Microelectronic array devices and techniques for electric field enhanced DNA hybridization in low-conductance buffers

A variety of electronic DNA array devices and techniques have been developed that allow electric field enhanced hybridization to be carried out under special low-conductance conditions. These devices include both planar microelectronic DNA array/chip devices as well as electronic microtiter plate-like devices. Such "active" electronic devices are able to provide controlled electric (electrophoretic) fields that serve as a driving force to move and concentrate nucleic acid molecules (DNA/RNA) to selected microlocation test-sites on the device. In addition to ionic strength, pH, temperature and other agents, the electric field provides another controllable parameter that can affect and enhance DNA hybridization. With regard to the planar microelectronic array devices, special low-conductance buffers were developed in order to maintain rapid transport of DNA molecules and to facilitate hybridization within the constrained low current and voltage ranges for this type of device. With regard to electronic microtiter plate type devices (which do not have the low current/voltage constraints), the use of mixed buffers (low conductance upper chamber/high conductance lower chamber) can be used in a unique fashion to create favorable hybridization conditions in a microzone within the test site location. Both types of devices allow DNA molecules to be rapidly and selectively hybridized at the array test sites under conditions where the DNA in the bulk solution can remain substantially denatured.     

Parallel analysis of biomolecules on a microfabricated capillary array chip

This paper focused on a self-developed microfluidic array system with microfabricated capillary array electrophoresis (mu-CAE) chip for parallel chip electrophoresis of biomolecules. The microfluidic array layout consists of two common reservoirs coupled to four separation channels connected to sample injection channel on the soda-lime glass substrate. The excitation scheme for distributing a 20 mW laser beam to separation channels in an array is achieved. Under the control of program, the sample injection and separation in multichannel can be achieved through six high-voltage modules' output. A CCD camera was used to monitor electrophoretic separations simultaneously in four channels with LIF detection, and the electropherograms can be plotted directly without reconstruction by additional software. Parallel multichannel electrophoresis of series biomolecules including amino acids, proteins, and nucleic acids was performed on this system and the results showed fine reproducibility.     

Transport of biomolecules in asymmetric nanofilter arrays

We propose a theoretical model for describing the electric-field-driven migration of rod-like biomolecules in nanofilters comprising a periodic array of shallow passages connecting deep wells. The electrophoretic migration of the biomolecules is modeled as transport of point-sized Brownian particles, with the orientational degree of freedom captured by an entropy term. Using appropriate projections, the formulation dimensionality is reduced to one physical dimension, requiring minimal computation and making it ideal for device design and optimization. Our formulation is used to assess the effect of slanted well walls on the energy landscape and resulting molecule mobility. Using this approach, we show that asymmetry in the well shape, such as a well with one slanted and one vertical wall, may be used for separation using low-frequency alternating-current fields because the mobility of a biomolecule is different in the two directions of travel. Our results show that, compared to methods using direct-current fields, the proposed method remains effective at higher field strengths and can achieve comparable separation using a significantly shorter device.     

Nonmonotonous variation of DNA angular separation during asymmetric pulsed field electrophoresis

Asymmetric pulsed field electrophoresis within crystalline arrays is used to generate angular separation of DNA molecules. Four regimes of the frequency response are observed, a low frequency rise in angular separation, a plateau, a subsequent decline, and a second plateau at higher frequencies. It is shown that the frequency response for different sized DNA is governed by the relation between pulse time and the reorientation time of DNA molecules. The decline in angular separation at higher frequencies has not previously been analyzed. Real‐time videos of single DNA molecules migrating under high frequency‐pulsed electric field show the molecules no longer follow the head to tail switching, ratchet mechanism seen at lower frequencies. Once the pulse period is shorter than the reorientation time, the migration mechanism changes significantly. The molecule reptates along the average direction of the two electric fields, which reduces the angular separation. A freely jointed chain model of DNA is developed where the porous structure is represented with a hexagonal array of obstacles. The model qualitatively predicts the variation of DNA angular separation with respect to frequency.     

