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.     

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser-printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift-off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120 degrees C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 microm width and 750 microm gap between them. The best results were recorded with a frequency of 400 kHz and 10 Vpp using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 microm wide x 6 microm deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13,000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 micromol/L, respectively. Besides the low-cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.     

Simple and rapid methods for the fabrication of polymeric and glass chips for using in analytical chemistry

This paper describes simple and rapid methods for the fabrication of glass and polymeric chips for routine analytical applications. The methods are easily interfaced to the general laboratory environment and do not require special clean room facilities or expensive instruments. Glass microchips were fabricated by etching with HF solution. Microfluidic channels were designed with CAD program and transferred onto a sheet of commercial polymeric self-adhesive (PSA) film by a cutter plotter. The PSA film was used as a mask for etching process. The etching rate was about 7 μm min⁻¹. A cover glass plate was sealed on the top of etched substrate by using polycellulose (cellophane). Polymeric microchips were fabricated by sawing with a jigsaw. Commercial polycarbonate (PC) was used as a substrate and two iron sheets were used as leader masks. While this restricts us to the fabrication of straight channels, it is however, much faster and less complicated than the other methods. The chip comprised three polymeric plates and the channels were created in the middle plate. Thermal bonding was used to bond three layers of the microfluidic chip. With this method, we could achieve simple channels with the width of about 200 μm. The channel depth depends on the polymeric plate thickness. Fabricated channels were accurate without any sinuosity or sideshow.     

A pressure driven injection system for an ultra-flat chromatographic microchannel

A pressure-actuated on-chip injection system has been developed that is compatible with shallow microchannels with a very large aspect ratio, i.e. 1 microm deep and up to 1000 microm wide. Such channels offer potential advantages in the miniaturisation of liquid chromatography and other separation methods as they allow high loadability and low sample dispersion at the same time. Computational fluid dynamics simulations were performed to predict the flow profiles and the transport of a sample in the system and to justify the injection principle. Based on these simulations, a prototype integrated into a chip for hydrodynamic chromatography has been realised and tested experimentally. The performance of the device is satisfactory and the results are in qualitative agreement with the numerical models.     

Comparison of capillary zone electrophoresis performance of powder-blasted and hydrogen fluoride-etched microchannels in glass

The applicability of glass chips with powder-blasted microchannels for electrophoretic separations was examined, and the performance was compared to microchannels etched with hydrogen fluoride (HF), using bicarbonate buffer and rhodamine B and fluorescein as model compounds. The measured electroosmotic mobilities in all chips were comparable, with values of ca. 7 x 10(-4) cm(2) V(-1)s(-1). The effect of electrical field strength and detection length on the separation efficiency was monitored. It was found that the main source of dispersion is of the Taylor-Aris type, which was discussed in relation to channel roughness differences. Although in powder-blasted channels with a separation length of 8.20 cm, 7-9 times lower plate numbers were obtained than in a HF-etched channel with similar dimensions, successful separation of five fluorescein isothiocyanate (FITC)-labeled amino acids was obtained on a powder-blasted chip within 80 s. Efficiencies of up to 360 000 plates/m were demonstrated on this chip, when a higher buffer concentration was used at a field strength of 664 V/cm. It can be concluded that powder-blasted microchannel chips, although they have a lower separation efficiency compared to HF-etched chips, perform well enough for many applications. Powder blasting can therefore be considered a low-cost and efficient alternative to HF etching, in particular because of the possibility to fabricate access holes through the glass with the same process.     

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.     

Poly(vinyl alcohol)-coated microfluidic devices for high-performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA-coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA-coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA-coated microchips for the separation of amines labeled with Alexa Fluor 350 even sub-second separations, utilizing a separation length of only 650 microm, could be obtained, while this was not possible using uncoated devices. By using PVA-coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA-coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA-coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.     

Packaging of microfluidic chips via interstitial bonding technique.

In this paper, we describe an interstitial bonding technique for packaging of microfluidic chips. The cover plate is first placed on top of the microfluidic chip, followed by dispensing the UV-curable resin into the resin-loading reservoirs. With the interstitial space between the cover plate and the microfluidic chip connecting to the loading reservoirs, the UV-curable resin wicks through capillary force action and hydrostatic pressure generated by the liquid level in the resin-loading reservoirs. When reaching the microchannels, the UV-curable resin stops flowing into the microchannels due to the force balance between the surface tension and hydrostatic pressure. The assembly is then placed under the UV light, followed by further curing in the thermal oven. It is found that there is no leakage from the bonded microfluidic chips and a good DNA separation result was obtained by using the microfluidic chips as fabricated. This bonding technique is relatively simple and fast, which can be applied to the packaging of microfluidic chips made from hybrid materials with complicated designs as long as the interstitial space connects to the loading reservoirs.     

