Automated electric valve for electrokinetic separation in a networked microfluidic chip

This paper describes an automated electric valve system designed to reduce dispersion and sample loss into a side channel when an electrokinetically mobilized concentration zone passes a T-junction in a networked microfluidic chip. One way to reduce dispersion is to control current streamlines since charged species are driven along them in the absence of electroosmotic flow. Computer simulations demonstrate that dispersion and sample loss can be reduced by applying a constant additional electric field in the side channel to straighten current streamlines in linear electrokinetic flow (zone electrophoresis). This additional electric field was provided by a pair of platinum microelectrodes integrated into the chip in the vicinity of the T-junction. Both simulations and experiments of this electric valve with constant valve voltages were shown to provide unsatisfactory valve performance during nonlinear electrophoresis (isotachophoresis). On the basis of these results, however, an automated electric valve system was developed with improved valve performance. Experiments conducted with this system showed decreased dispersion and increased reproducibility as protein zones isotachophoretically passed the T-junction. Simulations of the automated electric valve offer further support that the desired shape of current streamlines was maintained at the T-junction during isotachophoresis. Valve performance was evaluated at different valve currents based on statistical variance due to dispersion. With the automated control system, two integrated microelectrodes provide an effective way to manipulate current streamlines, thus acting as an electric valve for charged species in electrokinetic separations.     

Integrated microfluidic device for solid-phase extraction coupled to micellar electrokinetic chromatography separation

An integrated microdevice was utilized for the autonomous coupling of solid-phase extraction (SPE) to micellar electrokinetic chromatography (MEKC). Porous plugs of polymethacrylate polymer approximately 200 microm in length) were fabricated by ultraviolet irradiation in microchannels. Microcolumns of hydrophobic beads packed against the polymethacrylate plugs were utilized for the quantitative extraction of rhodamine B, yielding preconcentration factors over 200 for a 90-s extraction. The calculated detection limit for this dye was 60 fM. A sample of coumarin dyes were concentrated by SPE, eluted in a nonaqueous solvent from a separate on-chip reservoir, and injected by a gated valve onto a separate column for MEKC analysis. Using the integrated device, a completely automated sequence of extraction, elution, injection, separation, and detection were performed in less than 5 min. Observed separation efficiencies were high, with plate heights below 2 microm. The analysis was at least 3 times faster than semiautomated, conventional, solid-phase extraction, while requiring no user intervention. The design, fabrication, and autonomous operation of the device is discussed.     

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.     

Voltammetry on microfluidic chip platforms

Microfluidic chip devices are shown to be attractive platforms for performing microscale voltammetric analysis and for integrating voltammetric procedures with on-chip chemical reactions and fluid manipulations. Linear-sweep, square-wave, and adsorptive-stripping voltammograms are recorded while electrokinetically "pumping" the sample through the microchannels. The adaptation of voltammetric techniques to microfluidic chip operation requires an assessment of the effect of relevant experimental variables, particularly the high voltage used for driving the electroosmotic flow, upon the background current, potential window, and size or potential of the voltammetric signal. The exact potential window of the chip detector is dependent upon the driving voltage. Manipulation of the electroosmotic flow opens the door to hydrodynamic modulation (stopped-flow) and reversed-flow operations. The modulated analyte velocity permits compensation of the microchip voltammetric background. Reversal of the driving voltage polarity offers extended residence times in the detector compartment. Rapid square-wave voltammetry/flow injection operation allows a detection limit of 2 x 10(-12) mol (i.e., 2 pmol) of 2,4,6-trinitrotoluene (TNT) in connection with 47 nL of injected sample. The ability of integrating chemical reactions with voltammetric detection is demonstrated for adsorptive stripping measurements of trace nickel using the nickel-dimethylglyoxime model system. The voltammetric response is characterized using catechol, hydrazine, TNT, and nickel as test species. The ability to perform on-chip voltammertic protocols in advantageous over nanovial voltammetric operations that lack a liquid-handling capability. Coupling the versatility of microfluidic chips with the rich information content of voltammetry thus opens an array of future opportunities.     

Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification

An integrated platform is presented for rapid and sensitive protein identification by on-line protein digestion and analysis of digested proteins using electrospray ionization mass spectrometry or transient capillary isotachophoresis/capillary zone electrophoresis with mass spectrometry detection. A miniaturized membrane reactor is constructed by fabricating the microfluidic channels on a poly(dimethylsiloxane) substrate and coupling the microfluidics to a poly(vinylidene fluoride) porous membrane with the adsorbed trypsin. On the basis of he large surface area-to-volume ratio of porous membrane media, adsorbed trypsin onto the poly(vinylidene fluoride) membrane is employed for achieving ultrahigh catalytic turnover. The extent of protein digestion in a miniaturized membrane reactor can be directly controlled by the residence time of protein analytes inside the trypsin-adsorbed membrane, the reaction temperature, and the protein concentration. The resulting peptide mixtures can either be directly analyzed using electrospray ionization mass spectrometry or further concentrated and resolved by electrophoretic separations prior to the mass spectrometric analysis. This microfluidic system enables rapid identification of proteins in minutes instead of hours, consumes very little sample (nanogram or less), and provides on-line interface with upstream protein separation schemes for the analysis of complex protein mixtures such as cell lysates.     