Flow-based and sieving matrix-free DNA differentiation by a miniaturized field flow fractionation device

DNA separation is typically done by gel electrophoresis based on its charge property. In our previous work, we reported that dielectrophoresis could be used to manipulate polystyrene nanoparticles' motion by using a miniaturized electrical field flow fractionation device (micro-EFFF) with a segmented electrode operated under a pulsed voltage (PV). In this work, we report the manipulation and separation of DNA molecules using the micro-EFFF. DNA motion was in situ visualized inside the device. Results revealed that dielectrophoresis governed DNA motion, which was strongly correlated with the pulse frequency but not the duty cycle of a PV. A longer retention time of DNA molecules was measured under a PV. The retention time increased with the length of DNA molecules. As the micro-EFFF is flow-based and sieving-matrix-free, it has a potential to be applied to sample preparation in a micrototal analysis system or when fractionated molecules are needed for downstream analysis.     

Confinement effects on electromigration of long DNA molecules in an ordered cavity array

Confinement effects on the electromigration of long dsDNA molecules in an array of well-ordered, molecular sized cavities interconnected by nanopores are described. The array was prepared by replicating the structure of a colloidal crystal of microspheres in a polymer matrix. Both conformation and mobility studies show that the electrophoretic behavior of large DNA molecules is distinct from that in gels and other microfabricated sieves, showing a peak in mobility versus electric field, and an inversion in the separation order with field strength. A simple model was proposed to interpret this unexpected observation qualitatively. We conclude that the molecular behavior of DNA can be modified by the combination of entropic effect and electric trapping as a consequence of both unique geometry and conductivity of this cavity array material. This approach provides insights for design and optimization of on-chip molecular sieving structures for rapid separation of long DNA molecules. It may lead to a promising material for manipulation and size fractionation of other biological macromolecules.     

A decade of capillary electrophoresis

Since the introduction of the first commercial capillary electrophoresis (CE) instrument a decade ago, CE applications have become widespread. Today, CE is a versatile analytical technique which is successfully used for the separation of small ions, neutral molecules, and large biomolecules and for the study of physicochemical parameters. It is being utilized in widely different fields, such as analytical chemistry, forensic chemistry, clinical chemistry, organic chemistry, natural products, pharmaceutical industry, chiral separations, molecular biology, and others. It is not only used as a separation technique but to answer physicochemical questions. In this review, we will discuss different modes of CE such as capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, capillary isoelectric focusing, and capillary electrochromatography, and will comment on the future direction of CE, including array capillary electrophoresis and array microchip separations.     

Selective extraction of size-fractioned DNA samples in microfabricated electrophoresis devices

We have designed and constructed a microfabricated device for separation of double-stranded DNA fragments using a crosslinked sieving medium and spatially selective extraction of the desired fraction. Based on measuring the width and spacing of migrating bands, a narrow side channel is constructed perpendicular to the separation channel to collect the DNA fragments of interest. This selective collection technique was tested using a 100 base pair double-stranded DNA ladder. We successfully demonstrate selective extraction of the desired fragment with minimal interference from the adjacent bands in an electric field of 31 V/cm. We also achieve extraction of multiple DNA fragments using an array of microelectrodes in this side channel. The device uses cross-linked polyacrylamide gel matrix, allowing the separation to be performed in a distance of 1 cm or less and at a low electric field strength. Together with on-chip electrode, this design is amenable to integration with reaction chambers into a single device for portable genetic-based analysis.     

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.     

Detection of DNA and Protein Molecules Using an FET‐Type Biosensor with Gold as a Gate Metal

In this study, a field effect transistor (FET)‐type biosensor based on 0.5 μm standard complementary metal oxide semiconductor (CMOS) technology is proposed and its feasibility for detecting deoxyribonucleic acid (DNA) and protein molecules is investigated. Au, which has a chemical affinity with thiol by forming a self‐assembled monolayer (SAM), was used as the gate metal in order to immobilize DNA and protein molecules. A Pt pseudo‐reference electrode was employed for the detection of biomolecules. The sensor was fabricated as a p‐channel (P)MOSFET‐type because PMOSFET with positive surface potential is useful for detecting negatively charged biomolecules from the view point of its high sensitivity and fast response time. DNA and protein molecules were detected by measuring the variation of the drain current due to the variation of biomolecular charge and capacitance. DNA and protein molecules used in the experiment were 15mer–oligonucleotide probe and streptavidin‐biotin protein complexes, respectively. DNA was detected by both in situ and ex situ measurements. Additionally, to verify the interactions among SAM, streptavidin, and biotin, surface plasmon resonance (SPR) measurement was performed.     

