Millisecond kinetics on a microfluidic chip using nanoliters of reagents

This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.     

High-sensitivity miniaturized immunoassays for tumor necrosis factor alpha using microfluidic systems

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).     

Micro-patterned porous substrates for cell-based assays

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.     

Density, viscosity, and surface tension of n-octanol-phosphoric acid solutions in a temperature range 293.15–333.15 K

Densities, viscosities, and surface tensions have been measured for n-octanol-phosphoric acid solutions in the range from x ₁ = 0 to 0.0266 and temperature from 293.15 to 333.15 K at atmospheric pressure. The coefficient of thermal expansion, excess molar volumes and deviations of surface tension are calculated from the experimental data. The excess molar volumes and deviations of surface tensions are fitted to use the Redlich-Kister polynomial equation. According to the experimental data, the measured viscosities are well fitted to regression equation under the correlating temperature and mass fraction of phosphoric acid. The experimental and calculated thermodynamic properties can be used in research on the nature of mixing behavior of the solutions for molecular models and industrial applications.     

Monolayer-functionalized microfluidics devices for optical sensing of acidity

This paper describes the integration of opto-chemosensors in microfluidics networks. Our technique exploits the internal surface of the network as a platform to build a sensing system by coating the surface with a self-assembled monolayer and subsequently binding a fluorescent sensing molecule to the monolayer. Fluorescent molecules were used that can switch between a fluorescent and a non-fluorescent state, depending on the acidity of the surrounding solution. Two systems were investigated. The first employs surface confinement of a Rhodamine B dye in a glass micro channel that serves as a molecular switch in organic solutions. Upon rinsing the micro channels with acidic or basic solutions it was possible to switch between the fluorescent and non-fluorescent forms reversibly. Moreover, this system could be used to monitor the mixing of two solutions of different acidity along the micro channel. To widen the scope of optical sensing in micro channels an Oregon Green dye derivative was immobilized, which functions as a sensing molecule for pH differences in aqueous solutions. In this case, a hybrid system was used consisting of a glass slide and PDMS channels. The fluorescence intensity was found to be directly correlated to the pH of the solution in contact, indicating the possibility of using such a system as a pH sensor. These systems allow real-time measurements and can be easily implemented in micro- and nanofluidics systems thus enabling analysis of extremely small sample volumes in a fast and reproducible manner.     

Electroosmotic mixing in microchannels

Mixing is an essential, yet challenging, process step for many Lab on a Chip (LOC) applications. This paper presents a method of mixing for microfluidic devices that relies upon electroosmotic flow. In physical tests and in computer simulations, we periodically vary the electric field with time to mix two aqueous solutions. Good mixing is shown to occur when the electroosmotic flow at the two inlets pulse out of phase, the Strouhal number is on the order of 1, and the pulse volumes are on the order of the intersection volume.     

Simplest method for creating micropatterned nanostructures on PDMS with UV light

The fabrication of micropatterned structures on PDMS is a critical step in soft lithography, microfluidics, and many other PDMS-based applications. To substitute traditional mold-casting methods, we develop a simple method to create micropatterned nanostructures on PDMS in one step. After exposing a flat PDMS surface to a UV pen lamp through a photomask (such as a TEM grid), micropatterned nanostructures can be formed readily on the PDMS surface. We also demonstrate that fabricated PDMS can be used for the microcontact printing of protein immunoglobulin (IgG) on solid surfaces. This method is probably the simplest method of creating micropatterned nanostructures on PDMS reported so far because it does not need casting, surface coating, or chemical reagents. Only a UV pen lamp and a photomask are required, and this method can be performed under ambient conditions without vacuum. We expect that this method will greatly benefit researchers who use PDMS regularly in various applications such as soft lithography and microfluidics.     

Preparation,Micro-Patterning and Electrical Characterization of Functionalized Carbon-Nanotube Polydimethylsiloxane Nanocomposite Polymer

Summary : We present the preparation, improved micro-patterning, and electrical property characterization of COOH- functionalized mutli-walled carbon nanotube (MWCNT) and polydimethylsiloxane (PDMS) conductive nanocomposite polymers that can be employed for lab on a chip applications. The nanocomposites are prepared by mixing functionalized MWCNTs into an uncured PDMS matrix and employing high frequency ultrasonics (∼ 42-50 kHz) using a horn tip probe. The prepared nanocomposites are micromolded using soft lithography techniques down to a feature size of 25 µm against a micropatterned SU-8 polymer master. An array of peg like microstructures have been fabricated with a radii of 25 µm and height of 100 µm, that are embedded on a non-conductive PDMS substrate using novel and improved fabrication techniques. The percolation threshold of the prepared nanocomposite is achieved at 1.5 weight percentage (wt.%) of COOH- functionalized MWCNT in the PDMS matrix. Resistivity levels at 2 wt.% of functionalized MWCNTs are 62 Ω-cm or better, which is an improvement over our previously reported nanocomposite resistivity value of 100 Ω-cm at 2 wt.% of nonfunctionalized MWCNT's in a PDMS matrix. The nanocomposites also have fairly uniform dispersion and no agglomeration of COOH- functionalized MWCNT as shown by SEM analysis. Furthermore, the nanocomposites show a negative temperature coefficient of resistivity (NTCR), making them ideal candidates for micropatternable temperature microsensors for lab on a chip systems.     

