DNA separation by EFFF in a microchannel

This paper theoretically explores the application of electric field flow fractionation (EFFF) for the size-based separation of DNA strands in a microchannel. An axial electric field cannot separate DNA strands in solution because the electrical mobility of the strands is independent of the length. However, lateral electric fields coupled with an axial Poiseuille flow can separate the DNA strands of different sizes. By using regular perturbation analysis, we obtain the effective diffusivity and the mean velocity of the DNA molecules that are undergoing a pressure driven Poiseuille flow in a 2D channel in presence of a lateral electric field. The mean velocities and the dispersion coefficients are then utilized to determine the scaling for length of the channel and the time required for separation of DNA molecules in different parameter regimes. The results show that EFFF can separate DNA strands in the range of 10 kbp that differ in size by about 2.5 kbp in about half an hour in a 1 cm long channel. While DNA strands can be separated by EFFF, the performance of devices based on EFFF seems to be at best comparable to other techniques such as entropic trapping.     

Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices.

Single photon burst techniques were used to detect double-stranded DNA molecules in poly(methylmethacrylate) (PM MA) and polycarbonate (PC) microfluidic devices. A confocal epi-illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono-intercalating dye, TOPRO-5, which possessed an absorption maximum at 765 nm allowing excitation with a solid-state diode laser and fluorescence monitoring in the near-infrared (IR). Near-IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing lambda-DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 microm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PM MA-based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths > -120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 microm widths compared to those of 50 microm, indicating improved sampling efficiency for the narrower channels without significantly deteriorating detection efficiency. Attempts were made to do single molecule sizing of lambda-DNA, M13 (7.2 kbp) and pUC19 (2.7 kbp) using photon burst detection. While the average number of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To demonstrate the sensitivity of single molecule analysis in the near-IR using polymer microfluidic devices, the near-IR chromophore, NN382, wasanalyzed using ourconfocal imager. A detection efficiency of 94% for single NN382 molecules was observed in the PC devices.     

Simplex maximization of the correlation coefficient for DNA sizing analysis by capillary electrophoresis

The separation of DNA molecules in polymeric solution by capillary electrophoresis involves the optimization of several variables, such as polymer solution concentration, electric field separation, temperature, etc. The optimization of each variable individually usually is a time-consuming process and the results may reach a false optimum point. Chemometric methods are suitable to be applied in such cases in which a number of variables can be optimized simultaneously. The simplex is a chemometric method that can perform such a task easily and efficiently. In this study, a simplex method was carried out to maximize the correlation coefficient (r(2)) of a logarithmic plot of mobility (mu) vs. base pair (bp), which was obtained from the separation of DNA fragments of size between 75 and 4072 bp. The simplex showed three vertexes with r(2) > 0.98 and the vertex 21 showing the highest resolution. For the fragments between 201 and 2036 bp, the r(2) increased to 0.992 with and relative standard deviation (RSD) lower than 0.2% (inter- and intra-day variation). The precision of the method in determining the size of a PCR DNA fragment was carried out using a 1 kbp DNA ladder. With the addition of an internal standard to the sample, the precision could be further improved.     

Separation of long DNA fragments by inversion field capillary electrophoresis

This study reports improved pulsed field capillary electrophoresis (PFCE) for separation of large DNA ladders. Important analytical conditions, including gel polymer concentration, ratio of forward to backward pulse duration, and separation potential, were investigated for their effects on the separation performance of DNA ranging in size from 0.1 to 10.0 kilo base pairs (kbp). Results show that DNA fragments from 0.1 to 8.0 kbp can be resolved with high resolution, simultaneously, in a short time. The ratio of forward to backward pulse duration affects the separation performance for DNA fragments greater than 1.5 kbp, and 3 or 4 is the optimum value of the ratio for separation of DNA up to 10 kbp. Furthermore, the separations that were obtained with 74–19,329 bp λ-DNA restriction fragments clearly demonstrate a dramatic improvement in the separation time and resolution over the conventionally used square-wave PFCE. The inversion field capillary electrophoresis reported here may help enable future DNA analysis studies to be performed quickly and effectively.     

Mathematical model for DNA separation by capillary electrophoresis in entangled polymer solutions

A mathematical model of DNA separation by capillary electrophoresis in entangled polymer solution is presented. The mechanism is modeled as a DNA molecule moving through transient pores formed in polymer solutions and colliding with blobs of polymer molecules encountered during migration. By taking account of the average retardation time (t(c)) of DNA-blob collision and calculating the total collision number (N(c)), a quantitative mathematical equation was reported, leading to predictions for the DNA mobility as a function of the experimental conditions like the size of DNA, the polymer concentration and the electric field strength. For DNA fragments in frequent size range, the initial experimental data agree well with the model. The DNA shape function (f(E)) was suggested and then discussed by the experimental data. The relationship between f(E) and electric field strength E was empirically estimated. Then, the average retardation time t(c) was obtained as about (2 approximately 3)x10(-6)s in linear polyacrylamide (LPA) and hydroxyethylcellulose (HEC) solution.     

