Multilevel microfluidics via single-exposure photolithography

This communication reports a new method to form multilevel features in a single layer of SU-8 photoresist to facilitate the generation of 3D microfluidic chips. The method utilizes the spatial dependence of diffracted light intensity to selectively overexpose masked regions of photoresist and requires only a UV light source and a single transparency mask. 3D structures are formed within microfluidic channels using this selective overexposure method, with feature sizes being determined by the exposure dose and mask feature sizes. The dimensions of the internal features and the microfluidic channels can be varied independently according to these parameters, and any number of different heights can be obtained in a single exposure step. The method provides a simple means of forming 3D microfluidic structures with integrated features, including mixing structures, flow stabilization ridges, and separation weirs to increase the capabilities of microfluidic chips in a variety of microchemical applications.     

Contact printing of arrayed microstructures

A novel contact printing method utilizing a sacrificial layer of polyacrylic acid (PAA) was developed to selectively modify the upper surfaces of arrayed microstructures. The method was characterized by printing polystyrene onto SU-8 microstructures to create an improved substrate for a cell-based microarray platform. Experiments measuring cell growth on SU-8 arrays modified with polystyrene and fibronectin demonstrated improved growth of NIH 3T3 (93% vs. 38%), HeLa (97% vs. 77%), and HT1080 (76% vs. 20%) cells relative to that for the previously used coating method. In addition, use of the PAA sacrificial layer permitted the printing of functionalized polystyrene, carboxylate polystyrene nanospheres, and silica nanospheres onto the arrays in a facile manner. Finally, a high concentration of extracellular matrix materials (ECM), such as collagen (5 mg/mL) and gelatin (0.1%), was contact-printed onto the array structures using as little as 5 μL of the ECM reagent and without the formation of a continuous film bridge across the microstructures. Murine embryonic stem cells cultured on arrays printed with this gelatin hydrogel remained in an undifferentiated state indicating an adequate surface gelatin layer to maintain these cells over time.     

Fabrication of high-aspect-ratio polymer microstructures and hierarchical textures using carbon nanotube composite master molds

Scalable and cost effective patterning of polymer structures and their surface textures is essential to engineer material properties such as liquid wetting and dry adhesion, and to design artificial biological interfaces. Further, fabrication of high-aspect-ratio microstructures often requires controlled deep-etching methods or high-intensity exposure. We demonstrate that carbon nanotube (CNT) composites can be used as master molds for fabrication of high-aspect-ratio polymer microstructures having anisotropic nanoscale textures. The master molds are made by growth of vertically aligned CNT patterns, capillary densification of the CNTs using organic solvents, and capillary-driven infiltration of the CNT structures with SU-8. The composite master structures are then replicated in SU-8 using standard PDMS transfer molding methods. By this process, we fabricated a library of replicas including vertical micro-pillars, honeycomb lattices with sub-micron wall thickness and aspect ratios exceeding 50:1, and microwells with sloped sidewalls. This process enables batch manufacturing of polymer features that capture complex nanoscale shapes and textures, while requiring only optical lithography and conventional thermal processing.     

Simple photografting method to chemically modify and micropattern the surface of SU-8 photoresist

SU-8 has gained widespread acceptance as a negative photoresist. It is also finding increasing use as a structural material in microanalytical devices. Consequently, methods to tailor the surface properties of SU-8 as well as to micropattern coatings on the surface of SU-8 are needed. The SU-8 photoresist consists of EPON SU-8 resin mixed with the photoacid generator triarylsulfonium hexafluoroantimonate. This photoacid generator can also serve as a photoinitiator generating free radicals when illuminated with UV light. Under the appropriate conditions, sufficient triarylsulfonium hexafluoroantimonate remains within cured SU-8 to act as a source of free radicals and initiate UV-mediated grafting of polymers onto the surface of the SU-8. UV-mediated grafting was used to coat SU-8 surfaces with poly(acrylic acid) and other water-soluble monomers. The SU-8 surface was chemically micropatterned by placing a mask between the UV light and SU-8. The X-Y spatial resolution of micropatterned poly(acrylic acid) on the SU-8 surface was 2 mum. Three applications of these chemically modified SU-8 surfaces were demonstrated. In the first, poly(ethylene glycol) was used to protect the SU-8 from interactions with proteins, yielding a surface resistant to biofouling. In the second demonstration, the SU-8 surface was micropatterned with a cell-resistant layer to guide cellular attachment and growth. In the final application, SU-8 micropallets were encoded with polymer lines. The bar codes were read by either absorbance or fluorescence measurements. Thus, UV-mediated graft polymerization is an efficient and effective method to micropattern coatings onto the surface of SU-8.     

