Electrophoretic Transport of Single DNA Nucleotides through Nanoslits: A Molecular Dynamics Simulation Study.

There is potential for flight time based DNA sequencing involving disassembly into individual nucleotides which would pass through a nanochannel with two or more detectors. We performed molecular dynamics simulations of electrophoretic motion of single DNA nucleotides through 3 nm wide hydrophobic slits with both smooth and rough walls. The electric field (E) varied from 0.0 to 0.6 V/nm. The nucleotides adsorb and desorb from walls multiple times during their transit through the slit. The nucleotide-wall interactions differed due to nucleotide hydrophobicities and wall roughness which determined duration and frequency of nucleotide adsorptions and their velocities while adsorbed. Transient association of nucleotides with one, two, or three sodium ions occurred, but the mean association numbers (ANs) were weak functions of nucleotide type. Nucleotide-wall interactions contributed more to separation of nucleotide flight time distributions than ion association and thus indicate that nucleotide-wall interactions play a defining role in successfully discriminating between nucleotides on the basis of their flight times through nanochannels/slits. With smooth walls, smaller nucleotides moved faster, but with rough walls larger nucleotides moved faster due to fewer favorable wall adsorption sites. This indicates that roughness, or surface patterning, might be exploited to achieve better time-of-flight based discrimination between nucleotides.

Modeling of misalignment effects in microfluidic interconnects for modular bio-analytical chip applications.

Minimizing misalignments during the interconnection of microfluidic modules is extremely critical to develop a fully integrated microfluidic device. Misalignments arising during chip-to-chip or world-to-chip interconnections can be greatly detrimental to efficient functioning of microfluidic devices. To address this problem, we have performed numerical simulations to investigate the effect of misalignments arising in three types of interconnection methods: (i) end-to-end interconnection (ii) channel overlap when chips are stacked on top of each other, and (iii) tube-in-reservoir misalignment occurring due to the offset between the external tubing and the reservoir. For the case of end-to-end interconnection, the effect of misalignment was investigated for 0, 13, 50, 58, and 75% reduction in the available flow area at the location of geometrical misalignment. In the channel overlap interconnection method, various possible misalignment configurations were simulated by maintaining the same amount of misalignment (75% flow area reduction). The effect of misalignment in a tube-in-reservoir interconnection was investigated by positioning the tube at an offset of 164 µm from the reservoir center. All the results were evaluated in terms of the equivalent length of a straight pipe. The effect of Reynolds number (Re) was also taken into account by performing additional simulations of aforementioned cases at Re ranging between 0.075 ≤ Re ≤ 75. Correlations were developed and the results were interpreted in terms of equivalent length (Le ). Equivalent length calculations revealed that the effect of misalignment in tube-in-reservoir interconnection method was the least significant when compared to the other two methods of interconnection.

Influence of material transition and interfacial area changes on flow and concentration in electro-osmotic flows.

This paper presents a numerical study to investigate the effect of geometrical and material transition on the flow and progression of a sample plug in electrokinetic flows. Three cases were investigated: (a) effect of sudden cross-sectional area change (geometrical transition or mismatch) at the interface, (b) effect of only material transition (i.e. varying ζ-potential), and (c) effect of combined material transition and cross-sectional area change at the interface. The geometric transition was quantified based on the ratio of reduced flow area A2 at the mismatch plane to the original cross-sectional area A1. Multiple simulations were performed for varying degrees of area reduction i.e. 0-75% reduction in the available flow area, and the effect of dispersion on the sample plug was quantified by standard metrics. Simulations showed that a 13% combined material and geometrical transition can be tolerated without significant loss of sample resolution. A 6.54% reduction in the flow rates was found between 0% and 75% combined material and geometrical transition.

Distinguishing single DNA nucleotides based on their times of flight through nanoslits: a molecular dynamics simulation study.

