A method for DNA detection in a microchannel: fluid dynamics phenomena and optimization of microchannel structure

A simple DNA diagnosis method using microfluidics has been developed which requires simple and straightforward procedures such as injection of sample and probe DNA solutions. This method takes advantage of the highly accurate control of fluids in microchannels, and is superior to DNA microarray diagnosis methods due to its simplicity, highly quantitative determination, and high-sensitivity. The method is capable of detecting DNA hybridization for molecules as small as a 20 mer. This suggests the difference in microfluidic behavior between single strand DNA (ssDNA) and double stranded DNA (dsDNA). In this work, influence of both the inertial force exerted on DNA molecules and the diffusion of DNA molecules was investigated. Based on the determination of these parameters for both ssDNA and dsDNA by experiments, a numerical model describing the phenomena in the microchannel was designed. Computational simulation results using this model were in good agreement with previously reported experimental results. The simulation results showed that appropriate selection of the analysis point and the design of microchannel structure are important to bring out the diffusion and inertial force effects suitably and increase the sensitivity of the detection of DNA hybridization, that is, the analytical performance of the microfluidic DNA chip.     

Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel

A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described. A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy (SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising real-time detection method for label-free DNA targets in the solution phase. Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel     

Rapid screening of phenylketonuria using a CD microfluidic device

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5μL, and the limit-of-detection (LOD) of DNA template was 0.7ng/μL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.     

Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18?000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

Cationic polymer coatings for design of electroosmotic flow and control of DNA adsorption

A difficulty with the design and operation of an electrokinetically operated DNA hybridization microfluidic chip is the opposite direction of the electroosmotic flow and electrophoretic mobility of the oligonucleotides. This makes it difficult to simultaneously deliver targets and an appropriate hybridization buffer simultaneously to the probe sites. In this work we investigate the possibility of coating the inner walls of the microfluidic system with hexadimentrine bromide (polybrene, PB) and other cationic polymers in order to reverse the direction of electroosmotic flow so that it acts in the same direction as the electrophoretic transport of the oligonucleotides. The results indicated that the electroosmotic flow (EOF) in channels that were coated with the polymer could be reversed in 1× TBE buffer or 1× SSC buffer. Under these conditions, the DNA and EOF move in the same direction, and the flow can be used to deliver DNA to an area for selective hybridization within the channel. The effects of coating the surface of a nucleic acid microarray with polybrene were also studied to assess non-selective adsorption and stability. The polybrene coating significantly reduced the extent of non-selective adsorption of oligonucleotides in comparison to adsorption onto a glass surface, and the coating did not alter the extent of hybridization. The results suggest that use of the coating makes it possible to achieve semi-quantitative manipulation of nucleic acid oligomers for delivery to an integrated microarray or biosensor.     

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays

Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within single array microspots before and after target hybridization. Principal component analysis (PCA) applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with array spot nonuniformities (e.g., "halo" or "donut" effects often observed in fluorescence images). Chemically specific spot images were compared to conventional fluorescence scanned images in microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity.     

Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD–oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip.     

Polymer microfluidic chip for online monitoring of microarray hybridizations

A disposable single use polymer microfluidics chip has been developed and manufactured by micro injection molding. The chip has the same outer dimensions as a standard microscope slide (25 x 76 x 1.1 mm) and is designed to be compatible with existing microscope slide handling equipment like microarray scanners. The chip contains an inlet, a 10 microL hybridization chamber capable of holding a 1000 spot array, a waste chamber and a vent to allow air to escape when sample is injected. The hybridization chamber ensures highly homogeneous hybridization conditions across the microarray. We describe the use of this chip in a flexible setup with fluorescence based detection, temperature control and liquid handling by computer controlled syringe pumps. The chip and the setup presented in this article provide a powerful tool for highly parallel studies of kinetics and thermodynamics of duplex formation in DNA microarrays. The experimental setup presented in this article enables the on-chip microarray to be hybridized and monitored at several different stringency conditions during a single assay. The performance of the chip and the setup is demonstrated by on-line measurements of a hybridization of a DNA target solution to a microarray. A presented numerical model indicates that the hybridization process in microfluidic hybridization assays is diffusion limited, due to the low values of the diffusion coefficients D of the DNA and RNA molecules involved.     

Computer simulation study of probe-target hybridization in model DNA microarrays: effect of probe surface density and target concentration

We use lattice Monte Carlo simulations to study the thermodynamics of hybridization of single-stranded "target" genes in solution with complementary "probe" DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct, with each segment representing a sequence of nucleotides that interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how surface density (number of probes per unit surface area) and concentration of target molecules affect the extent of hybridization. For short probe lengths, as the surface density increases, the probability of binding long stretches of target segments increases at low surface density, reaches a maximum at an intermediate surface density, and then decreases at high surface density. Furthermore, as the surface density increases, the target is less likely to bind completely to one probe; instead, it binds simultaneously to multiple probes. At short probe lengths, as the target concentration increases, the fraction of targets binding completely to the probes (specificity) decreases. At long probe lengths, varying the target concentration does not affect the specificity. At all target concentrations as the probe length increases, the fraction of target molecules bound to the probes by at least one segment (sensitivity) increases while the fraction of target molecules completely bound to the probes (specificity) decreases. This work provides general guidelines to maximizing microarray sensitivity and specificity. Our results suggest that the sensitivity and specificity can be maximized by using probes 130-180 nucleotides long at a surface density in the range of 7 x 10(-5)- 3 x 10(-4) probe molecules per nm(2).     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.     

