微流控芯片检测技术进展

对近年来用于微流控芯片的光学检测(包括荧光、吸收光度和电化学发光检测等)、电化学检测(电导检测、电位检测及安培检测)的发展和其他一些检测方法的研究成果进行了综述,随着微加工技术的不断发展,高速多通道检测以及集成多种方法的高通用性微流控检测芯片都将成为未来的研究热点。     

基于双富集微流控技术的食品中蜡样芽孢杆菌快速检测

为实现食源性致病菌多场景即时检验,替换常规检验及快检技术中耗时的增菌培养过程,以缩短检验时间和降低实验环境及设备要求。研究以蜡样芽孢杆菌为研究对象,采用双层膜富集样品中菌体,磁珠提取纯化微量DNA的方式,取代增菌培养过程,同时将环介导等温扩增反应与微流控芯片相结合,有效避免扩增中气溶胶污染问题。实验结果显示：使用双富集微流控技术检测食品中蜡样芽孢杆菌,可将现行国标的检验时间由5～7 d缩短至1 h内,方法特异性良好,检出限为10 CFU/g(mL)等同于现行国标,Ct值变异系数<5%,显示重复性好。研究建立的双富集微流控技术快速检测食品中蜡样芽孢杆菌方法可实现食源性致病菌即时快速检验,为食源性致病菌引起的食品安全事件和舆情风险防控快速反应提供技术支撑。     

LAMP实时浊度法快速检测大肠杆菌方法的建立与优化研究

根据大肠杆菌的特异性基因（malB）,设计与筛查环介导等温扩增（LAMP）的引物,通过对甜菜碱用量、环引物的浓度、反应温度、dNTPs用量等反应条件的优化,得到较佳的LAMP反应体系,建立了LAMP实时浊度法快速检测大肠杆菌;在较优条件下,先后对9株非大肠杆菌菌株进行特异性检测和5株大肠杆菌的进行同源性检测。此外,结合课题组自行研制的多通道浊度仪,对LAMP扩增产物进行实时浊度监测。结果表明：LAMP实时浊度法检测大肠杆菌的灵敏度达16.010 ng/L,与实时荧光定量PCR的检测限相当。因此,建立与优化LAMP实时浊度法可为食品中大肠杆菌的现场快速检测提供了有力手段。     

微流控芯片表面修饰及在蛋白质富集中的应用

应用分子自组装技术，在SiO2表面衍生活泼醛基，在SiO2表面有125nm的衍生物，衍生的醛基和氨基发生共价反应而将入免疫球蛋白G固定在二氧化硅表面，抗原抗体反应显示固定的抗体有活性。应用微加工技术加工含交叉排列的椭圆形微柱阵列的微流控芯片，有效增加内表面积和流体接触机会，用同样修饰方法修饰微流控芯片内表面并固定人免疫球蛋白G，流经管道的相应抗抗体和其发生反应而被吸附在管道表面，实现对该抗抗体的亲和富集，富集后荧光密度增加15倍。表面修饰技术能实现蛋白质在二氧化硅表面的固定并保持其生物活性，结合微流控芯片能实现对相应蛋白质的微量富集。     

PMMA微流控检测芯片的设计与制作

设计并制作了一种PMMA（polymethyl methacrylate）材料的微流控检测芯片,将外界气体驱动液体用于实际水样的分析和检测．利用精密加工的方法加工出芯片的整体尺寸为86mm×60mm×4．5mm．采用溶胶-凝胶的改性方法对微通道管路进行亲水处理,正硅酸乙酯的水解缩合生成了一层溶胶．凝胶覆盖在PMMA表面,从而大大提高了亲水性．在室温下对芯片进行键合,溶剂为二氯乙烷和无水乙醇按1：1混合的混合液．该方法避免了微通道的坍塌,有效防止了堵塞．实验证明,芯片接触紧密,且冲击强度能够满足要求．同时,芯片上集成了多个阀．阀膜选用0．5mm厚的硅胶膜,采用硅橡胶做黏合剂     

