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].     

玻璃微流控芯片廉价快速制作方法的研究

研究了一种玻璃微流控芯片的快速、低成本制作工艺和方法. 该方法采用商品化的显微载玻片(soda-lime玻璃)作为芯片基质材料, 利用AZ 4620光刻胶代替传统工艺中的溅射金属层或多晶硅/氮化硅层作为玻璃刻蚀的掩膜层, 同时利用一种紫外光学胶键合方法代替传统熔融键合方法实现芯片的键合, 整个工艺对玻璃基质材料要求低, 普通微流控芯片(深度小于50 μm)制作流程仅需约3.5 h, 可降低制作成本, 缩短制作周期. 还系统地研究了光刻胶厚度、光刻胶硬烘时间和玻璃腐蚀液配比对玻璃微流控芯片制作的影响, 获得了优化的工艺参数.     

CO2激光-化学腐蚀制作金阵列微电极

微电极的制作是微流控芯片电化学检测的关键技术.本文提出CO2激光烧蚀结合化学腐蚀快速制作微流控芯片阵列微电极的方法.在溅射Au/Cr的玻璃基片上涂敷指甲油作牺牲层,利用CO2激光烧蚀开窗口,经化学腐蚀后获得阵列电极,电极宽度为100μm.考察了激光加工参数及牺牲层对电极加工质量的影响,对由键合包封制作的微流控芯片,循环伏安及流动注射分析测试表明,该电极芯片可用于微流控芯片的安培检测.     

纸芯片微流控技术的发展及应用

纸芯片微流控技术是一种新型微流控技术。相比于以玻璃、石英、高聚物等为基底的传统微流控芯片,纸芯片具有成本低、易操作、可携带、耗样量小等优点。该文介绍了纸芯片的发展及常用的制作方法,并举例说明了光度法、荧光法、化学发光及电化学发光法和电化学法在纸芯片检测中的应用;归纳了纸芯片技术在临床诊断、环境监控以及食品安全分析等方面的应用;最后对纸芯片微流控的应用前景进行了展望。     

功能型微流控芯片实验室的研究及其应用

功能型微流控芯片实验室由芯片、芯片工作站、功能芯片试剂盒等3部分组成. 开展了不同类型的芯片设计,研制了有不同集成度和不同结构的玻璃芯片以及PMMA、PDMS和SU-8等塑料芯片,其中注塑型PMMA塑料芯片已具备规模生产的能力.     

激光诱导荧光微流控芯片分析仪的研制

研制了一种基于激光诱导荧光检测方法的微流控芯片分析仪。该分析仪使用玻璃基质聚二甲基硅氧烷(PDMS)微流控芯片,可一次性进行12通道的电泳分离实验。仪器采用共聚焦式光路结构,并可通过检测由微流控芯片反射的激光信息,控制步进电机实现芯片的自动精确定位。实验结束自动保存数据,绘制分离图谱。。对9种不同长度的50 bp DNA Ladder片段进行电泳分离及数据分析,耗时在5 min内,且分离效果良好。     

自制玻璃微流控芯片及其基本性能考察

20世纪90年代初,自Manz等首次提出微全分析系统的概念以来,各种不同材料和功能的微流控芯片不断问世,其中应用得最多的是玻璃、石英及聚合物3种材料．玻璃和石英芯片因具有优异的电渗、光学和表面性质,其刻蚀加工技术和表面改性的化学方法均比较成熟,且传统毛细管电泳中各种成熟的分离方法可直接应用到玻璃芯片的制作中,因此在微全分析系统研究中具有十分重要的地位．     

磁控微流控芯片的研究现状及应用

微流控芯片作为一种现代分析方法,近年来得到迅速发展。而磁控微流控芯片是在微流控芯片中引入磁场调控,通过引入磁场丰富了微流控芯片的操控手段,同时结合了磁性材料的优势,使之成为微流控芯片研究的重要组成部分之一。本文重点介绍磁控微流控芯片的研究现状及应用。     

