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.     

Surface modification of poly(dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting

Poly(dimethylsiloxane) (PDMS)-based microfluidic devices are increasing in popularity due to their ease of fabrication and low costs. Despite this, there is a tremendous need for strategies to rapidly and easily tailor the surface properties of these devices. We demonstrate a one-step procedure to covalently link polymers to the surface of PDMS microchannels by ultraviolet graft polymerization. Acrylic acid, acrylamide, dimethylacrylamide, 2-hydroxylethyl acrylate, and poly(ethylene glycol)monomethoxyl acrylate were grafted onto PDMS to yield hydrophilic surfaces. Water droplets possessed contact angles as low as 45 degrees on the grafted surfaces. Microchannels constructed from the grafted PDMS were readily filled with aqueous solutions in contrast to devices composed of native PDMS. The grafted surfaces also displayed a substantially reduced adsorption of two test peptides compared to that of oxidized PDMS. Microchannels with grafted surfaces exhibited electroosmotic mobilities intermediate to those displayed by native and oxidized PDMS. Unlike the electroosmotic mobility of oxidized PDMS, the electroosmotic mobility of the grafted surfaces remained stable upon exposure to air. The electrophoretic resolution of two test peptides in the grafted microchannels was considerably improved compared to that in microchannels composed of oxidized PDMS. By using the appropriate monomer, it should be possible to use UV grafting to impart a variety of surface properties to PDMS microfluidics devices.     

Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection

Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization of a PDMS surface treatment that considerably decreases adsorption of low and high molecular mass substances to channel walls while maintaining a modest cathodic electroosmotic flow. Channels are modified with a three-layer biotin-neutravidin sandwich coating, made of biotinylated IgG, neutravidin, and biotinylated dextran. By replacing biotinylated dextran with any biotinylated reagent, the modified surface can be readily patterned with biochemical probes, such as antibodies. Combination of probe immobilization chemistry with low nonspecific binding enables affinity binding assays within channel networks. The example of an electrokinetic driven, heterogeneous immunoreaction for human IgG is described.     

Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices

This paper describes the compatibility of poly(dimethylsiloxane) (PDMS) with organic solvents; this compatibility is important in considering the potential of PDMS-based microfluidic devices in a number of applications, including that of microreactors for organic reactions. We considered three aspects of compatibility: the swelling of PDMS in a solvent, the partitioning of solutes between a solvent and PDMS, and the dissolution of PDMS oligomers in a solvent. Of these three parameters that determine the compatibility of PDMS with a solvent, the swelling of PDMS had the greatest influence. Experimental measurements of swelling were correlated with the solubility parameter, delta (cal(1/2) cm(-3/2)), which is based on the cohesive energy densities, c (cal/cm(3)), of the materials. Solvents that swelled PDMS the least included water, nitromethane, dimethyl sulfoxide, ethylene glycol, perfluorotributylamine, perfluorodecalin, acetonitrile, and propylene carbonate; solvents that swelled PDMS the most were diisopropylamine, triethylamine, pentane, and xylenes. Highly swelling solvents were useful for extracting contaminants from bulk PDMS and for changing the surface properties of PDMS. The feasibility of performing organic reactions in PDMS was demonstrated by performing a Diels-Alder reaction in a microchannel.     

Isoelectric focusing in a poly(dimethylsiloxane) microfluidic chip

This paper reports the application of ampholyte-based isoelectric focusing in poly(dimethylsiloxane) (PDMS) using methylcellulose (MC) to reduce electroosmosis and peak drift. Although the characteristics of PDMS make it possible to fabricate microfluidic chips using soft lithography, unstable electroosmotic flow (EOF) and cathodic drift are significant problems when this medium is used. This paper demonstrates that EOF is greatly reduced in PDMS by applying a dynamic coat of MC to the channel walls and that higher concentrations of MC can be used to increase the viscosity of the electrode solutions in order to suppress pH gradient drift and reduce "compression"of the pH gradient. To illustrate the effect of MC on performance, several fluorescent proteins were focused in microchip channels 5 microm deep by 300 microm wide by 2 cm long in 3-10 min using broad-range ampholytes at electric field strengths ranging from 25 to 100 V/cm.     

