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.     

Fabrication of poly(methyl methacrylate) capillary electrophoresis microchips by in situ surface polymerization

A novel method based on in situ surface polymerization of methyl methacrylate (MMA) has been developed for the rapid fabrication of poly(methyl methacrylate) (PMMA) capillary electrophoresis (CE) microchips. MMA containing both thermal and ultraviolet (UV) initiators was allowed to prepolymerize in a water bath to form a fast curing molding solution that was subsequently sandwiched between a nickel template and a PMMA plate. The images of the raised microchannels on the nickel template were precisely replicated into the synthesized PMMA substrates during the UV-initiated polymerization of the molding solution within 30 min under ambient temperature. The attractive performances of the novel PMMA microchips have been demonstrated in connection with amperometric detection for the separation and detection of several model analytes. The new approach significantly simplifies the process for fabricating PMMA devices and could be applied to other materials that undergo light-initiated polymerization.     

Near-infrared time-resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices

High aspect-ratio microstructures were hot-embossed in polymer substrates with a molding tool fabricated using lithography/electroplating/forming (LIGA). The resulting devices were used for the electrophoretic separation of oligonucleotides labeled with near-infrared (near-IR) dyes. Near-IR time-resolved fluorescence was used as an identification method for the labeling dyes. The detection apparatus consisted of a pulsed laser diode operating at 680 nm, a single-photon avalanche diode, an integrated microscope, and a PC-board incorporating time-correlated single photon counting electronics. Investigation of the optical quality and amount of autofluorescence generated from different polymer substrates was carried out in the near-IR region for determining compatibility with time-resolved fluorescence. Our results revealed that of several poly(methylmethacrylate)(PMMA) substrates, brand Plexiglas offered minimal replication errors in the embossed features using appropriate embossing conditions with low background fluorescence contributions to the observed decay. Near-IR dye-labeled oligonucleotides were separated to determine the applicability of fluorescence lifetime discrimination between Cy5.5 (tauf = 930 ps) and IRD700 (tauf = 851 ps) labeling dyes during the microchip separation. These dyes were used to label T-fragments (thymine) of an M13mp18 ssDNA template. The DNA ladders were electrophoresed at 130 V/cm in a 4% linear polyacrylamide gel (LPA) gel matrix in a 9.5 cm long serpentine channel heated to 50 degrees C. The electropherogram revealed that the lifetimes could be accurately read well beyond 450 bases, although single-base pair resolution in the electropherogram was difficult to achieve due to potential solute-wall interactions in the polymer microdevice or the electroosmotic flow (EOF) properties of the device. The relative standard deviations secured for individual bands in the electropherogram were similar to those obtained using capillary gel electrophoresis, in spite of the lower load volume.     

Towards disposable lab-on-a-chip: poly(methylmethacrylate) microchip electrophoresis device with electrochemical detection

A fully disposable microanalytical device based on combination of poly(methylmethacrylate) (PMMA) capillary electrophoresis microchips and thick-film electrochemical detector strips is described. Variables influencing the separation efficiency and amperometric response, including separation voltage or detection potential are assessed and optimized. The versatility, simplicity and low-cost advantages of the new design are coupled to an attractive analytical performance, with good precision (relative standard deviation RSD = 1.68% for n = 10). Applicability for assays of mixtures of hydrazine, phenolic compounds, and catecholamines is demonstrated. Such coupling of low-cost PMMA-based microchips with thick-film electrochemical detectors holds great promise for mass production of single-use micrototal analytical systems.     

Simple room temperature bonding of thermoplastics and poly(dimethylsiloxane)

We describe a simple and versatile method for bonding thermoplastics to elastomeric polydimethylsiloxane (PDMS) at room temperature. The bonding of various thermoplastics including polycarbonate (PC), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), and polystyrene (PS), to PDMS has been demonstrated at room temperature. An irreversible bonding was formed instantaneously when the thermoplastics, activated by oxygen plasma followed by aminopropyltriethoxysilane modification, were brought into contact with the plasma treated PDMS. The surface modified thermoplastics were characterized by water contact angle measurements and X-ray photoelectron spectroscopy. The tensile strength of the bonded hybrid devices fabricated with PC, COC, PMMA, and PS was found to be 430, 432, 385, and 388 kPa, respectively. The assembled devices showed high burst resistance at a maximum channel pressure achievable by an in-house built syringe pump, 528 kPa. Furthermore, they displayed very high hydrolytic stability; no significant change was observed even after the storage in water at 37 °C over a period of three weeks. In addition, this thermoplastic-to-PDMS bonding technique has been successfully employed to fabricate a relatively large sized device. For example, a lab-on-a-disc with a diameter of 12 cm showed no leakage when it spins for centrifugal fluidic pumping at a very high rotating speed of 6000 rpm.     