Micro magnetic stir-bar mixer integrated with parylene microfluidic channels

Previously, we reported a micro magnetic stir-bar mixer driven by an external rotating magnetic field and its rapid mixing performance in polydimethyl-siloxane (PDMS) channels. The PDMS piece with embedded fluid channels were manually aligned to a glass substrate and assembled. In this paper, we report the fabrication and testing results of a micro magnetic stir-bar monolithically integrated in parylene surface-micromachined channels with improved design features, including small tolerance of the stir-bar to channel wall (10 microm). Using of parylene based microchannels with improved design not only provides improved mixing, but also eliminates certain problems associated with PDMS-based channels. For example, porosity of PDMS causes evaporation and absorption of chemicals and thus channels made of PDMS are prone to cross-contamination. We have also demonstrated that the magnetic stir-bar can be used to pump liquid in micro channels.     

Numerical study of a novel induced-charge electrokinetic micro-mixer

A novel micro-mixer based on the induced-charge electrokinetic motion of an electrically conducting particle is proposed and numerically demonstrated in this paper. For most microfluidic applications, it is desired to mix different streams of solutions rapidly in a continuous flow mode. Therefore, in this work, we consider a mixing chamber containing an electrically conducting particle and the mixing chamber is located in the middle of a microchannel. Vortices are generated around the electrically conducting particle in an aqueous solution due to the interaction of the applied electric field and the induced surface charge on the particle. These vortices will enhance significantly the mixing of different solutions around the particle. The effectiveness of mixing the two streams entering the mixing chamber is numerically studied as functions of the applied electric field. Excellent mixing can be achieved in this system under two perpendicularly applied electric fields. The proposed micro-mixer is simple and easy to be fabricated for lab-on-a-chip applications.     

Microgram-scale testing of reaction conditions in solution using nanoliter plugs in microfluidics with detection by MALDI-MS

This paper describes a microfluidic system to screen and optimize organic reaction conditions on a submicrogram scale. The system uses discrete droplets (plugs) as microreactors separated and transported by a continuous phase of a fluorinated carrier fluid. Previously, we demonstrated the use of a microfabricated PDMS plug-based microfluidic system to perform assays and crystallization experiments in aqueous solutions with optical detection. Here, we developed an approach that does not require microfabrication of microfluidic devices, is applicable to synthetic reactions in organic solvents, and uses detection by MALDI-MS. As a demonstration, conditions for selective deacetylation of ouabain hexaacetate were tested, and the optimum conditions for mono-, bis-, or trisdeacetylation have been identified. These conditions were validated by scale-up reactions and isolating these potentially neurotoxic products. Mono- and bisdeacetylated products are unstable intermediates in the deacetylation and were isolated for the first time. This system enables no-loss handling of submicroliter volumes containing a few micrograms of a compound of interest. It could become valuable for investigating or optimizing reactions of precious substrates (e.g., products of long synthetic sequences and natural products that can be isolated only in small quantities).     

Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and ‘smart’ polymers

Microfluidic channels prepared from polydimethylsiloxane (PDMS) have been modified by UV-mediated graft polymerization of temperature-responsive polymers (poly[N-isopropyl acrylamide] or pNIPAAm), temperature- and pH-responsive copolymers (P[NIPAAm-co-acrylic acid (AAc)]), and a non-fouling hydrogel (polyethyleneglycol diacrylate, or PEGDA). This was done by presorbing a photosensitizer (PS) within the PDMS channel surface regions, contacting the different monomer solutions with the PS-containing surface under nitrogen, and irradiating with UV. The pNIPAAm-grafted surface was hydrophilic below its lower critical solution temperature (LCST), resisting non-specific adsorption, while it was hydrophobic above its LCST, now binding pNIPAAm-coated nanoparticles. Combined temperature- and pH-responsive surfaces were also prepared by UV radiation grafting a monomer mixture of pNIPAAm with AAc. The surfaces have been characterized by advancing water contact angle measurements. These smart microfluidic channels should be useful for many applications such as affinity separations and diagnostic assays.     

