微流控浓度梯度芯片在肺癌耐药性研究中的应用

微流控芯片系统是20世纪末开发的一种高效的微分析系统,可将样品制备、液体分离、生物与化学反应检测、细胞培养及蛋白分析等研究手段集成,具有高通量检测的特点,近年来已逐渐应用于化学、药物筛选及环境检测等多个领域,但在医学及分子生物学研究中的应用较少.本研究中利用该微流控芯片系统对人肺鳞状细胞癌细胞内葡萄糖调节蛋白78(GRP78)在肺癌对化疗耐药中的作用进行分析.     

心肌损伤标志物即时检测技术的应用进展

快速检测心肌损伤标志物可以为急性心肌梗死诊断提供重要依据。该文主要对纸基类检测、生物传感器、微流控芯片技术和可穿戴设备等即时检测技术在检测心肌损伤标志物中的应用作一介绍。     

基于微流控核酸等温扩增的 登革病毒现场快速检测技术研究

[摘要]?目的?结合环介导等温扩增技术（loop-mediated isothermal amplification, LAMP）和微流控芯片技术，建立一种适合现场快速检测登革病毒的方法。方法?利用RT-LAMP，针对登革病毒基因组中3’非编码区中特异性序列进行扩增，建立基于微流控芯片技术的LAMP检测方法，优化检测体系，并对该方法的灵敏性和特异性进行评估。结果?基于LAMP 和微流控芯片技术的登革病毒检测方法，通过对病毒模板进行扩增，发现与其他病毒无交叉反应，特异性良好；同时，结果显示该方法对登革病毒检测灵敏性可达61.2 pg/μl，与实时荧光定量PCR仪所达到的检测灵敏性一致。结论?基于LAMP和微流控芯片技术的登革病毒检测方法具有操作简单、快速、对设备要求低等优势,并且灵敏性、特异性均较好，是一种便于开展现场快速检测的方法。     

微流控芯片技术在心血管疾病研究中的应用

心血管疾病是威胁人类健康的重要疾病，具有很高的致死率和致残率，已成为现代医学研究的热点话题。将传统实验技术用于心血管疾病发生机制、诊断及治疗等方面的研究具有一定局限性。微流控芯片技术是一项新型实验研究手段，具有微型化、集成化、高通量、低消耗、高灵敏、分析速度快等特点，其在体外生物模型构建、生物分子检测、药物疗效评价及筛选等方面较传统技术具有优势，现已逐渐应用于医学生物学研究的多个领域。本文谨对微流控芯片技术在心血管疾病研究领域的应用进行简要综述。     

环介导等温扩增技术检测方法的研究进展

环介导等温扩增技术是一种可用于临床诊断的快速检测方法,可用于检测病毒、细菌、寄生虫等多种病原体。经不断完善,现已在临床检测过程中发挥重要作用。可用显色试剂对反应结果实现可视化,即结果的即时显示,无需后续验证。但是,无论是荧光染料还是显色指示剂,对反应过程都会产生或多或少的影响,目前研究表明羟基萘酚蓝是最好的显色试剂。同时,人们发现电化学技术也可以应用于此,使检测反应更快,操作更加简单。生物传感器可以对多种反应产物进行同步检测,而微流控芯片技术更是方便携带,有望成为实时检测技术的理想载体。     

HIV HCV和HBV核酸检测技术及策略在血筛应用中的研究进展

人类免疫缺陷病毒(HIV)、乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)是输血传播疾病的主要病原体。为防止因输血引起的HIV、HBV及HCV传播,国内外已有大量针对献血者血液筛查方法的研究。如抗原和/或抗体的酶联免疫吸附试验(ELISA),经过多年研究和应用成效显著。为降低"窗口期"导致的疾病传播,核酸检测(NAT)技术也不断地发展,同时血清学检测(SS)与NAT的互补和联合应用,形成了不同的血筛检测策略。同时,基于纳米材料的微流控技术对病原体多元快速检测,以及聚合酶链反应-酶联免疫吸附(PCR-ELISA)技术等新技术的不断探索研究,将会为血液筛查技术开辟新的途径。文章综述了血液中HIV、HBV和HCV的检测在血筛应用中的研究进展。     

浙江温州地区1020例乙型肝炎患者HBV基因分型研究

目的:了解浙江温州地区乙肝病毒基因型分布,初步评价微流芯片分析仪在乙肝病毒基因分型中的应用特点.方法:用HBV基因型特异引物经聚合酶链反应扩增温州地区1 020例慢性乙型肝炎患者血清HBV,其扩增产物应用微流芯片检测.结果:该地区1 020例患者中966例有基因分型结果,其中B型20.49%(198/966)、C型66.46%(642/966),B、C混合型4.87%(47/966),C、B混合型6.21%(60/966),D型1.97%(19/966).结论:浙江温州地区乙型肝炎病毒基因型分布具有北方流行特点,微流芯片在同类检测方法中具有灵敏、准确、快速优势.     

