On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation

Poly(dimethylsiloxane) (PDMS) membrane valves were utilized for diaphragm pumping on a PDMS-glass hybrid microdevice in order to couple infrared-mediated DNA amplification with electrophoretic separation of the products in a single device. Specific amplification products created during non-contact, infrared (IR) mediated polymerase chain reaction (PCR) were injected via chip-based diaphragm pumping into an electrophoretic separation channel. Channel dimensions were designed for injection plug shaping via preferential flow paths, which aided in minimizing the plug widths. Unbiased injection of sample could be achieved in as little as 190 ms, decreasing the time required with electrokinetic injection by two orders of magnitude. Additionally, sample stacking was promoted using laminar or biased-laminar loading to co-inject either water or low ionic strength DNA marker solution along with the PCR-amplified sample. Complete baseline resolution (Res = 2.11) of the 80- and 102-bp fragments of pUC-18 DNA marker solution was achieved, with partially resolved 257- and 267-bp fragments (Res = 0.56), in a separation channel having an effective length of only 3.0 cm. This resolution was deemed adequate for many PCR amplicon separations, with the added advantage of short separation time-typically complete in <120 s. Decreasing the amount of glass surrounding the PCR chamber reduced the DNA amplification time, yielding a further enhancement in analysis speed, with heating and cooling rates as high as 13.4 and -6.4 degrees C s(-1), respectively. With the time requirements greatly reduced for each step, it was possible to seamlessly couple IR-mediated amplification, sample injection, and separation/detection of a 278-bp fragment from the invA gene of <1000 starting copies of Salmonella typhimurium DNA in approximately 12 min on a single device, representing the fastest PCR-ME integration achieved to date.     

Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤ 3?psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ~ 100?pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (< 0.1?Hz to > 2?Hz) with good reproducibility (peak height relative standard deviation ≤ 3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC-labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥ 7.0 × 103 theoretical plates for the ~2.4-cm-long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample-limited bioanalyses are discussed.     

Microdevice for separation and quantitative fraction collection

A new microfluidic concept for quantitative whole-column fraction collection of electrophoretically separated zones was developed. The prototype device, fabricated on a polycarbonate disk by injection molding, integrated electrophoretic separation channels with fraction collection reservoirs distributed along the separation channel. The microdevice was designed in a CD-like format to use the centrifugal force for moving the liquid in the microchannels. A serpentine shape of the separation channel was selected to create segments for quantitative whole-column fraction collection. The operation was tested with visual monitoring of isotachophoretic separation and collection of cationic dyes.     

Sequencing of real-world samples using a microfabricated hybrid device having unconstrained straight separation channels

We describe a microfabricated hybrid device that consists of a microfabricated chip containing multiple twin-T injectors attached to an array of capillaries that serve as the separation channels. A new fabrication process was employed to create two differently sized round channels in a chip. Twin-T injectors were formed by the smaller round channels that match the bore of the separation capillaries and separation capillaries were incorporated to the injectors through the larger round channels that match the outer diameter of the capillaries. This allows for a minimum dead volume and provides a robust chip/capillary interface. This hybrid design takes full advantage, such as sample stacking and purification and uniform signal intensity profile, of the unique chip injection scheme for DNA sequencing while employing long straight capillaries for the separations. In essence, the separation channel length is optimized for both speed and resolution since it is unconstrained by chip size. To demonstrate the reliability and practicality of this hybrid device, we sequenced over 1000 real-world samples from Human Chromosome 5 and Ciona intestinalis, prepared at Joint Genome Institute. We achieved average Phred20 read of 675 bases in about 70 min with a success rate of 91%. For the similar type of samples on MegaBACE 1000, the average Phred20 read is about 550-600 bases in 120 min separation time with a success rate of about 80-90%.     

