样复合物从反应液中分离,以DTT溶液将信号探针从纳米金颗粒上洗脱.洗脱后的信号探针数量反映靶基因的多寡,信号探针一段与预先点样的基因芯片上的捕捉探针2结合,检测探针与信号探针另一段相结合,最后用银染液将检测探针显色从而得到靶目标DNA相对定量信息.结果表明,本检测方法的检测灵敏度达到10-15 mol/L水平.检测时间少于1.5 h,检测结果与HBV DNA水平呈现较好的线性关系且无假阳性结果;本方法有望用于乙肝病人血清中HBV DNA的快速筛测及其它微生物基因的检测.     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

Examination of interactions of oppositely charged proteins in gels

Understanding the interactions of proteins with one another serves as an important step for developing faster protein separation methods. To examine protein-protein interactions of oppositely charged proteins, fluorescently labeled albumin and poly-l-lysine were subjected to electrophoresis in agarose gels, in which the cationic albumin and the anionic poly-L-lysine were allowed to migrate toward each other and interact. Fluorescence microscopy was used to image fluorescently tagged proteins in the gel. The secondary structure of the proteins in solution was studied using conventional FTIR spectroscopy. Results showed that sharp interfaces were formed where FITC tagged albumin met poly-L-lysine and that the interfaces did not migrate after they had been formed. The position of the interface in the gel was found to be linearly dependent upon the relative concentration of the proteins. The formation of the interface also depended upon the fluorescent tag attached to the protein. The size of the aggregates at the interface, the fluorescence intensity modifications, and the mobility of the interface for different pore sizes of the gel were investigated. It was observed that the interface was made up of aggregates of about 1 microm in size. Using dynamic light scattering, it was observed that the size of the aggregates that formed due to interactions of oppositely charged proteins depended upon the fluorescent tags attached to the proteins. The addition of small amounts of poly-L-lysine to solutions containing FITC albumin decreased the zeta potential drastically. For this, we propose a model suggesting that adding small amounts of poly-L-lysine to solutions containing FITC -albumin favors the formation of macromolecular complexes having FITC albumin molecules on its surface. Although oppositely charged FITC tagged poly-L-lysine and FITC tagged albumin influence each other's migration velocities by forming aggregates, there were no observable secondary structural modifications when the proteins were mixed in solution.     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

Simultaneous sample washing and concentration using a "trapping-and-releasing" mechanism of magnetic beads on a microfluidic chip

Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 μL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 μL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.     

A NOVEL METAL CHELATE AFFINITY ADSORBENT FOR PROTEIN UPTAKE

1. INTRODUCTION Chitosan is a hydrolyzed derivative of chitin and belongs to a family of linear unbranched polysaccharides which contain large amounts of 1,4-linked-2-amino-2-deoxy-β-D-glucan residues. The presence of free amine groups in chitosan enhances the solubility and reactivity of this polymer. Interest in modifying chitosan by using glutaraldehyde has recently increased. The derivatized polymers have been employed for many applications [1~2], including protein immobilization…     

A New Fluorescein Isothiocyanate-Piperidine Self-ordered Ring Phosphorescent Probe for the Determination of Trace Protein