集成一次性微流控芯片的便携式荧光检测系统

本文研究了一种新型的生物化学分析系统,该系统包括便携式荧光检测仪和带光纤的微流控芯片．采用基于MEMS技术的微泵将待测物与荧光试剂的混合物导入微流控芯片,采用PMT检测受激发产生的荧光,荧光强度与待测物浓度成一定比例．激发光则通过光纤将光源LED光信号导入微沟道中．随着液体在微沟道中的流动,可连续分析和检测不同的样品．该系统检测1～1000μg／L浓度的荧光素具有0．966的相关系数．基于荧光猝灭原理,该系统还可检测浓度为5ng／μL的硝基化合物．该生化分析系统除具有便携式和一次性微流控芯片优点外,还具有成本低．试剂、样品消耗量少,且分析时间短等优点该系统能实现现场检测,可应用于临床诊断、环境检测及生物战剂检测等领域     

集成高梯度磁场分离结构的微流控芯片快速制作法

随着MEMS技术和免疫磁珠技术的不断发展,平面电磁线圈作为控制纳米磁珠在微流体中运动的关键部件,受到广泛关注和研究.但其复杂的加工工艺,较低的磁珠捕获效率以及电磁线圈的热效应,限制了它在微流控芯片中的进一步发展和应用.本文介绍了一种高梯度磁场分离微流控芯片,通过在芯片内部集成顺磁性的微柱结构,形成高磁场来捕获磁珠.采用基于SU-8多层模具和PDMS铸模工艺的快速加工方法,在芯片内部制作出顺磁性的微柱阵列.在外磁场磁化作用下,这些微柱能产生磁珠捕获所需的高梯度磁场,有效的进行磁珠操控和分离,通过蛋白捕获实验验证了芯片的可行性.该方法加工简单快捷,也不会带来电磁线圈的热效应问题.     

基于微流控芯片的一种蛋白检测方法

利用MEMS技术制作石英玻璃材料的微流控芯片,在自行研制的紫外可见吸收检测系统上,实现了芯片上对牛血清蛋白BSA、人免疫球蛋白IgG和人转铁蛋白TRF及它们的混合溶液的分离检测,结果重复性较好。实验提供了一种微流控芯片分离检测蛋白的手段,它是一种快速低成本的检测蛋白的方法。     

微流控技术在法医DNA快速检验方面的应用

微流控芯片技术因其微型化、自动化、高通量、集成化、快速等优势使得实验室研究产生革命性的变化,并在生物化学、医学等诸多领域得到广泛应用,但目前还没有基于微流控芯片技术的国产全集成自动化DNA分析仪。该文总结微流控技术在DNA提取、PCR扩增、电泳分离等DNA检验流程方面的研究现状与进展,尤其是在法医DNA快速检验方面的研究进展,同时介绍国内外全集成式DNA分析的研究状况。全集成与功能化是目前微流控技术研究的主流方向。未来,以微流控芯片为主导的全自动、便携式、集成化的DNA分析系统,将使得法医DNA检验从实验室走进案件现场甚至日常生活,实现真正的快速即时检验。     

微流控芯片制作及电特性研究

采用热压和键合的方法制作玻璃和有机聚合物(PMMA)芯片,对玻璃和PMMA芯片在高压直流电场作用下的伏安特性进行了研究和分析。实验表明,玻璃芯片的伏安线性区域为1100V,PMMA芯片为700V,由于玻璃的导热性能优于PMMA,所以玻璃芯片的伏安线性区域大于PMMA芯片。在此线性段内,根据基尔霍夫电流定律将芯片简化为等效电阻模型,研究了分离电压以及分离焦耳热对芯片分离效果的影响因素,为微流控芯片的优化设计提供了理论依据。     

Bubble-free operation of a microfluidic free-flow electrophoresis chip with integrated Pt electrodes