Electromechanical properties of pressure-actuated poly(dimethylsiloxane) microfluidic push-down valves

Pressure-actuated poly(dimethylsiloxane) (PDMS) valves have been characterized with respect to their electromechanical properties. Measurements of the valve opening and closing times, threshold pressures, and impedance spectra for closed valves can be used to assess the quality of the devices in general, determine their suitability for specialized applications, such as providing electrical isolated fluidic compartments for planar patch clamping, and specify ideal operating conditions. For our particular valve designs, we report valve opening times of the order of 10-100 micros, making them suitable for rapid buffer exchange applications. They can effectively provide reversible electrical isolation between adjacent fluidic compartments with typical resistances of 5 Gohms in the closed state, which meets the gigaohm requirement for patch clamping applications.     

Potentiometric titrations in a poly(dimethylsiloxane)-based microfluidic device

This paper describes a microfluidic device, fabricated in poly(dimethylsiloxane), that is used for potentiometric titrations. This system generates step gradients of redox potentials in a series of microchannels. These potentials are probed by microelectrodes that are integrated into the chip; the measured potentials were used to produce a titration curve from which the end point of a reaction was measured.     

An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.     

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.     

Surface modification of poly(dimethylsiloxane) for controlling biological cells’ adhesion using a scanning radical microjet

A scanning radical microjet (SRMJ) equipment using oxygen microplasma has been developed and successfully applied for controlling biological cells’ attachment on biocompatible polymer material, poly(dimethylsiloxane) (PDMS). The radical microjet has advantages in localized and high-rate surface treatment. Moreover, maskless hydrophilic patterning using SRMJ has been demonstrated to be applicable to patterned cell cultivation which is useful in emerging biotechnological field such as tissue engineering and cell-based biosensors. Since control of PDMS surface properties is an indispensable prerequisite for cells’ attachment, effects of oxygen flow rates and treatment time on localized hydrophilic patterning of PDMS surfaces were first investigated for controlling HeLa cells’ (human epitheloid carcinoma cell line) attachment. Relationships between surface conditions of treated PDMS films and attached cell density are also discussed based on surface properties analyzed using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS).     

Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device

Intracellular nitric oxide (NO) production in a microfluidic endothelium is detected using fluorescence microscopy. Bovine pulmonary artery endothelial cells (bPAECs) were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent fluorescent DAF-FM DA/NO adduct was measured. Solutions of bradykinin, a well-known stimulus of endothelium-derived NO, activated nitric oxide synthase (NOS) in the immobilized bPAECs. This activation was inhibited using l-nitro arginine methyl ester (L-NAME), a competitive inhibitor of NOS. Importantly, the NO production was also stimulated with adenosine triphosphate (ATP) using concentrations as low as 1 microM. Previous reports on stimulating NO production using an immobilized endothelium in microfluidic channels were limited by the requirement of ATP concentrations of at least 100 microM, a value that is not physiologically relevant. The ability to monitor NO production with ATP concentrations that are similar to in vivo levels of ATP in the microcirculation represents a major advance in the use of microfluidic technology as an in vitro model of the microcirculation.     

Fabrication of poly(methyl methacrylate) microfluidic chips by atmospheric molding

A greatly simplified method for fabricating poly(methyl methacrylate) (PMMA) separation microchips is introduced. The new protocol relies on UV-initiated polymerization of the monomer solution in an open mold under ambient pressure. Silicon microstructures are transferred to the polymer substrate by molding a methyl methacrylate solution in a sandwich (silicon master/Teflon spacer/glass plate) mold. The chips are subsequently assembled by thermal sealing of the channel and cover plates. The new fabrication method obviates the need for specialized replication equipment and reduces the complexity of prototyping and manufacturing. Variables of the fabrication process were assessed and optimized. The new method compares favorably with common fabrication techniques, yielding high-quality devices with well-defined channel and injection-cross structures, and highly smoothed surfaces. Nearly 100 PMMA chips were replicated using a single silicon master, with high chip-to-chip reproducibility (relative standard deviations of 1.5 and 4.7% for the widths and depths of the replicated channels, respectively). The relatively high EOF value of the new chips (2.12 x 10(-4) cm(2) x V(-1) x s(-1)) indicates that the UV polymerization process increases the surface charge and hence enhances the fluidic transport. The attractive performance of the new CE microchips has been demonstrated in connection with end-column amperometric and contactless-conductivity detection schemes. While the new approach is demonstrated in connection with PMMA microchips, it could be applied to other materials that undergo light-initiated polymerization. The new approach brings significant simplification of the process of fabricating PMMA devices and should lead to a widespread low-cost production of high-quality separation microchips.     

Integration of dialysis membranes into a poly(dimethylsiloxane) microfluidic chip for isoelectric focusing of proteins using whole-channel imaging detection

A poly(dimethylsiloxane) microfluidic chip-based cartridge is developed and reported here for protein analysis using isoelectic focusing (IEF)-whole-channel imaging detection (WCID) technology. In this design, commercial dialysis membranes are integrated to separate electrolytes and samples and to reduce undesired pressure-driven flow. Fused-silica capillaries are also incorporated in this design for sample injection and channel surface preconditioning. This structure is equivalent to that of a commercial fused-silica capillary-based cartridge for adapting to an IEF analyzer (iCE280 analyzer) to perform IEF-WCID. The successful integration of dialysis membranes into a microfluidic chip significantly improves IEF repeatability by eliminating undesired pressure-driven hydrodynamics and also makes sample injection much easier than that using the first-generation chip as reported recently. In this study, two microfluidic chips with a 100-microm-high, 100-microm-wide and a 200-microm-high, 50-microm-wide microchannel, respectively, were applied for qualitative and quantitative analysis of proteins. The mixture containing six pI markers with a pH range of 3-10 was successfully separated using IEF-WCID. The pH gradient exhibited a good linearity by plotting the pI value versus peak position, and the correlation coefficient reached 0.9994 and 0.9995 separately for the two chips. The separation of more complicated human hemoglobin control sample containing HbA, HbF, HbS, and HbC was also achieved. Additionally, for the quantitative analysis, a good linearity of IEF peak value versus myoglobin concentration in the range of 20-100 microg/mL was obtained.     

Electrokinetic protein preconcentration using a simple glass/poly(dimethylsiloxane) microfluidic chip

We discovered that a protein concentration device can be constructed using a simple one-layer fabrication process. Microfluidic half-channels are molded using standard procedures in PDMS; the PDMS layer is reversibly bonded to a glass base such as a microscope slide. The microfluidic channels are chevron-shaped, in mirror image orientation, with their apexes designed to pass within approximately 20 microm of each other, forming a thin-walled section between the channels. When an electric field is applied across this thin-walled section, negatively charged proteins are observed to concentrate on the anode side of it. About 10(3)-10(6)-fold protein concentration was achieved in 30 min. Subsequent separation of two different concentrated proteins is easily achieved by switching the direction of the electric field in the direction parallel to the thin-walled section. We hypothesize that a nanoscale channel forms between the PDMS and the glass due to the weak, reversible bonding method. This hypothesis is supported by the observation that, when the PDMS and glass are irreversibly bonded, this phenomenon is not observed until a very high E-field was applied and dielectric breakdown of the PDMS is observed. We therefore suspect that the ion exclusion-enrichment effect caused by electrical double layer overlapping induces cationic selectivity of this nanochannel. This simple on-chip protein preconcentration and separation device could be a useful component in practically any PDMS-on-glass microfluidic device used for protein assays.     

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).     

Surface modification of hydrogels based on poly(2-hydroxyethyl methacrylate) with extracellular matrix proteins

Infrared attenuated total reflection spectroscopy was used for in situ observation of the deposition of collagen I on poly(2-hydroxyethyl methacrylate-co-methacrylic acid, 2.9%) hydrogels and subsequent attachment of laminin or fibronectin on the collagen surface. While there was no adsorption of collagen dissolved in an acid solution on the hydrogel surface, it deposited on the surface at pH 6.5. The collagen layers with attached laminin or fibronectin were stable on hydrogel surface in physiological solution. The modification with collagen and particularly with collagen and laminin or fibronectin allowed the adhesion and growth of mesenchymal stromal cells and astrocytes on the hydrogel surface.     

