Stable Non‐Covalent Large Area Patterning of Inert Teflon‐AF Surface: A New Approach to Multiscale Cell Guidance

Micro‐ and nano‐patterning of cell adhesion proteins is demonstrated to direct the growth of neural cells, viz. human neuroblastoma SHSY5Y, at precise positions on a strongly antifouling substrate of technolological interest. We adopt a soft‐lithographic approach with oxygen plasma modified PDMS stamps to pattern human laminin on Teflon‐AF films. These patterns are based on the interplay of capillary forces within the stamp and non‐covalent intermolecular and surface interactions. Remarkably, they remain stable for several days upon cell culture conditions. The fabrication of substrates with adjacent antifouling and adhesion‐promoting regions allows us to reach absolute spatial control in the positioning of neuroblastoma cells on the Teflon‐AF films. This patterning approach of a technologically‐relevant substrate can be of interest in tissue engineering and biosensing.     

On-chip amperometric measurement of quantal catecholamine release using transparent indium tin oxide electrodes

Carbon-fiber amperometry has been extensively used to monitor the time course of catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis, but microfabricated devices offer the promise of higher throughput. Here we report development of a microchip device that uses transparent indium tin oxide (ITO) electrodes to measure quantal exocytosis from cells in microfluidic channels. ITO films on a glass substrate were patterned as 20-mum-wide stripes using photolithography and wet etching and then coated with polylysine to facilitate cell adherence. Microfluidic channels (100 mum wide by 100 mum deep) were formed by molding poly(dimethylsiloxane) (PDMS) on photoresist and then reversibly sealing the PDMS slab to the ITO-glass substrate. Bovine adrenal chromaffin cells were loaded into the microfluidic channel and adhered to the ITO electrodes. Cells were stimulated to secrete by perfusing a depolarizing "high-K" solution while monitoring oxidation of catecholamines on the ITO electrode beneath the cell using amperometry. Amperometric spikes with charges ranging from 0.1 to 1.5 pC were recorded with a signal-to-noise ratio comparable to that of carbon-fiber electrodes. Further development of this approach will enable high-throughput measurement of quantal catecholamine release simultaneously with optical cell measurements such as fluorescence.     

Plasma induced patterning of polydimethylsiloxane surfaces

We report on a plasma-based method to fabricate chemical and physical patterns on polydimethylsiloxane (PDMS) surfaces. A copper TEM grid was placed on cured, planar (2D) and periodic (3D) PDMS surfaces, and the samples exposed to a low-pressure “air” plasma. The pattern of the grid was precisely replicated, forming hydrophilic channels only where the grid wires contacted the PDMS surface. Exposed regions of the surface between the mesh wires were not chemically modified and retained their hydrophobic character. This plasma-based procedure provides a simple, fast, and inexpensive method for creating patterned chemical functionalities on 2D and 3D PDMS surfaces for directed assembly and for the development of micro-scale sensors and bio-chip devices.     

Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane)

This paper describes a procedure that makes it possible to design and fabricate (including sealing) microfluidic systems in an elastomeric material [Formula: see text] poly(dimethylsiloxane) (PDMS) [Formula: see text] in less than 24 h. A network of microfluidic channels (with width >20 μm) is designed in a CAD program. This design is converted into a transparency by a high-resolution printer; this transparency is used as a mask in photolithography to create a master in positive relief photoresist. PDMS cast against the master yields a polymeric replica containing a network of channels. The surface of this replica, and that of a flat slab of PDMS, are oxidized in an oxygen plasma. These oxidized surfaces seal tightly and irreversibly when brought into conformal contact. Oxidized PDMS also seals irreversibly to other materials used in microfluidic systems, such as glass, silicon, silicon oxide, and oxidized polystyrene; a number of substrates for devices are, therefore, practical options. Oxidation of the PDMS has the additional advantage that it yields channels whose walls are negatively charged when in contact with neutral and basic aqueous solutions; these channels support electroosmotic pumping and can be filled easily with liquids with high surface energies (especially water). The performance of microfluidic systems prepared using this rapid prototyping technique has been evaluated by fabricating a miniaturized capillary electrophoresis system. Amino acids, charge ladders of positively and negatively charged proteins, and DNA fragments were separated in aqueous solutions with this system with resolution comparable to that obtained using fused silica capillaries.     

