微流控芯片技术及其在天然产物活性成分筛选中的应用

微流控芯片技术在药物研究尤其是天然产物活性成分高通量筛选方面得到越来越广泛的应用,本文主要讨论了微流控芯片技术的发展以及在天然产物研究与开发方面的应用,并对其应用现状进行了分析。     

条形码微流控芯片在分泌蛋白检测方面的应用

微流控芯片具有液体流动可控、消耗试样少、分析速度快等特点,它可以在几分钟甚至更短的时间内进行上百个样品的同时分析,并且可以实现在线样品的预处理及分析全过程。一种条形码微流控芯片能够以高密度的单链DNA为模板,从而克服了传统蛋白质微流控芯片固定在固体表面容易变性的缺点,既解决了稳定性的要求,又满足芯片平行处理大量数据的要求,可以用来大量的、快速的定量检测细胞的分泌蛋白。条形码微流控芯片因其对样品要求简单、低耗高效、高通量等特点正在成为分泌蛋白检测的最具吸引力的分析工具,在样品分析与检测以及临床检测研究等领域得到了广泛的应用。     

基于微流控芯片的秀丽隐杆线虫的研究进展

微流控芯片技术作为近年来最前沿的分析技术之一,已经在化学、生物学、医药学等研究领域取得了突破性的进展.微流控芯片具有高通量、微型化和多功能集成化等独特优势,已经成为生物医学研究的新平台之一,被越来越多地应用于秀丽隐杆线虫的研究.综述了基于微流控芯片上的秀丽隐杆线虫在生物医学领域中的研究进展,侧重介绍了微流控芯片在线虫的自动化固定、行为学、衰老与发育学、神经学、药物筛选及基因筛选等六大方面所取得的最新进展,并展望了微流控芯片的应用前景.     

基于微流控芯片的细胞-细菌相互作用光电检测技术

细胞/细菌及其相互作用研究对于生命科学、药物研发、医学诊疗等领域的研究具有重要意义。微流控芯片分析技术因微环境可控、生物相容性好、检测并行性、微型化等特性，正发展成为细胞/细菌及其相互作用研究的高效手段。本文在简要介绍基于微流控芯片分析技术的细胞-细菌分析方法和技术基础之上，对微流控芯片上细胞-细菌相互作用模型的建立进行了讨论，重点针对细胞-细菌及其相互作用过程的芯片检测进行了综述，尤其对芯片集成的光电检测技术及其测试效果进行总结和比较。通过芯片集成微流体控制、多种光电传感监测模块，使微流控芯片分析技术成为细胞/细菌及其相互作用过程分析和检测的支撑平台和优势手段。最后，对微流控光电检测技术在细胞-细菌相互作用检测中面临的挑战及发展趋势进行了讨论和展望。     

基于微流控芯片的核酸等温扩增技术研究进展

核酸等温扩增技术是一种在恒温体系内对核酸进行高效扩增的分子扩增技术,它能够在短时间内实现目的基因的指数增长。微流控芯片(microfluidic chip)技术是把研究样品制备、核酸富集、纯化和检测等多个操作步骤集成到一块"微型化"的芯片上,经自动化处理,得出实验结果,即"样品进,结果出"。将核酸等温扩增技术与微流控芯片相结合,不仅可以实现核酸快速扩增,还可以降低对实验器材的依赖。在床边即时诊断、病原体快速筛查中具有广阔的应用前景。综合国内外相关研究报道,综述了各种等温扩增技术原理,以及基于微流控芯片的核酸等温扩增技术应用,展望了集成化微流控芯片的发展趋势和应用前景。     

微流控芯片在水环境污染分析中的应用

近年来,一种新型技术——微流控芯片技术因其分析速度快、消耗低、体积小、操作简单等特点而备受世界各国的广泛重视.该技术以微通道网络为基本特征,以微机电系统(MEMS)工艺为技术依托,将整个实验室的功能集成在微小芯片上,即构成所谓“芯片实验室”.本文从该技术的基本情况出发,介绍了微流控芯片的发展,并从仪器小型化、系统集成化、不同的芯片材料以及多种检测技术等方面,着重讨论了其在水环境污染分析方面的实际应用和发展前景,指出了它当前所面临的一些问题.随着微流控芯片的不断发展,高速多通道检测装置、低成本设备以及集成了多种方法的高通用性微流控检测芯片,都将成为未来研究的热点.     

