光纤倏逝波生物传感器及其应用

介绍光纤倏逝波生物传感器的基本原理、常用试验方法、基本仪器构建及应用进展。光纤倏逝波生物传感器是基于光波在光纤内以全反射方式传输时产生倏逝波的原理,以生物分子作为敏感元件进行检测的一类新兴传感器。光纤倏逝波生物传感器有望应用于环境监控、食品卫生监控、临床疾病监测、DNA检测和生物战剂检测。     

倏逝波光纤免疫传感器的光纤再生

倏逝波光纤免疫传感器作为一种新兴的检测技术,在环境检测、食品卫生检测及生物医学检测等领域具有广泛的应用前景。为了降低检测成本并达到快速检测,光纤再生问题就显得尤为重要。我们在对倏逝波光纤免疫传感器原理和光纤再生原理介绍的基础上,对光纤再生的酸性碱性溶液、高浓度盐溶液、去污剂等方法进行简要综述,探讨了光纤再生存在的问题,并展望了解决光纤重生后倏逝波光纤免疫传感器的应用前景。     

邻苯二甲酸酯类污染物雌激素结合活性筛查

以倏逝波光纤荧光生物传感器为平台,以邻苯二甲酸酯类(PAEs)污染物为靶标,优化了基于受体作用理论的生物传感分析技术,实现了PAEs雌激素结合活性的定性筛查。对7种典型邻苯二甲酸酯的雌激素结合活性测试结果为BBP>DBP>DIPP,其他4种物质几乎无雌激素结合活性,结果与文献广泛报道结论相一致,验证了所建立方法的准确性。在最优测试条件下,光纤传感界面可再生300次以上,为雌激素活性污染物的筛查提供了一种低成本、自动化方法。     

用于Escherichia coli O157∶H7直接快速检测的倏逝波荧光核酸适配体传感器研究

通过融合倏逝波荧光光纤传感器和特异性核酸适配体的优势,提出了一种基于倏逝波荧光原理及其与病原菌尺寸效应的Escherichia coli O157∶H7(E.coli O157∶H7)直接快速检测方法。基本原理是当一定浓度荧光标记E.coli O157∶H7核酸适配体加入样品检测池时,倏逝波激发荧光分子发出荧光,利用倏逝波全光纤生物传感器即可实现荧光信号的定量检测;当荧光标记的核酸适配体与E.coli O157∶H7混合后加入样品检测池,因倏逝波渗入深度仅为100 nm,导致特异性结合E.coli O157∶H7的核酸适配体标记荧光分子不能被激发,从而使得检测荧光信号降低;利用荧光信号强度与E.coli O157∶H7浓度的比例关系即可实现其定量检测。结果表明:该方法检测E.coli O157∶H7的检测限可达610 CFU/mL,线性检测区间为1.1×10~3-1.4×10~7 CFU/mL。实际水样加标回收率在40%-180%之间,相对标准偏差在10%之内,水样基质对E.coli O157∶H7的检测没有明显影响。本研究建立基于倏逝波荧光原理及其与病原菌尺寸效应的生物传感分析方法具有普适性,仅需使用不同荧光标记的生物识别分子即可实现其他病原菌的直接快速检测。     

光纤生物传感器及其在微生物检测中的应用

本文介绍了光纤生物传感器的原理,对光纤传感器制作中的工程学和生物学问题进行了探讨并概述了它的应用情况。     

消失波生物传感器及其在DNA与免疫分析中的应用

消失波光纤生物传感器是近年来发展很快的一项的分析技术。它现在已成为分了生物学领域的热门技术。本文叙述消失波生物传感器的识别元件,换能装置以及检测研究系统的研究进展。着重讨论消失波技术在ＤＮＡ检测与免疫检测中的应用。并对这些技术的应用价值做出评价。     

电场驱动微悬臂生物传感器及对DNA 的快速、高灵敏度检测

微悬臂列阵传感器在生物检测方面具有快速、痕量和非标记的特性. 我们以镀金并在其上固定了 DNA 探针的微悬臂为正极,在靶杂交液槽内引入另一电极作为负极,构成电场驱动微悬臂 DNA 生物传感器. 对该传感器系统施加静电场,驱动 DNA 分子朝正极迁移,使溶液中的 DNA 分子富集在微悬臂上,促进 DNA 分子的杂交. 结果表明： a. DNA 在微悬臂上的杂交时间仅需 3 min,加快了微悬臂生物传感器对 DNA 分子的检测速度; b. 提高了微悬臂生物传感器的灵敏度,可以检测到皮克级的 DNA 分子.     

