激光技术在聚合酶链式反应微流控芯片中的应用

评述了激光技术在聚合酶链式反应(polymerase chain reaction,PCR)微流控芯片领域中的应用进展,包括激光技术在PCR微流控芯片微加工、微流体物理参数测量、温度循环控制以及芯片上在线产物检测(包括荧光实时定量/终点和毛细管电泳检测)中的应用。最后,展望了激光技术在未来基于PCR的微全分析系统(micro-total analysis system,μTAS)中的新应用。     

液滴微流控系统在数字聚合酶链式反应中的应用研究进展

数字聚合酶链式反应( PCR)技术近年来发展迅速。与以实时荧光定量PCR为代表的传统PCR技术相比,数字PCR技术显著提高了定量分析的精确度和灵敏度。数字PCR的快速发展与近年来微流控技术在数字PCR技术中的广泛应用有着密切的联系。早期的研究和商业化产品使用的是大规模集成流路微流控芯片,加工过程复杂且价格高昂。近年来,液滴微流控芯片被应用到数字PCR技术中,它可以在短时间内产生102~107个微液滴,每一个微液滴都是最多只含有一个目的基因片段的PCR反应器。 PCR扩增后,通过对单个微液滴的观察计数,就可以获得绝对定量的分析数据。本文综述了不同种类的液滴微流控系统在数字PCR技术中的应用,以及液滴数字PCR微流控芯片在生物、医药、环境等领域的应用。     

复合式蝎形引物实时定量检测端粒酶延伸产物

针对端粒酶延伸产物中靶基因序列的特殊性,开发了一种可产生荧光的复合式蝎形引物,该引物的5'端带有可特异性检测靶基因的探针序列,PCR阻断剂将其与引物序列连接.当复合式蝎形引物延伸,探针序列与同一分子内的靶基因杂交,荧光信号产生.运用该技术,建立了定量检测端粒酶延伸产物的实时荧光PCR方法.该法可在快速PCR循环条件下,对0.15～1.50×10³ amol/μL范围内的样品进行定量检测,线性相关系数R²=0.9992.该法操作简便,无需PCR后额外的检测步骤.     

一种快速鉴别食用油的气相色谱指纹法

本发明公开了提供一种实时荧光定量PCR检测HC．MVgBn的分型方法,在PCR反应体系中加入荧光探针,利用荧光信号积累实时监测PCR进程,进行定量定性分析确定HCMVgBn型。本发明封闭反应,无需PCR后处理,避免污染;特异性强,灵敏度高采用对数期分析,摒弃终点数据,定量准确;实时荧光定量PCR技术既可对HCMVgB进行分型,又可对其进行定量;仪器在线式实时检测,结果直观,避免人为判断;可实现一管双检或多检;操作安全,缩短时间,     

基于微磁珠分离技术的适配体实时定量PCR检测方法

利用适配体的识别能力和可扩增性, 构建了基于微磁珠分离技术的适配体实时定量聚合酶链式反应(PCR)检测方法. 通过微磁珠偶联的互补链与适配体序列之间的碱基配对结合, 有效除去溶液中未与靶分子结合的适配体序列, 采用实时定量PCR技术测定上清液中结合态的适配体序列浓度, 从而间接实现对靶分子的定量检测. 分别选取代表生物大分子和有机小分子的凝血酶和ATP作为检测对象, 验证了该方法的普适性. 研究结果表明, 在获取特异性适配体序列后, 仅需简单优化其互补链序列, 即可对超低含量的凝血酶和ATP进行准确定量, 检出限分别为50 pmol/L和5 μmol/L. 该方法具有同时适用于高特异性和高灵敏度地检测生物大分子和有机小分子的优势.     

基于非接触电导检测的PCR液滴检测芯片的研究

数字PCR是继实时定量PCR之后新兴发展起来的一种绝对定量分析技术,广泛应用于疾病早期检测诊断和微生物检测分析等领域。然而,在数字PCR的微反应器检测环节,常规的光学检测方法需要复杂的光学设备,应用环境有限,成本高。因此研究了一种基于非接触电导检测的PCR液滴检测芯片,用电导检测取代传统的光学检测,有效降低了数字PCR结果读出系统的成本。通过优化电极结构与排布,采用三电极平行3D电极结构,减小了相对误差,提高了灵敏度,实现了无标记的PCR反应液滴阴性阳性比例检测。在DNA模板浓度为0.5×10~4～3×10~4 copies/μL内实现了良好的相关性(r~2=0.998),检测通量可达100个/秒。     