一种微流控芯片中大体积进样方法及专用芯片

本发明涉及微流控芯片系统的进样方法。在使用固定相的微流控芯片系统中,于芯片分离通道与流动相人口之间设置两个或两个以上的分流通道。通过控制其中流体的切换和截止,实现对微流控芯片系统的大体积进样。该方法适合于微流控芯片中电色谱、加压电色谱和液相色谱模式下的大体积进样。该方法的优点为：可实现微流控芯片中大体积样品的上样;克服了系统中的梯度延迟效应,     

农药残留快速检测芯片将实现产业化

芯片实验室是一项基于微流控芯片的技术．已被列入21世纪最为重要的前沿技术行列。不久前,留学法国的王晓东博士带着他的“基于微流控芯片技术的便携式农药残留快速检测系统的开发与产业化”项目来到杭州市萧山区．成功入选该区2014年第一批“5213”计划重点项目。     

通用型激光诱导荧光微流控芯片分析仪的研制与性能考察

设计和研制了一种通用型激光诱导荧光微流控芯片分析仪.检测部分按共聚焦检测原理设计,采用CCD(电荷耦合器件)监测通道,三维自动调节聚焦,发射波长滤光片可方便地更换以适应多种染料选择,能分别显示进样和分离通道2条电流-时间曲线.考察了该分析仪的检测灵敏度、检测极限和线性范围,显示了分析灵敏度高,检测限低和线性范围宽等特点,在自制注塑型PMMA塑料芯片上实现了φX174Haedi-gesT_dNA片段的分离测定和烟叶act基因PCR产物的分析     

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected psiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.     

Replica multichannel polymer chips with a network of sacrificial channels sealed by adhesive printing method

Replica microchips for capillary array electrophoresis containing 10 separation channels (50 microm width, 50 microm depth and 100 microm pitch) and a network of sacrificial channels (100 microm width and 50 microm depth) were successfully fabricated on a poly(methyl methacrylate) (PMMA) substrate by injection molding. The strategy involved development of moving mask deep X-ray lithography to fabricate an array of channels with inclined channel sidewalls. A slight inclination of channel sidewalls, which can not be fabricated by conventional deep X-ray lithography, is highly required to ensure the release of replicated polymer chips from a mold. Moreover, the sealing of molded PMMA multichannel chips with a PMMA cover film was achieved by a novel bonding technique involving adhesive printing and a network of sacrificial channels. An adhesive printing process enables us to precisely control the thickness of an adhesive layer, and a network of sacrificial channels makes it possible to remove air bubbles and an excess adhesive, which are crucial to achieving perfect sealing of replica PMMA chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to simultaneously monitor electrophoretic separations in ten micro-channels with laser-induced fluorescence detection. High-speed and high-throughput separations of a 100 bp DNA ladder and phi X174 Hae III DNA restriction fragments have been demonstrated using a 10-channel PMMA chip. The current work establishes the feasibility of mass production of PMMA multichannel chips at a cost-effective basis.     

Separation of double-stranded DNA fragments in plastic capillary electrophoresis chips by using E99P69E99 as separation medium.

The separation of double-stranded DNA (dsDNA) fragments in polymethylmethacrylate (PMMA) capillary electrophoresis (CE) chips by using E99P69E99 as a separation medium has been demonstrated. The PMMA CE chips were simply manufactured by micromachining and adhesive tape sealing. To make the separation channel compatible with the separation medium, a dynamic nonionic surfactant coating procedure was developed, which made the plastic separation channel sufficiently hydrophilic to allow the separation medium to fill the channel by capillary action. Subsequent separation of DNA fragments was successful with a separation efficiency of the order of 10(4) theoretical plates over an effective separation distance of 1.5 cm. By using an applied electric field strength of 200 V/cm, the separation of low DNA mass ladder was completed within 5 min. The simple coating procedure, together with the self-assembled viscosity-adjustable separation medium, should be useful to meet some of the essential requirements for developing single-use disposable CE chips. Coating the channels with polymer blends of PMMA and the separation medium also showed promise.     