Patterned solvent delivery and etching for the fabrication of plastic microfluidic devices

A very simple method for micropatterning flat plastic substrates that can be used to build microfluidic devices is demonstrated. Patterned poly(dimethylsiloxane) elastomer is used as a template to control the flow path of an etching solvent through a channel design to be reproduced on the plastic substrate. The etching solvent was a acetone/ethanol mixture for poly(methyl methacrylate) substrates or a dimethylformamide/acetone mixture for polystyrene. The method is extremely fast in that duplicate plastic substrates can be patterned in just a few minutes each. We identified conditions that lead to smooth channel surfaces and characterized the rate of etching under these conditions. We determined that, for sufficiently short etching times (shallow channel depths), the etch rate is independent of the linear flow rate. This is very important since it means that the etch depth is approximately constant even in complex channel geometries where there will be a wide range of etchant flow rates at different positions in the pattern to be reproduced. We also demonstrate that the method can be used to produce channels with different depths on the same substrate as well as channels that intersect to form a continuous fluid junction. The method provides a nice alternative to existing methods to rapidly fabricate microfluidic devices in rigid plastics without the need for specialized equipment.     

Shaping Metallic Nanolattices: Design by Microcontact Printing from Wrinkled Stamps

A method for the fabrication of well‐defined metallic nanostructures is presented here in a simple and straightforward fashion. As an alternative to lithographic techniques, this routine employs microcontact printing utilizing wrinkled stamps, which are prepared from polydimethylsiloxane (PDMS), and includes the formation of hydrophobic stripe patterns on a substrate via the transfer of oligomeric PDMS. Subsequent backfilling of the interspaces between these stripes with a hydroxyl‐functional poly(2‐vinyl pyridine) then provides the basic pattern for the deposition of citrate‐stabilized gold nanoparticles promoted by electrostatic interaction. The resulting metallic nanostripes can be further customized by peeling off particles in a second microcontact printing step, which employs poly(ethylene imine) surface‐decorated wrinkled stamps, to form nanolattices. Due to the independent adjustability of the period dimensions of the wrinkled stamps and stamp orientation with respect to the substrate, particle arrays on the (sub)micro‐scale with various kinds of geometries are accessible in a straightforward fashion. This work provides an alternative, cost‐effective, and scalable surface‐patterning technique to fabricate nanolattice structures applicable to multiple types of functional nanoparticles. Being a top‐down method, this process could be readily implemented into, e.g., the fabrication of optical and sensing devices on a large scale.     

Micro-contact printing of polydiacetylene liposomes using hydrophilic stamps

Micron-sized polydiacetylene (PDA) liposome patterns have been fabricated on titanium (Ti) substrates using a micro-contact printing (micro-CP) technique. Two types of stamps (PDMS and agarose) and inking methods ("soaking" and "dropping") are used for micro-CP, and we compare their effect on the morphology of the PDA patterns. The size and morphology of the patterned PDA liposomes are analysized by optical and fluorescence microscopies and atomic force microscopy (AFM). When the agarose stamp is inked by the "dropping" method, PDA patterns are most efficiently transferred to the Ti substrate. However, the thickness of the transferred PDA patterns is not homogeneous, with the edge of the transferred pattern being thicker than its center. In contrast, when the PDMS stamp is used for micro-CP, the center of the pattern is thicker than the edge. Red fluorescence patterns are readily obtained by heat treatment of the PDA-immobilized solid substrate. The intensity of the fluorescence of the samples is consistent with the results of optical microscopy and AFM experiments.     

Micropatterning functional groups on polymer surfaces via masked vapor-phase photografting

Controlling cell behavior on biomaterial surface is the ultimate goal of cell and tissue engineering. Fabrication of biomaterials with alternatively hydrophilic/hydrophobic surface of parallel nanopatterned groves can provide biomaterial surfaces for the study of cell-surface interactions. In the present communication, masked vapor-phase photografting was used in patterning functional groups on flat polymeric substrates using poly (dimethylsiloxane) (PDMS) channels. Surface patterns were fabricated by UV-initiated photografting in the presence of a patterned PDMS mask. The approach is exemplified by patterning maleic anhydride (MAH) and acrylamide (AAm) onto poly (methyl methacrylate) (PMMA). The method offers another means to chemically functionalize and pattern polymer surface at the same time.     

Hydrogel-based microreactors as a functional component of microfluidic systems

A simple two-step method for fabricating poly(ethylene glycol) (PEG) hydrogel-based microreactors and microsensors within microfluidic channels is described. The intrachannel micropatches contain either a dye, which can report the pH of a solution within a fluidic channel, or enzymes that are able to selectively catalyze specific reactions. Analytes present within the microfluidic channel are able to diffuse into the micropatches, encounter the enzymes, and undergo conversion to products, and then the products interact with the coencapsulated dye to signal the presence of the original substrate. The micropatches are prepared by photopolymerizing the PEG precursor within the channel of a microfluidic system consisting of a poly(dimethylsiloxane) mold and a glass plate. Exposure takes place through a slit mask oriented perpendicular to the channel, so the size of the resulting micropatch is defined by the channel dimensions and the width of the slit mask. Following polymerization, the mold is removed, leaving behind the micropatch(es) atop the glass substrate. The final microfluidic device is assembled by irreversibly binding the hydrogel-patterned glass slide to a second PDMS mold that contains a larger channel. Multiple micropatches containing the same or different enzymes can be fabricated within a single channel. The viability of this approach is demonstrated by sensing glucose using micropatches copolymerized with glucose oxidase, horseradish peroxidase, and a pH-sensitive dye.     