Monolithic integration of well-ordered nanoporous structures in the microfluidic channels for bioseparation

Gel electrophoresis and capillary gel electrophoresis are widely used for the separation of biomolecules. With increasing demand in the miniaturized devices such as lab-on-a-chip, it is necessary to integrate such a separation component into a chip format. Here, we describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels for the separation of DNA molecules. In our approach, the colloidal crystals were first grown inside the microchannel using evaporation assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist (SU-8). UV radiation was used to cure the photoresist at the desired area inside the microchannel. After subsequent development and nanoparticle removal, the well-ordered nanoporous structures inside the microchannel were obtained. Our results indicated that it was possible to construct periodic porous nanostructures inside the microchannels with cavity size around 300 nm and interconnecting pores around 30 nm. The mobility of large DNA molecules with different sizes was measured as a function of the applied electric field in the nanoporous materials. It was also demonstrated that 1 kilo-base pair (kbp) DNA ladders could be separated in such an integrated system within 10 min under moderate electric field.     

Pressure-induced transport of DNA confined in narrow capillary channels

Pressure-induced transport of double-stranded DNA (dsDNA) from 10 base pairs (bp) to 1.9 mega base pairs (Mbp) confined in a 750-nm-radius capillary was studied using a hydrodynamic chromatographic technique and four distinct length regions (rod-like, free-coiled, constant mobility, and transition regions) were observed. The transport behavior varied closely with region changes. The rod-like region consisted of DNA shorter than the persistence length (～150 bp) of dsDNA, and these molecules behaved like polymer rods. Free-coiled region consisted of DNA from ～150 bp to ～2 kilo base pairs (kbp), and the effective hydrodynamic radius R(HD) of these DNA scaled to L(0.5) (L is the DNA length in kbp), a characteristic property of freely coiled polymers. Constant mobility region consisted of DNA longer than ～100 kbp, and these DNA had a constant hydrodynamic mobility and could not be resolved. Transition region existed between free-coiled and constant mobility regions. The transport mechanism of DNA in this region was complicated, and a general empirical equation was established to relate the mobility with DNA length. Understanding of the fundamental principles of DNA transport in narrow capillary channels will be of great interest in the development of "lab-on-chip" technologies and nongel DNA separations.     

Do orientation effects contribute to the molecular weight dependence of the free solution mobility of DNA?

The free solution mobility of DNA increases with increasing molecular weight and then levels off and becomes constant at molecular weights above approximately 400 bp (Stellwagen, N. C., Gelfi, C., Righetti, P. G., Biopolymers 1997,42, 687-703). To investigate whether the increase in mobility could be attributed to an increased orientation of the larger DNA molecules in the electric field, the free solution mobility of DNA was measured by capillary electrophoresis as a function of electric field strength. Mixtures containing 20-, 118- and 422-bp DNA molecules, and 20-, 422- and 2116-bp DNAs, were studied. If the larger DNA molecules in each mixture were oriented by the electric field, their mobilities should increase with electric field strength faster than the mobility of the 20-bp oligomer, which is too small to be oriented by the electric fields used in this study. Instead, the ratios of the mobilities of the 118-, 422- and 2116-bp fragments to the mobility of the 20-bp oligomer were independent of electric field strength. Hence, orientation effects are not important for DNA molecules up to 2 kbp in size, in electric fields up to 500 V/cm in amplitude. An explanation is suggested.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA

Single-molecule free solution hydrodynamic separation (SML-FSHS) cohesively integrates cylindrical illumination confocal spectroscopy with free solution hydrodynamic separation. This technique enables single-molecule analysis of size separated DNA with 100% mass detection efficiency, high sizing resolution and wide dynamic range, surpassing the performance of single molecule capillary electrophoresis. Furthermore, SML-FSHS required only a bare fused silica microcapillary and simple pressure control rather than complex high voltage power supplies, sieving matrices, and wall coatings. The wide dynamic range and high sizing resolution of SML-FSHS was demonstrated by separating both large DNA (23 vs 27 kbp) and small DNA (100 vs 200 bp) under identical conditions. Separations were successfully performed with near zero sample consumption using as little as 5 pL of sample and 240 yoctomoles (～150 molecules) of DNA. Quantitative accuracy was predominantly limited by molecular shot noise. Furthermore, the ability of this method to analyze of single molecule nanosensors was investigated. SML-FSHS was used to examine the thermodynamic equilibrium between stochastically open molecular beacon and target-bound molecular beacon in the detection of E. coli 16s rRNA targets.     

Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: a design of experiments approach

Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of ～27 kDa and ～1 MDa were blended with a second class of polymer, high-molar mass (～7 MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (200-40,000 bp) was completed in 2 min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 and 10 kbp, and large dsDNA fragments above 10 kbp.     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

Influence of electric field strength and capillary dimensions on the separation of DNA

This work is a continuation of earlier studies on the influence of polymer concentration and polymer composition on the capillary electrophoretic separation of DNA. The focus is on the capillary dimensions and the electric field strength as factors influencing the resolution. The aim was to establish optimum conditions for the separation of single-stranded DNA in capillaries and derive strategies for the construction of micromachined separation devices.     