PDMS微流控电泳芯片的快速制备

采用Protel软件绘制微流控沟道的形状,利用电路板制作技术加工出模具.该芯片由PDMS基片和PDMS盖片组成,微流控沟道位于基片上,深度和宽度分别为75μm和100μm,由盖片对其进行密封.考察了有绝缘漆模具和无绝缘漆模具制作的芯片的电泳分离情况.在所制作的PDMS微流控电泳芯片上对用异硫氰酸酯荧光素标记的氨基酸进行了电泳分离,当信噪比S/N=3时,最小检测浓度达到0.8×10-11mol/L.     

Gateable nanofluidic interconnects for multilayered microfluidic separation systems

The extension of microfluidic devices to include three-dimensional fluidic networks allows complex fluidic and chemical manipulations but requires innovative methods to interface fluidic layers. Externally controllable interconnects, employing nuclear track-etched polycarbonate membranes containing nanometer-diameter capillaries, are described that produce hybrid three-dimensional fluidic architectures. Controllable nanofluidic transfer is achieved by controlling applied bias, polarity, and density of the immobile nanopore surface charge and the impedance of the nanocapillary array relative to the microfluidic channels. Analyte transport between vertically separated microchannels has three stable transfer levels, corresponding to zero, reverse, and forward bias. The transfer can even depend on the properties of the analyte being transferred such as the molecular size, illustrating the flexible character of the analyte transfer. In a specific analysis implementation, nanochannel array gating is applied to capillary electrophoresis separations, allowing selected separated components to be isolated for further manipulation, thereby opening the way for preparative separations at attomole analyte mass levels.     

Multistage isoelectric focusing in a polymeric microfluidic chip

This paper reports a protocol that improves the resolving power of isoelectric focusing (IEF) in a polymeric microfluidic chip. This method couples several stages of IEF in series by first focusing proteins in a straight channel using broad-range ampholytes and then refocusing segments of the first channel into secondary channels that branch from the first one at T-junctions. Experiments demonstrate that several fluorescent proteins that had focused within a segment of the straight channel in the first stage were refocused at significantly higher resolution due to the shallower pH gradient and higher electrical field gradient. Two variants of green fluorescent protein from the second-stage IEF fractionation were further separated in a third stage. Three stages of IEF were completed in less than 25 min at electric field strengths ranging from 50 to 214 V/cm.     

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。     

Magnetic hydrophobic affinity nanobeads for lysozyme separation

N-Methacryloyl-l-phenylalanine (MAPA) containing poly(2-hydroxyethylmethacrylate) based magnetic [mag-poly(HEMA–MAPA)] nanobeads was prepared for lysozyme purification form chicken egg white. MAPA was synthesized by reacting methacryloyl chloride with l-phenylalanine methyl ester and provided hydrophobic functionality to the nanobeads. Size of mag-poly(HEMA–MAPA) nanobeads was 386 nm and obtained by surfactant free emulsion polymerization of HEMA and MAPA having a specific surface area of 580 m²/g. Mag-poly(HEMA–MAPA) nanobeads were characterized by FTIR, AFM, TEM, ESR, and elemental analysis. Lysozyme adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, protein concentration, temperature, salt type). Lysozyme adsorption capacity of mag-poly(HEMA) and mag-poly(HEMA–MAPA) nanobeads from aqueous solutions was estimated as 24 and 517 mg/g, respectively. Lysozyme molecules were desorbed with 50% ethylene glycol solution with 98% recovery. It was observed that mag-poly(HEMA–MAPA) nanobeads can be used without significant decrease in lysozyme adsorption capacity after ten adsorption–desorption cycles. Mag-poly(HEMA–MAPA) nanobeads was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE.     

低电压驱动微流控芯片二氧化硅绝缘薄膜的研究

采用电子束蒸发法在玻璃基底上制备了二氧化硅薄膜,利用原子力显微镜（AFM）、台阶仪、X射线衍射仪（XRD）,分别对不同条件下制备的二氧化硅薄膜的表面形貌、膜厚、结构进行了表征,并采用金属/绝缘膜/金属（MIM）结构对薄膜的I-V电学特性进行了分析。结果表明玻璃基底温度在300℃条件下生长的4μm厚度的二氧化硅薄膜,其表面均匀平整,耐压能力〉200V,能够承受500kV/cm以上的场强,满足作为低电压驱动微流控芯片绝缘薄膜的要求,并在样品驱动的应用中得到验证。     

肿瘤微血管芯片的制备及其应用

肿瘤血管结构不同于正常组织血管,由内皮细胞松散地围成不完整的管道,呈高通透性.为建立体外简便、有效、低廉的肿瘤血管模型,本研究采用微加工技术,制作了肿瘤血管微流控芯片.芯片包括24 mm长50 μm宽40 μm深的微管道和一侧的微腔体以及两者之间的微缝.微管道中部设有膨胀区域以模拟肿瘤血管结构.在微管道内导入血管内皮细...     