Surface-directed, graft polymerization within microfluidic channels

We demonstrate a simple procedure to coat the surfaces of enclosed PDMS microchannels by UV-mediated graft polymerization. In prior applications, only disassembled channels could be coated by this method. This limited the utility of the method to coatings that could easily and tightly seal with themselves. By preadsorbing a photoinitiator onto the surface of PDMS microchannels, the rate of polymer formation at the surface was greatly accelerated compared to that in solution. Thus, a gel did not form in the lumen of enclosed microchannels. We demonstrate that the photoinitiator benzophenone remained on the surface of PDMS even after extensive washing. After addition of a variety of monomer solutions (acrylic acid, poly(ethylene glycol) monomethoxyl acrylate, or poly(ethylene glycol) diacrylate) and illumination with UV light, a stable, covalently attached surface coating formed in the microchannels. The electroosmotic mobility was stable in response to air exposure and to repeated cycles of hydration-dehydration of the coating. These surfaces also supported the electrophoretic separation of two model analytes. Placement of an opaque mask over a portion of the channel permitted photopatterning of the microchannels with a resolution of approximately 100 microm. By using an appropriate mixture of monomers combined with masks, it should be possible to fabricate PDMS microfluidic devices with distinct surface properties in different regions or channels.     

Fabrication of microfluidic devices containing patterned microwell arrays

A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 μm features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (≥20 μm) microchannel features and focused ion beam (FIB) milling for smaller (≤10 μm) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 μm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device.     

Controlling orientation of V(2)O(5) nanowires within micropatterns via microcontact printing combined with the gluing Langmuir-Blodgett technique

Previously, we suggested a facile method to transfer dioctadecyldimethylammonium bromide (DODAB)/V(2)O(5) nanowire hybrid patterns onto both hydrophobic and hydrophilic substrates via microcontact printing combined with the Langmuir-Blodgett (LB) technique (Park et al 2007 Nanotechnology 18 405301). Herein, we report on the delicate control of the orientation of V(2)O(5) nanowires within the micropatterns transferred via the gluing LB technique using a patterned polydimethylsilicate (PDMS) stamp. According to the orientation of the PDMS line patterns relative to the air-water interface, the aligned orientation of the nanowires, either parallel or perpendicular to the patterns, could be obtained and attributed to the moving direction of the water menisci formed between the PDMS stamp and water. In particular, addition of a small amount of ethanol in the subphase enhanced the dispersion of the DODAB at the air-water interface as well as the aggregation of V(2)O(5) nanowires, resulting in alignment of the V(2)O(5) nanowires via compression of the hybrid LB film by a barrier. Directional alignment of nanowires has potentially broad applications in the fabrication of aligned nanowire devices.     

Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing

A solid-object printer was used to produce masters for the fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS). The printer provides an alternative to photolithography for applications where features of > 250 microm are needed. Solid-object printing is capable of delivering objects that have dimensions as large as 250 x 190 x 200 mm (x, y, z) with feature sizes that can range from 10 cm to 250 microm. The user designs a device in 3-D in a CAD program, and the CAD file is used by the printer to fabricate a master directly without the need for a mask. The printer can produce complex structures, including multilevel features, in one unattended printing. The masters are robust and inexpensive and can be fabricated rapidly. Once a master was obtained, a PDMS replica was fabricated by molding against it and used to fabricate a microfluidic device. The capabilities of this method are demonstrated by fabricating devices that contain multilevel and tall features, devices that cover a large area (approximately 150 cm2), and devices that contain nonintersecting, crossing channels.     

Patterning enzymes inside microfluidic channels via photoattachment chemistry

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.     

Solution-phase surface modification in intact poly(dimethylsiloxane) microfluidic channels

An improved approach composed of an oxidation reaction in acidic H2O2 solution and a sequential silanization reaction using neat silane reagents for surface modification of poly(dimethylsiloxane) (PDMS) substrates was developed. This solution-phase approach is simple and convenient for some routine analytical applications in chemistry and biology laboratories and is designed for intact PDMS-based microfluidic devices, with no device postassembly required. Using this improved approach, two different functional groups, poly(ethylene glycol) (PEG) and amine (NH2), were introduced onto PDMS surfaces for passivation of nonspecific protein absorption and attachment of biomolecules, respectively. X-ray electron spectroscopy and temporal contact angle experiments were employed to monitor functional group transformation and dynamic characteristics of the PEG-grafted PDMS substrates; fluorescent protein solutions were introduced into the PEG-grafted PDMS microchannels to test their protein repelling characteristics. These analytical data indicate that the PEG-grafted PDMS surfaces exhibit improved short-term surface dynamics and robust long-term stability. The amino-grafted PDMS microchannels are also relatively stable and can be further activated for modifications with peptide, DNA, and protein on the surfaces of microfluidic channels. The resulting biomolecule-grafted PDMS microchannels can be utilized for cell immobilization and incubation, semiquantitative DNA hybridization, and immunoassay.     