Cross-linked coatings for electrophoretic separations in poly(dimethylsiloxane) microchannels

We have developed a strategy using ultraviolet light to polymerize mixed monomer solutions onto the surface of a poly(dimethylsiloxane) (PDMS) microdevice. By including monomers with different chemical properties, electrophoretic separations were optimized for a test set of analytes. The properties of surfaces grafted with a single neutral monomer, a neutral and a negative monomer, or a neutral, negative, and cross-linking monomer were assessed. The highest quality separations were achieved in channels with cross-linked coatings. The separation efficiency for biologically relevant peptides (kinase substrates) on these surfaces was as high as 18 600 theoretical plates in a 2.5 cm channel. The test peptides were fluorescein-AEEEIYGEFEAKKKK, fluorescein-GRPRAATFAEG, fluorescein-GRPRAA(T-PO(3))FAEG, fluorescein-DLDVPIP GRFDRRVSVAAE, and fluorescein-DLDVPIPGRFDRRV(S-PO(3))VAAE. Separations between two different peptides occurred in as little as 400 ms after injection into the separation channel. The simultaneous separation of five kinase and phosphatase substrates was also demonstrated. By carefully selecting mixtures of monomers with the appropriate properties, it may be possible to tailor the surface of PDMS for a large number of different electrophoretic separations.     

Microchip free-flow electrophoresis on glass substrate using laser-printing toner as structural material

In this work, a microfluidic free-flow electrophoresis device, obtained by thermal toner transferring on glass substrate, is presented. A microdevice can be manufactured in only 1 h. The layout of the microdevice was designed in order to improve the fluidic and electrical characteristics. The separation channel is 8 microm deep and presents an internal volume of 1.42 microL. The deleterious electrolysis effects were overcome by using a system that isolates the electrolysis products from the separation channel. The Joule heating dissipation in the separation channel was found to be very efficient up to a current density of 8.83 mA/mm(2) that corresponds to a power dissipation per unit volume of running electrolyte of 172 mW/microL. Promising results were obtained in the evaluation of the microdevices for the separation of ionic dyes. The microfluidic device can be used for a continuous sample pretreatment step for micro total analysis system.     

Flow-through sampling for electrophoresis-based microfluidic chips using hydrodynamic pumping

This work presents a novel electrophoretic microchip design which is capable of directly coupling with flow-through analyzers for uninterrupted sampling. In this device, a 3 mm wide sampling channel (SC) was etched on quartz substrate to create the sample inlet and outlet and the 75 microm wide electrophoretic channels were also fabricated on the same substrate. Pressure was used to drive the sample flow through the external tube into the SC and the flow was then split into outlet and electrophoretic channels. A gating voltage was applied to the electrophoretic channel to control the sample loading for subsequent separations and inhibit the sample leakage. The minimum gating voltage required to inhibit the sample leakage depended on the solution buffer and increased with the hydrodynamic flow-rate. A fluorescent dye mixture containing Rhodamine B and Cy3 was introduced into the sample stream at either a continuous or discrete mode via an on-line injection valve and then separated and detected on the microchip using laser-induced fluorescence. For both modes, the relative standard deviation of migration time and peak intensity for consecutive injections was determined to be below 0.6 and 8%, respectively. Because the SC was kept floating, the external sampling equipment requires no electric connection. Therefore, such an electrophoresis-based microchip can be directly coupled with any pressure-driven flow analyzers without hardware modifications. To our best knowledge, this is something currently impossible for reported electrophoretic microchip designs.     

Fabrication of a glass-implemented microcapillary electrophoresis device with integrated contactless conductivity detection

Glass microdevices for capillary electrophoresis (CE) gained a lot of interest in the development of micrototal analysis systems (microTAS). The fabrication of a microTAS requires integration of sampling, chemical separation and detection systems into a microdevice. The integration of a detection system into a microchannel, however, is hampered by the lack of suitable microfabrication technology. Here, a microfabrication method for integration of insulated microelectrodes inside a leakage-free microchannel in glass is presented. A combination of newly developed technological approaches, such as low-temperature glass-to-glass anodic bonding, channel etching, fabrication of buried metal interconnects, and deposition of thin plasma-enhanced chemical vapour deposition (PECVD) silicon carbide layers, enables the fabrication of a CE microdevice with an integrated contactless conductivity detector. The fabrication method of this CE microdevice with integrated contactless conductivity detector is described in detail. Standard CE separations of three inorganic cations in concentrations down to 5 microM show the viability of the new microCE system.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

