生物传感技术的新应用——生物分子相互作用分析技术（BIA）

BIA 技术(biomolecular interaction analysis)——生物分子相互作用分析技术是利用生物传感技术发展的全新概念.生物分子间的相互作用可在免标记的状态得到实时的追踪和分析.文章简单介绍BIA的原理和应用范围.在以后的几期杂志中,还将介绍一系列应用实例.结合已发表的近400篇应用文献,读者可以看到此新技术应用范围之广,得到的信息之多,堪称开创了生物技术的新纪元.     

SPR生物传感器的特点及其在生物特异性相互作用分析中的应用

利用表面等离子模共振技术（SPR）进行生物特异性相互作用分析（BIA）已成为现代基因工程技术中的一种先进的手段。与传统的研究方法如酶联免疫吸附测定（ELISA）相比,它具有方便快捷、灵敏度高,应用范围广,实时监控等多项特点,利用这种新型研究手段对于生命科学的基础研究。医学诊断以及治疗等方面有着十分重的意义。本粗略概括了近几年来利用SPR生物传感器进行基础研究的基本情况以及对其的改进,并简要分析了此项技术的优点以及发展前景。     

应用实时BIA研究酵母PHO4和PHO2的相互作用

应用实时生物分子相互作用分析技术（ＢＩＡ）分析了酵母ＰＨＯ４和ＰＨＯ２蛋白的相互作用。将ＰＨＯ４蛋白通过氨基耦联在感应片上。进样５μｍｏｌ／Ｌ重组的谷胱甘肽转移酶融合的ＰＨＯ２蛋白（ＧＳＴ－ＰＨＯ２）及其两种突变体融合蛋白。结果表明ＰＨＯ２的２３０位丝氨酸变为天冬氨酸的突变体（ＧＳＴ－２ＳＤ）与ＰＨＯ４有强的表面等离子体激元共振信号,而生型ＰＨＯ２和２３０位丝氨酸变为丙氨酸的突变体（ＧＳＴ－２ＳＡ     

解析用于环境监测的生物传感技术

随着科技的不断进步,企业的不断发展,我国的经济水平有了很大的提升。但在实际的发展中,一切的一切都是建立在破坏环境的基础上,因此在将来的工作中必须对环境进行有效的保护。现今的一般方法和手段已经没有办法对环境进行有效的监测,更不要说保护环境。鉴于这样的情况,我们需要采取有效的措施来监测环境,获得确切的资料,也就是生物传感技术。这种技术从研究到实施经历了较长的时间,但取得的效果却非常好。     

生物分子相互作用分析技术的一次突破

细胞生命活动的过程,无不涉及两个或多个生物分子之间相互作用,如信号传导、免疫反应、酶与底物作用等。因此,为了更好地研究生命过程,就迫切需要开发出一种能实时检测出生物分子间相互作用的系统,Phar－maca公司于90年代初就开发出了这样一种技术,即BM技术（BiornoecularInterationAnalssis）,该技术是基于表面等离子共振（Snd。PI。R。ce,简称SPR）的光物理现象［fi。在该现象中,所检测出的共振角与结合在传感片表面的生物分子的质量呈正比例关系。因此以时间对共振角（以共振单位表示）作图可记录相互作用的动态全过程,…     

生物分子相互作分析技术应用实例（一）：配体垂钓和抗体测定

ＢＩＡ技术－即生物分子相经作用分析技术的应用范围相当广泛,先介绍利用ＢＩＡｃｏｒｅ分离得到ＥＣＫ－酪氨酸激酶受体的蛋白配体。另一应用实例是利用ＢＩＡｃｏｒｅ直接从杂交瘤细胞上清液中测定单克隆抗体的活性、亲和力和动态常数。     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

实时荧光定量PCR技术及其在植物转基因产品检测中的应用

实时荧光定量PCR技术因其实时、快速、高效和准确定量的优点,已经广泛用于转基因产品定量检测。本文介绍荧光定量探针技术的原理、特点及其在植物转基因产品检测中的应用情况。     

生物芯片技术及其在水体环境生物监测中的应用

水源微生物污染严重威胁着人类的健康。为有效控制水体环境生物安全,水体环境中微生物快速而准确地监测是关键的技术基础之一。生物芯片(biochip)技术是20世纪90年代初期发展起来的一门新兴技术,能迅速检测出水中的微生物。本文阐述了生物芯片的基本概念,对基因芯片技术作了简介。重点叙述了生物芯片技术在水体环境生物监测方面的应用,并就其应用前景作了展望。     

