Electrokinetic focusing injection methods on microfluidic devices

This paper presents an experimental and numerical investigation into electrokinetic focusing injection on microfluidic chips. The valving characteristics on microfluidic devices are controlled through appropriate manipulations of the electric potential strengths during the sample loading and dispensing steps. The present study also addresses the design and testing of various injection systems used to deliver a sample plug. A novel double-cross injection microfluidic chip is fabricated, which employs electrokinetic focusing to deliver sample plugs of variable volume. The proposed design combines several functions of traditional sample plug injection systems on a single microfluidic chip. The injection technique uses an unique sequence of loading steps with different electric potential distributions and magnitudes within the various channels to effectuate a virtual valve.     

Organic transistor based circuit as drive for planar microfluidic devices

Organic transistor based circuits are a promising candidate for acting as drivers for microfluidic devices handling discrete droplets. The ease of fabrication along with the ability to generate desired voltage levels for performing electrowetting based actuation of liquids make them an ideal match for discrete droplet based microfluidic systems. In this article, we report the implementation of an organic transistor based complementary metal-oxide semiconductor (CMOS) inverter used to actuate microliter quantities of droplets on a simple planar microfluidic device. We also present two approaches for fabricating an open-structured device for different applications. The inverter is fabricated using Pentacene and N, N′- bis (n-octyl) dicyanoperylene-3, 4:9, 10-bis (dicarboxyimide) (PDI-8CN₂) (Northwestern University). The inverter output is stable and repeatable and is used to actuate droplets over adjacent electrodes as well as in merging of discrete droplets.     

High-efficiency, two-dimensional separations of protein digests on microfluidic devices

High-efficiency, two-dimensional separations of tryptic digests were achieved using glass microfluidic devices. Following micellar electrokinetic chromatography (MEKC) separations in a 19.6-cm-long serpentine channel, the peptides were rapidly sampled into a 1.3-cm-long second-dimension channel, where they were separated by capillary electrophoresis (CE). The turns in the serpentine channel were asymmetrically tapered to minimize geometrical contributions to band broadening and to provide ample channel length for high-efficiency chromatographic separations. Analysis of rhodamine B injections routinely produced plate numbers of 230000 and 40000 in the first (MEKC) and second (CE) dimensions, respectively, corresponding to plate heights of 0.9 and 0.3 microm. The electric field strengths were 200 V/cm for MEKC and 2400 V/cm for CE. In analysis times less than 15 min, two-dimensional separation of bovine serum albumin tryptic digest produced a peak capacity of 4200 (110 in the first dimension and 38 in the second dimension). The system was used to identify a peptide from a tryptic digest of ovalbumin using standard addition and to distinguish between tryptic digests of human and bovine hemoglobin.     

Electrokinetic injection techniques in microfluidic chips

The separation efficiency of a microfluidic chip is influenced to a significant degree by the flow field conditions within the injection microchannel. Therefore, an understanding of the physics of the flow within this channel is beneficial in the design and operation of such a system. The configuration of an injection system is determined by the volume of the sample plug that is to be delivered to the separation process. Accordingly, this paper addresses the design and testing of injection systems with a variety of configurations, including a simple cross, a double-T, and a triple-T configuration. This paper also presents the design of a unique multi-T injection configuration. Each injection system cycles through a predetermined series of steps, in which the electric field magnitude and distribution within the various channels is strictly manipulated, to effectuate a virtual valve. The uniquemulti-T configuration injection system presented within this paper has the ability to simulate the functions of the cross, double-T, and triple-T systems through appropriate manipulations of the electric field within its various channels. In other words, the proposed design successfully combines several conventional injection systems within a single microfluidic chip.     