A microfabricated hybrid device for DNA sequencing

We have created a hybrid device of a microfabricated round-channel twin-T injector incorporated with a separation capillary in order to extend the straight separation distance for high speed and long readlength DNA sequencing. Semicircular grooves on glass wafers are obtained using a photomask with a narrow line-width and a standard isotropic photolithographic etching process. Round channels are made when two etched wafers are face-to-face aligned and bonded. A two-mask fabrication process has been developed to make channels of two different diameters. The twin-T injector is formed by the smaller channels whose diameter matches the bore of the separation capillary, and the "usual" separation channel, now called the connection channel, is formed by the larger ones whose diameter matches the outer diameter of the separation capillary. The separation capillary is inserted through the connection channel all the way to the twin-T injector to allow the capillary bore flush with the twin-T injector channels. The total dead-volume of the connection is estimated to be approximately 5 pL. To demonstrate the efficiency of this hybrid device, we have performed four-color DNA sequencing on it. Using a 200 microm twin-T injector coupled with a separation capillary of 20 cm effective separation distance, we have obtained readlengths of 800 plus bases at an accuracy of 98.5% in 56 min, compared to about 650 bases in 100 min on a conventional 40 cm long capillary sequencing machine under similar conditions. At an increased separation field strength and using a diluted sieving matrix, the separation time has been reduced to 20 min with a readlength of 700 bases at 98.5% base-calling accuracy.     

Column switching in zone electrophoresis on a chip

This feasibility study deals with column switching in zone electrophoresis (ZE) separations on a column coupling (CC) chip. The column switching implemented into the ZE separations an on-chip sample clean up applicable for both the multicomponent and high salinity samples. In addition, complemented by different separation mechanisms in the coupled columns (channels), it provided benefits of two-dimensional separations. Properly timed column switching gave column-to-column transfers of the analytes, characterized by 99-102% recoveries, delivered to the second separation stage on the chip the analyte containing fractions contaminated only with minimum amounts of the matrix constituents. A diffusion driven transport of the matrix constituents to the second channel of the chip (due to direct contacts of the electrolyte solutions in the bifurcation region), representing 0.1-0.2% of the loaded sample constituents, was found to accompany the sample clean up performed on the CC chip. This source of potential disturbances to the separation in the second channel, however, is not detectable in a majority of practical situations. With respect to a 900 nl volume of the sample channel on the CC chip, the electric field and isotachophoresis (ITP) stackings were employed to minimize the injection dispersion in the separations and concentrate the analytes. Here, the column switching, removing a major part of the stacker from the separation system, provided a tool effective in a control of the destacking of analytes. Highly reproducible ZE separations as attained in this work also for the chip-to-chip and equipment-to-equipment frames can be ascribed, at least in part, to suppressions of electroosmotic and hydrodynamic flows of the solutions in which the separations were performed.     

Design and simulation of sample pinching utilizing microelectrodes in capillary electrophoresis microchips

The paper proposed novel designs to pinch the transverse diffusion of the sample in the injection mode using microelectrodes to generate the potential difference at the channel intersection in the capillary electrophoresis (CE) microchip. A pair of microelectrodes was used to conduct the injection channel and the separation channel, which directly provided the potential to pinch the sample without using a power supply. These new designs of the CE microchip simplify the electric circuitry and improve performance. Simulations were performed using the CFD-ACE[trade mark sign] software. The mechanisms of diffusion and electrophoresis were employed in the numerical simulation. The injection and separation processes of the sample were simulated and the parameters of the present design were investigated numerically.     

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.     