Combinatorial mixing of microfluidic streams

We have devised a microfluidic mixer design that produces all the mixture combinations of a given number of dilutions of the input compounds. As proof of the concept, we present a device that generates four titrations of two dye solutions, blue and yellow, and combinatorially mixes the blue titrations with the yellow titrations to deliver the sixteen mixture combinations in separate outlet microchannels. Our device features four different flow levels made by stacking nine laser-cut Mylar laminates. The fluidic network has a symmetric design that guarantees that the flow rates are the same at all the outlets, with deviations attributable to imperfections in the fabrication, assembly, or perfusion processes. Design rules for scaling up the number of compounds and/or dilutions are presented. The mixing scheme has broad applicability in high-throughput combinatorial testing applications such as drug screening, cell-based biochemical assays, lab-on-a-chip devices, and biosensors.     

Microfluidic devices for fluidic circulation and mixing improve hybridization signal intensity on DNA arrays

Reactions of biomolecules with surface mounted materials on microscope slides are often limited by slow diffusion kinetics, especially in low volumes where diffusion is the only means of mixing. This is a particular problem for reactions where only small amounts of analyte are available and the required reaction volume limits the analyte concentration. A low volume microfluidic device consisting of two interconnected 9 mm x 37.5 mm reaction chambers was developed to allow mixing and closed loop fluidic circulation over most of the surface of a microscope slide. Fluid samples are moved from one reaction chamber to the other by the rotation of a magnetic stirring bar that is driven by a standard magnetic stirrer. We demonstrate that circulation and mixing of different reagents can be efficiently accomplished by this closed loop device with solutions varying in viscosity from 1 to 16.2 centipoise. We also show by example of a microarray hybridization that the reaction efficiency can be enhanced 2-5 fold through fluid mixing under conditions where diffusion is rate limiting. For comparison, similar results were achieved with a disposable commercial device that covers only half of the reaction area of the closed loop device.     

Solvent-resistant photocurable liquid fluoropolymers for microfluidic device fabrication [corrected

We report the first fabrication of a solvent-compatible microfluidic device based on photocurable "Liquid Teflon" materials. The materials are highly fluorinated functionalized perfluoropolyethers (PFPEs) that have liquidlike viscosities that can be cured into tough, highly durable elastomers that exhibit the remarkable chemical resistance of fluoropolymers such as Teflon. Poly(dimethylsiloxane) (PDMS) elastomers have rapidly become the material of choice for many recent microfluidic device applications. Despite the advantages of PDMS in relation to microfluidics technology, the material suffers from a serious drawback in that it swells in most organic solvents. The swelling of PDMS-based devices in organic solvents greatly disrupts the micrometer-sized features and makes it impossible for fluids to flow inside the channels. Our approach to this problem has been to replace PDMS with photocurable perfluoropolyethers. Device fabrication and valve actuation were accomplished using established procedures for PDMS devices. The additional advantage of photocuring allows fabrication time to be decreased from several hours to a matter of minutes. The PFPE-based device exhibited mechanical properties similar to those of Sylgard 184 before and after curing as well as remarkable resistance to organic solvents. This work has the potential to expand the field of microfluidics to many novel applications.     

Fabrication of circular microfluidic channels by combining mechanical micromilling and soft lithography

The fabrication of microfluidic channels with complex three-dimensional (3D) geometries presents a major challenge to the field of microfluidics, because conventional lithography methods are mainly limited to rectangular cross-sections. In this paper, we demonstrate the use of mechanical micromachining to fabricate microfluidic channels with complex cross-sectional geometries. Micro-scale milling tools are first used to fabricate semi-circular patterns on planar metallic surfaces to create a master mold. The micromilled pattern is then transferred to polydimethylsiloxane (PDMS) through a two-step reverse molding process. Using these semi-circular PDMS channels, circular cross-sectioned microchannels are created by aligning and adhering two channels face-to-face. Straight and serpentine-shaped microchannels were fabricated, and the channel geometry and precision of the metallic master and PDMS molds were assessed through scanning electron microscopy and non-contact profilometry. Channel functionality was tested by perfusion of liquid through the channels. This work demonstrates that micromachining enabled soft lithography is capable of fabricating non-rectangular cross-section channels for microfluidic applications. We believe that this approach will be important for many fields from biomimetics and vascular engineering to microfabrication and microreactor technologies.     