Reagent-loaded cartridges for valveless and automated fluid delivery in microfluidic devices

An important problem in the life sciences and in health care is simple and rapid detection of biomarkers. Although microfluidic devices are potentially useful in addressing this problem, current techniques for automating fluid delivery--which include valves and electroosmosis--require sophisticated microfabrication of the chip, bulky instrumentation, or both. In this paper, we describe a simple and reliable technique for storing and delivering a sequence of reagents to a microfluidic device. The technique is low-cost, requires minimal user intervention, and can be performed in resource-poor settings (e.g., outside of a laboratory) in the absence of electricity and computer-controlled equipment. In this method, cartridges made of commercially available tubing are filled by sequentially injecting plugs of reagents separated by air spacers. The air spacers prevent the reagents from mixing with each other during cartridge preparation, storage, and usage. As an example, we used this "plug-in cartridge" technology to complete a solid-phase immunoassay in a microchannel in 2 min with low-nanomolar sensitivity and demonstrate the diagnosis of HIV in 13 min.     

High-pressure microfluidic control in lab-on-a-chip devices using mobile polymer monoliths

We have developed a nonstick polymer formulation for creating moving parts inside of microfluidic channels and have applied the technique to create piston-based devices that overcome several microfluidic flow control challenges. The parts were created bycompletely filling the channels of a glass microfluidic chip with the monomer/ solvent/initiator components of a nonstick photopolymer and then selectively exposing the chip to UV light in order to define mobile pistons (or other quasi-two-dimensional shapes) inside the channels. Stops defined in the substrate prevent the part from flushing out of the device but also provide sealing surfaces so that valves and other flow control devices are possible. Sealing against pressures greater than 30 MPa (4,500 psi) and actuation times less than 33 ms are observed. An on-chip check valve, a diverter valve, and a 10-nL pipet are demonstrated. This valving technology, coupled with high-pressure electrokinetic pumps, should make it possible to create a completely integrated HPLC system on a chip.     

Micropreparative fraction collection in microfluidic devices

Micropreparative fraction collection following microchip-based electrophoretic analysis of biomolecules is of major importance for a variety of biomedical applications. In this paper, we present a microfabricated device-based fraction collection system. Various size DNA fragments were separated and collected by simply redirecting the desired portions of the detected sample zones to corresponding collection wells using appropriate voltage manipulations. The efficiency of sampling and collection of the fractions was enhanced by placing a cross channel at or downstream of the detection point. Following the detection of the band of interest, the potentials were reconfigured to sampling/collection mode, so that the selected sample zone migrated to the appropriate collection well of the microdevice. The potential distribution assured that the rest of the analyte components in the separation column was retarded, stopped, or reversed, increasing in this way the spacing between the sample zone being collected and the immediately following one. By this means, a precise collection of spatially close consecutive bands could be facilitated. Once the target sample fraction reached the corresponding collection well, the potentials were switched back to separation mode. Alternation of the separation/detection and sampling/collection cycles was repeated until all required sample zones were physically isolated. The integrated device consists of a sample introduction, separation, fraction sampling, and fraction collection compartments. The feasibility of the fraction collection technique was tested on a mixture of dsDNA fragments. The amounts of DNA collected in this way were enough for further downstream sample processing, such as conventional PCR-based analysis.     

Minimizing the number of voltage sources and fluid reservoirs for electrokinetic valving in microfluidic devices

A microchip gated valve is demonstrated that uses a single voltage source and three fluid reservoirs. The fluidic valve is a cross intersection, and the channels are dimensioned to perform the appropriate voltage division, simplifying the voltage control hardware. A single voltage source is applied directly to the sample reservoir and through a high-voltage relay to the buffer reservoir, and the waste reservoir is grounded. The volume of sample dispensed is determined by the duration that the high-voltage relay is open. Volumetric reproducibility is demonstrated to be <0.5% relative standard deviation for volumes of ≥20 pL. The valve is tested for the minimum applied voltage necessary for leakage-free operation, i.e., sample diffusing from the cross intersection into the analysis channel. Moreover, appropriate channel dimensions are used to minimize the number of fluid reservoirs allowing effluent from the analysis and waste channels to be combined into a single reservoir.     

