标签谷胱甘肽S-转移酶的二价亲和标记试剂的制备与应用

设计合成融合表达标签谷胱甘肽S-转移酶(GST)的二价亲和标记试剂,用于功能化磁珠后位点选择性固定化标签GST,为磁分离筛选配体混合物库提供固定化融合靶蛋白的候选方案。 为减少疏水配体在标签GST活性位点的结合,需同时占据标签GST双活性中心内疏水结合位点并发生共价修饰的二价亲和标记试剂。以双苯环为疏水定位基、溴乙酰基为巯基修饰基团、羧基为连接官能团得单价标记试剂,以二乙基三胺为连接臂将单价标记试剂与连接臂两端伯胺连接得标签GST的对称二价亲和标记试剂,再以线性三胺连接臂中间的氨基与羧基磁珠偶联得功能化磁珠。 表征目标化合物对标签GST的标记动力学、结合比;功能化磁珠对标签GST的不可逆固定化动力学和固载容量,及将磁珠表面二价亲和标记试剂转变成还原型谷胱甘肽(GSH)加合物后对标签GST可逆固定化的效果;以碱性磷酸酶及疏水荧光配体为模型考察磁珠固定化标签GST后的非特异结合。 目标化合物对标签GST半抑制浓度为(22±0.2) μmol/L,其与GSH的饱和加合物半抑制浓度为(0.41±0.06) μmol/L,二者与标签GST二聚体结合比接近1：1。 功能化磁珠对标签GST不可逆及可逆固定化的容量均接近25 mg/g磁珠。 偶联GST的磁珠对蛋白非特异吸附很弱,再进一步用单价亲和标记试剂和GSH加合物封闭固定化标签GST剩余的活性位点后对疏水小分子也无显著结合。 结果表明,所设计二价亲和标记试剂功能化磁珠适合用于标签GST及其融合表达蛋白的位点选择性固定化。     

荧光量子点免疫标记法检测炭疽芽孢杆菌

建立了荧光量子点标记-免疫分析技术联用检测炭疽芽孢杆菌的方法.通过抗原抗体反应,结合生物素与亲和素间的特异性相互作用,将QDs特异性标记在炭疽芽孢杆菌上,并利用荧光显微镜和荧光分光光度计进行了验证.采用实验室自制的便携式荧光检测系统对标记QDs的炭疽芽孢杆菌样品进行定量检测.结果表明,在炭疽芽孢杆菌浓度在100～1×1...     

罗丹明B硅纳米粒子探针及其在蛋白芯片检测中的应用

本工作将罗丹明B分子通过共价结合的方式成功地包裹在二氧化硅纳米粒子中,制备的纳米粒子荧光强度和罗丹明B分子相比提高了1000倍.对此硅纳米荧光粒子进一步进行了链亲和素修饰,成功制备了可特异性结合生物素修饰蛋白的纳米荧光检测探针.以反相蛋白质芯片检测为模式,研究了此探针对微量蛋白的检测性能.实验中将不同微量浓度的人IgG固定于醛基修饰玻璃片表面,并加入生物素标记的抗人IgG,结果显示在800fg～100pg含量的微量蛋白检测中此纳米荧光探针具有良好的线性关系,最小蛋白检测量可达100fg.与商品化亲和素偶联cy3荧光探针对比分析发现,本方法制备的荧光探针对蛋白的检测灵敏度可提高8倍,且具有成本低,生物修饰简单等优点.     

量子点标记链霉亲和素及其生物活性检测

选用无机盐为前驱体,在水相中合成CdTe量子点,并用此量子点标记链霉亲和素,通过SephadexG-100层析分离纯化量子点标记的链霉亲和素,采用磁颗粒标记的链霉亲和素与量子点标记的链霉亲和素竞争结合辣根过氧化酶标记的生物素,即酶联免疫竞争抑制分析法检测链霉亲和素标记量子点后的生物活性,计算约70.3%的链霉亲和素标记到量子点上,且具有生物活性。每毫克量子点大约可偶联0.14 mg的链霉亲和素。采用荧光光谱研究量子点标记前后的荧光变化,标记后量子点的最大发射波长蓝移了8 nm,而发射光谱的半峰宽基本不变,说明量子点与链霉亲和素结合后粒子没有团聚,分散性好。     