Sol-gel modified poly(dimethylsiloxane) microfluidic devices with high electroosmotic mobilities and hydrophilic channel wall characteristics

Using a sol-gel method, we have fabricated poly(dimethylsiloxane) (PDMS) microchips with SiO2 particles homogeneously distributed within the PDMS polymer matrix. These particles are approximately 10 nm in diameter. To fabricate such devices, PDMS (Sylgard 184) was cast against SU-8 molds. After curing, the chips were carefully removed from the mold and sealed against flat, cured pieces of PDMS to form enclosed channel manifolds. These chips were then solvated in tetraethyl orthosilicate (TEOS), causing them to expand. Subsequently, the chips were placed in an aqueous solution containing 2.8% ethylamine and heated to form nanometer-sized SiO2 particles within the cross-linked PDMS polymer. The water contact angle for the PDMS-SiO2 chips was approximately 90.2 degrees compared to a water contact angle for Sylgard 184 of approximately 108.5 degrees . More importantly, the SiO2 modified PDMS chips showed no rhodamine B absorption after 4 h, indicating a substantially more hydrophilic and nonabsorptive surface than native PDMS. Initial electroosmotic mobilities (EOM) of (8.3+/-0.2)x10(-4) cm2/(V.s) (RSD=2.6% (RSD is relative standard deviation); n=10) were measured. This value was approximately twice that of native Sylgard 184 PDMS chips (4.21+/-0.09)x10(-4) cm2/(V.s) (RSD=2.2%; n=10) and 55% greater than glass chips (5.3+/-0.4)x10(-4) cm2/(V.s) (RSD=7.7%; n=5). After 60 days of dry storage, the EOM was (7.6+/-0.3)x10(-4) cm2/(V.s) (RSD=3.9%; n=3), a decrease of only 8% below that of the initially measured value. Separations performed on these devices generated 80,000-100,000 theoretical plates in 6-14 s for both tetramethylrhodamine succidimidyl ester and fluorescein-5-isothiocyanate derivatized amino acids. The separation distance was 3.5 cm. Plots of peak variance vs analyte migration times gave diffusion coefficients which indicate that the separation efficiencies are within 15% of the diffusion limit.     

Long-term affinity modification on poly(dimethylsiloxane) substrate and its application for ELISA analysis

Poly(dimethylsiloxane) (PDMS) possesses many advantages, such as biocompatibility and high oxygen permeability, which makes it an attractive material for fabricating biodevices. Creating an affinity surface with long-term stability and reactivity for biomolecular interactions on a PDMS substrate, however, is difficult due to its inherent hydrophobicity. In this study, an affinity surface on a PDMS substrate with long-term hydrophilicity and affinity reactivity is reported. This modification is composed of two parts. The bottom part is made of polyelectrolyte multilayers and is capable of providing long-term hydrophilic stability. The top part consists of three protein layers, bovine serum albumin (BSA), anti-BSA, and protein G, and offers an affinity surface for antibody binding and, more importantly, provides favorable orientation and minimum nonspecific binding. The chemical modification for the different stages was monitored by atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR), and contact angle and fluorescence measurements. A long-term PDMS immunodevice (LPID) based on polyelectrolyte multilayers and protein layers was fabricated and applied to the detection of transforming growth factor beta (TGF-beta) protein in mouse serum by the enzyme-linked immunosorbent assay (ELISA) method. Results show that a linear calibration curve was obtained in the concentration range from 500 to 15.125 pg/mL, and the relative standard deviation was less than 3%. Also, the amount of TGF-beta spiked in mouse serum was precisely determined. Results indicate that the modified surface was hydrophilic and reactive to biospecies up to more than 7 days in its dry form. Moreover, the blocking reagent used to reduce nonspecific binding was found to be not necessary for the LPID. Thus, the reported method is expected to hold a great potential for fabricating PDMS-based affinity devices such as protein chips.     

Method for microfluidic whole-chip temperature measurement using thin-film poly(dimethylsiloxane)/rhodamine B

A novel method is presented for on-chip temperature measurements using a poly(dimethylsiloxane) (PDMS) thin film dissolved with Rhodamine B dye. This thin film is sandwiched between two glass substrates (one of which is 150 microm thick) and bonded to a microchannel molded in a PDMS substrate. Whole-chip (liquid and substrate) temperature measurements can be obtained via fluorescent intensity visualization. For verification purposes, the thin film was tested with a tapered microchannel subjected to Joule heating, with resulting axial temperature gradients comparing well with numerical simulations. Errors induced by the definite film thickness are discussed and accounted for during experimental and analytical analysis. Alternative validation using the traditional in-channel Rhodamine B injection method was also attempted. The thin film has several advantages over traditional methods. First, false intensity readings due to adsorption and absorption of Rhodamine B into PDMS channels are eliminated. Second, whole-chip temperature measurements are possible. Third, separation of working liquid from Rhodamine B dye prevents possible electrophoresis effects.