Fully enclosed microfluidic paper-based analytical devices

This article introduces fully enclosed microfluidic paper-based analytical devices (microPADs) fabricated by printing toner on the top and bottom of the devices using a laser printer. Enclosing paper-based microfluidic channels protects the channels from contamination, contains and protects reagents stored on the device, contains fluids within the channels so that microPADs can be handled and operated more easily, and reduces evaporation of solutions from the channels. These benefits extend the capabilities of microPADs for applications as low-cost point-of-care diagnostic devices.     

Enzymatic reactions in microfluidic devices: Michaelis-Menten kinetics

Kinetic rate constants for enzymatic reactions are typically measured with a series of experiments at different substrate concentrations in a well-mixed container. Here we demonstrate a microfluidic technique for measuring Michaelis-Menten rate constants with only a single experiment. Enzyme and substrate are brought together in a coflow microfluidic device, and we establish analytically and numerically that the initial concentration of product scales with the distance x along the channel as x5/2. Measurements of the initial rate of product formation, combined with the quasi-steady rate of product formation further downstream, yield the rate constants. We corroborate the x5/2 scaling result experimentally using the bioluminescent reaction between ATP and luciferase/luciferin as a model system.     

Thermoelectric manipulation of aqueous droplets in microfluidic devices

This article describes a method for manipulating the temperature inside aqueous droplets, utilizing a thermoelectric cooler to control the temperature of select portions of a microfluidic chip. To illustrate the adaptability of this approach, we have generated an "ice valve" to stop fluid flow in a microchannel. By taking advantage of the vastly different freezing points for aqueous solutions and immiscible oils, we froze a stream of aqueous droplets that were formed on-chip. By integrating this technique with cell encapsulation into aqueous droplets, we were also able to freeze single cells encased in flowing droplets. Using a live-dead stain, we confirmed the viability of cells was not adversely affected by the process of freezing in aqueous droplets provided cryoprotectants were utilized. When combined with current droplet methodologies, this technology has the potential to both selectively heat and cool portions of a chip for a variety of droplet-related applications, such as freezing, temperature cycling, sample archiving, and controlling reaction kinetics.     

The role of feedback in microfluidic flow-focusing devices

A model was developed for the analysis of steady and unsteady bubble generation frequencies in a microfluidic flow-focusing device. In both cases, the generation frequency depends on the downstream influence of bubbles created by the flow-focusing device, which provides a source of feedback. For steady-state generation of bubbles, this feedback explains the relationships among frequency, gas pressure and liquid flow rate in our experiments as well as gives a physical explanation for previously observed frequency relations. The role of feedback is also exploited to explain unsteady behaviour; we develop a numerical model and analytical expressions that agree with experimental measurements.     

Plastic microfluidic devices modified with polyelectrolyte multilayers

Control of the polymer surface chemistry is a crucial aspect of development of plastic microfluidic devices. When commercially available plastic substrates are used to fabricate microchannels, differences in the EOF mobility from plastic to plastic can be very high. Therefore, we have used polyelectrolyte multilayers (PEMs) to alter the surface of microchannels fabricated in plastics. Optimal modification of the microchannel surfaces was obtained by coating the channels with alternating layers of poly(allylamine hydrochloride) and poly(styrene sulfonate). Polystyrene (PS) and poly(ethylene terephthalate) glycol (PETG) were chosen as substrate materials because of the significant differences in the polymer chemistries and in the EOF of channels fabricated in these two plastic materials. The efficacy of the surface modification has been evaluated using XPS and by measuring the EOF mobility. When microchannels prepared in both PS and PETG are modified with PEMs, they demonstrate very similar electroosmotic mobilities. The PEMs are easily fabricated and provide a means for controlling the flow direction and the electroosmotic mobility in the channels. The PEM-coated microchannels have excellent wettability, allowing facile filling of the channels. In addition, the PEMs produce reproducible results and are robust enough to withstand long-term storage.     