Rapid prototyping of poly(methyl methacrylate) microfluidic systems using solvent imprinting and bonding

We have developed a method for rapid prototyping of hard polymer microfluidic systems using solvent imprinting and bonding. We investigated the applicability of patterned SU-8 photoresist on glass as an easily fabricated template for solvent imprinting. Poly(methyl methacrylate) (PMMA) exposed to acetonitrile for 2 min then had an SU-8 template pressed into the surface for 10 min, which provided appropriately imprinted channels and a suitable surface for bonding. After a PMMA cover plate had also been exposed to acetonitrile for 2 min, the imprinted and top PMMA pieces could be bonded together at room temperature with appropriate pressure. The total fabrication time was less than 15 min. Under the optimized fabrication conditions, nearly 30 PMMA chips could be replicated using a single patterned SU-8 master with high chip-to-chip reproducibility. Relative standard deviations were 2.3% and 5.4% for the widths and depths of the replicated channels, respectively. Fluorescently labeled amino acid and peptide mixtures were baseline separated using these PMMA microchips in <15s. Theoretical plate numbers in excess of 5000 were obtained for a approximately 3 cm separation distance, and the migration time relative standard deviation for an amino acid peak was 1.5% for intra-day and 2.2% for inter-day analysis. This new solvent imprinting and bonding approach significantly simplifies the process for fabricating microfluidic structures in hard polymers such as PMMA.     

Large-area patterning of coinage-metal thin films using decal transfer lithography

We describe two new procedures that appear to hold significant promise as means for patterning thin-film microstructures of the coinage metals (Cu, Ag, Au). A feature central to both is the modification of their surfaces to promote the adhesive transfer of PDMS thin-film microstructures, a material suitable for use as resist layers in large-area patterning, using Decal Transfer Lithography (DTL). The present work provides a significant extension of the capabilities of DTL patterning, providing general protocols that can be used to transfer decal resists to essentially any substrate surface. The first method involves the functionalization of a surface, specifically those of gold and silver films with a thiol-terminated silane coupling agent, (mercaptopropyl)trimethoxysilane. This self-assembled monolayer, when hydrolyzed to its silanol form, provides a robust adhesion-promoting layer suitable for use in DTL patterning. The second method exploits the surface chemistry provided by the deposition of a nanoscale silicon dioxide thin-film capping layer using e-beam evaporation. This procedure provides an exceptional method for patterning large-area, thin-film microstructures of Cu-one compatible with micrometer-scale design rules-that are essentially defect free. Both surface modification strategies enable high-quality poly(dimethylsiloxane) decal transfers, and as the current work shows, these structures are suitable for large-area micrometer-sized patterning of gold, silver, and copper thin films via both wet-etching and lift-off procedures.     

Direct immobilization of cholesteryl-TEG-modified oligonucleotides onto hydrophobic SU-8 surfaces

We introduce a rapid, simple one-step procedure for the high-yield immobilization of cholesteryl-tetraethyleneglycol-modified oligonucleotides (chol-DNA) at hydrophobic sites made of SU-8 photoresist. Topographic structures of SU-8 were microfabricated on microscope glass coverslips sputtered with a Ti/Au layer. Upon application, chol-DNA adsorbed to the SU-8 structures from solution, leaving the surrounding gold surface free of chol-DNA. chol-DNA immobilization is complete within 15 min and yields a surface coverage in the range of 20-95 pmol/cm(2), which corresponds to a film density of 10(12)-10(13) molecules/cm(2). chol-DNA immobilization is stable and can be sustained despite rinsing, drying, dry storage for several hours, and rehydration of chips. Furthermore, complementary DNA in solution hybridizes efficiently to immobilized chol-DNA.     

Nano/microstructural effect of hydroxyapatite nanocrystals on hepatocyte cell aggregation and adhesion

Hepatocyte cell aggregation and adhesion to HAp nanocrystals covered with SU-8 polymer micropatterns by nano/microfabrication techniques is demonstrated. The surface roughness and wettability of the HAp nanocrystals are significantly different from those of the SU-8 polymer. QCM-D and microscopic observation clearly reveal that the cells realize the surface properties to form aggregation and preferentially adhere to the HAp nanocrystals at 2 h after seeding, indicating the importance of the microstructures as well as the interfacial phenomena at a nanometer scale.     