Thermodynamics of polyether solutions

Measurements are reported of the vapor pressures and the heats and volumes of mixing of solutions of poly(ethylene glycol dimethyl ether) in chloroform and in carbon tetrachloride. The measurements have been used to calculate the thermodynamic mixing functions for mixing with zero volume change. These values have been compared with the predictions of the lattice theory of mixtures at three different levels of approximation, viz., the generalized “first approximation” theory of Barker, the “zeroth approximation” theory, and the Flory theory. In the hydrogen-bonding chloroform solutions the Barker theory is more successful than the other forms of the lattice theory. The results have also been used, in conjunction with those for solutions of poly(propylene glycol dimethyl ether) in the same two solvents, to test the ability of the Barker theory to correlate the properties of related systems. Good correlation is found between the two carbon tetrachloride solutions but not between the chloroform solutions. It is probable that the side-chain methyl groups in the propylene oxide repeating unit cause the hydrogen-bonding interaction to be weaker in this polymer than it is in the polymer with the ethylene oxide repeating unit.     

A simple method for patterning poly(dimethylsiloxane) barriers in paper using contact-printing with low-cost rubber stamps

This paper presents a simple and low-cost method for patterning poly(dimethylsiloxane) (PDMS) barriers in porous support such as paper for the construction of flexible microfluidic paper-based analytical devices (μPADs). The fabrication method consisted of contact-printing a solution of PDMS and hexane (10:1.5 w/w) onto chromatographic paper using custom-designed rubber stamps containing the patterns of μPADs. After penetrating the paper (∼30 s), the PDMS is cured to form hydrophobic barriers. Under optimized conditions, hydrophobic barriers and hydrophilic channels with dimensions down to 949 ± 88 μm and 771 ± 90 μm (n = 5), respectively, were obtained. This resolution is well-suitable for most applications in analytical chemistry. Chemical compatibility studies revealed that the PDMS barriers were able to contain some organic solvents, including acetonitrile and methanol, and aqueous solutions of some surfactants. This find is particularly interesting given that acetonitrile and methanol are the most used solvents in chromatographic separations, non-aqueous capillary electrophoresis and electroanalysis, as well as aqueous solutions of surfactants are suitable mediums for cell lyses assays. The utility of the technique was evaluated in the fabrication of paper-based electrochemical devices (PEDs) with pencil-drawn electrodes for experiments in static cyclic voltammetry and flow injection analysis (FIA) with amperometric detection, in both aqueous and organic mediums.     

Rapid and inexpensive method for the simple fabrication of PDMS‐based electrochemical sensors for detection in microfluidic devices

The fabrication of PDMS microfluidic structures through soft lithography is widely reported. While this well‐established method gives high precision microstructures and has been successfully used for many researchers, it often requires sophisticated instrumentation and expensive materials such as clean room facilities and photoresists. Thus, we present here a simple protocol that allows the rapid molding of simple linear microchannels in PDMS substrates aiming microfluidics‐based applications. It might serve as an alternative to researchers that do not have access to sophisticated facilities such as clean rooms. The method developed here consists on the use of pencil graphite leads as template for the molding of PDMS channels. It yields structures that can be used for several applications, such as housing support for electrochemical sensors or channels for flow devices. Here, the microdevices produced through this protocol were employed for the accommodation of carbon black paste, which was utilized for the first time as amperometric sensor in microchip electrophoresis. This platform was successfully used for the separation and detection of model analytes. Ascorbic acid and iodide were separated within 45 s with peak resolution of 1.2 and sensitivities of 198 and 492 pA/μM, respectively. The background noise was ca. 84 pA. The analytical usefulness of the system developed was successfully tested through the quantification of iodide in commercial pharmaceutical formulations. It demonstrates good efficiency of the microfabrication protocol developed and enables its use for the easy and rapid prototyping of PDMS structures over a low fabrication cost.     

Silver nanowire-embedded PDMS with high electrical conductivity: nanowires synthesis,composite processing and electrical analysis

A simple, fast, and ecofriendly polyol method for the synthesis of silver nanowires (AgNWs) with high aspect ratios is presented. Besides, the effects of reaction parameters on the synthesis process, namely reaction temperature, PVP molecular weight, sodium chloride content, as well as solution mixing rate on the morphology and dimensions of silver nanowires, are thoroughly investigated. AgNWs were fully characterized using FE-SEM, TEM, XRD, FT-IR, and UV–Vis analysis and showed an average diameter of 50 nm. Polymer nanocomposites were fabricated through mixing and curing of the AgNWs, PDMS, and curing agents. The prepared nanocomposites were analyzed microscopically, and their electrical conductivity was measured. PDMS containing 25 wt% AgNWs was considered as an optimal composite, mainly because it possessed a combination of flexibility and transparency, accompanied by the high electrical conductivity of 5840.4 S/cm. This composite can be deemed as a potential candidate in lab-on-a-chip and biosensing applications.