Microcontact printing-based fabrication of digital microfluidic devices

Digital microfluidics is a fluid manipulation technique in which discrete droplets are actuated on patterned arrays of electrodes. Although there is great enthusiasm for the application of this technique to chemical and biological assays, development has been hindered by the requirement of clean room fabrication facilities. Here, we present a new fabrication scheme, relying on microcontact printing (microCP), an inexpensive technique that does not require clean room facilities. In microCP, an elastomeric poly(dimethylsiloxane) stamp is used to deposit patterns of self-assembled monolayers onto a substrate. We report three different microCP-based fabrication techniques: (1) selective etching of gold-on-glass substrates; (2) direct printing of a suspension of palladium colloids; and (3) indirect trapping of gold colloids from suspension. In method 1, etched gold electrodes are used for droplet actuation; in methods 2 and 3, colloid patterns are used to seed electroless deposition of copper. We demonstrate, for the first time, that digital microfluidic devices can be formed by microCP and are capable of the full range of digital microfluidics operations: dispensing, merging, motion, and splitting. Devices formed by the most robust of the new techniques were comparable in performance to devices formed by conventional methods, at a fraction of the fabrication time. These new techniques for digital microfluidics device fabrication have the potential to facilitate expansion of this technology to any research group, even those without access to conventional microfabrication tools and facilities.     

Electrophoretic analysis of N-glycans on microfluidic devices

We report rapid and efficient electrophoretic separations of N-glycans on microfluidic devices. Using a separation length of 22 cm and an electric field strength of 750 V/cm, analysis times were less than 3 min, and separation efficiencies were between 400,000 and 655,000 plates for the N-glycans and up to 960,000 plates for other sample components. These high efficiencies were necessary to separate N-glycan positional isomers derived from ribonuclease B and linkage isomers from asialofetuin. Structural isomers of N-glycans derived from a blood serum sample of a cancer patient were also analyzed to demonstrate that clinically relevant, complex samples could be separated on-chip with efficiencies similar to those derived from model glycoproteins. In addition, we compared microchip and capillary electrophoresis under similar separation conditions, and the microchips performed as well as the capillaries. These results confirmed that the noncircular cross section of the microchannel did not hamper separation performance. For all experiments, the glycan samples were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid to impart needed charge for electrophoresis and a fluorescent label for detection.     

Rapid prototyping of thermoset polyester microfluidic devices

This paper presents a simple procedure for the fabrication of thermoset polyester (TPE) microfluidic systems and discusses the properties of the final devices. TPE chips are fabricated in less than 3 h by casting TPE resin directly on a lithographically patterned (SU-8) silicon master. Thorough curing of the devices is obtained through the combined use of ultraviolet light and heat, as both an ultraviolet and a thermal initiator are employed in the resin mixture. Features on the order of micrometers and greater are routinely reproduced using the presented procedure, including complex designs and multilayer features. The surface of TPE was characterized using contact angle measurements and X-ray photoelectron spectroscopy (XPS). Following oxygen plasma treatment, the hydrophilicity of the surface of TPE increases (determined by contact angle measurements) and the proportion of oxygen-containing functional groups also increases (determined by XPS), which indicates a correlated increase in the charge density on the surface. Native TPE microchannels support electroosmotic flow (EOF) toward the cathode, with an average electroosmotic mobility of 1.3 x 10(-4) cm(2) V(-1) s(-1) for a 50-microm square channel (20 mM borate at pH 9); following plasma treatment (5 min at 30 W and 0.3 mbar), EOF is enhanced by a factor of 2. This enhancement of the EOF from plasma treatment is stable for days, with no significant decrease noted during the 5-day period that we monitored. Using plasma-treated TPE microchannels, we demonstrate the separation of a mixture of fluorescein-tagged amino acids (glycine, glutamic acid, aspartic acid). TPE devices are up to 90% transparent (for approximately 2-mm-thick sample) to visible light (400-800 nm). The compatibility of TPE with a wide range of solvents was tested over a 24-h period, and the material performed well with acids, bases, alcohols, cyclohexane, n-heptane, and toluene but not with chlorinated solvents (dichloromethane, chloroform).