Regulating oxygen levels in a microfluidic device

Microfluidic devices made from poly(dimethylsiloxane) (PDMS) are gas permeable and have been used to provide accurate on-chip oxygen regulation. However, pervaporation in PDMS devices can rapidly lead to dramatic changes in solution osmotic pressure. In the present study, we demonstrate a new method for on-chip oxygen control using pre-equilibrated aqueous solutions in gas-control channels to regulate the oxygen content in stagnant microfluidic test chambers. An off-chip gas exchanger is used to equilibrate each control solution prior to entering the chip. Using this strategy, problems due to pervaporation are considerably reduced. An integrated PDMS-based oxygen sensor allows accurate real-time measurements of the oxygen within the microfluidic chamber. The measurements were found to be consistent with predictions from finite-element modeling.     

A Flexible Piezoelectric Sensor for Microfluidic Applications Using Polyvinylidene Fluoride

A flexible technology for microfluidic applications using piezoelectric polyvinylidene fluoride (PVDF) and polydimethylsiloxane (PDMS) was developed. The flexible piezoelectric PVDF detects the flow rates and impulse pressure signal using piezoelectric characteristics. This study uses microelectromechanical systems (MEMS) technology to fabricate the sensing patterns on PVDF sheets, designs a molding transfer to form the microfluidic channels of the PDMS, and integrates them together. Experimental results show that PVDF films has good piezoelectricity at stretching ratio of 4, the flow rates ranged from 100 to 450 mL/min at dynamic controlling sensing, the miniature curvature radius is about 3 cm, and the cross section of the flexible microchannels is about 200 times 200 mum². The feasibility studies show that molding transfer is an appropriate low-cost technology for fabricating the flexible piezoelectric channels. The PVDF can be easily manufactured using MEMS process because it has a good mechanical strength and electrochemical stability in polymers.     

Formation of Single Micro‐ and Nanowires with Extreme Aspect Ratios in Microfluidic Channels

Shown here is the site‐specific formation of single extraordinarily long metal–organic micro‐ and nanowires using a microfluidic device made of poly(dimethylsiloxane) (PDMS). This approach exploits two concepts, i) the diffusion of organic precursor molecules through PDMS and ii) the use of microfluidic channels as a growth template. To initiate wire formation, metal and organic precursor solutions are filled into different supply channels that are separated by PDMS. As the precursor diffuses through PDMS, and thereby infiltrates the adjacent channel, the growth of micro‐ and nanowires starts at the side walls of this adjacent channel. The formation yields single wires with sizes ranging from several hundreds of micrometers to millimeters at diameters of 0.5–2 µm. The principles of this formation pathway are demonstrated with the reaction of tetrathiafulvalene (TTF) and gold(III) ions that yields Au‐TTF wires. The influence of various reaction parameters including the choice of solvents and the chip fabrication protocol on the reaction are evaluated. Based on these findings, a further microfluidic device design with orthogonally arranged channels is developed, and the formation of single wires in a channel‐defined pattern is demonstrated. Moreover, the possibility of pulsed precursor supply allows for advanced control over the growth of the wires.     

Addressable Microfluidic Polymer Chip for DNA‐Directed Immobilization of Oligonucleotide‐Tagged Compounds

A microfluidic polymer chip for the self‐assembly of DNA conjugates through DNA‐directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica‐casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS–glass microfluidic chip is equivalent to standard microscope slides (76 × 26 mm²). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA‐directed immobilization (DDI) of DNA‐modified gold nanoparticles (AuNPs) and DNA‐conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface‐modification process. In the case of the DNA–enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi‐enzyme microreactors.     

Electrodeposition in capillaries: bottom-up micro- and nanopatterning of functional materials on conductive substrates

A cost-effective and versatile methodology for bottom-up patterned growth of inorganic and metallic materials on the micro- and nanoscale is presented. Pulsed electrodeposition was employed to deposit arbitrary patterns of Ni, ZnO, and FeO(OH) of high quality, with lateral feature sizes down to 200-290 nm. The pattern was defined by an oxygen plasma-treated patterned PDMS mold in conformal contact with a conducting substrate and immersed in an electrolyte solution, so that the solid phases were deposited from the solution in the channels of the patterned mold. It is important that the distance between the entrance of the channels, and the location where deposition is needed, is kept limited. The as-formed patterns were characterized by high resolution scanning electron microscope, energy-dispersive X-ray analysis, atomic force microscopy, and X-ray diffraction.