Patterning microbeads inside poly(dimethylsiloxane) microfluidic channels and its application for immobilized microfluidic enzyme reactors

We propose a convenient and reliable approach for immobilizing microbeads on poly(dimethylsiloxane) (PDMS) microchips. It is built upon a simple fabrication procedure of PDMS chip through directly printing the master with an office laser printer which was described in our previous work (J. Chromatogr. A 2005, 1089, 270-275). On the printed toners used as the positive relief of the master, microbeads were immobilized by a thermal treatment and then transferred to the surface of the microchip by direct molding of the prepolymer on the master. With this approach, the region-selective immobilization of microbeads and the fabrication of PDMS microchips can be accomplished at the same time. Then, using these microbeads as supports, further modification with enzyme was achieved. Surface characteristics of the microbeads-modified PDMS microchannels were investigated with scanning electron microscope, atomic force microscope, and inverse fluorescence microscope. The electrokinetic properties of the native PDMS and the modified PDMS chips were also compared. Based on this approach, an immobilized glucose oxidase (GOD) reactor was constructed and the reaction using glucose as substrate was studied. All these experiments aim to show that the proposed approach may have a good potential in the study of biochemistry and other related areas.     

Immunosensing of Staphylococcus enterotoxin B (SEB) in milk with PDMS microfluidic systems using reinforced supported bilayer membranes (r-SBMs)

A versatile and novel method has been developed for microfluidic immunosensing of the food-borne pathogen Staphylococcus enterotoxin B (SEB) in poly(dimethylsiloxane) (PDMS) chips. Supported bilayer membranes (SBMs) were generated by vesicle fusion in oxidized PDMS microchannels for minimizing non-specific adsorption of biomolecules. The stability of SBMs was strengthened with a streptavidin layer to make them air-stable and allow for subsequent display of the biotin-functionalized antibodies. The reinforced supported bilayer membranes (r-SBMs) are fluid, exhibiting a lateral diffusion coefficient of approximately 1.9 microm(2) s(-1), and no detectable change of mobility was found after dehydration/rehydration. This is a substantial improvement over phosphatidylcholine (PC) membranes on PDMS, which suffered a roughly 10% reduction in the mobile fraction and 30% decrease in mobility after dehydration. Non-specific protein adsorption in the membrane-treated channels was reduced 100-1000 fold as compared to PDMS surfaces without a membrane coating. A flow-based microfluidic immunosensor for SEB was developed using antibodies linked to the r-SBMs in PDMS channels, and a detection limit of 0.5 ng mL(-1) was obtained from the linear portion of the calibration curve. The microchip was applied to detection of SEB in milk, and similar response and sensitivity were obtained, demonstrating the sensor's remarkable performance for real world samples. The r-SBMs overcome the stability hurdle in SBM-modified surfaces, opening up possibilities for transport and storage of membrane-functionalized microchips in the dehydrated form without compromising the performance, and facilitating the commercialization of disposable SBM-based microdevices.     

Faster and improved microchip electrophoresis using a capillary bundle

Joule heating generated in CE microchips is known to affect temperature gradient, electrophoretic mobility, diffusion of analytes, and ultimately the efficiency and reproducibility of the separation. One way of reducing the effect of Joule heating is to decrease the cross-section area of microchannels. Currently, due to the limit of fabrication technique and detection apparatus, the typical dimensions of CE microchannels are in the range of 50-200 microm. In this paper, we propose a novel approach of performing microchip CE in a bundle of extremely narrow channels by using photonic crystal fiber (PCF) as separation column. The PCF was simply encapsulated in a poly(methyl methacrylate) (PMMA) microchannel right after a T-shaped injector. CE was simultaneously but independently carried out in 54 narrow capillaries, each capillary with diameter of 3.7 microm. The capillary bundle could sustain high electric field strength up to 1000 V/cm due to efficient heat dissipation, thus faster and enhanced separation was attained.     

Microdevices for manipulation and accumulation of micro- and nanoparticles by dielectrophoresis

Microfluidic devices with three-dimensional (3-D) arrays of microelectrodes embedded in microchannels have been developed to study dielectrophoretic forces acting on synthetic micro- and nanoparticles. In particular, so-called deflector structures were used to separate particles according to their size and to enable accumulation of a fraction of interest into a small sample volume for further analysis. Particle velocity within the microchannels was measured by video microscopy and the hydrodynamic friction forces exerted on deflected particles were determined according to Stokes law. These results lead to an absolute measure of the dielectrophoretic forces and allowed for a quantitative test of the underlying theory. In summary, the influence of channel height, particle size, buffer composition, electric field, strength and frequency on the dielectrophoretic force and the effectiveness of dielectrophoretic deflection structures were determined. For this purpose, microfluidic devices have been developed comprising pairs of electrodes extending into fluid channels on both top and bottom side of the microfluidic channels. Electrodes were aligned under angles varying from 0 to 75 degrees with respect to the direction of flow. Devices with channel height varying between 5 and 50 microm were manufactured. Fabrication involved a dedicated bonding technology using a mask aligner and UV-curing adhesive. Particles with radius ranging from 250 nm to 12 microm were injected into the channels using aqueous buffer solutions.     