Two-dimensional nitrosylated protein fingerprinting by using poly (methyl methacrylate) microchips

S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (PTM) of cysteine, plays an important role in cellular functions and cell signalling pathways. Nitrosylated proteins are considered as biomarkers of aging and Alzheimer's disease (AD). Microfluidics has been widely used for development of novel tools for separation of protein mixtures. Here we demonstrate two-dimensional micro-electrophoresis (2D μ-CE) separations of nitrosylated proteins from the human colon epithelial adenocarcinoma cells (HT-29) and AD transgenic mice brain tissues. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimensional separations, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection was used with field strength at 200 V cm(-1). After 80 s separation in the first CGE dimension, fractions were successfully transferred to a second MEEKC dimension for a short 10 s separation. We first demonstrate this 2D μ-CE separation by resolving five standard proteins with molecular weight (MW) ranging from 20 to 64 kDa. We also present a high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following menadione (MQ) treatment to induce oxidative stress. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and their age matched controls were also generated. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in biological samples on a microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.     

Pulsed amperometric detection with poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips for the determination of EPA priority pollutants

A miniaturized analytical system for separation and detection of three EPA priority phenolic pollutants, based on a poly(dimethylsiloxane)-fabricated capillary electrophoresis microchip and pulsed amperometric detection is described. The approach offers a rapid (less than 2 min), simultaneous measurement of three phenolic pollutants: phenol, 4,6-dinitro-o-cresol and pentachlorophenol. The highly stable response (RSD = 6.1%) observed for repetitive injections (n > 100) reflects the effectiveness of Au working electrode cleaned by pulsed amperometric detection. The effect of solution conditions, separation potential and detection waveform were optimized for both the separation and detection of phenols. Under the optimum conditions (5.0 mM phosphate buffer pH = 12.4, detection potential: 0.7 V, separation potential: 1200 V, injection time: 10 s) the baseline separation of the three selected compounds was achieved. Limits of detection of 2.2 microM (2.8 fmol), 0.9 microM (1.1 fmol), and 1.3 microM (1.6 fmol) were achieved for phenol, 4,6-dinitro-o-cresol and pentachlorophenol, respectively. A local city water sample and two over-the-counter sore-throat medicines were analyzed in order to demonstrate the capabilities of the proposed technique to face real applications.     

Plasticizer-assisted bonding of poly(methyl methacrylate) microfluidic chips at low temperature

As an important phthalate plasticizer, dibutyl phthalate (DBP) was employed to decrease the bonding temperature of poly(methyl methacrylate) (PMMA) microfluidic chips in this work based on the fact that it can lower the glass transition temperature of PMMA. The channel plates of the PMMA microchips were fabricated by the UV-initiated polymerization of prepolymerized methyl methacrylate between a silicon template and a PMMA plate. Prior to bonding, DBP solution in isopropanol was coated on PMMA covers. When isopropanol in the coating was allowed to evaporate in air, DBP was left on the PMMA covers. Subsequently, the DBP-coated covers were bonded to the PMMA channel plates at 90 °C for 10 min under pressure. The channels in the complete microchips had been examined by optical microscope and scanning electron microscope. The results indicated that high quality bonding was achieved below the glass transition temperature of PMMA (∼105 °C). The performance of the PMMA microfluidic chips sealed by plasticizer-assisted bonding has been demonstrated by separating and detecting ionic species by capillary electrophoresis in connection with contactless conductivity detection.     

Ultraviolet sealing and poly(dimethylacrylamide) modification for poly(dimethylsiloxane)/glass microchips

Simple sealing methods for poly(dimethylsiloxane) (PDMS)/glass-based capillary electrophoresis (CE) microchips by UV irradiation are described. Further, we examined the possibility to modify the inner surface of separation channels, using polymethylacrylamide (PDMA) as a dynamic coating reagent. The surface properties of native PDMS, UV-irradiated PDMS, and PDMA-coated PDMS were systematically studied by atomic force microscopy (AFM), infrared absorption by attenuated total reflection infrared (ATR-IR) spectroscopy, and contact angle measurement. We found that PDMA forms a stable coating on PDMS and glass surfaces, eliminating the nonhomogeneous electroosmotic flow (EOF) in channels on PDMS/glass microchips, and improving the hydrophilicity of PDMS surfaces. Mixtures of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and fluorescein were separated in 35 s using PDMA-coated PDMS/glass microchips. A high efficiency of theoretical plates with at least 1365 (105 000 N/m) and a good reproducibility with relative standard deviations (RSD) below 4% in five successive separations were achieved.     