生物芯片进展*

生物芯片是近二十年来生物技术领域发展迅速和获得重大突破的高新技术,该综述介绍了经典的平铺生物芯片（DNA和蛋白质芯片）之后,重点介绍了近十年来发展的新的生物芯片的概念和内容：质谱为读出机制的表面增强激光解析离子化飞行时间质谱（SELDI）芯片、高通量DNA测序技术、基于胶体光子晶体的微球高通量生物检测技术、三维阵列生物芯片技术、和微流控等生物芯片技术。描述了各种生物芯片技术的优点、应用范围、和有待解决的问题,最后展望了生物芯片技术的未来。     

集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

基于功能纳米材料的液相生物芯片检测技术

近年来基于编码微球的液相生物芯片技术在对单一样本进行高通量和多指标检测中发挥巨大的作用。由于其快速的动力学结合速率、高通量、高灵敏和多元检测的优势,因此基于编码微球的液相芯片对基因分析、蛋白表达、疾病的早期诊断、预后等也是一种强有力的检测工具。受益于纳米技术和纳米材料的快速发展,特别是功能纳米颗粒/聚合物复合微球的应用,液相生物芯片在提高其多元分析能力、分析灵敏度和自动化检测等多方面取得了巨大进展。本文将分别从液相生物芯片概述、功能纳米颗粒编码微球、功能纳米颗粒编码微球的制备、基于编码微球的液相生物芯片的设计及性能调控等方面来介绍近年来基于功能纳米材料的液相生物芯片技术的研究进展。最后,我们对液相生物芯片技术存在的挑战和可能的解决方案进行总结,并对其技术发展方向及应用前景进行展望。希望通过本文的系统介绍可助力液相生物芯片检测技术及其相关研究领域的发展。     

连续流动式PCR芯片相关技术研究进展

微电子机械系统(microeletromechanical system,MEMS)技术的兴起及其在生物化学领域的广泛应用,推动了聚合酶链式反应(polymerase chain reaction,PCR)热循环装置越来越微型化,各种PCR微芯片／装置被开发。本文主要介绍了基于MEMS技术的连续流动式PCR(continuous—flow PCR)芯片／微装置的相关技术,包括基底材料的选择、通道表面钝化技术、微细加工技术、封接技术以及系统检测技术等,最后简单介绍目前实验室的研究工作。     

Miniature Continuous-Flow Polymerase Chain Reaction: A Breakthrough?

A time–space conversion enables the polymerase chain reaction (PCR) to be carried out in a continuous-flow process: the mobile reaction mixture is pumped continuously through a glass microchip and passes many times through three constant temperature zones (see picture). The flow rate can be varied to obtain an amplification time of only 90 s. When combined with other continuous-flow microdevices this micromachine may prove useful for routine medical applications and biochemical research.     

Methods for detection of GMOs in food and feed

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.     

Polymeric microfluidic system for DNA analysis

The application of micro total analysis system (μTAS) has grown exponentially in the past decade. DNA analysis is one of the primary applications of μTAS technology. This review mainly focuses on the recent development of the polymeric microfluidic devices for DNA analysis. After a brief introduction of material characteristics of polymers, the various microfabrication methods are presented. The most recent developments and trends in the area of DNA analysis are then explored. We focus on the rapidly developing fields of cell sorting, cell lysis, DNA extraction and purification, polymerase chain reaction (PCR), DNA separation and detection. Lastly, commercially available polymer-based microdevices are included.     

Advanced Immobilization and Amplification for High Performance Protein Chips

With the success of high-throughput DNA microarrays, protein biochips have been intensively investigated and broadly used in bioscience research, clinic diagnosis, drug discovery, and other applications. However, there is great need to significantly improve the sensitivity of protein chips, especially in early diagnosis. A major challenge of improving sensitivity is that protein detection does not have an effective amplification method, such as PCR for DNA microarrays. Construction of unique biofilms for efficient immobilization of protein probes and innovation of new amplification schemes could play a critical role in performance improvement of protein biochips. With dramatic developments in microfabrication, nanotechnologies, and biotechnologies, enormous progress has been made, particularly in improving biosensing sensitivity. This article reviews new advances in protein biochip technologies with emphasis on novel approaches for efficient probe immobilization and nanomaterials-assisted signal amplification for high performance protein chips. Prominent progress in integration of protein microarrays with microfluidic platforms is briefly discussed. The major challenges and perspectives on the future of protein biochips are also addressed.     

A Review of Multifunctions of Dielectrophoresis in Biosensors and Biochips for Bacteria Detection

This paper reviews the functions of dielectrophoresis (DEP) that have been applied to biosensor and biochip platforms for bacteria detection, including concentration of bacterial cells from continuous flows, separation of target bacterial cells from non-target cells, as well as the enhancement of antibody capture efficiency on biosensor and biochip surfaces. DEP could provide effective concentration and separation simultaneously in well-designed microfluidic biosensor and biochip systems. The integration of DEP with a detection system allows the integration of sample preparation and enrichment steps with detection, which has the potential to eliminate the traditionally used time-consuming culture-based enrichment steps and other multiple off-chip sample preparation steps. DEP is also useful in biosensor and biochips platforms for enhancing antibody capture efficiency in both flow-through and non-flow-through microdevices. The enhanced antibody capture efficiency could allow the sensor capture more cells and to be detected by the sensor, particularly in dealing with low number of cells. The integration of multifunctions of DEP into biosensor and biochip platform has the potential to improve the detection of bacterial cells.     