基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌

建立了基于免疫磁分离的荧光微球免疫层析法,检测猪霍乱沙门氏菌.待检样品经免疫磁分离富集和热洗脱处理后,用荧光微球免疫层析试纸条进行检测.每毫克纳米磁珠标记30μg抗体制备的免疫磁珠,对浓度为102 ～ 106 CFU/mL的猪霍乱沙门氏菌的捕获率均大于90％,特异性好;在pH=6时,以300μ,g/mg猪霍乱沙门氏菌单抗11D8-D4标记荧光微球,制备免疫荧光微球;以2.0 mg/mL猪霍乱沙门氏菌单抗5F11-B11喷涂检测线(T线),以1.0 mg/mL驴抗鼠IgG喷涂质控线(C线),制备免疫层析试纸条.采用建立的基于免疫磁分离的荧光微球免疫层析方法检测猪霍乱沙门氏菌,在PBS缓冲液中检出限为1.5×105 CFU/mL,牛奶中检出限为7.6×105 CFU/mL,与直接采用荧光微球免疫层析方法检测相比,检出限分别降低了10倍和200倍.本方法可有效富集牛奶中的沙门氏菌,避免了基质干扰,灵敏度大大提高,具有较好的应用前景.     

亲和毛细管电泳测定孕酮与其单克隆抗体的结合常数

采用亲和毛细管电泳的配体分离模式,以激光诱导荧光作为检测手段,测定了荧光素标记的孕酮与孕酮我隆抗体之间的结合常数,并研究了温育时间、电泳条件等因素对测定的影响。     

NaYF_4：Yb,Er上转换荧光纳米粒子的表面修饰及其在免疫分析中的应用

稀土掺杂上转换荧光纳米材料因其近红外区激发,可见光区发射的特殊发光性能,在生物标记方面具有独特优势,可大幅度降低荧光背景.β-NaYF4：Yb,Er是目前已知的发光效率最高的上转换荧光纳米材料之一,已在生命分析及生物成像分析领域展现出了广阔的应用前景.然而,由于现有β-NaYF4：Yb,Er制备工艺多是在高温条件下于高沸点有机溶剂中反应制得,所得产品在水溶液中的分散性差,限制了其在生命分析中的广泛应用.本文采用聚丙烯酸（PAA）配体交换反应,对表面包覆油酸基团的疏水β-NaYF4：Yb,Er纳米粒子进行了有效的表面修饰.表面修饰后,上转换荧光纳米粒子表面的PAA具有众多游离羧基,使其在水溶液中具有良好的分散性.同时,由于羧基的存在,使得带有氨基的生物分子能够通过化学交联反应结合到纳米粒子表面.本文以PAA表面修饰后的β-NaYF4：Yb,Er纳米粒子为荧光探针,以磁珠作为免疫反应的载体,成功构建了一种新型免疫传感器,对模型靶标分子羊抗人IgG进行了灵敏检测.磁珠表面固定兔抗羊IgG,PAA修饰的β-NaYF4：Yb,Er纳米粒子表面连接人IgG,当样品中存在羊抗人IgG时,便会在磁珠表面形成（兔抗羊IgG-羊抗人IgG-上转换荧光纳米粒子标记的人IgG）三明治式免疫复合体,通过磁分离除去未反应的组分,在980nm激光激发下测定免疫复合体的上转换荧光强度,即可实现靶标分子的高灵敏分析,可检测到低至0.1ng/mL的羊抗人IgG.同时,以磁珠为载体的免疫复合体也可通过激光扫描共聚焦荧光显微镜进行荧光成像分析,背景荧光信号低,成像质量高.实验结果表明,PAA修饰的β-NaYF4：Yb,Er上转换荧光纳米粒子是一种理想的生物标记材料,有望在生物传感及生物成像分析领域获得广泛应用.     

