Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters

A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000-80,000 N/m), excellent precision, good detection limit (450 nM) and resolution (0.90-1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices.     

Fabrication of SU-8 multilayer microstructures based on successive CMOS compatible adhesive bonding and releasing steps

This paper describes a novel fabrication process based on successive wafer-level bonding and releasing steps for stacking several patterned layers of the negative photoresist EPON SU-8. This work uses a polyimide film to enhance previous low temperature bonding technology. The film acts as a temporary substrate where the SU-8 is photopatterned. The poor adhesion between the polyimide film and SU-8 allows the film to be released after the bonding process, even though the film is still strong enough to carry out photolithography. Using this technique, successive adhesive bonding steps can be carried out to obtain complex 3-D multilayer structures. Interconnected channels with smooth vertical sidewalls and freestanding structures are fabricated. Unlike previous works, all the layers are photopatterned before the bonding process yielding sealed cavities and complex three-dimensional structures without using a sacrificial layer. Adding new SU-8 layers reduces the bonding quality because each additional layer decreases the thickness uniformity and increases the polymer crosslinking level. The effect of these parameters is quantified in this paper. This process guarantees compatibility with CMOS electronics and MEMS. Furthermore, the releasing step leaves the input and the output of the microchannels in contact with the outside world, avoiding the usual slow drilling process of a cover. Hence, in addition to the straightforward integration of electrodes on a chip, this fabrication method facilitates the packaging of these microfluidic devices.     

Design and fabrication of integrated solid-phase extraction-zone electrophoresis microchip

Integrated solid-phase extraction-zone electrophoresis (SPE-ZE) device has been designed and fabricated on microchip. The structures were fabricated by using multiple layers of SU-8 polymer with a novel technique that enables easy alignment and high yield of the chips. SU-8 adhesive bonding has two major advantages: it enables bonding of high aspect ratio pillars and it results in fully SU-8 microchannels with uniform electrokinetic flow properties. The SPE-ZE device has a fluidic reservoir with 15:1 high aspect ratio pillars for bead filters that act as a SPE part in the chip structure. The separation unit is a 25 mm long electrophoresis channel starting from the outlet of SPE reservoir. Argon laser-induced fluorescence (LIF) detector was used to monitor simultaneously the SPE reservoir and the detection site at the end of the electrophoresis channel. Flow characteristics and electric field distributions were simulated with Femlab software. Fluorescein was used as the analyte for detecting the operational performance of the chip. Adsorption, bead rinsing, elution and detection were tested to verify functioning of the chip design.     

Design and fabrication of a dielectrophoresis-based cell-positioning and cell-culture device for construction of cell networks

In this paper, we describe the design and fabrication of a dielectrophoresis (DEP)-based cell-positioning and cell-culture device for the construction of cell networks. This device enables both individual cell positioning and cell culture. Titanium electrodes were fabricated by deposition. Furthermore, microchambers and microchannels composed of SU-8, which is a negative photoresist, were used to carry out cell culture and enable cell differentiation. Using our device, N1E-115 cells were individually positioned in the microchambers, and the positioning yield was 45%. After positioning, the cells could be continuously cultured in the microchambers. Furthermore, the cells differentiated, and their neurites extended through the microchannels after cultivation for several days. These results indicate that our device greatly increases the prospects for individual cell positioning and can be used to construct cell networks that have several applications in the medical field, for example, in drug screening.     

Microchannel protein separation by electric field gradient focusing

A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel.     

Effect of exposure dose on the replication fidelity and profile of very high aspect ratio microchannels in SU-8

We are interested in using SU-8 dense gratings with very high aspect ratio microchannels as the master mold for fabrication of child molds needed for replication. For such applications, the sidewall taper angle and mask replication fidelity of SU-8 are very important. Increasing the exposure time was experimentally observed to decrease the width of the microchannel and the sidewall angle of SU-8 bars. A new diffraction-refraction-reflection model was also developed. The calculated microchannel width and sidewall angle at high exposure dose agreed well with the experimentally observed values indicating that reflection at the silicon substrate was significant. The larger than calculated actual microchannel width for low exposure dose was shown to be due to leaching of unreacted SU-8 in the developer. Dense gratings of high aspect ratio SU-8 bars separated by high aspect ratio (19.1) microchannels were also demonstrated.     