Siloxane photopolymer to replace polydimethylsiloxane in microfluidic devices for polymerase chain reaction

Herein, we describe the preparation and characterization of a material suitable for the fabrication of microfluidic devices. The material is a silicone acrylic polymer, obtained by photopolymerization. It is polymerase chain reaction (PCR) compatible, resistant to temperature, optically transparent, and dimensionally stable; it has a better water and solvent resistance if compared with polydimethylsiloxane. Production of microfluidic layouts is successfully tested: a simple photolithographic approach allows to accurately control the pattern transfer and to produce PCR compatible microfluidic devices. The polymer characterization suggests that the proposed material satisfies all the characteristics required for an ideal PCR chip, without further treatment. Moreover, the possibility for fast, accurate, and cheap reproducibility of microdevices by liquid phase photopolymerization increases the polymer attractiveness. The material is a good alternative with respect to polydimethylsiloxane for the fabrication of microfluidic chips for biological analysis purposes. Copyright © 2013 John Wiley & Sons, Ltd.     

Laser processing for bio-microfluidics applications (part II)

This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips, in particular for applications in which silicon is not the preferred material base. This review addresses applications and devices based on ablation using femtosecond lasers, infrared lasers as well as laser-induced micro-joining, and the laser-assisted generation of micro-replication tools, for subsequent replication of polymeric chips with a technique like laser LIGA.     

"Smart" biopolymer for a reversible stimuli-responsive platform in cell-based biochips

The rapid response of a smart material surface to external stimuli is critical for application to cell-based biochips. The sharp and controllable phase transition of elastin-like polypeptide (ELP) enabled reversible cell adhesion on the surface by changing the temperature or salt concentration in the system. First, ELP micropatterns were prepared on a glass surface modified into aldehyde. The lysine-containing ELP (ELP-K) was genetically synthesized from E. coli for conjugation with the aldehyde on the glass surface. The phase transition of ELP was monitored in PBS and cell culture media using UV-visible spectroscopy, and a significant difference in transition temperature (Tt) was observed between the two solution systems. The micropatterning of ELP on the glass surface was performed by microcontact printing a removable polymeric template on the aldehyde-glass followed by incubation in ELP-K aqueous solution. The ELP micropatterns were imaged with atomic force microscopy and showed a monolayer thickness of approximately 4 nm. Imaging from time-of-flight secondary ion mass spectroscopy confirmed that the ELP molecules were successfully immobilized on the highly resolved micropatterns. Cell attachment and detachment could be reversibly controlled on the ELP surfaces by external stimuli. The hydrophobic phase above Tt resulted in the adhesion of fibroblasts, while the detachment of cells was induced by lowering the incubation temperature below Tt. The smart properties of ELP were reliable and reproducible, demonstrating potential applications in cell-based microdevices.     

Integration of sample pretreatment, μPCR,and detection for a total genetic analysis microsystem

Since the emergence of lab-on-a-chip technology, a variety of chemical and biochemical assays were successfully implemented on microdevice platforms. Among the chip-based applications, genetic analysis based on the polymerase chain reaction (PCR) has been extensively developed in order to accomplish the goal of cheap, rapid, high-throughput, and point-of-care DNA testing. We are summarizing here several formats of the miniaturized PCR systems including the integration of units for sample pretreatment and downstream analytical detection. The various sections cover (a) miniaturized PCR systems, (b) integrated sample pretreatment-PCR microsystems, (c) integrated PCR-detection microsystems, and (d) integrated sample pretreatment-PCR-detection microsystems. Respective microdevices were successfully introduced recently in the form of a fully integrated microsystem for genetic analysis with sample-in-answer-out capability. Contains 120 references. Figure

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Microfluidic channel fabrication in dry film resist for production and prototyping of hybrid chips

Microfluidic networks are patterned in a dry film resist (Ordyl SY300/550) that is sandwiched in between two substrates. The technique enables fabrication of complex biochips with active elements both in the bottom and the top substrate (hybrid chips). The resist can be double bonded at relatively low temperatures without the use of extra adhesives. A postbake transfers the resist into a rigid structure. The resist is qualified in terms of resolution, biocompatibility and fluidic sealing. Fabrication in both a fully equipped cleanroom setting as well as a minimally equipped laboratory is described. The technique is applied for dielectrophoresis-based cell separation systems and a fuel cell reaction chamber with micropillars. The dry film resist can be considered a cheap and fast alternative to SU-8.