Crystallization of isotactic poly(methylmethacrylate) in monolayers and thin films

Langmuir-Blodgett monolayers of isotactic PMMA exhibit a pressure-induced transition upon compression, that can be described in terms of a two-dimensional crystallization process, analogous to a normal melt crystallization. These water surface crystallized monolayers can be used to prepare highly crystalline thin films of isotactic PMMA with tailor-made orientational characteristics.     

Incorporation of pyrimidine fluorophores into poly(methylmethacrylate) polymer structures

Three polymethyl methacrylate based copolymers bearing arylvinylpyrimidine chromophores have been synthesized in four steps. Spectral properties of the copolymers have been studied in solution, powder and thin films and compared with the corresponding free chromophores. The copolymers exhibit strong emission solvatochromism and act as polarity sensors. They also exhibit halochromism: when adding acid, a bathochromic shift is observed in absorption whereas a red-shifted fluorescence is observed with increased intensity for the methoxy-substituted macromolecule.     

Chemical force titrations of antigen- and antibody-modified poly(methylmethacrylate)

Poly(methylmethacrylate) (PMMA) is a versatile polymer that displays desirable properties for development of cheap and disposable microfluidic devices for sensing biomolecular interactions. Atomic force microscopy (AFM) and chemical force titrations were used to determine the efficacy of surface modifications made to accommodate protein-substrate linkage. AFM images show the effects on surface morphology of carboxylated-, amine-, hCG antigen- and anti-hCG antibody-modified PMMA substrates. Confocal microscopy was used to determine the fluorescent intensity of labeled antibody species on the PMMA substrate, confirming the success of surface antigen/antibody immobilization. Surface pK(1/2) value for carboxylic acid and amine species grafted on PMMA were determined. When carboxylic acid or amine-terminated tips were titrated against PMMA samples terminated with the hCG antigen and anti-hCG antibody, peaks appeared in the force titration curve consistent with the pI range of the antigen or antibody species. Strong adhesive forces were present at pH values above 7.0 when the antigen was present on the PMMA substrate, and these were attributed to hydrophobic interactions between the antigen and the alkane "linker" chain attaching the amine or carboxylate group to the AFM tip. Such hydrophobic interactions were not observed with the carboxylic acid or amine/antibody combinations suggesting that the surface-linked antibody was more resistant to denaturation under higher pH. The results demonstrated the feasibility of using AFM approaches for interrogating protein grafting strategies in the fabrication of PMMA-based microsystems.     

Covalent immobilization of proteases and nucleases to poly(methylmethacrylate)

The increased popularity of microfabricated devices formed from plastics such as poly(methylmethacrylate) (PMMA) will benefit from approaches adding (bio)chemical functionality to such surfaces. Here, various proteases and nucleases have been covalently immobilized to PMMA surfaces and shown to retain their enzymatic activity as monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Immobilized enzymes yield structural information at a level equivalent to or exceeding that obtained from conventional homogeneous solution-based approaches. Such an approach could be used to expand the functionality of polymer-based microfabricated devices for biological mass spectrometry.     

Macroporous poly(methyl methacrylate) produced by phase separation during polymerisation in solution

Polymethyl methacrylate (PMMA) sponges were obtained by polymerization in a solution with monomer/ethanol ratios up to 20:80. The material obtained after the elimination of the solvent present a homogeneous distribution of dispersed pores up to a monomer/ethanol ratio lower than 40:60. For higher ethanol contents in the reacting mixture, the morphology of the sponge corresponds to a network of PMMA microparticles, leaving large empty spaces leading to highly porous structure. The monomer/ethanol ratio during polymerization has a large influence on the porosity, thermal, and mechanical properties of the material and, for large solvent contents, on the size of the polymer microparticles.     

Atmospheric molded poly(methylmethacrylate) microchip emitters for sheathless electrospray

Disposable poly(methylmethacrylate) (PMMA) sheathless electrospray microchip emitters were prepared for the first time using the atmospheric molding fabrication protocol. A sheathless electrospray from uncoated channel outlets, machined to cone-shaped three-dimensional tips, is demonstrated utilizing a simple cross design with an on-chip liquid junction to obviate the need for external unions to voltage electrodes, thus reducing the dead volume effects as well as the complexity of fabrication. The fast replication of microchip emitters was performed by molding prepolymeric methylmethacrylate solutions into silicon-master/aluminum-spacer/glass-plate molds followed by UV-initiated free radical polymerization. The performance of the new microchip emitters was demonstrated for mass spectral measurements of methionine enkephalin, adrenocorticotropic hormone and insulin peptide/protein mixtures. The samples were infused through capillary connections using hydrodynamic pumping. The polymeric emitters prepared by this flexible fabrication route offer an easy way of operation and high stability, without a need for attachment of external voltage unions or metallizing the emitter tips. The new approach should provide a useful low-cost tool for widespread coupling of mass spectrometry to chip systems.     

