微流控芯片分选临床样品中循环肿瘤细胞的研究进展

癌症作为常见病正严重威胁着我国乃至全球居民的健康。循环肿瘤细胞(CTCs)是一类由癌变部位释放并进入血液中的癌细胞,其在癌症的早期诊断、个体化及肿瘤转移机制研究等方面的作用正逐渐被发现和认可,但由于血液中的CTCs含量极少,对其分选极具挑战。微流控芯片作为一种微型化、高通量、集成化平台,在CTCs研究中彰显了独特的优势,相关报道也越来越多。随着研究的深入,微流控芯片技术不再局限于基于模型样品的方法学开发,而是更注重于能否用于临床实际样品中CTCs的检测,但目前未见该角度的综述报道。为此,文章综述了近年来用于临床实际样品CTCs分析的微流控芯片分选技术,并探讨了微流控芯片用于CTCs分选的发展趋势。     

微流控可控分步组装制备透明质酸/聚乙烯亚胺/DNA纳米复合物

阳离子聚合物/DNA形成的复合物纳米颗粒呈正电性,因此表面必须遮盖一层电中性或负电性的聚合物才能在体内应用,但如何控制遮蔽层在复合物纳米颗粒上组装,形成结构可控、尺寸均一的基因输送系统是关键.本文以基因输送系统中常见的透明质酸(HA)/聚乙烯亚胺(PEI)/DNA系统为例,探索了利用微流控芯片进行可控分步的层层自组装,制备尺寸大小均一、表面电势为负的HA/PEI/DNA纳米复合物的方法.将PEI与DNA通过第一个微流控芯片自组装得到PEI/DNA纳米复合物,该复合物颗粒在第二个微流控芯片内与HA再次组装得到HA/PEI/DNA纳米复合物.考察了微流控芯片管道的尺寸、溶液流速及流速比R、PEI与DNA氮磷比(N:P)、HA与DNA质量比(HA:DNA)等参数与所形成的纳米复合物的尺寸、均一性及表面电势的关系,并与涡旋振荡法制备的复合物进行比较.结果表明,传统的涡旋振荡法制备的HA/PEI/DNA纳米复合物尺寸偏大(340～490 nm)、均一度低(PDI,0.506～0.863);而用微流控法制备的复合物尺寸较小(190 nm)、分布更为均一(PDI=0.316).     

磁场控制技术在微流控芯片中的应用*

磁场作为除了电场和力场之外的另一个有力的驱动和控制手段,由于不需与溶液接触即可实现对被分析物的操纵,极大降低污染的可能,近年来被越来越广泛地用于微流控芯片系统,尤其在细胞、病毒甚至单分子的捕获、分选以及操纵等方面显示出较大优势。本文综述了微流控芯片系统中磁场控制技术的最新进展,分别从理论分析,磁场加工技术、泵阀的实现,微流体控制和磁分离等方面介绍了该领域近几年的发展状况,并重点分析了微流控芯片磁场操控技术在临床分析和现场检测方面的应用,及其未来发展趋势和需解决的主要问题。     

顺序注射-液芯波导H-通道芯片联用系统DNA片段的毛细管电泳分离激光诱导荧光检测

毛细管电泳微流控芯片分离-激光诱导荧光(LIF)检测DNA片段是近年来微流控分析系统中研究得较为成功的领域,该方向的研究成果极大地促进了微流控分析系统的发展.在相关的报道中,待分析样品和系统运行溶液仍然主要使用手工操作.     