A simple DNA Characterization method using fiber-fluorescence in situ hybridization performed without DNA fragmentation

We performed high-resolution fluorescence imaging of lambda phage DNA molecules hybridized with fluorescent-labeled DNA and peptide nucleic acid probes. In this method, the target DNA and probe were mixed, rapidly denatured and then subjected to liquid hybridization conditions. The hybridized DNA sample was then spotted onto a nontreated glass substrate and subjected to molecular combing. The resultant continuous fluorescence signal of intact lambda DNA shows that the fluorescent-labeled probes bound to the predicted sites but in a pattern that was clearly different to the beads-on-a-string pattern typical for fiber-fluorescence in situ hybridization. The key changes to the conventional method are hybridization of the free target DNA in liquid and lowering the denaturation temperature. The method described here allows the rapid and direct visualization of the specific binding sites of intact DNA molecules without damaging the DNA fibers and causing fragmentation of the fluorescence signal. This technique should be a useful tool in studies of genetics and also large-scale DNA sequencing projects.     

A rapid method for detection of genetically modified organisms based on magnetic separation and surface-enhanced Raman scattering

In this study, a new method combining magnetic separation (MS) and surface-enhanced Raman scattering (SERS) was developed to detect genetically modified organisms (GMOs). An oligonucleotide probe which is specific for 35 S DNA target was immobilized onto gold coated magnetic nanospheres to form oligonucleotide-coated nanoparticles. A self assembled monolayer was formed on gold nanorods using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and the second probe of the 35 S DNA target was immobilized on the activated nanorod surfaces. Probes on the nanoparticles were hybridized with the target oligonucleotide. Optimization parameters for hybridization were investigated by high performance liquid chromatography. Optimum hybridization parameters were determined as: 4 μM probe concentration, 20 min immobilization time, 30 min hybridization time, 55 °C hybridization temperature, 750 mM buffer salt concentration and pH: 7.4. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. The correlation between the target concentration and the SERS signal was found to be linear within the range of 25-100 nM. The analyses were performed with only one hybridization step in 40 min. Real sample analysis was conducted using Bt-176 maize sample. The results showed that the developed MS-SERS assay is capable of detecting GMOs in a rapid and selective manner.     

A single-bead analysis on a disk-shaped microfluidic device using an antigen-immobilized bead.

A disk-shaped microfluidic device (lab-on-a-Disk) was developed to allow the evaluation of mental stress. As a standard sample, secretory immunoglobulin A (sIgA), which is a candidate marker of mental stress, was measured by a heterogeneous enzyme immunoassay (EIA) on the lab-on-a-Disk. Centrifugal force provided a microfluidic control on the lab-on-a-Disk. We examined the relationship between the rotational speed, the channel profile, and the position of the microfluidic chambers from the center of rotation to manipulate sample solutions into each reaction reservoir through microchannels sequentially, i.e., retain in a reservoir or flow into a subsequent reservoir. A single glass bead with immobilized sIgA on its surface was injected into a reservoir for a competitive antigen-antibody reaction, and applied to a specific surface in a heterogeneous assay. It is expected that the lab-on-a-Disk would be suitable for miniaturization and automation of the processes in EIA compared with a conventional EIA using a titer plate.     

Packaging of microfluidic chips via interstitial bonding technique.

In this paper, we describe an interstitial bonding technique for packaging of microfluidic chips. The cover plate is first placed on top of the microfluidic chip, followed by dispensing the UV-curable resin into the resin-loading reservoirs. With the interstitial space between the cover plate and the microfluidic chip connecting to the loading reservoirs, the UV-curable resin wicks through capillary force action and hydrostatic pressure generated by the liquid level in the resin-loading reservoirs. When reaching the microchannels, the UV-curable resin stops flowing into the microchannels due to the force balance between the surface tension and hydrostatic pressure. The assembly is then placed under the UV light, followed by further curing in the thermal oven. It is found that there is no leakage from the bonded microfluidic chips and a good DNA separation result was obtained by using the microfluidic chips as fabricated. This bonding technique is relatively simple and fast, which can be applied to the packaging of microfluidic chips made from hybrid materials with complicated designs as long as the interstitial space connects to the loading reservoirs.     

Improved understanding of the effect of electrical double layer on pressure-driven flow in microchannels

Traditionally, the effects of electrical double layer on pressure-driven flow in microchannels were modeled by using the Poisson-Boltzmann equation and the fluid momentum equation with a flow-induced body force term. Such a model, however, usually underestimate the electrical double layer effects on the flow. In this study, a theoretical model of the electrical double layer field is developed to provide a better understanding of the electrical double layer effects. The electrical potential and ionic concentration distribution in dilute solutions in small microchannels are investigated by numerically solving this new model. This newly developed model predicted the deficit of counter-ions in the bulk liquid region due to the accumulation of counter-ions in the EDL region, and the surplus of co-ions in the bulk liquid region due to rejection of the co-ions in the EDL region. The presence of the net charges in the bulk liquid region is responsible for the strong electroviscous effects in dilute solutions in small microchannels.     

Label-free detection of DNA with interdigitated micro-electrodes in a fluidic cell

We investigate the analytical performance of an interdigitated electrode sensor for the label-free detection of DNA, by monitoring the complex impedance of 5 microm wide interdigitated Pt microelectrodes on a glass substrate. We detect the hybridization of unlabeled 38-mer target ssDNA with a complementary probe that is bound on the glass in between the electrodes by a disuccinimidyl terephtalate and aminosilane immobilization procedure. The sensor is mounted in a microfluidic flow cell, in which hybridization is monitored and in situ compared with a reference. After hybridization, the cell is perfused with deionised water and the dependence of the measured conductance due to the immobilized target DNA layer, to target DNA concentrations down to 1 nM is demonstrated. Subsequently, we apply our sensor to the detection of pathogen DNA from Salmonella choleraesuis in dairy food.