Differential transport of DNA by a rectified Brownian motion device

An interdigitated electrode array (IDEA) device has been designed and used to transport DNA based on a Brownian ratchet mechanism. This migration is produced by the periodic formation of an asymmetric sawtooth electric field in the device. Oligonucleotides of 25, 50, and 100 bases in length were tested using two different array geometries. DNA transport as a function of DNA size, electric field frequency, and array geometry is shown to be in qualitative agreement with theory. Such a device could provide for DNA separations over a broad size range, and can be readily scaled as a component in a microfabricated DNA analysis 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.     

Functionalized Nanogap for DNA Read-Out: Nucleotide Rotation and Current-Voltage Curves

Functionalized nanogaps embedded in nanopores show a strong potential for enhancing the detection of biomolecules, their length, type, and sequence. This detection is strongly dependent on the features of the target biomolecules, as well as the characteristics of the sensing device. In this work, through quantum-mechanical calculations, we elaborate on representative such aspects for the specific case of DNA detection and read-out. These aspects include the influence of single DNA nucleotide rotation within the nanogap and the current-voltage (I-V) characteristics of the nanogap. The results unveil a distinct variation in the electronic current across the functionalized device for the four natural DNA nucleotides with the applied voltage. These also underline the asymmetric response of the rotating nucleotides on this applied voltage and the respective variation in the rectification ratio of the device. The electronic tunneling current across the nanogap can be further enhanced through the proper choice of an applied bias voltage. We were able to correlate the enhancement of this current to the nucleotide rotation dynamics and a shift of the electronic transmission peaks towards the Fermi level. This nucleotide specific shift further reveals the sensitivity of the device in reading-out the identity of the DNA nucleotides for all different configurations in the nanogap. We underline the important information that can be obtained from both the I-V curves and the rectification characteristics of the nanogap device in view of accurately reading-out the DNA information. We show that tuning the applied bias can enhance this detection and discuss the implications in view of novel functionalized nanopore sequencers.     

DNA separations in microfabricated devices with automated capillary sample introduction

A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention.     

On-chip electrophoretic accumulation of DNA oligomers and streptavidin

A micro-chamber for electrophoretic accumulation of charged biomolecules has been designed and evaluated. The system is based on a chip with an array of planar focusing electrodes. Particular attention was devoted to a design which enables penetration of a large sample volume by the electric field of the focusing electrodes. General design principles for a cylindrically symmetrical arrangement of the focusing electrodes were derived. Accumulation of DNA oligomers and streptavidin in aqueous solution was demonstrated. The concentration of biomolecules in the centre of the chip was enhanced by up to a factor of 200. The major fraction of the total charge delivered during electrophoretic accumulation results from Faradaic processes. The maximum charge density deliverable without visible gas formation was determined. By careful control of the voltage and current density applied to the electrodes, evolution of gas bubbles could be avoided for the time required to accumulate analyte molecules in the centre of the micro-chamber. On-chip electrophoretic accumulation of biomolecules can be applied to sample pre-conditioning in lab-on-a-chip devices for analysis of DNA and protein samples.     

Design of a microfabricated magnetic cell separator.

A new magnetic separation idea utilizing several ideas from microfabrication and nanomagnetics is presented. The basic idea comes from our earlier work using asymmetry in obstacles and Brownian motion to effect separation of objetcs [10] by moving them in streams whose angle to the hydrodynamic average velocity is a function of the diffusion coefficient of the object. The device we propose here is not technically a Brownian ratchet device but uses the idea of force which acts at angle to the hydrodynamic flow. In our case, the force is generated by a magnetic field gradient which comes from an array of magnetized wires which lie at an angle 0 to a hydrodynamic field flow. The sum of the hydrodynamic force and the magnetic force create a new vector which as in the case of the Brownian ratchet moves the cell out of the main stream direction.     