Characterization of SU-8 for electrokinetic microfluidic applications

The characterization of SU-8 microchannels for electrokinetic microfluidic applications is reported. The electroosmotic (EO) mobility in SU-8 microchannels was determined with respect to pH and ionic strength by the current monitoring method. Extensive electroosmotic flow (EOF), equal to that for glass microchannels, was observed at pH > or =4. The highest EO mobility was detected at pH > or =7 and was of the order of 5.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer. At pH < or =3 the electroosmotic flow was shown to reverse towards the anode and to reach a magnitude of 1.8 x 10(-4) cm(2) V(-1) s(-1) in 10 mM phosphate buffer (pH 2). Also the zeta-potential on the SU-8 surface was determined, employing lithographically defined SU-8 microparticles for which a similar pH dependence was observed. SU-8 microchannels were shown to perform repeateably from day to day and no aging effects were observed in long-term use.     

Fabrication of enclosed SU-8 tips for electrospray ionization-mass spectrometry

We describe a novel electrospray tip design for MS which is fabricated completely out of SU-8 photoepoxy. A three-layer SU-8 fabrication process provides fully enclosed channels and tips. The tip shape and alignment of all SU-8 layers is done lithographically and is therefore very accurate. Fabrication process enables easy integration of additional fluidic functions on the same chip. Separation channels can be made with exactly the same process. Fluidic inlets are made in SU-8 during the fabrication process and no drilling or other postprocessing is needed. Channels have been fabricated and tested in the size range of 10 microm x 10 microm-50 microm x 200 microm. Mass spectrometric performance of the tips has been demonstrated with both pressure-driven flow and EOF. SU-8 microtips have been shown to produce stable electrospray with EOF in a timescale of tens of minutes. With pressure driven flow stable spray is maintained for hours. Taylor cone was shown to be small in volume and well defined even with the largest channel cross section. The spray was also shown to be well directed with our tip design.     

Microfabricated solenoids and Helmholtz coils for NMR spectroscopy of mammalian cells

NMR-microprobes based on solenoids and Helmholtz coils have been microfabricated and NMR-spectra of mammalian cells have successfully been taken. The microfabrication technology developed for these probes consists of three electroplated copper levels for low resistance coils and three SU-8 layers for the integration of microchannels. This technology allows fabricating solenoids, Helmholtz and planar coils on the same wafer. The coils have inner diameters in the range of 160 to 400 microm and detection volumes of 5 to 22 nL. The solenoid and Helmholtz coils show improved RF-field characteristics compared to a planar coil fabricated with the same process. The fabricated solenoid has a particularly low resistance of only 0.46 Omega at 300 MHz. Moreover, it is very sensitive and has a very uniform RF-field, but shows large line width. The Helmholtz coils are slightly less sensitive, but display a far narrower line width, and are therefore a good compromise. With a Helmholtz coil, a SNR of 620 has been measured after one scan on 9 nL pure water. An NMR-microprobe based on a Helmholtz coil has also been used to take spectra of CHO cells that have been concentrated in the sensitive region of the coil with a mechanical filter integrated into the channel.     

Comparison between capillary and polyacrylamide gel electrophoresis for identification of Lolium species and cultivars.

In recent years variety discrimination has been achieved in a range of agricultural crops by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Previous works on genus Lolium have shown the effectiveness of this technique for the topic of cultivar identification. In the present research the potential of capillary gel electrophoresis (CGE) for identification of Italian (Lolium multiflorum Lam.), annual (Lolium rigidum Gaud.) and perennial (Lolium perenne L.) ryegrass cultivars in comparison with SDS-PAGE was investigated. Separation conditions of SDS-CGE were chosen in order to obtain electrophoretic data comparable with those of SDS-PAGE, at the expense of analysis speed. Both peak area and migration time of SDS-CGE electropherograms were reproducible. In the examined cultivars, a total of 27 (16.8-96.8 kDa) and 28 (13.2-111.5 kDa) protein subunits were detected by SDS-PAGE and SDS-CGE, respectively. The variability in seed storage protein composition was processed by numerical taxonomy. All cultivars were clearly identified by both electrophoresis systems. The orthogonality between SDS-PAGE and SDS-CGE was suggested by the lack of correlation among protein profiles obtained with the two separation systems. The nonredundant information from these analytical systems should provide a relevant benefit for identification of Lolium cultivars and wild biotypes that are extremely uniform phenotypically.     