Analysis of DNA fragments by microchip electrophoresis fabricated on poly(methyl methacrylate) substrates using a wire-imprinting method

Microfluidic devices were fabricated on poly(methyl methacrylate) (PMMA) substrate using two small-diameter (79 microm) wires to create a cross impression in plastics softened by low-temperature heating. The resulting channels had a rounded shape and 75 microm in depth. The variability of the channel dimensions was found to be less than 6% from different locations of the same channel and less than 10% between chips. Moreover, the fabricated PMMA chip appeared to sustain an electric field strength up to 300 V/cm without significant Joule heating. The function of resulting devices for electrophoretic injection and separation of a DNA size marker, HaeIII digest of (phiX174, was also characterized. Results indicated that all of the 11 DNA fragments of the size marker could be identified in less than 3 min with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. Moreover, with the use of near infrared (IR) dye, fluorescence signals of the higher molecular weight fragments (> 603 bp in length) could be detected at total DNA concentrations as low as 0.1 microg/mL (S/N = 4.2). In conclusion, the performance of wire-imprinted devices on PMMA substrate were comparable to those fabricated by other professional means.     

聚甲基丙烯酸甲酯微流控分析芯片的简易热压制作法

提出聚甲基丙烯酸甲酯(PMMA)微流控分析芯片的一种简易热压制作法,研究了镍基、单晶硅和玻璃3种阳模制备芯片及芯片的封合条件.采用扫描电镜(SEM)和电荷耦合检测器(CCD)对PMMA芯片的微通道及其横截面形貌进行了表征.SEM图和CCD图表明实现了热压封接.测定了PMMA芯片的伏安曲线和电渗流,其电渗流值与文献报道值基本一致.本法制作的PMMA芯片用于电泳分离Cy5荧光染料,峰高RSD为2.2%(n=11),理论塔板数7.4×10⁴m^-1.     

Rapid prototyping of poly(methyl methacrylate) microfluidic systems using solvent imprinting and bonding

We have developed a method for rapid prototyping of hard polymer microfluidic systems using solvent imprinting and bonding. We investigated the applicability of patterned SU-8 photoresist on glass as an easily fabricated template for solvent imprinting. Poly(methyl methacrylate) (PMMA) exposed to acetonitrile for 2 min then had an SU-8 template pressed into the surface for 10 min, which provided appropriately imprinted channels and a suitable surface for bonding. After a PMMA cover plate had also been exposed to acetonitrile for 2 min, the imprinted and top PMMA pieces could be bonded together at room temperature with appropriate pressure. The total fabrication time was less than 15 min. Under the optimized fabrication conditions, nearly 30 PMMA chips could be replicated using a single patterned SU-8 master with high chip-to-chip reproducibility. Relative standard deviations were 2.3% and 5.4% for the widths and depths of the replicated channels, respectively. Fluorescently labeled amino acid and peptide mixtures were baseline separated using these PMMA microchips in <15s. Theoretical plate numbers in excess of 5000 were obtained for a approximately 3 cm separation distance, and the migration time relative standard deviation for an amino acid peak was 1.5% for intra-day and 2.2% for inter-day analysis. This new solvent imprinting and bonding approach significantly simplifies the process for fabricating microfluidic structures in hard polymers such as PMMA.     