微流控芯片与基因诊断关系的研究进展

<正>微流控芯片(MC)也称为芯片实验室,其已经广泛于医学、生物、电子、流体、化学等领域~([1,2]),且MC可把样品制备、反应、分离、检测、扩增、分析等集成到一块几微米至几百微米尺度的芯片上并自动完成所有基本过程。目前,MC已经广泛地应用到医学基因诊断(GD)方面,例如基因多态性检测~([3])、基因高效性测序~([4])、基因快速性扩增~([5,6])等,为此,本文主要对MC与GD关系进行综述。1 MC简介1.1 MC相关概念MC主要是指在几微米至     

基于微流控平台的胶质母细胞瘤干细胞中miRNA-874表达的研究

目的通过微流控检测平台,研究miRNA-874（miR-874）表达在胶质母细胞瘤干细胞（GSCs）生长中的作用及意义。 方法应用高通量、多样本、低成本的微流控技术,对GSCs和神经干细胞（NSCs）进行miR-874转染,并通过荧光定量PCR检测细胞内miR-874和mRNA表达等一系列细胞分子生物实验,研究miRNA-874对GSCs生长和凋亡的作用。 结果miR-874在GSCs中的表达显著降低,约为NSCs中表达量的2%~40%（P<0.05）。利用树状分子将miR-874转染入GSCs中后,GSCs的细胞数量显著下降,凋亡细胞的数量显著上升,提示miR-874对GSCs具有杀伤作用。 结论miR-874在GSCs中表达显著降低,在GSCs中过表达miR-874会导致显著的细胞生长抑制和死亡,有助于开发基于miRNA的新型脑癌药物和疗法。     

再测序芯片在北京首例人禽流感病例病原学检测中的应用

目的利用再测序芯片对北京市首例人禽流感病例进行病原筛查和验证。方法采集病例咽拭子和气管抽吸物标本,利用real-ti me RT-PCR进行禽流感病毒H5N1亚型核酸检测;应用病原体再测序芯片对其进行复核,并对其它呼吸道病原体和流感病毒其它亚型进行筛查。结果气管抽吸物标本经real-ti me RT-PCR检测为禽流感病毒H5N1核酸阳性;再测序芯片检测的结果是获得了H5N1的非结构蛋白基因(NS)特异序列,通过与GenBank进行序列比对,确定为禽流感病毒H5N1核酸,并排除了30种流感病毒亚型和其它33种呼吸道病原体的感染。结论病原体再测序芯片具有高灵敏性和特异性,在北京市首例人禽流感病例的病原学筛查和验证中发挥了重要作用。     

Whole-Teflon microfluidic chips

Although microfluidics has shown exciting potential, its broad applications are significantly limited by drawbacks of the materials used to make them. In this work, we present a convenient strategy for fabricating whole-Teflon microfluidic chips with integrated valves that show outstanding inertness to various chemicals and extreme resistance against all solvents. Compared with other microfluidic materials [e.g., poly(dimethylsiloxane) (PDMS)] the whole-Teflon chip has a few more advantages, such as no absorption of small molecules, little adsorption of biomolecules onto channel walls, and no leaching of residue molecules from the material bulk into the solution in the channel. Various biological cells have been cultured in the whole-Teflon channel. Adherent cells can attach to the channel bottom, spread, and proliferate well in the channels (with similar proliferation rate to the cells in PDMS channels with the same dimensions). The moderately good gas permeability of the Teflon materials makes it suitable to culture cells inside the microchannels for a long time.     

The State of the Art in Biodefense Related Bacterial Pathogen Detection Using Bacteriophages: How It Started and How It’s Going

Accurate pathogen detection and diagnosis is paramount in clinical success of treating patients. There are two general paradigms in pathogen detection: molecular and immuno-based, and phage-based detection is a third emerging paradigm due to its sensitivity and selectivity. Molecular detection methods look for genetic material specific for a given pathogen in a sample usually by polymerase chain reaction (PCR). Immuno-methods look at the pathogen components (antigens) by antibodies raised against that pathogen specific antigens. There are different variations and products based on these two paradigms with advantages and disadvantages. The third paradigm at least for bacterial pathogen detection entails bacteriophages specific for a given bacterium. Sensitivity and specificity are the two key parameters in any pathogen detection system. By their very nature, bacteriophages afford the best sensitivity for bacterial detection. Bacteria and bacteriophages form the predator-prey pair in the evolutionary arms race and has coevolved over time to acquire the exquisite specificity of the pair, in some instances at the strain level. This specificity has been exploited for diagnostic purposes of various pathogens of concern in clinical and other settings. Many recent reviews focus on phage-based detection and sensor technologies. In this review, we focus on a very special group of pathogens that are of concern in biodefense because of their potential misuse in bioterrorism and their extremely virulent nature and as such fall under the Centers for Disease and Prevention (CDC) Category A pathogen list. We describe the currently available phage methods that are based on the usual modalities of detection from culture, to molecular and immuno- and fluorescent methods. We further highlight the gaps and the needs for more modern technologies and sensors drawing from technologies existing for detection and surveillance of other pathogens of clinical relevance.     