用于药物筛选的微流控细胞阵列芯片

细胞区域分布培养以及如何有效地对微流体进行操控是微流控阵列芯片在细胞药物研究中的关键技术。本研究介绍了一种利用SU-8负性光刻胶模具和PDMS制作双层结构的微流控细胞阵列芯片的方法,该芯片通过C型的坝结构将进样细胞拦截在芯片的细胞培养的固定区域,键合双层PDMS构成阀控制层,阀网络的开关作用成功实现了芯片通道内微流体的操控,同时芯片设计了药物浓度梯度网络,产生6个不同浓度的药物刺激细胞。通过对芯片3种共培养细胞活性的检测和药物伊立替康(CTP-11)对肝癌细胞的浓度梯度刺激等实验结果验证该芯片在细胞研究和药物筛选等方面的可行性。     

单细胞机械性能检测方法与应用研究进展

细胞机械性能与细胞的生理状态与功能存在密切联系。早期对于细胞机械性能的研究受制于技术条件,只能获得细胞群的弹性或剪切模量,使得少量异质细胞的机械表型被淹没。近年来,单细胞机械性能检测技术得到了蓬勃发展。原子力显微镜、微吸管技术、光镊与光学拉伸、磁扭转流变仪与磁镊等单细胞机械性能检测技术展现出非常高的检测精度,但检测通量相对较低。新型微流控高通量检测方法的出现使检测通量呈几何式增长,有望解决大样本快速检测的需求。本文首先综述原子力显微镜、微吸管、光镊与光学拉伸和磁扭转流变仪与磁镊等单细胞机械性能检测技术。在此基础上,重点介绍细胞过孔、剪切诱导细胞变形和拉伸诱导细胞变形3种新兴微流控高通量检测技术的工作原理及最新研究进展,探讨各类方法的优缺点。最后,本文展望单细胞机械性能检测技术的未来发展方向。     

微流控器件-质谱联用接口技术研究进展与应用

生物分析是生命科学研究中的重要环节,分析仪器的小型化是提高生物分析灵敏度、速度、通量和降低成本的有效途径之一.微流控技术能够方便地操纵微量样品,具有集成度高、样品耗量小、污染少等诸多其他常量流控技术难以具备的优点,适用于进行多通道样品处理和高通量分析.除广泛采用的光学和电化学检测手段外,质谱也被用作这些微流控器件的检测器,并逐渐形成了微流控器件-质谱联用技术专门研究领域,进一步促进了自动化程度好、灵敏度高、特异性强的高通量生物分析方法的迅速发展.在大量调研国内外文献的基础上,对微流控器件-质谱联用领域的研究背景和现状进行了综述,不但介绍了微流控器件的制造技术还着重介绍了微流控器件-质谱联用技术在蛋白质组学等生物质谱分析方面的应用和新近进展,评述了可能的发展趋势.     

微流控芯片分析技术在生化研究中的应用进展

综述了微流控芯片分析技术在生物和化学领域中进展,主要从药物筛选、PCR、细胞研究和微流控芯片电泳4个方面总结目前的进展。     

Microfluidic bio-particle manipulation for biotechnology

Microfluidics and lab-on-a-chip technology offers unique advantages for the next generation devices for diagnostic therapeutic applications. For chemical, biological and biomedical analysis in microfluidic systems, there are some fundamental operations such as separation, focusing, filtering, concentration, trapping, detection, sorting, counting, washing, lysis of bio-particles, and PCR-like reactions. The combination of these operations led to the complete analysis systems for specific applications. Manipulation of the bio-particles is the key ingredient for these applications. Therefore, microfluidic bio-particle manipulation has attracted a significant attention from the academic community. Considering the size of the bio-particles and the throughput of the practical applications, manipulation of the bio-particles is a challenging problem. Different techniques are available for the manipulation of bio-particles in microfluidic systems. In this review, some of the techniques for the manipulation of bio-particles; namely hydrodynamic based, electrokinetic-based, acoustic-based, magnetic-based and optical-based methods have been discussed. The comparison of different techniques and the recent applications regarding the microfluidic bio-particle manipulation for different biotechnology applications are presented. Finally, challenges and the future research directions for microfluidic bio-particle manipulation are addressed.     

