基于微流控技术的蛋白质结晶及其筛选方法的研究进展

微流控技术以其高通量、 低消耗和集成化等优点成为蛋白质结晶微型化研究的重要手段. 本文综述了基于微流控技术的蛋白质结晶技术和方法, 主要包括微泵微阀、 液滴(Droplet)、 滑动芯片(SlipChip)以及液滴实验室(DropLab)等技术. 此外, 还针对当前膜蛋白在结构生物学研究中的重要地位, 综述了应用于膜蛋白结晶的微流控技术的研究进展.     

微液滴微流控芯片:微液滴的形成、操纵和应用

微流控芯片中形成的微液滴粒径均一、可控,与传统的连续流体系相比,具有能实现试剂的快速混合、通量更高等优点.本文介绍了微流控芯片中由微通道控制的微液滴的形成、分裂、合并、混合、分选和捕获等微液滴操纵技术,以及微液滴技术在纳米粒子、聚合物微粒的合成、纳米粒子自组装、蛋白质结晶研究和DNA、细胞分析等领域的研究进展.     

微流控液滴技术及其应用的研究进展

微液滴具有体积小、比表面积大,速度快、通量高,大小均匀、体系封闭,内部稳定等特性,在药物控释、病毒检测、颗粒材料合成、催化剂等领域中均有重要应用.微流控技术的发展为微液滴生成中实现尺寸规格、结构形貌和功能特性等的可控设计和精确操控提供了全新平台.本文概述了微流控液滴技术的基本原理、液滴生成方式及其基本操控,比较分析了微液滴的传统制备法与微流控合成法的异同,介绍了近年来微流控液滴技术在功能材料合成、生物医学和食品加工等领域中的研究新进展,探讨并展望了微流控液滴技术的潜在价值和未来发展方向.     

微流控技术应用于蛋白质结晶的研究*

随着微电子微机械等技术的不断进步,微流控(microfluidics)技术成为目前迅速发展的前沿领域之一,是化学科学和生命科学分析研究的重要技术平台。微流控技术高通量、低消耗和低成本的特点使其在蛋白质结晶条件筛选和优化方面展示了良好的应用前景。本文对应用于蛋白质结晶的各种微流控芯片技术的原理和方法进行了综述,并对目前几种商业化和文献报道的典型蛋白质结晶微流控系统进行了介绍和比较。     

基于液滴技术的微流控芯片实验室

液滴微流控系统是微流控芯片领域的一个新的分支,由于其诸多独特的优势而得到了广泛的研究和报道。本文对液滴的制备和相关的操控技术,包括液滴的分裂、融合、混合、分选、存储和编码等进行了介绍,对液滴技术近年来在化学与生物化学分析等领域中的应用进行了综述,并展望了液滴微流控技术的发展前景。     

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。     

液滴微流控技术在生物医学中的应用进展

液滴微流控是微流控芯片领域的一个重要分支,由于其诸多独特优势而得到了广泛的应用与研究.本文将概述液滴微流控的特点和基本原理,同时对近年来其在生物医学中的应用进行了简要综述,并展望了液滴微流控技术的发展前景.     

基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法

基于微流体脉冲驱动控制技术搭建了电化学微流控芯片的制备系统.首先将纳米银墨水和甘油溶液分别微喷射到玻璃基底表面形成微电极图形和微流道液体阳模图形;然后分别进行烧结和聚二甲基硅氧烷(PDMS)模塑工艺制得微电极和微流道;最后将微电极和微流道键合形成电化学微流控芯片.研究了系统参量对液滴产生的影响以及液滴直径和重叠率对液滴成线的影响,制得的微电极最小线宽为45 μm、厚度为2.2 μm、电阻率为5.2 μΩ·cm,制得的微流道最小线宽为35 μm,流道表面光滑.采用制得的电化学微流控芯片进行了葡萄糖浓度的电化学流动检测.结果表明,葡萄糖溶液的浓度与响应电流具有较高的线性关系,可对一定浓度范围内的葡萄糖溶液进行定量检测.基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法具有微喷射精度高、重复性好,制备系统结构简单、成本低廉等优点,可用于生化分析、生物传感器等领域的芯片制备.     

基于渗透脱水的自动化蛋白质结晶高通量筛选芯片

构建了一种基于渗透脱水模式的自动进样微流控结晶芯片. 该芯片通过真空预脱气将包含蛋白质和结晶剂的液滴自动分配至结晶微腔阵列中, 然后利用集成的一排包含不同浓度盐溶液的透析管道, 通过渗透脱水方式经一层聚二甲基硅氧烷(PDMS)膜实现液滴的逐渐浓缩, 使之趋于过饱和状态, 进而形成结晶. 此芯片可一次筛选较宽范围的过饱和状态, 实现蛋白质结晶的快速优化. 利用模式蛋白溶菌酶的结晶实验验证了该芯片的性能.     

微流控液滴软模板制备二氧化钛中空微球

应用微流控液滴技术合成功能材料已发展成为一个新兴领域。本文以夹流结构微流控芯片产生的微液滴作为软模板,以液滴模板界面处发生的水解反应生成二氧化钛球壳,并经后续脱核处理,制备二氧化钛中空微球。采用激光诱导荧光成像、扫描电镜等手段对微球形貌结构进行了分析表征。结果表明,通过控制微流控芯片液滴合成条件,可以得到壁厚约2 μm的二氧化钛中空微球。这种以微流控液滴为模板的合成方法简单灵活,若与其他材料改性方法相结合,有望实现对更多元、更复杂功能微球材料的制备,并进一步拓宽其在光电和催化剂材料领域的应用。     

Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

Detergent-associated solution conformations of helical and beta-barrel membrane proteins

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.     

高通量、自动化蛋白质结晶筛选技术以及相关仪器的研究进展

基于X射线晶体学的蛋白质结构解析主要依赖于大规模结晶条件筛选获得的高衍射分辨率的蛋白质晶体。近年来,自动化、高通量的液体操控技术和相关仪器的快速发展为蛋白质结晶筛选提供了高效、可靠的研究手段,显著推动了蛋白质结构生物学的研究。文章综述了蛋白质结晶筛选的自动化液体处理技术的发展,包括移液器、注射泵、同步纳升定量吸取注射、喷墨打印、超声喷射以及微流控等技术。文章详细介绍了各技术所对应的典型商品化仪器及其在蛋白质结晶筛选中的应用。此外,文章还介绍了集成多孔板的储存和操控、编码扫描、环境控制和软件管理等诸多功能的一体化液体操纵平台。     

Colloid-chemical aspects of protein crystallization

Problems and prospects of the crystallization of water-soluble and membrane proteins are discussed. The main focus is on colloid-chemical aspects of crystallization processes and the use of surfactants and various structured dispersions (surfactant-based gels and lyotropic phases). Advantages of the protein crystallization on solid supports of different nature, including biopolymer supports, are considered. Apparently, we are witnesses to the rise of a new field, nanocrystallography, dealing with the determination of protein structures from the investigation of nanocrystals.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Phase Behavior of a Designed Cyclopropyl Analogue of Monoolein: Implications for Low‐Temperature Membrane Protein Crystallization

Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low‐temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small‐angle X‐ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high‐temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP‐based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 °C as well as 4 °C.