A蛋白定向固定抗体用于椭偏光学生物传感器免疫检测

椭偏光学生物传感器是在椭偏光学显微成像技术的基础上发展的一项生物传感技术。它能够直接观测固体表面上的生物分子面密度,毋需任何标记辅助,适合发展成为一种无标记免疫检测技术。研究了在硅片表面上通过A蛋白定向固定抗体分子用于椭偏光学生物传感器免疫检测的可能性。实验结果表明,通过A蛋白固定抗体得到的抗体膜层的均一性和固定量的重复性能够保证椭偏光学生物传感器免疫检测结果的质量。通过A蛋白定向固定的抗体的抗原结合位点趋向一致,显著提高了抗体与抗原结合的能力。此外,通过蛋白A固定的免疫球蛋白G分子能够结合更多的多克隆抗体分子说明通过A蛋白固定的蛋白质分子能够较好地保持其空间构象。     

基于DNA分子导线的纳米生物传感器

DNA分子导线具有独特的导电性能和塞贝克(Seebeck)效应,它是构筑电化学纳米生物传感器和热电偶生物传感器的理想材料。文章简要介绍了DNA分子导线的制备方法及导电机理,以及基于DNA分子导线的纳米生物传感器的分子识别机制,着重分析了基于DNA分子导线的纳米生物传感器的传感原理。文章还介绍了基于DNA分子导线的纳米生物传感器在基因分析、单碱基突变检测等方面的应用。     

阵列电化学生物传感器研究进展

阵列生物传感器技术作为一种高通量、快速、选择性高和集成化的分析技术,已在基因组学和蛋白质组学的研究和药物筛选、环境分析,食品分析,临床诊断等领域中得到广泛的应用.阵列生物传感器主要有阵列光学生物传感器和阵列电化学生物传感器.阵列电化学生物传感器是将生物分子识别物质如酶、抗原/抗体、DNA等固定在阵列电极上,以阵列中每根电极产生的电化学信号作为检测信号的电化学分析器件.阵列电化学生物传感器以灵敏度高、分析速度快、选择性好、易于微型化和集成化以及仪器价格低廉等特点受到了研究工作者的极大关注.本文简单介绍了阵列电化学生物传感器的原理和特点,重点评述了2005年以来阵列电化学生物传感器在单组份检测和多组份同时检测两方面的研究进展,简单讨论了阵列电化学生物传感器研究中存在的问题.     

Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.     

Characterization of methods for determining sterilization efficacy and reuse efficiency of oxygen biosensor multiwell plates

High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System™ (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.     

A novel fluorescence-based array biosensor: principle and application to DNA hybridization assays

A novel fluorescence-based array biosensor targeted for field applications, such as environmental monitoring, has been developed, and successfully applied to DNA hybridization assays. The purpose was to meet the demand for automated, portable but easy-to-maintain systems allowing continuous flow monitoring of surface reactions. The biosensor presented here can be distinguished from the existing systems by the optical method used, which provides an enhanced simplicity and robustness, and enables a simple maintenance by potentially unskilled personnel. The system is based on a conventional microscope slide which acts both as transducer and biological array sensor. The excited fluorescence is guided by total internal reflection into the slide to the detector which is directly interfaced to the slide. Each region of the sensor array is successively optically interrogated, and the detection of the corresponding fluorescent emission synchronized. A real-time three-analyte analysis is thus feasible without any mechanical scanning movement or optical imaging systems as generally used in the existing instruments. The ability of the biosensor to operate in continuous flow for several tens of hours has been demonstrated. The biosensor has been assessed in terms of stability, and slide-to-slide reproducibility, which is found to be less than 3.7%, thus far below the standard biological reproducibility. DNA hybridization assays were performed to estimate a limit of detection, which was found to be 16 mol/microm(2), and to determine the reaction kinetics associated to the DNA model used. The developed biosensor is thus shown to be able to predict reaction kinetics, and to monitor in real time surface reactions between targets and probes.     

A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations

The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element. ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum. At 24 h following cell addition, calibration curves were constructed relating the steady state Δf and ΔR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface. We then utilized this EC QCM biosensor for the detection of the effect of [nocodazole] on the steady state Δf and ΔR shift values. Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells. At the doses used in these studies (0.11–15 μM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells. This leads a monolayer of well spread ECs to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape. We observed the negative Δf shift values and the positive ΔR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM. Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action.     

Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T. gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T. gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion. METHODS: We present here Laser Scanning Cytometer (LSC)-based assays of T. gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment. RESULTS: The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms. CONCLUSIONS: The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T. gondii, and can be readily adapted to study similar processes in other host-pathogen systems.     

Transfection of cells attached to selected cell based biosensor surfaces

Mammalian cell attachment studies were conducted on a variety of common microchip surfaces for potential use in cell based biosensors. COS-7 cell attachment to Au, Pt or ITO, per unit area was greater than to SiO(2) surfaces. The number of cells that would attach was essentially maximized 3 h after cell seeding. HL-1 cells attached more readily to surfaces precoated with fibronectin, but by 3 h equivalent number of cells had attached independent of fibronectin precoating. Inclusion of serum in media during the initial period of attachment decreased the number of COS-7 cells attached to SiO(2) surfaces, but no dependence on serum was seen for ITO surfaces. The number of cells attached per unit area varied with the composition of the surface. However, no differences were observed in the percentage of cells transfected with a green fluorescent protein gene, or in the level of reporter gene expression over the population of transfected cells on ITO, SiO(2), Pt, Ag, or Au surfaces. Similar FACS analysis of transfected Hep G2 cells revealed lower levels of both transfection efficiency and levels of GFP fluorescence. Hep G2 cells plated on Ag did not remain attached for analysis, but there were no significant differences between tissue culture plastic and the other biosensor surfaces in the percentage of cells transfected. This suggests that, in general, cells will attach to the various conducting and nonconducting biosensor surfaces studied and will provide comparable data in reporter gene expression assays.     

Label-free assays on the BIND system

Screening of biochemical interactions becomes simpler, less expensive, and more accurate when labels, such as fluorescent dyes, radioactive markers, and colorimetric reactions, are not required to quantify detected material. SRU Biosystems has developed a biosensor technology that is manufactured on continuous sheets of plastic film and incorporated into standard microplates and microarray slides to enable label-free assays to be performed with high throughput, high sensitivity, and low cost per assay. The biosensor incorporates a narrow band guided-mode resonance reflectance filter, in which the reflected color is modulated by the attachment/detachment of biochemical material to the surface. The technology offers 4 orders of linear dynamic range and uniformity within a plate, with a coefficient of variation of 2.5%. Using conventional biochemical immobilization surface chemistries, a wide range of assay applications are enabled. Small molecule screening, cell proliferation/cytotoxicity, enzyme activity screening, protein-protein interaction, and cell membrane receptor expression are among the applications demonstrated.     

Microplate-based, label-free detection of biomolecular interactions: applications in proteomics

This review describes a new type of label-free optical biosensor that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard format microplates to enable highly sensitive, high-throughput detection of small molecules, proteins and cells. The biosensor and associated detection instrumentation are applied to review two fundamental limiting issues for assays in proteomics research and drug discovery: requirement for quantitative measurement of protein concentration and specific activity, and measurements made with complex systems in highly parallel measurements. SRU BIosystems, Inc.'s BIND label-free detection will address these issues using data examples for hybridoma screening, epitope binning and mapping, small-molecule screening, and cell-based functional assays. The review describes several additional applications that are under development for the system, and the key issues that will drive adoption of the technology over the next 5 years.     

The role of motility in the in vitro attachment of Pseudomonas putida PaW8 to wheat roots

The attachment of motile and non-motile strains of Pseudomonas putida PaW8 to sterile wheat roots was assessed in both non-competitive and intra-specific competitive assays. The motile strain showed significantly greater attachment to wheat roots than non-motile strains in phosphate buffer. Overall, the motile strain attached better than the non-motile strain at 10(6), 10(7) and 10(8) cfu ml(-1) in competitive assays and at 10(6) and 10(7) cfu ml(-1) in non-competitive assays. When attachment was studied in Luria broth no significant difference between motile and non-motile strains was detected. P. putida PaW8 cells marked with the luxAB genes were used to compare direct detection of attached cells by luminometry with indirect detection by dilution plate counts following extraction from root material. Although direct detection permitted a rapid assessment (60 s) of attachment to surfaces, dilution plate counts provided a more sensitive method for quantification of bacteria. The detection limits were approximately 10 cfu root(-1) using dilution plate counts compared with 1000 cfu root(-1) using luminometry. All results highlighted the importance of motility for the attachment of P. putida to plant roots in simple model systems. To take this work further, studies to assess the role of motility using complex non-sterile systems are needed.     

Biosensor analysis of the interleukin-2 receptor complex

Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure-activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd.