新简易采集装置联接质谱实现雾霾微生物分析

<正>复旦大学和上海海洋大学的研究团队发明了一种简易的采集装置,可用于雾霾、气溶胶中的微生物样品采集,结合质谱分析技术,实现了对雾霾传播的微生物分析与鉴定。该成果发布在美国化学会《Analytical Chemistry》(分析化学)杂志上。该简易采样器,由微流控芯片、微型气泵、可充电锂电池、稳流阀等部件组成,利用气流在双螺旋通道的离心力对微生物样品进行分离捕获。螺旋式的微通道给气流施加了较强的离心力,有利于气溶胶中的微生物附着在芯片通道中。鱼骨形的结构设计     

基于流体动力单细胞高效捕获的微流控芯片结构研究进展

单细胞分析对于重大疾病的早期诊断及治疗、药物筛选和生理病理过程的研究具有重要意义。微流控芯片能够精确控制单细胞的微环境,实时监测单细胞的行为,已成为单细胞分析的强大工具。单细胞捕获是单细胞分析的重要步骤。目前已报道了多种微流控芯片用于单细胞捕获的方法,其中基于流体动力的微流控芯片单细胞捕获方法具有操作方便、单细胞捕获效率高等优点,受到研究人员的广泛关注及使用。为了全面了解基于流体动力的微流控芯片单细胞捕获方法的研究现状,掌握单细胞高效捕获的微流控芯片结构设计,实现单细胞精准快速分析,本文综述了基于流体动力的单细胞高效捕获（>70%）原理及微流控芯片结构,根据结构设计不同分为微井结构、微柱结构和旁路通道结构,介绍了单细胞高效捕获的微流控芯片优化过程,总结了微流控芯片的材质、结构特点及单细胞捕获效率等,对不同单细胞捕获结构的优势及不足进行了分析。最后,对基于流体动力的微流控芯片单细胞捕获方法的发展趋势进行了展望。     

高效细胞电融合芯片中的电场分析

细胞电融合芯片内的电场分布对细胞的控制及细胞融合效率有非常重要的意义,它是该类芯片设计的主要因素。电场分布主要由芯片内微通道和微电极的结构决定。在一个新研制的融合芯片中,采用大量微电极构成的阵列来提高融合效率。由于电极数量很多,微通道和微电极的结构和形状复杂,理论计算芯片内部电场分布具有较大难度。利用ANSYS有限元分析软件,对细胞电融合芯片中的电场分布进行模拟分析,得到其强度分布及变化梯度。通过不同设计的对比分析,提出了更加适合于细胞电融合的电极阵列结构模型——矩形梳状交叉微电极阵列,为高效细胞电融合芯片的实现奠定了基础。在矩形梳状交叉微电极阵列原型芯片的实验研究中,细胞融合(植物原生质体融合)效率约为40%,超过了传统的化学融合(小于1%)、电融合(小于10%),以及最初所采用的矩形对称梳状电极(小于20%)。表明在该融合芯片上可以实现高效的细胞电融合。     

基于DNA四面体的微流控芯片用于致病性大肠杆菌O157∶H7的检测

设计了一种微流控芯片,在其通道表面修饰DNA四面体,并通过生物素-链霉亲和素反应连接适配体作为捕获探针,用于大肠杆菌O157∶H7(Escherichia coli O157∶H7,E.coli O157∶H7)的检测研究。微流控芯片鱼骨形结构的设计降低了细菌捕获时受到的剪切力;在其表面修饰DNA四面体,可进一步调节探针之间的距离,提高探针对细菌的识别效率。琼脂糖凝胶电泳表征结果证实了DNA四面体纳米结构的成功制备和DNA四面体-适配体捕获体系的构建。采用荧光显微镜对检测结果进行进一步成像分析,并将此微流控芯片检测平台用于实际样品的检测。结果表明,不需要大型仪器或设备及其它信号放大技术的辅助,在普通光学显微镜下,利用此检测系统即能实现浓度为10 CFU/mL的E.coli O157∶H7的检测,且操作简便,检测耗时少于2 h。实际样品的检测回收率为88.3%～108.3%。本研究基于DNA四面体纳米结构构建的微流控平台,不仅为食源性致病菌的检测提供了一种有效的检测方法,在其它食品安全隐患、疾病早期诊断等研究领域也具有潜在应用价值。     

新简易采集装置联接质谱实现雾霾微生物分析

<正>复旦大学和上海海洋大学的研究团队发明了一种简易的采集装置,可用于雾霾、气溶胶中的微生物样品采集,结合质谱分析技术,实现了对雾霾传播的微生物分析与鉴定。该成果发布在美国化学会《Analytical Chemistry》(分析化学)杂志上。该简易采样器,由微流控芯片、微型气泵、可充电锂电池、稳流阀等部件组成,利用气流在双螺旋通道的离心力对微生物样品进行分离捕获。螺旋式的微通道给气流施加了较强的离心力,有利于气溶胶     

新简易采集装置联接质谱实现雾霾微生物分析

<正>复旦大学和上海海洋大学的研究团队发明了一种简易的采集装置,可用于雾霾、气溶胶中的微生物样品采集,结合质谱分析技术,实现了对雾霾传播的微生物分析与鉴定。该成果发布在美国化学会《Analytical Chemistry》(分析化学)杂志上。该简易采样器,由微流控芯片、微型气泵、可充电锂电池、稳流阀等部件组成,利用气流在双螺旋通道的离心力对微生物样品进行分离捕获。螺旋式的微通道给气流施加了较强的离心力,有利于气溶胶     

阵列式对电极介电电泳芯片及其用于细胞分离富集研究

基于介电电泳原理, 设计并制作了一种新型的能够用于细胞分离和富集的微流控介电电泳芯片. 该芯片由沉积有金电极的石英基片和带有微管道的聚二甲基硅氧烷(PDMS)盖片组成. 通过在管道底部布置间距不同的对电极阵列, 增大了正介电电泳力在管道中的有效作用范围, 能够在降低施加电压的同时, 实现对流动体系中细胞样品的捕获. 在3 V和3 MHz条件下, 该DEP芯片对人血红细胞的捕获效率达到83%; 进一步通过将肝癌细胞捕获在芯片电极上可实现对红细胞和肝癌细胞混合样品的分离, 在5 V和400 kHz条件下对肝癌细胞的捕获效率达到86%.     

基于薄膜电极的细胞电融合芯片

构建了一种薄膜电极阵列结构的细胞电融合芯片, 通过多聚物微通道底/顶层凸齿状的微电极, 以及多聚物微通道侧壁上溅射形成的一层离散式金属薄膜电极, 共同形成离散式"三明治"微电极结构. 该微电极结构可在微通道内部形成与传统凸齿状电极相似的非均匀分布的梯度电场, 通过介电电泳效应进行细胞控制及排队. 利用多聚物在芯片上填充了传统凸齿状电极的凹陷区, 克服了细胞在凹陷区无法有效排队与融合的缺点. 在芯片上利用K562细胞开展了基于介电电泳效应的细胞排队实验及基于可逆性电穿孔效应的电融合实验, 结果表明该芯片能够较好地实现细胞排队及融合, 融合所需控制电压低至10 V左右. 细胞排队率达99%以上, 几乎无细胞在绝缘物填充区(传统凸齿电极芯片的凹陷区)滞留, 细胞两两排队高于60%, 细胞融合效率约为40%, 比传统的细胞电融合方法和凸齿电极芯片有较大提高.     

多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

玻璃微-纳流控芯片的制备及在蛋白质电动富集中的应用

发展了一种以"二次刻蚀"技术制备玻璃微-纳流控芯片的新方法. 首先, 采用紫外光刻和化学湿法刻蚀技术在玻璃基片上加工微米深度的微通道; 去除剩余的光胶后, 在刻有微通道的基片上旋涂一层新的光胶; 再通过二次紫外光刻和湿法刻蚀在该基片上加工深度小于100 nm的纳通道; 最后, 采用室温键合技术, 将带有微纳结构的基片与盖片封合制成玻璃微-纳流控复合芯片. 利用本方法可以在普通化学实验室以简易的设备制得具有微-纳米复合结构的玻璃芯片. 将此玻璃微-纳流控复合芯片成功地应用于以电动离子捕集技术富集荧光素钠异硫氰酸酯(FITC)标记的人血清蛋白(HSA). 结果表明, 对于0.5 mg/mL的FITC-HSA, 30 s内富集倍率可达到200倍以上.     

A smart and portable micropump for stable liquid delivery

Precise and reliable liquid delivery is vital for microfluidic applications. Here, we illustrate the design, fabrication, characterization, and application of a portable, low cost, and robust micropump, which brings solution to stable liquid delivery in microfluidic environment. The pump is designed with three optional speeds of different pumping flow rates, and it can be simply actuated by spring‐driven mechanism. The different flow rates of the pump are realized via passive microvalves in a compact microfluidic chip, which is installed in the pump. Importantly, the membrane structures of the microvalves allow accurate liquid control, and stable flow rates can be achieved via a spring setup. The proposed pump is applied to continuously and stably infuse microbead suspension into an inertial microfluidic chip, and good particle focusing is realized in the spiral channel of the inertial microfluidic chip. The proposed portable, self‐powered, and cost‐efficient pump is crucial for microfluidic lab‐on‐a‐chip system integration, which may facilitate microfluidic application for precise liquid delivery, control, measurement, and analysis.     

Laminar flow mediated continuous single-cell analysis on a novel poly(dimethylsiloxane) microfluidic chip

A novel microfluidic chip with simple design, easy fabrication and low cost, coupled with high-sensitive laser induced fluorescence detection, was developed to provide continuous single-cell analysis based on dynamic cell manipulation in flowing streams. Making use of laminar flows, which formed in microchannels, single cells were aligned and continuously introduced into the sample channel and then detection channel in the chip. In order to rapidly lyse the moving cells and completely transport cellular contents into the detection channel, the angle of the side-flow channels, the asymmetric design of the channels, and the number, shape and layout of micro-obstacles were optimized for effectively redistributing and mixing the laminar flows of single cells suspension, cell lysing reagent and detection buffer. The optimized microfluidic chip was an asymmetric structure of three microchannels, with three microcylinders at the proper positions in the intersections of channels. The microchip was evaluated by detection of anticancer drug doxorubicin (DOX) uptake and membrane surface P-glycoprotein (P-gp) expression in single leukemia K562 cells. An average throughput of 6–8 cells min⁻¹ was achieved. The detection results showed the cellular heterogeneity in DOX uptake and surface P-gp expression within K562 cells. Our researches demonstrated the feasibility and simplicity of the newly developed microfluidic chip for chemical single-cell analysis.     

A new floating electrode structure for generating homogeneous electrical fields in microfluidic channels

In this article a new parallel electrode structure in a microfluidic channel is described that makes use of a floating electrode to get a homogeneous electrical field. Compared to existing parallel electrode structures, the new structure has an easier production process and there is no need for an electrical connection to both sides of the microfluidic chip. With the new chip design, polystyrene beads suspended in background electrolyte have been detected using electrical impedance measurements. The results of electrical impedance changes caused by beads passing the electrodes are compared with results in a similar planar electrode configuration. It is shown that in the new configuration the coefficient of variation of the impedance changes is lower compared to the planar configuration (0.39 versus 0.56) and less dependent on the position of the beads passage in the channel as a result of the homogeneous electrical field. To our knowledge this is the first time that a floating electrode is used for the realization of a parallel electrode structure. The proposed production method for parallel electrodes in microfluidic channels can easily be applied to other applications.     

用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证

设计并验证了一种用于细胞三维培养的集成微柱阵列的微流控芯片.芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃盖片组成, 在PDMS沟道片上集成了一个由两排微柱阵列围成的细胞培养室和两条用于输送培养基的侧沟道.微柱间距直接影响了芯片的使用性能, 是整个芯片设计的关键.基于数值模拟和实验验证, 本研究对微柱间距进行了优化设计.优化后的微流控芯片可以很好地实现细胞与细胞外基质模拟材料混合液的稳定注入、培养基中营养物质向培养室内的快速扩散和细胞代谢物的及时排出.在芯片上进行了神经干细胞的三维培养, 证明了芯片上构建的细胞体外微环境的稳定性.     

Portable Microfluidic Chip Based Surface-Enhanced Raman Spectroscopy Sensor for Crystal Violet

This paper describes the development of a portable microfluidic chip based on a surface-enhanced Raman spectroscopy (SERS) sensor for crystal violet analysis. A Y-shape microfluidic chip with a staggered herringbone structure was designed to efficiently mix the analyte and SERS active silver colloid. The subsequent detection of the analyte was performed on the microfluidic chip by a portable Raman system. Compared with other methods, this sensor is easy to operate and is expected to have applications for rapid and sensitive on-site analysis. A good linear correlation over the concentration range of 10 to 750 nM of crystal violet with a correlation coefficient of 0.992 was obtained. The recovery was between 98.6% and 102.9% for crystal violet in river water with relative standard deviations between 2.43% and 4.26%.     

A radial microfluidic concentration gradient generator with high-density channels for cell apoptosis assay

An integrated microfluidic concentration gradient chip was developed for generating stepwise concentrations in high-density channels and applied to high-throughput apoptosis analysis of human uterine cervix cancer (HeLa) cells. The concentration gradient was generated by repeated splitting-and-mixing of the source solutions in a radial channel network which consists of multiple concentric circular channels and an increasing number of branch channels. The gradients were formed over hundreds of branches with predictable concentrations in each branch channel. This configuration brings about some distinctive advantages, e.g., more compact and versatile design, high-density of channels and wide concentration ranges. This concentration gradient generator was used in perfusion culture of HeLa cells and a drug-induced apoptosis assay, demonstrated by investigating the single and combined effects of two model anticancer drugs, 5-fluorouracil and Cyclophosphamide, which were divided into 65 concentrations of the two drugs respectively and 65 of their combinatorial concentrations. The gradient generation, the cell culture/stimulation and staining were performed in a single chip. The present device offers a unique platform to characterize various cellular responses in a high-throughput fashion.     

Enrichment of viable bacteria in a micro-volume by free-flow electrophoresis

Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free flow electrophoresis and is demonstrated for Gram positive bacteria. Bacteria from a suspension flow are trapped on a gel interface that separates the trapping location from integrated actuation electrodes in order to enable non-destructive trapping. The microfluidic chip contains integrated electrolytic gas expulsion structures and phaseguides for gel and liquid handling. Trapping efficiency is systematically optimized to reach 25 times the initial concentration from a theoretical maximum of 30. Finally, enrichment from analytically relevant concentrations down to 3 × 10(2) colony forming units per millilitre is demonstrated with a trapping efficiency of 80% which represents the most important parameter in enrichment.     

Counting bacteria on a microfluidic chip

This paper reports a lab-on-a-chip device that counts the number of bacteria flowing through a microchannel. The bacteria number counting is realized by a microfluidic differential Resistive Pulse Sensor (RPS). By using a single microfluidic channel with two detecting arm channels placed at the two ends of the sensing section, the microfluidic differential RPS can achieve a high signal-to-noise ratio. This method is applied to detect and count bacteria in aqueous solution. The detected RPS signals amplitude for Pseudomonas aeruginosa ranges from 0.05 V to 0.17 V and the signal-to-noise ratio is 5-17. The number rate of the bacteria flowing through the sensing gate per minute is a linear function of the sample concentration. Using this experimentally obtained correlation curve, the concentration of bacteria in the sample solution can be evaluated within several minutes by measuring the number rate of the bacteria flowing through the sensing gate of this microfluidic differential RPS chip. The method described in this paper is simple and automatic, and have wide applications in determining the bacteria and cell concentrations for microbiological and other biological applications.     

Three‐dimensional microfluidic chip with twin‐layer herringbone structure for high efficient tumor cell capture and release via antibody‐conjugated magnetic microbeads

Harvesting rare circulating tumor cells (CTCs) from human blood is distinctly substantial to monitor tumor stage and evaluate therapeutic efficacy. As a proof‐of‐concept study, a microfluidic chip with twin‐layer herringbone grooves was developed to isolate and recover tumor cells with high efficiency based on the immunoreaction between cells and antibody‐conjugated microbeads (MBs) under local magnetic field. Functional MBs were initially localized onto the internal channel wall through the magnetic guidance. Then, infused tumor cells were deviated into the herringbone groove via passive microvortex and were further trapped through an irreversible interaction with MBs. Upon the removal of magnet, the captured cells and residual MBs were released from the channel and collected for further analysis in cell adhesion and proliferation in vitro. Capture efficiency of tumor cells reached up to ∼90% and limit of detection was down to 50 cells per mL based on this approach. Furthermore, recovery rate of tumor cells was as high as ∼94%, and potencies of cell attachment and proliferation was well maintained in retrieved cells. Hence, the present technique has a great potential for the isolation, quantitation and recovery of CTCs for cancer theranostic guidance and biomolecular analysis.     

在微流控芯片上合成对甲氧基苯甲醛肟

本文用负压进样的方法, 在自制的玻璃微流控芯片中进行了对甲氧基苯甲醛和盐酸羟胺合成对甲氧基苯甲醛肟的相转移反应. 测定了不同反应时间的产率, 并与常规方法进行了比较. 讨论了相接触面积和塞流对产率的影响.