纳米粒子毛细管电泳/微流控芯片新技术及其在手性分离中的应用

纳米粒子因其具有较大的比表面积和良好的生物相容性等特点,已广泛应用于分离科学领域。纳米粒子毛细管电泳/微流控芯片技术是纳米材料技术与毛细管电泳/微流控芯片技术相结合的产物。纳米粒子可以被吸附或键合到毛细管壁作为固定相与被分析物发生相互作用;也可以作为假固定相参与样品在柱内的分配和保留,从而提高柱效,改善分离。手性是自然界的本质属性之一,开发新的快速、高效、灵敏的手性分离分析方法对于对映体的立体选择性合成、药理研究、手性纯度检测和环境检测都具有重要的意义。本文主要综述了近些年来几种不同类型纳米粒子(聚合物纳米粒子、磁性纳米粒子、金纳米粒子、碳纳米管和其他类型纳米粒子)用于毛细管电泳/微流控芯片进行手性分离的现状,并对该领域今后的发展进行了展望。     

聚甲基丙烯酸甲酯微流控芯片分离DNA片段的初步研究

自从1995年Mathies^[1]首次将微流控芯片毛细管凝胶电泳用于基因测序研究以来,DNA片段的分离已成为微流控芯片应用的重要领域之一.最早应用于DNA分析的微流控芯片是玻璃芯片,聚合物微流控芯片以其品种多、成本低、易于加工,与玻璃芯片相比具有封接温度大大降低,微通道内电渗流显著减小等特点,已被成功应用于DNA片段的分离^[2,3].     

微流控芯片中颗粒/细胞磁操控的研究进展

微流控芯片中颗粒/细胞的磁操控是当前的热点研究领域.本文详细介绍了微流控芯片中颗粒/细胞磁操控原理及几种主要操控方式,包括分离、集中、捕获与排列组装.其中,基于颗粒/细胞大小、形状以及有无磁性对分离方法展开详述.此外,本文还比较了通道几何结构、磁场强度及分布、磁性液体种类(顺磁盐溶液和磁流体)对操控性能的影响.最后,针对微流控芯片中颗粒/细胞磁操控的前景进行了展望.     

微流控芯片电泳的研究进展

该文综述了微流控芯片电泳的制备、结构和应用,比较了不同材料微流控芯片电泳的制备机理、表面改性和性能特点,归纳和总结了不同结构微流控芯片电泳的进样、分离和检测系统以及不同类型微流控芯片电泳在荧光物质、金属离子、糖、药物、核酸、DNA、氨基酸、多肽和蛋白质分析中的应用,并对微流控芯片电泳的未来发展方向做了展望.     

微流控芯片技术在精细胞研究中的应用

具有多维网络微通道结构的微流控芯片可在微纳尺度上集成细胞进样、培养、分选、裂解和分离检测等多种功能单元,不仅在尺寸上与精细胞匹配,还可为精细胞提供相对封闭的接近生理状态的生长微环境。研究者已利用此系统的层流、微通道特殊几何结构等特点对精子进行了多方面研究。该文对微流控芯片技术在精细胞的培养、分选、胞内成分分析和人工授精中的应用进行了综述,介绍了用于精细胞研究的多种微流控芯片系统,并讨论了精细胞分选的各种方法。     

微流控芯片上同工酶的孵育及活性检测

建立了一种在微流控芯片上进行同工酶孵育及活性检测的方法. 该方法在集成温控装置的微流控芯片上实现对同工酶与辅酶反应进程的控制, 完成同工酶的进样、孵育反应、电泳分离和活性检测的实验步骤. 建立了基于微流控芯片的同工酶荧光检测系统, 使用360 nm光源激发辅酶产生荧光, 在460 nm处选择性采集荧光信号. 在微流控芯片上实现了同工酶样品的快速活性检测, 酶活性检测限达到0.5 U/L.     

Enhancing DNA Detection Sensitivity Through a Two-Step Enrichment Method with Magnetic Beads and Droplet Evaporation

A simple and sensitive DNA detection method has been developed through a two-step enrichment process. Trace amount of target DNA in a large volume (1 mL) is selectively separated and condensed with DNA-modified magnetic beads into a small volume (5 μL) by denaturalization. Then, the pre-enriched target DNA solution (1 μL) is transferred onto a smooth hydrophobic surface, where the target DNA is further-enriched by natural sessile droplet evaporation. Using this method, the fluorescence detection sensitivity of the target DNA can be enhanced by 3 orders of magnitude and as low as 3.91 pM of the target DNA can be detected within 2 hours.     

ssDNA-QDs/GO multicolor fluorescence system for synchronous screening of hepatitis virus DNA