In order to ensure a stable and efficient separation in microfluidic free-flow electrophoresis (FFE) devices, various methods and chips have been presented until now. A major concern hereby is the generation of gas bubbles caused by electrolysis and the resulting disturbances in the position of the separated analyte lanes. Instable lane positions would lead to a decreased resolution in sample collection over time which certainly would be problematic when incorporating a stationary detector system. In contrast to our previous publications, in which we implemented laborious semipermeable membranes to keep bubbles outside the separation region, here we describe an electrochemical approach to suppress the electrolysis of water molecules and therefore bubble formation. This approach allowed a simpler and additionally a closed chip device with integrated platinum electrodes. With the use of this chip, the successful separation of three fluorescent compounds was demonstrated. Quinhydrone, which is a complex of hydroquinone and p-benzoquinone, was added only to the local flow streams along the electrodes, preventing mixing with the separation media and sample. The electrical current was generated via the oxidization and reduction of hydroquinone and p-benzoquinone up to a certain limit of the electrical current without gas formation. The separation stability was investigated for the chip with and without quinhydrone, and the results clearly indicated the improvement. In contrast to the device operating without quinhydrone, a 2.5-fold increase in resolution was achieved. Furthermore, separation was demonstrated within tens of milliseconds. This chemical approach with its high miniaturization possibilities offers an interesting alternative, in particular for low-current miniaturized FFE systems, in which large and open electrode reservoirs are not tolerable.     

应用微流体混合器芯片于食品中甲醛含量快速检测

本研究利用微流体芯片配合雷射激发荧光侦测法,侦测食品中不当添加物一甲醛,实验中以4-amino-3-penten-2-one(Fluoral-P)和甲醛作用,并于微流体混合器中进行反应及荧光衍生化以快速侦测.在微流体芯片中过高浓度甲醛荧光衍生物,因荧光分子基团讯号集中,易干扰全波长与荧光的侦测,因此在微流体芯片的检测以100 ppm以下为主.而在微流体混合器方面,本研究利用1:7型式不对称管道之微流体混合器,甲醛于此芯片中行荧光侦测法测定,随着甲醛浓度的降低,荧光讯号愈平缓,易出现噪声,侦测极限可达0.4 ppm且其线性度为R2=0.9954,而浓度低于1 ppm时,其S/N比表现度随之降低,此系统可提供较短分析时间、低试剂与成本消耗.     

环烯烃聚合物(COP)微流控芯片的制备及其与聚甲基丙烯酸甲酯(PMMA)微流控芯片的性能对比

采用热压方法制备了环烯烃聚合物(COP)微流控芯片.考虑到温度对微结构热压成形的质量影响最大,基于材料的粘弹性特性,通过变温准蠕变实验获得了热压参考温度Tr.实验证明,在该温度下热压成形,宽度和深度方向的复制精度分别达到了97.6%和94.3%.为了研究制备的COP微流控芯片的性能,将其和同一模具制备的PMMA微流控芯片进行了性能对比实验.通过背景荧光实验、电泳实验和DNA分析实验三方面的研究表明,与PMMA芯片相比,COP芯片背景荧光低,电泳效率高,检测重现性相对标准偏差小于2.5%,适用于生化分析.     

Velocity measurement of particles flowing in a microfluidic chip using Shah convolution Fourier transform detection

A noninvasive radiative technique, based on Shah convolution Fourier transform detection, for velocity measurement of particles in fluid flows in a microfluidic chip, is presented. It boasts a simpler instrumental setup and optical alignment than existing measurement methods and a wide dynamic range of velocities measurable. A glass-PDMS microchip with a layer of patterned Cr to provide multiple detection windows which are 40 microns wide and 70 microns apart is employed. The velocities of fluorescent microspheres, which were electrokinetically driven in the channel of the microfluidic chip, were determined. The effects of increasing the number of detection windows and sampling period were investigated. This technique could have wide applications, ranging from the determination of the velocity of particles in pressure-driven flow to the measurement of electrophoretic mobilities of single biological cells.     