Transport of single molecules in nanochannels or nanoslits might be used to identify them via their transit (flight) times. In this paper, we present molecular dynamics simulations of transport of single deoxynucleotide 5'-monophoshates (dNMP) in aqueous solution under pressure-driven flow, to average velocities between 0.4 and 1.0 m/s, in 3 nm wide slits with hydrophobic walls. The simulation results show that, while moving along the slit, the mononucleotides are adsorbed and desorbed from the walls multiple times. For the simulations, the estimated minimum slit length required for separation of the dNMP flight time distributions is about 5.9 µm, and the minimum analysis time per dNMP is about 10 µs. These are determined by the nature of the nucleotide-wall interactions, channel width, and by the flow characteristics. A simple analysis using realistic dNMP velocities shows that, in order to reduce the effects of diffusional broadening and keep the analysis time per dNMP reasonably small, the nucleotide velocity should be relatively high. Tailored surface chemistry could lead to further reduction of the analysis time toward its minimum value for a given driving force.

A vertically stacked, polymer, microfluidic point mutation analyzer: rapid high accuracy detection of low-abundance K-ras mutations.

Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user base for these types of molecular assays, a vertically stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employs a primary polymerase chain reaction (PCR) coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers, and ExoSAP-IT purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and were assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in approximately 1h, an 80% reduction in time compared with conventional benchtop instrumentation. Purifying the post-PCR products with the ExoSAP-IT enzyme led to optimized LDR performance, minimizing false-positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25-µl sample, equivalent to DNA from 42 mutant cells.

Surface modification of droplet polymeric microfluidic devices for the stable and continuous generation of aqueous droplets.

Droplet microfluidics performed in poly(methyl methacrylate) (PMMA) microfluidic devices resulted in significant wall wetting by water droplets formed in a liquid-liquid segmented flow when using a hydrophobic carrier fluid such as perfluorotripropylamine (FC-3283). This wall wetting led to water droplets with nonuniform sizes that were often trapped on the wall surfaces, leading to unstable and poorly controlled liquid-liquid segmented flow. To circumvent this problem, we developed a two-step procedure to hydrophobically modify the surfaces of PMMA and other thermoplastic materials commonly used to make microfluidic devices. The surface-modification route involved the introduction of hydroxyl groups by oxygen plasma treatment of the polymer surface followed by a solution-phase reaction with heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane dissolved in fluorocarbon solvent FC-3283. This procedure was found to be useful for the modification of PMMA and other thermoplastic surfaces, including polycyclic olefin copolymer (COC) and polycarbonate (PC). Angle-resolved X-ray photoelectron spectroscopy indicated that the fluorination of these polymers took place with high surface selectivity. This procedure was used to modify the surface of a PMMA droplet microfluidic device (DMFD) and was shown to be useful in reducing the wetting problem during the generation of aqueous droplets in a perfluorotripropylamine (FC-3283) carrier fluid and could generate stable segmented flows for hours of operation. In the case of PMMA DMFD, oxygen plasma treatment was carried out after the PMMA cover plate was thermally fusion bonded to the PMMA microfluidic chip. Because the appended chemistry to the channel wall created a hydrophobic surface, it will accommodate the use of other carrier fluids that are hydrophobic as well, such as hexadecane or mineral oils.

Titer-plate formatted continuous flow thermal reactors: Design and performance of a nanoliter reactor.