Viral hepatitis is a common infectious disease caused by five viruses (hepatitis virus A, B, C, D, and E). Given the diversity of hepatitis virus, rapid screening and accurate typing of viral hepatitis are the prerequisites for hepatitis therapy. Here, a multicolor fluorescence system was constructed by combining with the multi-color fluorescence properties of CdSe/ZnS quantum dots (QDs, emission wavelengths: 525 nm, 585 nm and 632 nm) and the broad-spectrum fluorescence quenching performance of GO. Taking advantage of the specific recognition of ssDNA modified CdSe/ZnS QDs to target hepatitis virus DNA, the constructed system could effectively distinguish hepatitis A virus DNA (HAV-DNA), hepatitis B virus DNA (HBV-DNA), and hepatitis C virus DNA (HCV-DNA) in a homogeneous solution. Based on the different adsorption property of GO for ssDNA and dsDNA, the fluorescence Forster resonance energy transfer (FRET) process between ssDNA modified QDs and GO could be regulated. The fluorescence signal of the constructed system presented a sensitive response to HAV-DNA, HBV-DNA, and HCV-DNA content in the range of 1.0–192 nM, 8.0–192 nM, and 1.0–128 nM, respectively. The limit of detection for HAV-DNA, HBV-DNA, and HCV-DNA is 0.46 nM, 1.53 nM, and 0.58 nM. The constructed system can be used to screen hepatitis virus DNA in real samples, which provides an alternative strategy for rapid screening and diagnosis of viral hepatitis.     

FluMag-SELEX as an advantageous method for DNA aptamer selection

Aptamers are ssDNA or RNA oligonucleotides with very high affinity for their target. They bind to the target with high selectivity and specificity because of their specific three-dimensional shape. They are developed by the so-called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. We have modified this method in two steps—use of fluorescent labels for DNA quantification and use of magnetic beads for target immobilization. Thus, radioactive labelling is avoided. Immobilization on magnetic beads enables easy handling, use of very small amounts of target for the aptamer selection, rapid and efficient separation of bound and unbound molecules, and stringent washing steps. We have called this modified SELEX technology FluMag-SELEX. With FluMag-SELEX we have provided a methodological background for our objective of being able to select DNA aptamers for targets with very different properties and size. These aptamers will be applied as new biosensor receptors. In this work selection of streptavidin-specific aptamers by FluMag-SELEX is described. The streptavidin-specific aptamers will be used to check the surface occupancy of streptavidin-coated magnetic beads with biotinylated molecules after immobilization procedures.     

基于Fe3O4@SiO2/Ni-NTA磁性微球的His-tag融合蛋白纯化体系的建立

合成了表面共价结合Ni-氨基三乙酸(Ni-NTA)基团的Fe3O4@ SiO2微球,这种磁性微球可用于分离含有His-tag标签的融合蛋白.微球中心由尺寸约402 nm的Fe3O4微粒组成,赋予了微球极好的磁性分离和离心分离的特性.应用Fe3O4@ SiO2/Ni-NTA磁性微球对含有6×His-tag(6聚组氨酸)标签的蛋白进行了分离纯化,结果表明,10 mg Fe3O4@ SiO2/Ni-NTA微球能够从10mL重组蛋白裂解液中纯化出约1 mg带有6×His-tag标签的融合蛋白.微球的高效分离效果使其能够用于含量较低的带有6×His-tag标签蛋白的分离纯化.     

DNA separation by EFFF in a microchannel

This paper theoretically explores the application of electric field flow fractionation (EFFF) for the size-based separation of DNA strands in a microchannel. An axial electric field cannot separate DNA strands in solution because the electrical mobility of the strands is independent of the length. However, lateral electric fields coupled with an axial Poiseuille flow can separate the DNA strands of different sizes. By using regular perturbation analysis, we obtain the effective diffusivity and the mean velocity of the DNA molecules that are undergoing a pressure driven Poiseuille flow in a 2D channel in presence of a lateral electric field. The mean velocities and the dispersion coefficients are then utilized to determine the scaling for length of the channel and the time required for separation of DNA molecules in different parameter regimes. The results show that EFFF can separate DNA strands in the range of 10 kbp that differ in size by about 2.5 kbp in about half an hour in a 1 cm long channel. While DNA strands can be separated by EFFF, the performance of devices based on EFFF seems to be at best comparable to other techniques such as entropic trapping.     