SU-8 as a structural material for labs-on-chips and microelectromechanical systems

Since its introduction in the nineties, the negative resist SU-8 has been increasingly used in micro- and nanotechnologies. SU-8 has made the fabrication of high-aspect ratio structures accessible to labs with no high-end facilities such as X-ray lithography systems or deep reactive ion etching systems. These low-cost techniques have been applied not only in the fabrication of metallic parts or molds, but also in numerous other micromachining processes. Its ease of use has made SU-8 to be used in many applications, even when high-aspect ratios are not required. Beyond these pattern transfer applications, SU-8 has been used directly as a structural material for microelectromechanical systems and microfluidics due to its properties such as its excellent chemical resistance or the low Young modulus. In contrast to conventional resists, which are used temporally, SU-8 has been used as a permanent building material to fabricate microcomponents such as cantilevers, membranes, and microchannels. SU-8-based techniques have led to new low-temperature processes suitable for the fabrication of a wide range of objects, from the single component to the complete lab-on-chip. First, this article aims to review the different techniques and provides guidelines to the use of SU-8 as a structural material. Second, practical examples from our respective labs are presented.     

Pressure-driven sample injection with quantitative liquid dispensing for on-chip electrophoresis.

A novel pressure-driven sample injection method was developed as an alternative to electrokinetic injection, and electrophoretic separation was carried out on a microfabricated device employing this method. This method enables a defined volume of liquid dispensing, followed by instantaneous injection driven by pneumatic pressure, greatly simplifying the injection procedure. A particular microstructure, called a "metering chamber", has been designed for the quantitative dispensing of an ultra-low volume of sample liquid; a "hydrophobic passive valve" equipped with an air vent channel is employed for injecting a dispensed sample into the separation channel. The reproducibility of dispensing was 3.3% (n = 15), expressed by the variation of dispensed volumes. The electrophoretic separation of DNA fragments was performed using this injection method, varying the injection volumes from 0.45 to 4.0 nL, and the separation efficiencies were compared. This precise injection method, easily variable in injection volumes, is highly suitable for quantitative as well as qualitative electrophoretic analyses.     

Development of novel microscale system as immobilized enzyme bioreactor

This study involves a novel method for immobilized enzyme catalysis. The focus of the work was to design and construct a microscale bioreactor using microfabrication techniques traditionally employed within the semiconductor industry. Enzymes have been immobilized on the microreactor walls by incorporating them directly into the wall material. Fabricated microchannels have cross-sectional dimensions on the order of hundreds of micrometers, constructed using polydimethylsiloxane cast on silicon/SU-8 molds. The resulting ratio of high surface area to volume creates an efficient, continuous-flow reaction system. Transverse features also containing enzymes were molded directly into the channels.     

高聚物微流控芯片镍阳模的简易加工技术

提出了一种简便快速制作高聚物微流控芯片镍阳模的新方法。采用抛光镍片作为电铸基底,涂覆SU-8光胶层后,光刻得到SU-8微结构。以镍基片作为阳极,用16～30A/dm2的电流密度电解刻蚀5min,清除SU-8微结构间隙底部镍片表面的氧化物,并刻蚀得到10～20μm深的凹坑,有效地提高了随后电沉积镍结构和基底镍片间结合力。利用SU-8微结构作为电铸模板,以镍基片作为阴极,电铸5h后制得了微结构倾角为83°深宽比较大的镍阳模。实现了在普通化学实验室中长寿命镍阳模的制作。用热压法制得500多片聚甲基丙烯酸甲酯(PMMA)聚合物芯片,并成功用于DNA片段的分离。     

Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies

This review describes microfluidic systems in poly(dimethylsiloxane) (PDMS) for biological studies. Properties of PDMS that make it a suitable platform for miniaturized biological studies, techniques for fabricating PDMS microstructures, and methods for controlling fluid flow in microchannels are discussed. Biological procedures that have been miniaturized into PDMS-based microdevices include immunoassays, separation of proteins and DNA, sorting and manipulation of cells, studies of cells in microchannels exposed to laminar flows of fluids, and large-scale, combinatorial screening. The review emphasizes the advantages of miniaturization for biological analysis, such as efficiency of the device and special insights into cell biology.     