Lensfree sensing on a microfluidic chip using plasmonic nanoapertures

We demonstrate lensfree on-chip sensing within a microfluidic channel using plasmonic nanoapertures that are illuminated by a partially coherent quasimonochromatic source. In this approach, lensfree diffraction patterns of metallic nanoapertures located at the bottom of a microfluidic channel are recorded using an optoelectronic sensor-array. These lensfree diffraction patterns can then be rapidly processed, using phase recovery techniques, to back propagate the optical fields to an arbitrary depth, creating digitally focused complex transmission patterns. Cross correlation of these patterns enables lensfree on-chip sensing of the local refractive index surrounding the near-field of the plasmonic nanoapertures. Based on this principle, we experimentally demonstrate lensfree sensing of refractive index changes as small as ～2×10(-3). This on-chip sensing approach could be quite useful for development of label-free microarray technologies by multiplexing thousands of plasmonic structures on the same microfluidic chip, which can significantly increase the throughput of sensing.     

Ultrasonic mixing in microfluidic channels using integrated transducers

This paper presents a microfluidic mixer that uses acoustic stirring created by ultrasonic waves. The ultrasound is introduced into the channel by integrated piezoelectric transducers. The transducers are made of a zinc oxide thin film, which is deposited on the bottom surface of a quartz substrate. The poly(dimethylsiloxane) channel is aligned to the transducers on the top surface of the substrate. The transducers are designed for operation around 450 MHz. The main mechanism of the mixing is the acoustic stirring of the fluid perpendicular to the flow direction. The radiation pressure that is generated by the transducer causes the stirring inside the microfluidic channel. The performance of the mixer is characterized by mixing phenolphthalein solution and sodium hydroxide dissolved in ethyl alcohol. Flow rates on the order of 1-100 microL/min are used. The transducers are driven by 1.2 V(rms) sinusoidal voltages at 450 MHz.     

Cytometry and velocimetry on a microfluidic chip using polyelectrolytic salt bridges

This paper reports a polyelectrolytic salt bridge-based electrode (PSBE), which is a key embedded unit in a microchip device that can size-selectively count microparticles and measure their velocities. The construction of salt bridges at specific locations within a microfluidic chip enables dc-driven electrical detection to be performed successfully. This is expected to be a competitive alternative to the optical methods currently used in conventional cell sorters. The PSBEs were fabricated by irradiating ultraviolet light over a patterned mask on the parts of interest, which were filled with an aqueous monomer solution containing diallyldimethylammonium chloride. A pair of such PSBEs was easily formed at the two lateral branches perpendicular to the main microchannel and was found to be very useful for dc impedometry. The human blood cells as well as the fluorescent microbeads passing between the two PSBEs produced impedance signals in proportional to their size. The information about the velocity of a microparticle was extracted from a doublet of the dc impedance signals, which were generated when cells or microbeads sequentially passed through two PSBE pairs separated from each other by a fixed distance. The plot of peak amplitude versus velocity of the moving microbeads and cells indicated only a slight correlation between the size and the velocity, which means that the peak amplitude of the dc impedance signals alone can provide information about the size of the cells in a mixture. The experimental results showed a screening rate of over 1000 cells s(-1) and a velocity of the cells of over 100 mm s(-1). Compared with the previously suggested electrical detection system based on metal electrodes, the sensitivity and selectivity in cell detection were remarkably improved. In addition, the detection unit including the operating circuit became innovatively simple and the whole device could be miniaturized.     

Quantitative polymerase chain reaction using infrared heating on a microfluidic chip

The IR-mediated polymerase chain reaction (IR-PCR) in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of the IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR long-pass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 templates at different starting copy numbers were amplified, and the threshold cycle showed an increase for decreasing amounts of starting DNA. The amplification efficiency was 80%, and the gel separation indicated no detectable nonspecific product. A melting curve was generated using IR heating, and this indicated a melting temperature of 85 °C for the 304 bp amplicon, which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification, and in highly integrated systems that combine pre- and post-PCR processes.     

Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 microL of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in mu-total analysis systems (mu-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.