Novel potentiometric immunosensor for determination of diphtheria antigen based on compound nanoparticles and bilayer two-dimensional sol–gel as matrices

A novel method for fabrication of a diphtheria potentiometric immunosensor has been developed by means of self-assembling compound nanoparticles to a thiol-containing sol–gel network. A cleaned gold electrode was first immersed in a 3-mercaptopropyltrimethoxysilane (MPS) sol–gel solution to assemble a silica sol–gel monolayer. The silane entities were then polymerized into a two-dimensional sol–gel network (2D network) by dipping into aqueous NaOH. The second silane layer was formed by re-immersion in the MPS sol–gel solution overnight. The compound nanoparticles (nanocompounds) containing gold nanoparticles and silver nanoparticles were then chemisorbed on to the thiol groups of the second silane layer. Finally, diphtheria antibody (anti-Diph) was adsorbed on to the surface of the compound nanoparticles. The modified process was characterized by cyclic voltammetry (CV). Detection is based on the change in the potentiometric response before and after the antigen–antibody reaction. A direct potentiometric response to diphtheria antigen (Diph) was obtained from the immobilized diphtheria antibody. The potentiometric response of the resulting immunosensor was rapid and the linear range was from 22 to 800 ng mL^–1 with the linear regression equation E=–79.5+69.4 log [Diph] and a detection limit of 3.7 ng mL^–1 (at 3). Up to 19 successive assay cycles with retention of sensitivity were achieved for probes regenerated with 0.2 mol L^–1 glycine–hydrochloric acid (Gly–HCl) buffer solution. Moreover, analytical results from several serum samples obtained using the developed technique were in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtheria antigen in clinical diagnosis.     

Surface Functionalization of Micro Mechanical Cantilever Sensors by Organic Capped TiO2 and Fe2O3 Nanocrystals

A convenient and rapid procedure has been achieved to immobilize densely packed nanoporous 3D arrays of oleic acid (OLEA)-capped rod-shaped TiO₂ nanocrystals (NCs) and nearly spherical Fe₂O₃ NCs on the surface of micro mechanical cantilever sensors on SU-8. The NCs have been immobilized at room temperature and in the dark on the micro cantilevers before their release. AFM, SEM and XPS investigations attest for an effective and attachment of the NCs on the SU-8 which occurs with not modifying the original morphology and chemical composition of the nano-objects allowing for an effective accomplishment of the cantilever fabrication.     

Quantitative measurement of the near-field enhancement of nanostructures by two-photon polymerization

The quantitative determination of the strength of the near-field enhancement in and around nanostructures is essential for optimizing and using these structures for applications. We combine the gaussian intensity distribution of a laser profile and two-photon-polymerization of SU-8 to a suitable tool for the quantitative experimental measurement of the near-field enhancement of a nanostructure. Our results give a feedback to the results obtained by finite-difference time-domain (FDTD) simulations. The structures under investigation are gold nanotriangles on a glass substrate with 85 nm side length and a thickness of 40 nm. We compare the threshold fluence for polymerization for areas of the gaussian intensity profile with and without the near-field enhancement of the nanostructures. The experimentally obtained value of the near-field intensity enhancement is 600 ± 140, independent of the laser power, irradiation time, and spot size. The FDTD simulation shows a pointlike maximum of 2600 at the tip. In a more extended area with an approximate size close to the smallest polymerized structure of 25 nm in diameter, we find a value between 800 and 600. Using our novel approach, we determine the threshold fluence for polymerization of the commercially available photopolymerizable resin SU-8 by a femtosecond laser working at a wavelength of 795 nm and a repetition rate of 82 MHz to be 0.25 J/cm(2) almost independent of the irradiation time and the laser power used. This finding is important for future applications of the method because it enables one to use varying laser systems.     

Polymer microcantilever biochemical sensors with integrated polymer composites for electrical detection

Micro fabricated sensors based on nanomechanical motion with piezoresistive electrical readout have become a promising biochemical sensing tool. The conventional microcantilever materials are mostly silicon-based. The sensitivity of the sensor depends on Young's modulus of the structural material, thickness of the cantilever as well as on the gauge factor of the piezoresistor. UV patternable polymers such as SU-8 have a very low Young's modulus compared to the silicon-based materials. Polymer cantilevers with a piezoresistive material having a large gauge factor and a lower Young's modulus are therefore highly suited for sensing applications. In this work, a spin coatable and photopatternable mixture of carbon black (CB) and SU-8, with proper dispersion characteristics, has been demonstrated as a piezoresistive thin film for polymer microcantilevers. Results on percolation experiments of SU-8/CB composite and fabrication of piezoresistive SU-8 microcantilevers using this composite are presented. With our controlled dispersion experiments, we could get a uniform piezoresistive thin film of thickness less than 1.2 μm and resistivity of 2.7 Ω cm using 10 wt% of CB in SU-8. The overall thickness of the SU-8/composite/SU-8 is approximately 3 μm. We further present results on the electromechanical characterization and biofunctionalization of the cantilever structures for biochemical sensing applications. These cantilevers show a deflection sensitivity of 0.55 ppm/nm. Since the surface stress sensitivity is 4.1 × 10⁻³ [N/m]⁻¹, these cantilevers can well be used for detection of protein markers for pathological applications.     