Recent advances in microfluidic cell sorting techniques based on both physical and biochemical principles

Microfluidic technologies for isolating cells of interest from a heterogeneous sample have attracted great attentions, due to the advantages of less sample consumption, simple operating procedure, and high separation accuracy. According to the working principles, the microfluidic cell sorting techniques can be categorized into biochemical (labeled) and physical (label‐free) methods. However, the inherent drawbacks of each type of method may somehow influence the popularization of these cell sorting techniques. Using the multiple complementary isolation principles is a promising strategy to overcome this problem, therefore there appears to be a continuing trend to integrate two or more sorting methods together. In this review, we focus on the recent advances in microfluidic cell sorting techniques relied on both physical and biochemical principles, with emphasis on the mechanisms of cell separation. The biochemical cell sorting techniques enhanced by physical principles and the physical cell sorting techniques enhanced by biochemical principles, are first introduced. Then, we highlight on‐chip magnetic‐activated cell sorting, on‐chip fluorescence‐activated cell sorting, multi‐step cell sorting and multi‐principle cell sorting techniques, which are based on both physical and biochemical separation mechanisms. Finally, the challenges and future perspectives of the integrated microfluidics for cell sorting are discussed.     

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.     

Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid

Pure separation and sorting of microparticles from complex fluids are essential for biochemical analyses and clinical diagnostics. However, conventional techniques require highly complex and expensive labeling processes for high purity separation. In this study, we present a simple and label-free method for separating microparticles with high purity using the elasto-inertial characteristic of a non-Newtonian fluid in microchannel flow. At the inlet, particle-containing sample flow was pushed toward the side walls by introducing sheath fluid from the center inlet. Particles of 1 μm and 5 μm in diameter, which were suspended in viscoelastic fluid, were successfully separated in the outlet channels: larger particles were notably focused on the centerline of the channel at the outlet, while smaller particles continued flowing along the side walls with minimal lateral migration towards the centerline. The same technique was further applied to separate platelets from diluted whole blood. Through cytometric analysis, we obtained a purity of collected platelets of close to 99.9%. Conclusively, our microparticle separation technique using elasto-inertial forces in non-Newtonian fluid is an effective method for separating and collecting microparticles on the basis of size differences with high purity.     

Different methods for the fractionation of magnetic fluids

 Magnetic fluids are used in many fields of application, such as material separation and biomedicine. Magnetic fluids consist of magnetic nanoparticles, which commonly display a broad distribution of magnetic and nonmagnetic parameters. Therefore, upon application only a small number of particles contribute to the desired magnetic effect. In order to optimize magnetic fluids for applications preference is given to methods that separate magnetic nanoparticles according to their magnetic properties. Hence, a magnetic method was developed for the fractionation of magnetic fluids. Familiar size-exclusion chromatography of two different magnetic fluids was carried out for comparison. The fractions obtained and the original samples were also magnetically characterized by magnetic resonance and magnetorelaxometry, two biomedical applications. The size-exclusion fractions are similar to those of magnetic fractionation, despite the different separation mechanisms. In this respect, magnetic fractionation has several advantages in practical use over size-exclusion chromatography: the magnetic method is faster and has a higher capacity. The fractions obtained by both methods show distinctly different magnetic properties compared to the original samples and are therefore especially suited for applications such as magnetorelaxometry. Received: 12 July 1999/Accepted in revised form: 9 November 1999     

具有光导性的磁性高分子微球——反应性铁酞菁与苯乙烯共聚

合成出了带有反应性基团的铁酞菁单体 ,并实现了与苯乙烯单体在磁流体存在下的分散聚合而得磁性高分子微球 .研究了微球结构、组成 ,测定了其磁响应性和光导性