Capillary electrophoresis microchip for direct amperometric detection of DNA fragments

Detection and quantitation of nucleic acids have gained much importance in the last couple of decades, especially in the post-human genome project era. Such processes are tedious, time consuming and require expensive reagents and equipment. Therefore, in the present study, we demonstrated a simple process for the separation and analysis of small DNA fragments using capillary electrophoretic amperometric detection on an inexpensive disposable glass microchip. The device used polydimethylsiloxane engraved microchannel and Au/Ti in-channel microelectrodes for sample detection. The DNA fragments were separated under low electric field (20 V/cm) for improved detection sensitivity and to retain the biomolecules in their native conformation. With a low sample requirement (as low as 1 μL) and high reproducibility, the proposed microchip device was successful in resolution and detection of DNA fragments of various lengths.     

Methods to electrophoretically stretch DNA: microcontractions, gels, and hybrid gel-microcontraction devices

The ability to controllably and continuously stretch large DNA molecules in a microfluidic format is important for gene mapping technologies such as Direct Linear Analysis (DLA). We have recently shown that electric field gradients can be readily generated in a microfluidic device and the resulting field is purely elongational. We present a single molecule fluorescence microscopy analysis of T4 DNA (169 kbp) stretching in the electric field gradients in a hyperbolic contraction microchannel. In addition, we are able to selectively pattern a crosslinked gel anywhere inside the microchannel. With an applied electric field, DNA molecules are forced to reptate through the gel and they moderately stretch as they exit the gel. By placing a gel immediately in front of the hyperbolic contraction, we bypass "molecular individualism" and achieve highly uniform and complete stretching of T4 DNA.     

Continuum transport model of Ogston sieving in patterned nanofilter arrays for separation of rod-like biomolecules

This article proposes a simple computational transport model of rod-like short dsDNA molecules through a microfabricated nanofilter array. Using a nanochannel consisting of alternate deep wells and shallow slits, it is demonstrated that the complex partitioning of rod-like DNA molecules of different sizes over the nanofilter array can be well described by continuum transport theory with the orientational entropy and anisotropic transport parameters properly quantified. In this model, orientational entropy of the rod-like DNA is calculated from the equilibrium distribution of rigid cylindrical rod near the solid wall. The flux caused by entropic differences is derived from the interaction between the DNA rods and the solid channel wall during rotational diffusion. In addition to its role as an entropic barrier, the confinement of the DNA in the shallow channels also induces large changes in the effective electrophoretic mobility for longer molecules in the presence of EOF. In addition to the partitioning/selectivity of DNA molecules by the nanofilter, this model can also be used to estimate the dispersion of separated peaks. It allows for fast optimization of nanofilter separation devices, without the need of stochastic modeling techniques that are usually required.     

Microfluidic system for dielectrophoretic separation based on a trapezoidal electrode array

This paper presents a novel microfluidic device for dielectrophoretic separation based on a trapezoidal electrode array (TEA). In this method, particles with different dielectric properties are separated by the device composed of the TEA for the dielectrophoretic deflection of particles under negative dielectrophoresis (DEP) and poly(dimethylsiloxane)(PDMS) microfluidic channel with a sinuous and expanded region. Polystyrene microparticles are exposed to an electric field generated from the TEA in the microfluidic channel and are dielectrophoretically focused to make all of them line up to one sidewall. When these particles arrive at the region of another TEA for dielectrophoretic separation, they are separated having different positions along the perpendicular direction to the fluid flow due to their different dielectrophoretic velocities. To evaluate the separation process and performance, both the effect of the flow rate on dielectrophoretic focusing and the influence of the number of trapezoidal electrodes on dielectrophoretic separation are investigated. Now that this method utilizes the TEA as a source of negative DEP, non-specific particle adhering to the electrode surface can be prevented; conventional separation approaches depending on the positive DEP force suffer from this problem. In addition, since various particle types are continuously separated, this method can be easily applicable to the separation and analysis of various dielectric particles with high particle recovery and selectivity.