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.     

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.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Analysis of passive mixing behavior in a poly(dimethylsiloxane) microfluidic channel using confocal fluorescence and Raman microscopy

Confocal fluorescence microscopy (CFM) and confocal Raman microscopy (CRM) have been applied to monitor the laminar flow mixing behavior in a poly(dimethylsiloxane) (PDMS) microfluidic channel. Two passive PDMS micromixing devices were fabricated for this purpose: a two-dimensional round-wave channel and a three-dimensional serpentine channel. The microscale laminar flow mixing of ethanol and isopropanol was evaluated using the CFM and CRM at various flow rates. The mixing behavior of confluent streams in the microchannel was assessed by determining the degree of color change in Rhodamine 6G dye on mixing using the CFM. However, it was also possible to quantitatively evaluate the mixing process without employing a fluorescence label using the CRM. The results show a strong potential for CRM as a highly sensitive detection tool to measure fundamental fluid mixing processes and to provide detailed information on chemical changes of non-fluorescent reaction mixtures in a PDMS microfluidic channel.     

Design and analysis of single-cell capture microfluidic device

The aim of our study was to develop microfluidic devices using microchannel technology with the capability of capturing single cells. We analyzed and compared the cell-capturing efficiencies of series-loop microchannel and parallel-loop microchannel devices that were produced using polydimethylsiloxane (PDMS). Each set of microchannels was composed of a main flow channel and several branch channels with capturing zones. The microfluidic devices were designed to use the differences in flow rates between the main flow channel and the branch channels as a means of capturing single cells based on size and sequestering them within the microstructure of multiple capture zones. The data indicated that the flow medium encountered significant resistance in the series-loop microchannel device, which resulted in an inability to hold the captured cells within any of the capture zones. Flow resistance was, however, greatly reduced in the parallel-loop microchannel device compared to the series-loop device, and single cells were captured in all the capturing zones of the device. Our data suggest that the parallel-loop microchannel technology has significant potential for development toward high-throughput platforms capable of capturing single cells for physiological analyses at the single-cell level.     

Characterization and resolution of evaporation-mediated osmolality shifts that constrain microfluidic cell culture in poly(dimethylsiloxane) devices

Evaporation is a critical problem when handling submicroliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2, or 0.1 mm) were measured over 96 h. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of approximately 10-9 m2/s, accounts for these evaporation and osmolality shifts. To overcome this problem, a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformation-based microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single-cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells under dynamic recirculation of submicroliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures.     

Microfluidic characterization and continuous separation of cells and particles using conducting poly(dimethyl siloxane) electrode induced alternating current-dielectrophoresis

This paper presents a poly(dimethyl siloxane) (PDMS) polymer microfluidic device using alternating current (ac) dielectrophoresis (DEP) for separating live cells from interfering particles of similar sizes by their polarizabilities under continuous flow and for characterizing DEP behaviors of cells in stagnant flow. The ac-DEP force is generated by three-dimensional (3D) conducting PDMS composite electrodes fabricated on a sidewall of the device main channel. Such 3D PDMS composite electrodes are made by dispersing microsized silver (Ag) fillers into PDMS gel. The sidewall AgPDMS electrodes can generate a 3D electric field that uniformly distributes throughout the channel height and varies along the channel lateral direction, thereby producing stronger lateral DEP effects over the entire channel. This allows not only easy observation of cell/particle lateral motion but also using the lateral DEP force for manipulation of cells/particles. The former feature is used to characterize the frequency-dependent DEP behaviors of Saccharomyces cerevisiae (yeast) and Escherichia coli (bacteria). The latter is utilized for continuous separation of live yeast and bacterial cells from similar-size latex particles as well as live yeast cells from dead yeast cells. The separation efficiency of 97% is achieved in all cases. The demonstration of these functions shows promising applications of the microfluidic device.     

Electrohydrodynamic generation and delivery of monodisperse picoliter droplets using a poly(dimethylsiloxane) microchip

We developed a drop-on-demand microdroplet generator for the discrete dispensing of biosamples into a bioanalytical unit. This disposable PDMS microfluidic device can generate monodisperse droplets of picoliter volume directly out of a plane sidewall of the microfluidic chip by an electrohydrodynamic mechanism. The droplet generation was accomplished without using either an inserted capillary or a monolithically built-in tip. The minimum droplet volume was approximately 4 pL, and the droplet generation was repeatable and stable for at least 30 min, with a typical variation of less than 2.0% of drop size. The Taylor cone, which is usually observed in electrospray, was suppressed by controlling the surface wetting property of the PDMS device as well as the surface tension of the sample liquids. A modification of the channel geometry right before the opening of the microchannel also enhanced the continuous droplet generation without applying any external pumping. A simple numerical simulation of the droplet generation verified the importance of controlling the surface wetting conditions for the droplet formation. Our microdroplet generator can be effectively applied to a direct interface of a microfluidic chip to a biosensing unit, such as AMS, MALDI-MS or protein microarray-type biochips.     

Poly(dimethylsiloxane)-based protein preconcentration using a nanogap generated by junction gap breakdown

Simple and efficient sample concentration tools are the key to the application of proteomics in a biological system. In this paper, we developed a method to realize a nanofluidic preconcentrator on a poly(dimethylsiloxane) (PDMS)-based microfluidic channel. The originality of our preconcentration device is the simple nanogap formation using the junction gap breakdown phenomenon between two PDMS microchannels, without using any photolithography and etching techniques. From the dc current measurement, we confirm that the nanogap formed between two microchannel junctions with approximately 80 nm depth. Using this device, we achieve the concentration volume of beta-phycoerythrin protein as high as 70 pL, which is 120-fold larger than that from our previous reports, with a concentration factor as high as 10(4) within 1 h. Also we show the availability of protein preconcentration under several different buffers (phosphate, acetate) at several different pH values (pH 5 to pH 9).     

Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.     

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.     

On-chip electric field driven electrochemical detection using a poly(dimethylsiloxane) microchannel with gold microband electrodes

An external electric field driven in-channel detection technique for on-chip electrochemical detection in micro fabricated devices is described based on a microfluidic system containing an array of 20 microband electrodes. It is shown that an external electric field induces a potential difference between two gold microband electrodes in a poly(dimethylsiloxane) (PDMS) microchannel, and that this enables the electrochemical detection of electroactive species such as ascorbic acid and Fe(CN) 6 (4-). The results, which are supported by simulations of the behavior of the microband electrodes in the microfluidic system, show that the induced potential difference between the electrodes can be controlled by altering the external electric field or by using different microbands in the microband array. As the obtained currents depend on the concentrations of electroactive species in the flowing solution and the detection can be carried out anywhere within the channel without interference of the external electric field, the present approach significantly facilitates electrochemical detection in capillary electrophoresis. This approach consequently holds great promise for application in inexpensive portable chip-based capillary electrophoresis (CE) devices.     

Microfluidic immunoassay for bacterial toxins with supported phospholipid bilayer membranes on poly(dimethylsiloxane)

We report a heterogeneous immunoassay for cholera toxin (CT) using supported bilayer membranes (SBMs) in a poly(dimethylsiloxane) (PDMS) microfluidic device. Phosphatidylcholine membranes assembled on plasma-oxidized PDMS by vesicle fusion bring about favorable surface properties, such as improved wettability and protein resistance. Contact angle measurements show that the lipid membranes can preserve hydrophilic surfaces for hours, whereas untreated substrates rapidly undergo hydrophobic recovery. Fluorescence recovery after photobleaching performed in situ reveals that the membranes have relatively high lateral mobility. Experimental data-fitting to theoretical models yields diffusion coefficients of 1.8 +/- 0.7 microm(2)/s on PDMS and 3.4 +/- 0.8 microm(2)/s on glass. Fluorescence studies utilizing tagged proteins show that SBMs reduce nonspecific adsorption of avidin and BSA on PDMS by 2-3 orders of magnitude, as compared to that on plasma oxidized surfaces. SBMs and their protein-resistant properties are not significantly affected by long flow times, indicating good membrane stability. These studies increase our understanding of the relationship between molecular level interactions and membrane properties, allowing for development of a rapid heterogeneous immunoassay for CT in PDMS microchips with cell surface receptor molecules. Using optimized sample injection and buffer washing conditions, microfluidic immunoassay of CT is complete within 25 min, and a dynamic range over 3 orders of magnitude with a detection limit of 8 fmol of toxin is achieved.     

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.     

Leakage-free bonding of porous membranes into layered microfluidic array systems

The integration of semiporous membranes into poly(dimethylsiloxane) (PDMS) microfluidic devices is useful for mass transport control. Several methods such as plasma oxidation and manual application of PDMS prepolymer exist to sandwich such membranes into simple channel structures, but these methods are difficult to implement with reliable sealing and no leakage or clogging for devices with intricate channel features. This paper describes a simple but robust strategy to bond semiporous polyester and polycarbonate membranes between layers of PDMS microchannel structures effectively without channel clogging. A thin layer of PDMS prepolymer, spin-coated on a glass slide, is transferred to PDMS substrates with channel features as well as to the edges of the semiporous membrane by stamping. This thin PDMS prepolymer serves as "mortar" to strongly bond the two PDMS layers and seal off the crevices generated from the thickness of the membranes. This bonding method enabled the fabrication of an 8x12 criss-crossing microfluidic channel array with 96 combinations of fluid interactions. The capability of this device for bioanalysis was demonstrated by measuring responses of cells to different color fluorescent reagents.     

Self-sealed vertical polymeric nanoporous-junctions for high-throughput nanofluidic applications

We developed a reliable but simple integration method of polymeric nanostructure in a poly(dimethylsiloxane) (PDMS)-based microfluidic channel, for nanofluidic applications. The Nafion polymer junction was creased by infiltrating polymer solution between the gaps created by mechanical cutting, without any photolithography or etching processes. The PDMS can seal itself with the heterogeneous polymeric nanoporous material between the PDMS/PDMS gap due to its flexibility without any (covalent) bonding between PDMS and the polymer materials. Thus, one can easily integrate the nanoporous-junction into a PDMS microchip in a leak-free manner with excellent repeatability. We demonstrated nanofluidic preconcentration of proteins (beta-phycoerythrin) using the device. Because the polymeric junction spans across the entire microchannel height, the preconcentration was achieved with high-pressure field or even in large channels, with the dimensions of 1000 microm width x 100 microm depth.     

Recirculation of nanoliter volumes within microfluidic channels

A microfluidic device is described, capable of recirculating nanoliter volumes in restricted microchannel segments. The device consists of a PDMS microfluidic structure, reversibly sealed to a glass substrate with integrated platinum electrodes. The integrated electrodes generate electroosmotic flow locally, which results in a cycling flow in the channel segment between the two electrodes in case one channel exit is closed (dead-end channel). This cycling flow is a consequence of the counterbalancing hydrodynamic pressure against the electroosmotically generated flow. Acid-base indicators were employed to study the formation of H(+) and OH(-) at both the in-channel electrodes. The formation of acid can locally change the zeta-potential of the channel wall, which will affect the flow profile. Using this method, small analyte volumes can be mixed for prolonged times within well-defined channel segments and/or exposed to in-channel sensor surfaces.     

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.     

Thermoswitchable electrokinetic ion-enrichment/elution based on a poly(N-isopropylacrylamide) hydrogel plug in a microchannel

This paper reports a novel protocol consisting of the thermomodulated electrokinetic enrichment, elution, and separation of charged species based upon a thermoswitchable swelling-shrinking property of a poly(N-isopropylacrylamide), PNIPAAm, hydrogel. A 0.2-1 mm long PNIPAAm hydrogel plug was photopolymerized inside a glass microfluidic channel to produce a composite device consisting of the PNIPAAm hydrogel plug and the glass microchannel (abbreviated as plug-in-channel). After voltage was applied to the composite device, anions, such as FITC, could be enriched at the cathodic end of the PNIPAAm plug when the temperature of the plug was kept below its lower critical solution temperature (LCST, ～32 °C). The concentrated analytes could then be eluted by electroosmotic flow when the temperature of the plug was heated above the LCST. The mechanism of the thermoswitchable ion enrichment/elution process was studied with the results presented. The analytical potential of the composite device was demonstrated for the temperature-modulated preconcentration, elution, and separation of FITC-labeled amino acids.     

A prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane)

A method for carrying out 2D gel electrophoresis in a capillary format is presented. In this method, separation in the first dimension is carried out in a 1D capillary, with this system physically isolated from the capillaries that provide the separation in the second dimension. After completion of the first separation, the 1D channel is physically connected to the 2D capillaries, and a second separation is carried out in an orthogonal set of parallel capillaries. The ability of poly(dimethylsiloxane) (PDMS) to support the fabrication of 3D microfluidic systems makes it possible to produce membranes that both enclose the gel used in the first separation in a capillary and provide passages for the proteins to migrate into the array of orthogonal capillaries. The elastomeric nature of PDMS makes it possible to make reversible connections between pieces of PDMS. The feasibility of this system is demonstrated using a protein mixture containing fluorescein-conjugated carbonic anhydrase, fluorescein-conjugated BSA, and Texas Red-conjugated ovalbumin. This work suggests one type of design that might form the basis for a microfabricated device for 2D capillary electrophoresis.