PCR技术应用于寄生虫分类鉴定的研究进展

PCR技术对寄生虫病原体的检测和鉴定提供了一种强有力的工具。PCR分子分类方法具有操作简便、特异性高和信息量丰富等优点,弥补了形态学分类上的不足。本文介绍了PCR-RFLP、RAPD和PCR-SSCP等技术的基本原理及用于寄生虫分类鉴定的研究进展。     

蛋白芯片法检测TORCH-IgM抗体的临床应用研究

目的评价TORCH蛋白芯片诊断致畸病原体的临床应用价值。方法采用TORCH蛋白芯片法与ELISA法对230份孕妇血清标本作平行检测;用蛋白芯片法对卫生部临床检验中心室问质评样本进行检测。结果TORCH蛋白芯片法与ELISA法各项指标检测结果均具有较好的一致性（P〉0.05）,其中TOX的阴、阳性符合率均为100％;RV的阳性符合率为85.7％,阴性符合率为99.6％;CMV的阳性符合率为91.7％,阴性符合率为99.1％;Hsv-Ⅱ的阳性符合率为93.8％,阴性符合率为99.1％。室间质评样本的检测结果与卫生部临床检验中心公布结果一致。结论蛋白芯片法检测TORCH-IgM抗体具有简便、快速,敏感性较高和特异性强等优点,是临床优生优育辅助诊断的有效方法,值得推广使用。     

等温扩增技术在寄生虫及其他病原体检测中的应用

以聚合酶链式反应（Polymerase chain reaction, PCR）为基础的体外核酸扩增技术已成功应用于科学研究各方面。近年来等温扩增技术（Isothermal amplification technology）凭借恒温、高效、耗时短、不过分依赖设备仪器等优点越来越多地被应用于分子诊断及疾病检测中。本文对现有的部分等温扩增技术原理、特点以及在寄生虫病病原体及其他病原体检测方面的应用进行综述,并展望其应用前景。     

Microfluidic approaches to malaria detection

Microfluidic systems are under development to address a variety of medical problems. Key advantages of micrototal analysis systems based on microfluidic technology are the promise of small size and the integration of sample handling and measurement functions within a single, automated device having low mass-production costs. Here, we review the spectrum of methods currently used to detect malaria, consider their advantages and disadvantages, and discuss their adaptability towards integration into small, automated micro total analysis systems. Molecular amplification methods emerge as leading candidates for chip-based systems because they offer extremely high sensitivity, the ability to recognize malaria species and strain, and they will be adaptable to the detection of new genotypic signatures that will emerge from current genomic-based research of the disease. Current approaches to the development of chip-based molecular amplification are considered with special emphasis on flow-through PCR, and we present for the first time the method of malaria specimen preparation by dielectrophoretic field-flow-fractionation. Although many challenges must be addressed to realize a micrototal analysis system for malaria diagnosis, it is concluded that the potential benefits of the approach are well worth pursuing.     

Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides

The development of a robust and portable biosensor for the detection of pathogenic bacteria could impact areas ranging from water-quality monitoring to testing of pharmaceutical products for bacterial contamination. Of particular interest are detectors that combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic readout. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of robustness. Here, we report selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays. The semiselective antimicrobial peptide magainin I--which occurs naturally on the skin of African clawed frogs--was immobilized on gold microelectrodes via a C-terminal cysteine residue. Significantly, exposing the sensor to various concentrations of pathogenic Escherichia coli revealed detection limits of approximately 1 bacterium/μL, a clinically useful detection range. The peptide-microcapacitive hybrid device was further able to demonstrate both Gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of E. coli and Salmonella. Finally, we report a simulated "water-sampling" chip, consisting of a microfluidic flow cell integrated onto the hybrid sensor, which demonstrates real-time on-chip monitoring of the interaction of E. coli cells with the antimicrobial peptides. The combination of robust, evolutionarily tailored peptides with electronic read-out monitoring electrodes may open exciting avenues in both fundamental studies of the interactions of bacteria with antimicrobial peptides, as well as the practical use of these devices as portable pathogen detectors.     

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.     

Automated parallel DNA sequencing on multiple channel microchips

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.