Microdevice for separation and quantitative fraction collection

A new microfluidic concept for quantitative whole-column fraction collection of electrophoretically separated zones was developed. The prototype device, fabricated on a polycarbonate disk by injection molding, integrated electrophoretic separation channels with fraction collection reservoirs distributed along the separation channel. The microdevice was designed in a CD-like format to use the centrifugal force for moving the liquid in the microchannels. A serpentine shape of the separation channel was selected to create segments for quantitative whole-column fraction collection. The operation was tested with visual monitoring of isotachophoretic separation and collection of cationic dyes.     

Application of magnetohydrodynamic actuation to continuous flow chemistry

Continuous flow microreactors with an annular microchannel for cyclical chemical reactions were fabricated by either bulk micromachining in silicon or by rapid prototyping using EPON SU-8. Fluid propulsion in these unusual microchannels was achieved using AC magnetohydrodynamic (MHD) actuation. This integrated micropumping mechanism obviates the use of moving parts by acting locally on the electrolyte, exploiting its inherent conductive nature. Both silicon and SU-8 microreactors were capable of MHD actuation, attaining fluid velocities of the order of 300 microm s(-1) when using a 500 mM KCl electrolyte. The polymerase chain reaction (PCR), a thermocycling process, was chosen as an illustrative example of a cyclical chemistry. Accordingly, temperature zones were provided to enable a thermal cycle during each revolution. With this approach, fluid velocity determines cycle duration. Here, we report device fabrication and performance, a model to accurately describe fluid circulation by MHD actuation, and compatibility issues relating to this approach to chemistry.     

High intensity light emitting diode array as an alternative exposure source for the fabrication of electrophoretic microfluidic devices

A commercially available array of light emitting diodes (LEDs), namely a UV Shark series LED high flux array, was evaluated as a light source for photolithographic patterning of SU-8 photoresist for the fabrication of templates suitable to make poly(dimethylsiloxane) (PDMS) microchips for electrophoresis. At a distance of 15 cm from the substrate, a relatively even intensity of 0.76+/-0.05 mW/cm(2) was obtained over an area sufficient for patterning a 10 cm (4 in.) silicon wafer. The exposure source was evaluated using a spiral mask design covering a 10 cm wafer. PDMS replicates of this template made in a 25 microm thick layer of SU-8 3025 showed little variation in width over the surface of the substrate, with a variation of 3.2% RSD (n=36) and a maximum range in widths of 7.8% of the mean channel width. The use of the optional metal reflector available with the LED array provided partial collimation of the light allowing near vertical structures to be produced across the entire wafer, something which was not possible without the reflector. SU-8 masters prepared using the LED array were compared to masters made using an alternative cheap lithographic source, namely a gel crosslinker. The SU-8 features were much narrower with the LED array than the crosslinker due to the multiple light sources in the crosslinker. A PDMS microchip made using a SU-8 template created using the Shark UV LED array was used for the electrophoretic separation of three anionic fluorescent dyes, with efficiencies up to 32,000 plates. Given that the LED array can be purchased and assembled for less than US$ 500, the Shark UV LED array is a promising alternative to more expensive lithographic light sources and will have significant appeal to many researchers wishing to undertake research in microfluidics around the world.     

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.     

Polyimide and SU-8 microfluidic devices manufactured by heat-depolymerizable sacrificial material technique

The following paper describes a sacrificial layer method for the manufacturing of microfluidic devices in polyimide and SU-8. The technique uses heat-depolymerizable polycarbonates embedded in polyimide or SU-8 for the generation of microchannels and sealed cavities. The volatile decomposition products originating from thermolysis of the sacrificial material escape out of the embedding material by diffusion through the cover layer. The fabrication process was studied experimentally and theoretically with a focus on the decomposition of the sacrificial materials and their diffusion through the polyimide or SU-8 cover layer. It is demonstrated that the sacrificial material removal process is independent of the actual channel geometry and advances linearly with time unlike conventional sacrificial layer techniques. The fabrication method provides a versatile and fast technique for the manufacturing of microfluidic devices for applications in the field of microTAS and Lab-on-a-Chip.