超支化聚酰胺酯改性聚甲基丙烯酸甲酯微流控芯片的制备及其在生物分子分离检测中的应用

利用亲水性超支化聚酰胺酯通过化学键合的方法对聚甲基丙烯酸甲酯(PMMA)微流控芯片的表面进行改性。对改性后PMMA微流控芯片的表面进行了接触角的测定,利用扫描电子显微镜(SEM)和体视显微镜观察了改性后芯片的表面形貌。结果表明,改性后的PMMA微流控芯片表面形成了一层均匀、致密、连续的亲水性涂层,芯片表面的亲水性得到了明显提高,接触角由未改性时的89.9°降低到29.5°。改性后芯片的电渗流较之改性前明显降低。利用芯片对腺苷和L-赖氨酸两种生物分子进行了分离检测。两种生物分子实现了完全分离,所得到的检测峰峰形尖锐,分离清晰。对腺苷和L-赖氨酸的分离柱效(理论塔板数)分别高达8.44×104 塔板/m和9.82×104 塔板/m,分离度(Rs)达到5.31,均远远高于未改性的芯片。改性后的芯片具有良好的分离时间重现性。本研究为提高PMMA微流控芯片的亲水性和应用范围提供了一种新的有效方法。     

Surface modification of poly(methyl methacrylate) for improved adsorption of wall coating polymers for microchip electrophoresis

The development of rapid and simple wall coating strategies for high-efficiency electrophoretic separation of DNA is of crucial importance for the successful implementation of miniaturized polymeric DNA analysis systems. In this report, we characterize and compare different methods for the chemical modification of poly(methyl methacrylate) (PMMA) surfaces for the application of wall coating polymers. PMMA surfaces coated with 40 mol% diethylacrylamide and 60 mol% dimethylacrylamide are compared to the PMMA surfaces first oxidized and then coated with hydroxypropylmethyl-cellulose or poly(vinyl alcohol) (PVA). PMMA oxidation was accomplished with UV/ozone or an aqueous solution of HNO(3) to yield hydrogen-bond donors for the spontaneous adsorption of the coating polymers. Contact angle measurements of UV/ozone exposed PMMA surfaces indicate increase in hydrophilicity, and polymer coated surfaces show a strong dependence on the coating polymer and the oxidation method. Fast and repeatable electrophoretic separations of a 10-base and 20-base DNA ladder were performed in PMMA micro CE devices. All analyses were completed in less than 10 min, resulting in the number of theoretical plates as high as 583 000 in a 7.7 cm long separation channel. The duration of UV/ozone treatment was found to have a considerable impact on separation performance. The microchips irradiated with UV for 10 min and coated with PVA as well as the microchips treated with HNO(3) and coated with HPMC were found to have the best separation performance. These results demonstrate facile and robust methods for the surface modification of PMMA enabling low-cost single use devices for electrophoretic DNA separations.     

Microfluidic devices fabricated in poly(methyl methacrylate) using hot-embossing with integrated sampling capillary and fiber optics for fluorescence detection

High-aspect-ratio microstructures have been prepared using hot-embossing techniques in poly(methyl methacrylate) (PMMA) from Ni-based molding dies prepared using LIGA (Lithographie, Galvanoformung, Abformung). Due to the small amount of mask undercutting associated with X-ray lithography and the high energy X-ray beam used during photoresist patterning, deep structures with sharp and smooth sidewalls have been prepared. The Ni-electroforms produced devices with minimal replication errors using hot-embossing at a turn around time of approximately 5 min per device. In addition, several different polymers (with different glass transition temperatures) could be effectively molded with these Ni-electroforms and many devices (>300) molded with the same master without any noticeable degradation. The PMMA devices consisted of deep and narrow channels for insertion of a capillary for the automated electrokinetic loading of sample into the microfluidic device and also, a pair of optical fibers for shuttling laser light to the detection zone and collecting the resulting emission for fluorescence analysis. Electrophoretic separations of double-stranded DNA ladders Phi X174 digested with Hae III) were performed with fluorescence detection accomplished using near-IR excitation. It was found that the narrow width of the channels did not contribute significantly to electrophoretic zone broadening and the plate numbers generated in the extended length separation channel allowed sorting of the 271/281 base pair fragments associated with this sizing ladder when electrophoresed in methylcellulose entangled polymer solutions. The dual fiber detector produced sub-attomole detection limits with the entire detector, including laser source, electronics and photon transducer, situated in a single box measuring 3' x 10" x 14".