Structure and dynamics of single DNA molecules manipulated by magnetic tweezers and or flow

Here we describe the use of magnetic tweezers and or microfluidics to manipulate single DNA molecules. We describe experiment which employ magnetic tweezers coupled to an inverted microscope as well as the use of a magnetic tweezers setup with an upright microscope. Using a chamber prepared via soft lithography, we also describe a microfluidic device for the manipulation of individual DNA molecules. Finally, we present some past successful examples of using these approaches to elucidate unique information about protein–nucleic acid interactions.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

Single-molecule manipulation of macromolecules on GUV or SUV membranes using optical tweezers

Despite their wide applications in soluble macromolecules, optical tweezers have rarely been used to characterize the dynamics of membrane proteins, mainly due to the lack of model membranes compatible with optical trapping. Here, we examined optical trapping and mechanical properties of two potential model membranes, giant and small unilamellar vesicles (GUVs and SUVs, respectively) for studies of membrane protein dynamics. We found that optical tweezers can stably trap GUVs containing iodixanol with controlled membrane tension. The trapped GUVs with high membrane tension can serve as a force sensor to accurately detect reversible folding of a DNA hairpin or membrane binding of synaptotagmin-1 C2AB domain attached to the GUV. We also observed that SUVs are rigid enough to resist large pulling forces and are suitable for detecting protein conformational changes induced by force. Our methodologies may facilitate single-molecule manipulation studies of membrane proteins using optical tweezers.     

Microfluidic-integrated biosensors: Prospects for point-of-care diagnostics

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics-integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro- to nano-liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point-of-care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic-integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.     

A New Tool for Routine Testing of Cellular Protein Expression: Integration of Cell Staining and Analysis of Protein Expression on a Microfluidic Chip-Based System

The key benefits of Lab-on-a-Chip technology are substantial time savings via an automation of lab processes, and a reduction in sample and reagent volumes required to perform analysis. In this article we present a new implementation of cell assays on disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow in microfluidic channels and two-color fluorescence detection of single cells. This new technology allows for simple flow cytometric studies of cells in a microfluidic chip-based system. In addition, we developed staining procedures that work “on-chip,” thus eliminating time-consuming washing steps. Cells and staining-reagents are loaded directly onto the microfluidic chip and analysis can start after a short incubation time. These procedures require only a fraction of the staining reagents generally needed for flow cytometry and only 30,000 cells per sample, demonstrating the advantages of microfluidic technology. The specific advantage of an on-chip staining reaction is the amount of time, cells, and reagents saved, which is of great importance when working with limited numbers of cells, e.g., primary cells or when needing to perform routine tests of cell cultures as a quality control step. Applications of this technology are antibody staining of proteins and determination of cell transfection efficiency by GFP expression. Results obtained with microfluidic chips, using standard cell lines and primary cells, show good correlation with data obtained using a conventional flow cytometer.     

A nanoliter-scale nucleic acid processor with parallel architecture

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.     

Using Optics to Measure Biological Forces and Mechanics

Spanning all size levels, regulating biological forces and transport are fundamental life processes. Used by various investigators over the last dozen years, optical techniques offer unique advantages for studying biological forces. The most mature of these techniques, optical tweezers, or the single-beam optical trap, is commercially available and is used by numerous investigators. Although technical innovations have improved the versatility of optical tweezers, simple optical tweezers continue to provide insights into cell biology. Two new, promising optical technologies, laser-tracking microrheology and the optical stretcher, allow mechanical measurements that are not possible with optical tweezers. Here, I review these various optical technologies and their roles in understanding mechanical forces in cell biology.     

Protein micro- and macroarrays: digitizing the proteome

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.