PDMS free-flow electrophoresis chips with integrated partitioning bars for bubble segregation

In this work, a microfluidic free-flow electrophoresis device with a novel approach for preventing gas bubbles from entering the separation area is presented. This is achieved by integrating partitioning bars to reduce the channel depth between electrode channels and separation chamber in order to obtain electrical contact and simultaneously prevent bubbles from entering the separation area. The three-layer sandwich chip features a reusable carrier plate with integrated ports for fluidic connection combined with a softlithographically cast microfluidic PDMS layer and a sealing glass slide. This design allows for a straightforward and rapid chip prototyping process. The performance of the device is demonstrated by free-flow zone electrophoretic separations of fluorescent dye mixtures as well as by the separation of labeled amines and amino acids with separation voltages up to 297 V.     

Integrated moulded polymer electrodes for performing conductivity detection on isotachophoresis microdevices

The feasibility of using integrated injection moulded polymer electrodes as drive and detection electrodes for performing miniaturised isotachophoresis (ITP) separations with conductivity detection has been demonstrated. Injection moulded electrodes were produced from three different grades of carbon-filled polymer. Two of the electrode designs were found to be suitable for performing on-chip conductivity detection. The high-voltage characteristics of the microdevices were found to be suitable for performing ITP, with a power dissipation up to 1.4 W m⁻¹ being achieved. Three model separations are presented to demonstrate the separation capability of the miniaturised injection moulded devices. Three anionic dyes, two inorganic anions and a mixture of eight alkaline earth, transition and lanthanide metal cations were analysed.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

Microchip separations of protein biotoxins using an integrated hand-held device

We report the development of a hand-held instrument capable of performing two simultaneous microchip separations (gel and zone electrophoresis), and demonstrate this instrument for the detection of protein biotoxins. Two orthogonal analysis methods are chosen over a single method in order to improve the probability of positive identification of the biotoxin in an unknown mixture. Separations are performed on a single fused-silica wafer containing two separation channels. The chip is housed in a microfluidic manifold that utilizes o-ring sealed fittings to enable facile and reproducible fluidic connection to the chip. Sample is introduced by syringe injection into a septum-sealed port on the device exterior that connects to a sample loop etched onto the chip. Detection of low nanomolar concentrations of fluorescamine-labeled proteins is achieved using a miniaturized laser-induced fluorescence detection module employing two diode lasers, one per separation channel. Independently controlled miniature high-voltage power supplies enable fully programmable electrokinetic sample injection and analysis. As a demonstration of the portability of this instrument, we evaluated its performance in a laboratory field test at the Defence Science and Technology Laboratory with a series of biotoxin variants. The two separation methods cleanly distinguish between members of a biotoxin test set. Analysis of naturally occurring variants of ricin and two closely related staphylococcal enterotoxins indicates the two methods can be used to readily identify ricin in its different forms and can discriminate between two enterotoxin isoforms.     

Fabrication and performance of a microfluidic traveling-wave electrophoresis system

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ～25 μm, provides high electric fields of ～200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 μm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

Peak compression and resolution for electrophoretic separations in diverging microchannels

We report the results of experiments and simulations on electrokinetic flow in diverging microchannels (with cross-sectional area that increases with distance along the channel). Because of conservation of mass and charge, the velocity of an analyte in the channel decreases as the channel cross-section increases. Consequently, the leading edge of a band of sample moves more slowly than the trailing edge and the sample band is compressed. Sample peak widths, rather than increasing diffusively with time, can then be controlled by the geometry of the channel and can even be made to decrease with time. We consider the possibility of using this peak compression effect to improve the resolution of electrophoretic separations. Our results indicate that for typical separations that are dispersion limited, this peak compression effect is more than offset by the decreased distance between peaks, and the separation resolution in diverging channels is worse than that found for straight channels at the same applied voltage. For separations in very short channels or at very high field strengths, however, when the separation efficiency is injection limited, the peak compression effect is dominant and diverging channels can then be used to achieve improved separation resolution.