A polymeric microfluidic chip for CE/MS determination of small molecules

A polymeric microfluidic chip made of Zeonor 1020 was fabricated using conventional embossing techniques to perform capillary electrophoresis for selected ion monitoring and selected reaction monitoring mass spectrometric detection of small molecules. A silicon master was microfabricated using photolithographic and dry etching processes. The microfluidic channel was embossed in the plastic from a silicon master. The embossed chip was thermally bonded with a Zeonor 1020 cover to form an enclosed channel. This channel (60-microm width, 20-microm depth, 2.0- and 3.5-cm length) provided capillary electrophoresis (CE) separation of polar small molecules without surface treatment of the polymer. A microsprayer coupled via a microliquid junction provided direct electrospray mass spectrometric detection of CE-separated components. An electric field of 0.5-2 kV/cm applied between the microsprayer and a separation buffer reservoir produced a separation of carnitine, acylcarnitine, and butylcarnitine with separation efficiencies ranging from 1,650 to 18,000 plates. Injection quantities of 0.2 nmol of these compounds produced a separation of the targeted polar small molecules without surface treatment of the polymer-abundant ion current signals and baseline separation of these compounds in less than 10 s. These results suggest the feasibility of polymeric chip-based devices for ion spray CE/MS applications.     

Development of a low-cost magnetic microfluidic chip for circulating tumour cell capture

The authors have developed a novel fabrication process for a selective micro-magnetic activated cell sorting (MACS) chip based on ferromagnetic material encapsulated micropillars (FMEMs), which is technically simple and low cost. The FMEM produces a high field gradient to magnetically attract, capture and hold cells on its interface. System test simulations were carried out to predict the efficacy of target capture and verify that the actual magnetic particles behaviour agreed well with model predictions. To determine the ability of the novel microMACS chip to capture circulating tumour cells (CTCs), SW620 human colon cancer cells were used in an in vitro flow model system and were able to be captured with the efficiency of 72.8%. The obvious accumulation of CTCs at a certain location on the chip suggested shear stress events at the pillar boundary may influence efficacy, and should be considered in further optimisation efforts.     

电泳芯片的低电压分离模型及讨论

电泳芯片是一种重要的微型分析仪器。针对目前电泳芯片分离电压高、不利于一体化集成等问题,利用在分离通道上分段、交替、循环施加电压的方法,提出电泳芯片的一种低电压分离模型,以降低电泳芯片的分离电压。同时,对影响电压降低的相关因素进行了详细的讨论,为这种低电压电泳芯片的设计提供了一定的理论基础。     

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.     

Passively driven integrated microfluidic system for separation of motile sperm

This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.     

Selective ion extraction: a separation method for microfluidic devices

A separation concept, selective ion extraction (SIE), is proposed on the basis of the combination of hydrodynamic and electrokinetic flow controls in microfluidic devices. Using a control system with multiple pressure and voltage sources, the hydrodynamic flow and electric field in any section of the microfluidic network can be set to desired values. Mixtures of compounds sent into a T-junction on a chip can be completely separated into different channels on the basis of their electrophoretic mobilities. A simple velocity balance model proved useful for predicting the voltage and pressure settings needed for separation. SIE provides a highly efficient separation with minimal additional dispersion. It is an ideal technique for high-throughput screening systems and demonstrates the power of lab-on-a-chip systems.     

Incubated protein reduction and digestion on an electrowetting-on-dielectric digital microfluidic chip for MALDI-MS

Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based techniques by developing a multifunctional electrode with closed-loop temperature control, while minimizing overall system complexity and addressing challenges associated with rapid evaporation. For the EWOD chip design, we discuss the performance of multifunctional surface electrodes for actuation, localized Joule heating, and thermistic temperature sensing. Furthermore, a hydrophilic pattern is formed in the multifunctional electrode to control the location of an evaporating droplet on the electrode. To demonstrate the capabilities and limitations of this technique, we performed three experiments and measured the results using MALDI-MS: (i) insulin disulfide reductions in dithiothreitol (DTT) over a range of heater temperatures (22-70 °C) to show how reaction rates can be affected by thermal control, (ii) insulin disulfide reductions at 130 °C in dimethyl sulfoxide (DMSO) to demonstrate a reaction in a high boiling point solvent, and (iii) tryptic digestions of cytochrome c at 22 and 40 °C to show that heated droplets can yield reasonably higher peptide sequence coverage than unheated droplets. Although they do not decouple the effects of changing temperatures and concentrations, these experiments verified that thermal cycling by EWOD electrodes accelerates reaction rates in liquid droplets in air.