The ? COOH in fluorescein isothiocyanate (FITC) reacted with ? NH? in piperidine (P) to form FITC‐P on the center of indentation of polyamide membrane (PAM) when drying for 2 min at (92±1)°C. Then, the FITC‐P diffused outward from the indentation center and formed the round SOR‐P‐FITC (containing the FITC‐P self‐ordered rings). Thus, multi‐FITC accumulated on SOR‐P‐FITC, leading to the enhancement of RTP signal on bio‐target, whose I_p increased 2.0 times compared with non‐generated SOR. When bovine serum albumin (BSA) was added to the center of SOR‐P‐FITC, ? NCS of FITC in SOR‐P‐FITC reacted with ? NH₂ of BSA to form SOR‐P‐FITC‐BSA, which caused the RTP signal of FITC to enhance sharply. The ΔI_p of the system was 3.4 times higher than that without β‐CD and 4.0 times higher than that without SOR‐P‐FITC formed. Its ΔI_p was linear to the content of BSA. Therefore, a new solid substrate‐room temperature phosphorimetry (SS‐RTP) for the determination of trace protein was established using SOR‐P‐FITC as a phosphorescent probe. Under the optimum condition, the linear range of this method was 0.040–16.0 ag·spot^?1 with a detection limit (LD) of 8.5 zg·spot^?1 (0.40 µL sample solution per spot, the corresponding concentration was 2.1×10^?17 g·mL^?1), and the regression equation of working curve was ΔI_p=3.848+4.240m_BSA (ag·spot^‐1), n=6, correlation coefficient (r) was 0.9993. This method with high sensitivity had been applied to determining the content of trace protein in the water samples, and the results coincided well with those obtained with pyrocatechol violet‐Mo(VI) method (P.V.M.M.). At the same time, the mechanism of SS‐RTP using SOR‐P‐FITC as a phosphorescent probe (SOR‐P‐FITC‐SS‐RTP) was discussed.     

Grayscale surface patterning using electrophoretic motion through a heterogeneous hydrogel material

Chemical surface patterning can be an incredibly powerful tool in a variety of applications, as it enables precise spatial control over surface properties. But the equipment required to create functional surface patterns—especially “grayscale” patterns where independent control over species placement and density are needed—is often expensive and inaccessible. In this work, we leveraged equipment and methods readily available to many research labs, namely 3D printing and electroblotting, to generate controlled grayscale surface patterns. Three-dimensional-printed molds were used to cast polyacrylamide hydrogels with regions of variable polymer density; regions of low polymer density within the hydrogels served as reservoirs for proteins that were later driven onto a target surface using electrophoresis. This mechanism was used to deposit grayscale patterns of fluorescently labeled proteins, and the fluorescent intensity of these patterns was measured and compared to a theoretical analysis of the deposition mechanism.     

Rapid imaging, using a cooled charge-coupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first-dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone

A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.     

A flow cytometric assay technology based on quantum dots-encoded beads

A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QDs-encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs’ unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement.     

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine‐based or three rhodamine‐based dyes

Despite all remarkable progress in gel‐based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in‐gel staining) or covalent coupling prior to gel electrophoresis (in‐solution staining). Here we report a multiplex analysis of protein samples using maleimide‐activated cyanine‐based (Cy3 and Cy5) and rhodamine‐based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol‐groups of cysteine‐containing proteins. The detection limits in SDS‐PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 μg protein of a mouse brain homogenate were analyzed by 2‐DE. Both cyanine‐ and rhodamine‐based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS‐PAGE and the Dy‐fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505‐, Dy555‐, and Dy635‐labeled Escherichia coli lysates were separated by 2‐DE in a single gel and the three spot patterns relatively quantified.     

1,10-邻菲啰啉衍生物钌配合物的合成、 表征及与DNA和BSA的相互作用

通过2-(4-吡啶)-咪唑[4,5-f]-1,10-邻菲啰啉(L₁)与[Ru(η⁶-cymene)(μ-Cl)Cl]₂反应合成了3种新型芳基钌配合物, 并利用新配体2-(4-咪唑基苯基)咪唑[4,5-f]-1,10-邻菲啰啉(L₂)与RuCl₃反应合成了配合物4. 利用核磁共振波谱、 质谱等对配合物进行了表征. 通过紫外光谱和圆二色谱研究了配合物在缓冲溶液中的稳定性及与CT-DNA的相互作用, 利用荧光光谱研究了配合物与牛血清蛋白的作用, 用乌氏黏度计测试了配合物对DNA黏度的影响, 并通过荧光光谱、 凝胶电泳研究了配合物4在不同pH条件下的荧光响应及与pBR322 DNA的作用. 结果表明, 配合物通过嵌入的方式与DNA作用, 并对DNA的二级结构产生影响; 配合物1~4均可与牛血清蛋白的一个位点发生相互作用并使其发生静态荧光猝灭. 配合物4在光照条件下有活性氧生成, 可以使pBR322 DNA断裂并在酸性溶液中荧光增强.