Electrochromatography in poly(dimethyl)siloxane microchips using organic monolithic stationary phases

This paper shows the in situ synthesis of an hexyl acrylate monolith in PDMS microfluidic devices and its subsequent use as stationary phase for electrochromatography on chip. To overcome the ability of PDMS material to absorb organic monomers, surface modification of the enclosed channels was realized by UV-mediated graft polymerization. This grafting procedure is based on the preliminary adsorption of a photoinitiator onto the PDMS surface and polymerization of charged monomers. Next, hexyl acrylate monoliths were cast in situ using photopolymerization process. The chromatographic behavior of the monolithic column was confirmed by the successful separation of derivatized catecholamines in the PDMS device using a 30 mm effective separation length (100 microm x 100 microm section). Efficiencies reached up to 200,000 plates per meter.     

Electrokinetic control of fluid in plastified laser-printed poly(ethylene terephthalate)-toner microchips

The application of plastified laser-printed poly(ethylene terephthalate)(PET)-toner microchips to capillary electrophoresis was investigated. Electroosmotic flow was observed in the direction of the cathode for the buffer system studied (phosphate, pH 3–10). Average electroosmotic mobilities of 1.71×10^–4 to 4.35×10^–4 cm² V^–1 s^–1 were observed from pH 3 to 10. This variation suggests that silica fillers in the toner and on the surface of the polymer dominate the zeta potential of the material, which is also confirmed by XPS measurements. Dopamine and catechol were used as model analytes for microchip electrophoresis in combination with electrochemical detection. Results show that these two analytes can be efficiently separated and detected electrochemically with the plastified laser-printed PET-toner microchips.     

Study on the separation of amino acids in modified poly(dimethylsiloxane) microchips

A mixture of five amino acids including arginine, histidine, phenylalanine, serine and glutamic acid was successfully separated in microchip capillary electrophoresis and detected with laser-induced fluorescence (LIF) detector. These amino acids were labeled with 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein (DTAF). The analyses were performed on two kinds of modified poly(dimethylsiloxane) (PDMS) microchips. One kind of chip was simply treated with oxygen plasma (OP-chip), and the other was further modified by coating double layers of non-ionic polymer poly(vinyl alcohol) (PVA) after plasma oxidization (PVA-chip). The derivatization condition of amino acids by DTAF was optimized. The properties of the two modified PDMS microchips were studied and separation conditions, such as the buffer pH, buffer concentration and separation voltage, were also optimized. The column efficiencies of the two microchips were in the range of 193,000–1,370,000 plates/m. The DTAF-labeled amino acids were sufficiently separated within 50 s and 90 s in 2.5 cm channels on OP-chip and PVA-chip, respectively.     

Blends of poly(carbonate) with poly(styrene) and poly(methylmethacrylate): phase morphology,mechanical, and thermal properties

This paper is concerned with the dependence of mechanical and thermal properties of heterogeneous blends of poly(carbonate) (PC) with poly(methyl-methacrylate) (PMMA) and with poly(styrene) (PS) on the concentration of the components. PS displays a very weak phase coupling in blends with PC, whereas PMMA is characterized by a strong coupling to PC. Experimental results as well as predictions based on composite theories are reported. The general finding is that mechanical properties, such as the tensile modulus and the dynamic shear modulus, as well as thermal properties, such as thermal expansion, are (i) only weakly affected by the occurrence of a phase inversion and of a continuous phase morphology, (ii) vary continuously with the concentration of the components, and (iii) are rather insensitive to the strength of the phase coupling. The theoretical predictions on the concentration—property relationship for these properties, based on a self-consistent approach, agree very well with those observed experimentally. The elongation at break as well as the yield stress, on the other hand, are strongly influenced by the nature of the phase coupling: a discontinuous variation of these properties with the composition is observed for PS/PC blends but not for PMMA/PC blends. The general conclusion is that a set of mechanical and thermal properties of heterogeneous blends can satisfactorily be predicted on the basis of rather simple composite theories.