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。     

Surface modification of SU-8 for enhanced biofunctionality and nonfouling properties

SU-8 is a chemically amplified, epoxy-based negative photoresist typically used for producing ultrathick resist layers during device manufacturing in the semiconductor industry. As a simple resist, SU-8 has garnered attention as a possible material for a variety of biomedical applications, including tissue engineering, drug delivery, as well as cell-based screening and sensing. However, as a hydrophobic material, the use of SU-8 is limited due to a high degree of nonspecific adsorption of biomolecules, as well as limited cell attachment. In this work, surface chemistry is utilized to modify the SU-8 surface by covalently attaching poly(ethylene glycol) (PEG) to increase biofunctionality and improve its nonfouling properties. Different molecular weights and concentrations of PEG were used to form films of various grafting densities on SU-8 surfaces. X-ray photoelectron spectroscopy (XPS) was used to verify the presence of PEG moieties on the SU-8 surface. High-resolution C1s spectra show that, with an increase in concentration and immobilization time, the grafting density of PEG also increases. Further, a standard overlayer model was used to calculate the thickness of the PEG films formed. The effect of PEG-modified SU-8 was examined in terms of protein adsorption on the surface and fibroblast-surface interactions.     

Functional lipid microstructures immobilized on a gold electrode for voltammetric biosensing of cholera toxin

Redox functionalized microstructures of diacetylene lipids containing cell surface ligand GM1 have been prepared for the construction of an electrochemical biosensor for cholera toxin from Vibrio cholerae. Incorporation of lipid molecules with disulfide functionality into the microstructures allows for firm attachment of the microstructures on a gold surface to form a sensing interface. The observed morphology of the microstructures is platelet, with size around 240 nm as determined by dynamic light scattering and transmission electron microscopy. The electrochemical response stems from electron transfer between the electrode and the redox sites on the microstructures, and the Faradaic current is influenced by the binding events of protein toxins to the ligands displayed on the crystalline surface. Electrochemical characterization indicates that electron transfer of surface ferrocene on the gold electrode is facile. Differential pulse voltammetry was used to measure the current magnitude as a function of toxin concentration, and a working range expanding from 1.0 x 10(-8) to 5.0 x 10(-7) M was obtained. Bovine serum albumin (BSA) was used as a control agent with which no interference to Faradaic response was found in the same concentration range. Atomic force microscopy (AFM) was used to characterize the morphology and distribution of microstructures on the gold surface. The effectiveness of the design for bypassing surface fouling of proteins in electrochemical detection has been demonstrated, and a binding regulated electron hopping mechanism for the observed electrochemical behavior has been proposed.     

A photopatternable silicone for biological applications

We show the application of a commercially available photopatternable silicone (PPS) that combines the advantageous features of both PDMS and SU-8 to address a critical bioMEMS materials deficiency. Using PPS, we demonstrate the ability to pattern free-standing mechanically isolated elastomeric structures on a silicon substrate: a feat that is challenging to accomplish using soft lithography-based fabrication. PPS readily integrates with many cell-based bioMEMS since it exhibits low autofluorescence and cells easily attach and proliferate on PPS-coated substrates. Because of its inherent photopatternable properties, PPS is compatible with standard microfabrication processes and easily aligns to complex featured substrates on a wafer scale. By leveraging PPS' unique properties, we demonstrate the design of a simple dielectrophoresis-based bioMEMS device for patterning mammalian cells. The key material properties and integration capabilities explored in this work should present new avenues for exploring silicone microstructures for the design and implementation of increasingly complex bioMEMS architectures.     

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.     

Simple fabrication of patterned gold nanoparticle arrays on functionalized block copolymer thin films

Patterned arrays of gold nanoparticles were fabricated using a simple dipping method that makes use of their specific interactions with nano-domains of carboxylic acid on a block copolymer template. Polystyrene-block-poly(tert-butyl acrylate) on the SU-8 photoresist pattern was selectively transformed to polystyrene-block-poly(acrylic acid). Au nanoparticles are selectively immobilized on the resulting carboxylic acid patterns to produce well-defined patterned Au nanoparticle arrays. This stable and robust template can be used to obtain any patterned nonaggregated metal or inorganic nanoparticle arrays.     

Plasmonic enhancement of the two photon absorption cross section of an organic chromophore using polyelectrolyte-coated gold nanorods

The effect of plasmonic enhancement on the two-photon absorption cross section of organic chromophores attached to polyelectrolyte-coated gold nanorods was investigated. The magnitudes of such enhancements were confirmed using single and two photon excitations of the chromophore molecules bound to polyelectrolyte-coated gold nanorods. By synthesizing two-, four-, six-, and eight-polyelectrolyte layer coated nanorods of a particular aspect ratio, the distance dependence of the evanescent electromagnetic field on molecular two-photon absorption was observed. Enhancements of 40-fold were observed for the chromophores nearest to the surface.     

Feasibility of SU-8-based capillary electrophoresis-electrospray ionization mass spectrometry microfluidic chips for the analysis of human cell lysates

Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, β-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.