Microchip capillary electrophoresis–electrospray ionization–mass spectrometry of intact proteins using uncoated Ormocomp microchips

We present rapid (<5 min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis–electrospray ionization (CE–ESI) microchips. The microchips are fabricated fully of commercial inorganic–organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp–Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8–9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ∼10⁴ theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6–5.9% RSD, n = 4) and intact proteins (1.3–7.5% RSD, n = 3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology.     

A rejuvenation method for poly(N,N-dimethylacrylamide)-coated glass microfluidic chips

As microfluidic chips come to integrate the higher levels of functionality required for the implementation of advanced bioanalytical protocols, a crucial factor is that of cost. Although glass chips provide advantages in multilayer integrations, their cost is far higher than that of polymer chips. However, a simple and effective rejuvenation protocol for glass microchips may enable higher levels of integration and functionality on glass microchips. Here we present a method to rejuvenate glass microchips that had been used for capillary electrophoresis to the extent that their performance was degraded. This degradation was due to one of the two mechanisms: (i) a deterioration of the polymer coating on the inner surface of the microchannel or (ii) an aging of the glass substrate. Using the method presented here, we have rejuvenated more than 50 such "aged" microchips. The performance of these microchips was fully restored after the rejuvenation and lasted for hundreds of DNA separation runs. Our experiments indicate that the loss of resolution in microchip separations was not associated with glass aging, but was due to the degradation of the polymer coating on the inner surface of microchannels. This suggests that it is possible to extend the microchip lifetime "forever" using the rejuvenation protocol and that the exploration of higher levels of integration and functionality on glass microchips (or of hybrid structures involving materials capable of withstanding the reagents and elevated temperatures used) is feasible.     

一种可逆键合电泳微芯片的制作及在蛋白质分离中的应用

阐述了一种可逆键合电泳微芯片的制作方法, 以及电泳微芯片在蛋白质分离、临床尿蛋白检测方面的应用. 用标准光刻腐蚀技术在石英基片上腐蚀泳道, 清洗腐蚀好的基片和盖片后, 在真空条件下实现键合. 此种方法键合制作的电泳微芯片可重复键合使用, 制得的电泳微芯片成功地用于标准蛋白质分离以及临床尿蛋白分析.     

微流控芯片操纵传输及实时监测单细胞量子释放

微流控芯片技术用于细胞生化分析已引起了广泛关注.Harrison等首次在微流控芯片上对细胞群体进行操纵、传输及反应.yang等在微流控芯片上操纵细胞群体的排列,并用荧光检测细胞群体摄取钙的反应.至今还未见到微流控芯片对单个细胞进行操纵传输、定位及实时监测的报道.单细胞受激释放的监测对探索生物体神经传导具有重要意义.     

"Overpass" at the junction of a crossed microchannel: An enabler for 3D microfluidic chips

Reported here is the design and fabrication of three-dimensional (3D) "overpass" microstructures at the junction of crossed microfluidic channels by femtosecond laser direct writing of photopolymers. The post-integrated overpass could be used for guiding different microfluids across the junction without mixing; therefore it is proposed as an enabler for achieving 3D microfluidic chips based on conventional two-dimensional (2D) microchannels. As representative examples, bi-crossed and tri-crossed microchannels have been equipped with bi-connected and tri-connected overpasses, respectively. Flow tests confirm 3D flowing capability. The integration of such overpass structures at the microchannel junction provides an opportunity to impart 3D capability to conventional 2D microchips, thus the method may hold great promise for both functionalization and miniaturization of Lab-on-a-Chip systems.     

Velocity measurement of particulate flow in microfluidic channels using single point confocal fluorescence detection.

This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (0.93 microm diameter) passing through a focused laser beam at a variety of flow rates (50 nL min(-1)-8 microL min(-1)). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate velocity data for the flowing particles. Fluid manipulation is achieved by hydrodynamically pumping fluid through microchannels (150 microm wide and 50 microm deep) structured in a polydimethylsiloxane (PDMS) substrate. The mean fluorescence burst frequency is shown to be directly proportional to flow speed. Furthermore, the Poisson recurrence time and width of recovered autocorrelation curves is demonstrated to be inversely proportional to flow speed. The component-based confocal fluorescence detection system is simple and can be applied to a diversity of planar chip systems. In addition, velocity measurement only involves interrogation of the fluidic system at a single point along the flow stream, as opposed to more normal multiple-point measurements.     

Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.     

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.