常见生物传感芯片及其应用进展

生物传感芯片是一类综合了生物芯片和生物传感器的优点的新型生物芯片,在保持传统生物芯片的高通量、可寻址、并行处理等特点的基础上,与生物传感器技术相结合,进一步提高了芯片检测的灵敏度和特异性。常见的生物传感芯片主要有光纤传感芯片、表面等离子体共振传感芯片、热生物传感芯片、压电晶体传感芯片等,可用于各种生物大分子,如蛋白质、核酸等的检测,金属离子的测定,病原体的检测,药物筛选等。     

Mechanisms of Action of Carbamazepine and Its Derivatives,Oxcarbazepine, BIA 2-093, and BIA 2-024

Carbamazepine (CBZ) has been extensively used in the treatment of epilepsy, as well as in the treatment of neuropathic pain and affective disorders. However, the mechanisms of action of this drug are not completely elucidated and are still a matter of debate. Since CBZ is not very effective in some epileptic patients and may cause several adverse effects, several antiepileptic drugs have been developed by structural variation of CBZ, such as oxcarbazepine (OXC), which is used in the treatment of epilepsy since 1990. (S)-(–)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), which were recently developed by BIAL, are new putative antiepileptic drugs, with some improved properties. In this review, we will focus on the mechanisms of action of CBZ and its derivatives, OXC, BIA 2-093 and BIA 2-024. The available data indicate that the anticonvulsant efficacy of these AEDs is mainly due to the inhibition of sodium channel activity.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays

The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.     

Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/MS)

Ongoing, worldwide efforts in genomic and protein sequencing, and the ability to readily access corresponding sequence databases, have emphatically driven the development of high‐performance bioanalytical instrumentation capable of characterizing proteins and protein–ligand interactions with great accuracy, speed and sensitivity. Two such analytical techniques have arisen over the past decade to play key roles in the characterization of proteins: surface plasmon resonance biomolecular interaction analysis (SPR‐BIA) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF). SPR‐BIA is used in the real‐time investigation of biomolecular recognition events, and is thereby capable of providing details on the association and dissociation kinetics involved in the interaction, information ultimately leading to the determination of dissociation constants involved in the event. MALDI‐TOF is used in the structural characterization, identification and sensitive detection of biomolecules. Although the two techniques have found many independent uses in bioanalytical chemistry, the combination of the two, to form biomolecular interaction analysis mass spectrometry (BIA/MS), enables a technique of analytical capabilities greater than those of the component parts. Reviewed here are issues of concern critical to maintaining high‐levels of performance throughout the multiplexed analysis, as well as examples illustrating the potential analytical capabilities of BIA/MS. Copyright © 1999 John Wiley & Sons, Ltd.     

Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry

Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.     

基于波长寻址的表面等离子体共振传感技术

本文提出了基于光谱扫描技术的非机械扫描的表面等离子体共振(SPR)传感技术,采用白光为SPR激发光源,通过单色仪控制入射光的波长实现光谱寻址,在保证灵敏度和动态范围的同时,使系统在整个动态范围内具有较好的线性,简化了传感器结构。理论分析了光谱扫描SPR传感技术的灵敏度和动态范围,搭建了实验系统,并测量了不同浓度的酒精水混合溶液的SPR信号变化。结果表明:系统折射率测量范围为1.30-1.38,灵敏度可达3.1×105RIU。     

TILLING技术及其应用

定向诱导基因组局部突变(targetinginducedlocallesionsingenomes,TILLING)可快速、有效地鉴定和定向筛选突变,是一种全新的反向遗传学技术。现对TILLING的技术流程、核心与特点,及其在突变研究、反向遗传学及功能基因组学、SNP检测、资源创新与分析以及作物遗传改良等方面的应用进行了综述。     

高通量测序技术及其应用

高通量测序技术是DNA测序发展历程的一个里程碑,它为现代生命科学研究提供了前所未有的机遇。详细介绍了以454、Solexa和SOLiD为代表的第二代高通量测序技术,以HeliScope TIRM和Pacific Biosciences SMRT为代表的单分子测序技术,以及最近Life Science公司推出的Ion Personal Genome Machine (PGM)测序技术等高通量测序技术的最新进展。在此基础上,阐述了高通量测序技术在基因组测序、转录组测序、基因表达调控、转录因子结合位点的检测以及甲基化等研究领域的应用。最后,讨论了高通量测序技术在成本和后续数据分析等方面存在的问题及其未来的发展前景。