悬浮芯片法与常规酶联免疫吸附法检测3种兽药残留的比较

建立氯霉素,克伦特罗和雌二醇 3种兽药残留同时检测的悬浮芯片法.通过将3种兽药的BSA蛋白结合物偶联于悬浮芯片的固相载体--聚苯乙烯荧光微球上作为检测探针,采用间接竞争法,在液相反应体系中,3种小分子兽药抗原和微球上的兽药结合物共同竞争液相中各自特异性的生物素化单抗,再加入藻红蛋白标记的链霉亲和素,反应后检测获得荧光信号,绘制出3种兽药残留检测的标准曲线.同时进行3种兽药的常规酶联免疫吸附法标准曲线的测定.在检测技术、检出限、检测区间、特异性、盲样测定和多元分析等方面对两种方法进行比较.除了特异性外的其它指标的比较中,悬浮芯片法均具有明显优势.两种方法的特异性检测具有良好的一致性.高通量悬浮芯片技术,具有操作简单、灵敏快速和成本低廉等优点,为多种兽药残留的快速检测提供了新方法.     

信号核酸识体用于药物托普霉素的高灵敏度检测

提出了一种利用RNA核酸识体和光开关化合物[Ru(phen)2dppz\]2+ (RU)高灵敏检测药物分子的荧光分析新方法. 以托普霉素(TOB)为靶分子, 检测限可达到10 nmol/L, 同时该方法有较好的选择性, 能在溶液中实现无需分离的实时检测. 这种无标记检测方法有利于不稳定RNA核酸识体在分析检测中的应用, 简化实验操作, 为其它药物分子的检测提供了新思路.     

基于适配体亲和毛细管电泳-激光诱导荧光检测的凝血酶分析

以一种高亲和力适配体作为亲和荧光探针,以自建的毛细管电泳-激光诱导荧光(CE-LIF)检测装置为基础,建立了一种高灵敏、快速测定人凝血酶的方法。荧光标记的凝血酶适配体特异性地与凝血酶结合并形成稳定的凝血酶-适配体复合物,采用CE-LIF对复合物进行分离检测,从而测定凝血酶浓度。探讨了盐离子种类及浓度对适配体与凝血酶结合的影响,并在选定的电泳条件下对凝血酶检测的线性范围、检出限和重现性进行了测定。结果表明,盐离子存在的条件下适配体与凝血酶的亲和力降低,不利于两者的结合;人血清溶液中,凝血酶浓度在0.25～10 nmol/L范围内与复合物峰面积具有良好的线性相关性(r20.991),检出限(S/N3)为55.6 pmol/L;精密度和回收率测定结果均能满足分析的要求。     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Magnetic bead-based label-free electrochemical detection of DNA hybridization.

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.     

New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces

Up to now, the development of the electrochemical DNA hybridization sensors relied on solid electrodes, on which both the hybridization and detection steps have been performed. Here we propose a new method in which the DNA hybridization is performed at commercially available magnetic beads and electrochemical detection on detection electrodes (DE). Due to minimum nonspecific DNA adsorption at the magnetic beads, very high specificity of the DNA hybridization is achieved. Optimum DE can be chosen only with respect to the given electrode process. It is shown that high sensitivity and specificity in the detection of relatively long target DNAs can be obtained (a) by using cathodic stripping voltammetry at mercury or solid mercury amalgam DEs for the determination of purine bases, released from DNA by acid treatment, and (b) by enzyme-linked immunoassay of target DNA modified by osmium tetroxide,2,2'-bipyridine (Os,bipy) at carbon DEs. Direct determination of Os,bipy at mercury and carbon electrodes is also possible.     

Nanoparticle-based electrochemical DNA detection

Nanoscale architectures of DNA-linked particle networks are attractive for electrical detection of DNA hybridization. This article reviews a variety of new nanoparticle/polynucleotide assemblies for advanced electrical detection of DNA sequences. Recent activity has led to innovative and powerful nanoparticle-based electrochemical DNA hybridization assays based on a variety of detection schemes. Such protocols rely on the use of colloidal gold tags, semiconductor quantum dot tracers, polymeric carrier (amplification) beads, or magnetic (separation) beads. Particularly useful have been protocols based on capturing of metal nanoparticle tracers followed by dissolution and anodic-stripping voltammetric measurement of the metal tag. Remarkable sensitivity is achieved by coupling particle-based amplification units and various amplification processes. The use of nanoparticle tracers for designing multi-target electrochemical coding protocols will also be documented.     

Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system

An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N = 3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.     

Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

Detection of single-base mutations using 1-D microfluidic beads array

The application of a 1-D microfluidic beads array that is composed of individually addressable functionalized SiO2 beads has been demonstrated for detection of single-base mutations based on "sandwich" hybridization assay without additional sample labeling and PCR amplification. We concentrated on detection of mutations in the human p53 tumor suppressor gene with more than 50% mutation frequency in the known human cancers. Using a microinjection system, functionalized beads could be selectively and linearly arrayed in a single microfluidic channel comprising many periodic chambers. This 1-D microfluidic beads array was sufficiently sensitive to identify single-nucleotide mutations in 40 pM quantities of DNA targets and could discriminate the mutated alleles in an excess of nonmutated alleles at a level of one mutant in 100 wild-type sequences. The surface of beads was regenerated and rehybridized up to six times without obvious loss of signal. The entire reaction process was done at room temperature within minutes, and only 2-10 microL sample solution was needed to complete the whole detection process. The p53 genotypes of A549, CNE2, and SKBr-3 cell lines were also correctly evaluated by using mRNA extracts as target without need for sample labeling and amplification. Thus, this platform enabled rapid and exact discrimination of gene mutations with the advantages of reusability, simple handling of liquid, low cost, and little reagent consumption.     

Microfluidic-based electrochemical genosensor coupled to magnetic beads for hybridization detection

This paper describes the development of a rapid and sensitive enzyme-linked electrochemical genosensor using a novel microfluidic-based platform. In this work, hybridization was performed on streptavidin-coated paramagnetic micro-beads functionalized with a biotinylated capture probe. The complementary sequence was then recognized via sandwich hybridization with a capture probe and a biotinylated signaling probe. After labeling the biotinylated hybrid with a streptavidin-alkaline phosphatase conjugate, the beads were introduced in a disposable cartridge composed of eight parallel microchannels etched in a polyimide substrate. The modified beads were trapped with a magnet addressing each microchannel individually. The presence of microelectrodes in each channel allowed direct electrochemical detection of the enzymatic product within the microchannel. Detection was performed in parallel within the eight microchannels, giving rise to the possibility of performing a multiparameter assay. Quantitative determinations of the analyte concentrations were obtained by following the kinetics of the enzymatic reaction in each channel. The chip was regenerated after each assay by removing the magnet and thus releasing the magnetic beads. The system was applied to the analytical detection of PCR amplified samples with a RSD% = 6. A detection limit of 0.2 nM was evaluated.     

Development of a facile and sensitive HPLC‐FLD method via fluorescence labeling for triterpenic acid bioavailability investigation

Triterpenic acids are widely distributed in many fruits and are known for their medicinal benefits. The study of bioavailability has been an important task for a better understanding of the triterpenic acids. Although many methods based on fluorescence labeling for triterpenic acid determination have been established, these reported methods needed anhydrous conditions, which are not suitable for the convenient study of triterpenic acid bioavailability. Inspired by that, a versatile method, which overcomes the difficulty of the reported methods, has been first developed in this study. The novel method using 2‐[12‐benzo[b ]acridin‐5‐ (12H)‐yl]‐acetohydrazide (BAAH) as the fluorescence labeling reagent coupled with high‐performance liquid chromatography with fluorescence detection was first developed for the study of triterpenic acid bioavailability. Furthermore, the labeling conditions have been optimized in order to achieve the best fluorescence labeling yield. Under the optimal conditions, the quantitative linear range of analytes was 2–1000 ng mL⁻¹, and the correlation coefficients were >0.9998. The detection limits for all triterpenic acid derivatives were achieved within the range of 0.28–0.29 ng mL⁻¹. The proposed method was successfully applied to the study of triterpenic acid bioavailability with excellent applicability and good reproducibility.