Systematic study of field and concentration effects in capillary electrophoresis of DNA in polymer solutions

A systematic study of the separation of double-stranded DNA in hydroxypropylcellulose (HPC) with a molecular mass of 10⁶ was undertaken, using a variety of concentrations (from 0.1 to 1%) and different electric fields (from 6 to 540 V/cm). The data show that a high polymer concentrations (0.4%) and low fields, the separation mechanism is similar to that occurring in gels. The results are in good agreement with theoretical models, and in particular with a recently proposed theory for gels with a pore size smaller than the persistence length of DNA. For more dilute solutions and high fields, however, the separation pattern cannot be explained by existing theories. The existence of an original mechanism was confirmed by the direct observation of the conformation of double-stranded DNA molecules in the polymer solution by fluorescence videomicroscopy. Practical conclusions for the capillary electrophoretic separation of duplex DNA are drawn.     

Capillary electrophoresis of double-stranded DNA in an untreated capillary.

Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.     

Non-orthogonal micro-free flow electrophoresis: From theory to design concept

Micro-free flow electrophoresis (μFFE) is a technique that facilitates continuous separation of molecules in a shallow channel with a hydrodynamic flow and an electric field at an angle to the flow. We recently developed a general theory of μFFE that suggested that an electric field non-orthogonal to the flow could improve resolution. Here, we used computer modeling to study resolution as a function of the electric field strength and the angle between the electric field and the hydrodynamic flow. In addition we used our general theory of μFFE to investigate other important influences on resolution, which include the velocity of the hydrodynamic flow, the height of the separation channel, and the magnitude and direction of the electroosmotic flow. Finally, we propose four designs that could be used to generate non-orthogonal electric fields and discuss their relative merits.     

Fast DNA sequencing up to 1,000 bases by capillary electrophoresis using poly(N,N-dimethylacrylamide) as a separation medium

Poly(N,N-dimethylacrylamide) (PDMA) with a molecular mass of 5.2 x 10(6) g/mol has been synthesized and used in DNA sequencing analysis by capillary electrophoresis (CE). A systematic investigation is presented on the effects of different separation conditions, such as injection amount, capillary inner diameter, polymer concentration, effective separation length, electric field and temperature, on the resolution. DNA sequencing up to 800 bases with a resolution (R) limit of 0.5 (and 1,000 bases with a resolution limit of 0.3) and a migration time of 96 min was achieved by using 2.5% w/v polymer, 150 V/cm separation electric field, and 60 cm effective separation length at room temperature on a DNA sample prepared with FAM-labeled--21M13 forward primer on pGEM3Zf(+) and terminated with ddCTP. Ultrafast and fast DNA sequencing up to 420 and 590 bases (R > or = 0.5) were also achieved by using 3% w/v polymer and 40 cm effective separation length with a separation electric field of 525 and 300 V/cm, and a migration time of 12.5 and 31.5 min, respectively. PDMA has low viscosity, long shelf life and dynamic coating ability to the glass surface. The unique properties of PDMA make it a very good candidate as a separation medium for large-scale DNA sequencing by capillary array electrophoresis (CAE).     

Identifying the orientation of DNA fragment in recombinant plasmid by capillary electrophoresis with a non-gel sieving solution.

It was demonstrated that a capillary electrophoresis (CE) method with a non-gel sieving solution has been developed to identify the orientation of DNA fragments in recombinant plasmids in molecular biology. The influences of the concentration of sieving polymer HEC, the applied electric field strength and sampling on CE separation were analyzed concerning the optimization of separation. YO-PRO-1 was used as a DNA intercalating reagent to facilitate fluorescence detection. Under the chosen conditions (buffer, 1 x TBE containing 1 microM YO-PRO-1 and 1.2% HEC; applied electric field strength, 200 V/cm; electrokinetic sampling: time, 5 s; voltage, -6 kV), three DNA markers (phi 174/HaeIII, pBR322/HaeIII and lambda DNA/HindIII) were tested for further evaluating the relationship between the DNA size and the mobility. The established CE method conjugated with the enzymatic approach was successfully applied to identifying the DNA orientation of recombinant plasmid in transgene operations of a newly cloned gene from Arabidopsis Thaliana.     

Effect of temperature on the separation of long DNA fragments in polymer solution

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of lambda-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.     

Improving the length-fractionation of DNA during capillary electrophoresis

The present study develops a path-lengthening strategy for capillary electrophoresis of short double-stranded DNA molecules, in an aqueous solution of neutral polymer (hydroxypropylmethylcellulose). Tests of the dependence of fractionations on pulse times reveal the operation of at least one mechanism in addition to increase in effective path length. Electrophoresis is performed in the following two-stage cycles (cyclic electrophoresis): The first analysis-stage of each cycle is a constant field (forward) capillary electrophoresis. This analysis-stage reveals the length distribution of the shortest DNA molecules not previously analyzed. The second, enhancement-stage of each cycle is zero-integrated field electrophoresis (ZIFE). The enhancement-stage improves the DNA length-fractionation for the next DNA molecules to be analyzed. A slight reverse migration occurs in the enhancement-stage. Increase in both peak separation and peak sharpness contribute to improvement in the length-fractionation of DNA molecules.