Arrays of continuous flow thermal reactors were designed, configured, and fabricated in a 96-device (12 × 8) titer-plate format with overall dimensions of 120 mm × 96 mm, with each reactor confined to a 8 mm × 8 mm footprint. To demonstrate the potential, individual 20-cycle (740 nL) and 25-cycle (990 nL) reactors were used to perform the continuous flow polymerase chain reaction (CFPCR) for amplification of DNA fragments of different lengths. Since thermal isolation of the required temperature zones was essential for optimal biochemical reactions, three finite element models, executed with ANSYS (v. 11.0, Canonsburg, PA), were used to characterize the thermal performance and guide system design: (1) a single device to determine the dimensions of the thermal management structures; (2) a single CFPCR device within an 8 mm × 8 mm area to evaluate the integrity of the thermostatic zones; and (3) a single, straight microchannel representing a single loop of the spiral CFPCR device, accounting for all of the heat transfer modes, to determine whether the PCR cocktail was exposed to the proper temperature cycling. In prior work on larger footprint devices, simple grooves between temperature zones provided sufficient thermal resistance between zones. For the small footprint reactor array, 0.4 mm wide and 1.2 mm high fins were necessary within the groove to cool the PCR cocktail efficiently, with a temperature gradient of 15.8°C/mm, as it flowed from the denaturation zone to the renaturation zone. With temperature tolerance bands of ±2°C defined about the nominal temperatures, more than 72.5% of the microchannel length was located within the desired temperature bands. The residence time of the PCR cocktail in each temperature zone decreased and the transition times between zones increased at higher PCR cocktail flow velocities, leading to less time for the amplification reactions. Experiments demonstrated the performance of the CFPCR devices as a function of flow velocity, fragment length, and copy number. A 99 bp DNA fragment was successfully amplified at flow velocities from 1 mm/s to 3 mm/s, requiring from 8.16 minutes for 20 cycles (24.48 s/cycle) to 2.72 minutes for 20 cycles (8.16 s/cycle), respectively. Yield compared to the same amplification sequence performed using a bench top thermal cycler decreased nonlinearly from 73% (at 1 mm/s) to 13% (at 3 mm/s) with shorter residence time at the optimal temperatures for the reactions due to increased flow rate primarily responsible. Six different DNA fragments with lengths between 99 bp and 997 bp were successfully amplified at 1 mm/s. Repeatable, successful amplification of a 99 bp fragment was achieved with a minimum of 8000 copies of the DNA template. This is the first demonstration and characterization of continuous flow thermal reactors within the 8 mm × 8 mm footprint of a 96-well micro-titer plate and is the smallest continuous flow PCR to date.

Temperature distribution effects on micro-CFPCR performance.

Continuous flow polymerase chain reactors (CFPCRs) are BioMEMS devices that offer unique capabilities for the ultra-fast amplification of target DNA fragments using repeated thermal cycling, typically over the following temperature ranges: 90 degrees C-95 degrees C for denaturation, 50 degrees C-70 degrees C for renaturation, and 70 degrees C-75 degrees C for extension. In CFPCR, DNA cocktail is pumped through the constant temperature zones and reaches thermal equilibrium with the channel walls quickly due to its low thermal capacitance. In previous work, a polycarbonate CFPCR was designed with microchannels 150 microm deep, 50 microm wide, and 1.78 m long-including preheating and post-heating zones, fabricated with LIGA, and demonstrated. The high thermal resistance of the polycarbonate led to a high temperature gradient in the micro-device at steady-state and was partly responsible for the low amplification yield. Several steps were taken to ensure that there were three discrete, uniform temperature zones on the polycarbonate CFPCR device including: reducing the thickness of the CFPCR substrate to decrease thermal capacitance, using copper plates as heating elements to ensure a uniform temperature input, and making grooves between temperature zones to increase the resistance to lateral heat conduction between zones. Finite element analyses (FEA) were used to evaluate the macro temperature distribution in the CFPCR device and the micro temperature distribution along a single microchannel. At steady-state, the simulated CFPCR device had three discrete temperature zones, each with a uniform temperature distribution with a variation of +/-0.3 degrees C. An infrared (IR) camera was used to measure the steady-state temperature distribution in the prototype CFPCR and validated the simulation results. The temperature distributions along a microchannel at flow velocities from 0 mm/s to 6 mm/s were used to estimate the resulting temperatures of the DNA reagents in a single microchannel. A 500 bp DNA fragment was generated from a bacteriophage lambda-DNA target using 20 cycles of PCR. The amplification efficiencies compared to a commercial thermal cycler were 72.7% (2 mm/s), 44% (3 mm/s), and 29.4% (4 mm/s). The amplification efficiency with the modified CFPCR device increased by 363% at 2 mm/s and 440% at 3 mm/s compared to amplification obtained using a CFPCR device with the same fluidic layout, (Hashimoto et al., Lab Chip 4:638, 2004) strictly due to the improved temperature distribution.

Rapid PCR in a continuous flow device.

Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 degrees C) and renaturation (55 degrees C-68 degrees C) zones; above 6 mm s(-1) the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from lambda-DNA. Amplifications at velocities ranging from 1 mm s(-1) to 20 mm s(-1) were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle(-1)) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle(-1)). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).