Exploiting the benefits of miniaturization for the enhancement of DNA microarrays

The present study demonstrates that the best way to enhance DNA microarray assays, both in terms of analysis speed and in final spot intensity, is to dissolve the available molar amount of sample in the smallest possible buffer volume and to subsequently convect this solution continuously across the surface of the array. The presently proposed shear-driven flow system is pre-eminently suited for this task, as it allows to induce strongly enhanced lateral transport rates, independently of the degree of miniaturization of the hybridization chamber. This transport enhancement method, however, only increases the hybridization rate and not the final spot intensity, as neither can any of the other transport enhancement methods already proposed in literature. A series of experiments with synthetic single-stranded (ssDNA) samples and an accompanying mass balance analysis are presented to demonstrate these points.     

A DNA hybridization assay using nanogold-streptavidin,electro-microchip and silver enhancement to analyze impedance signals

This paper reports a novel DNA hybridization detection method using gold-streptavidin conjugates as a reporter, the silver enhancement reaction to magnify the detection signal, a commercial LCR meter to detect the hybridization signal. The relationships between sample concentration and detection signal are discussed and the detection limit for the DNA sample is 0.825 ng/mL. Moreover, the probe specific tests are discussed for different species of bacteria, for same species but different strains, and for same genus but different species.     

An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules

A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically. The critical parameters for electrostretching of encapsulated DNA were investigated and optimum stretching was achieved by using 1.5 kHz pulses with electric field strength in the order of 10(3) V cm(-1) in 7% linear polyacrylamide (LPA) solution. LPA was adopted to minimize the adverse effects of ionic thermal agitation on molecular dielectrophoretic elongation in the microchamber. In our experiments, as the fragments were not immobilized throughout the entire protocol, it was found from repeated tests that digestion always occurred, producing the expected number of cleaved fragments. This versatile microchamber approach realized direct observation of these biological reactions on real-time basis at a single-molecule level. Furthermore, with the employment of porous polyacrylamide gel, the effective manipulation of DNA assays and the ability to combine conventionally independent bioanalytical processes have been demonstrated.     

A biological sensor platform using a pneumatic-valve controlled microfluidic device containing Tetrahymena pyriformis

In this study, we introduce a microfluidic device equipped with pneumatically actuated valves, generating a linear gradient of chemoeffectors to quantify the chemotactic response of Tetrahymena pyriformis, a freshwater ciliate. The microfluidic device was fabricated from an elastomer, poly(dimethylsiloxane) (PDMS), using multi-layer soft lithography. The components of the device include electronically controlled pneumatic microvalves, microchannels and microchambers. The linear gradient of the chemoeffectors was established by releasing a chemical from a ciliate-free microchamber into a microchamber containing the ciliate. The ciliate showed chemotactic behaviours by either swimming toward or avoiding the gradient. By counting the number of ciliates residing in each microchamber, we obtained a precise time-response curve. The ciliates in the microfluidic device were sensitive enough to be attracted to 10 pmol glycine-proline, which indicates a 10(5) increase in the ciliate's known sensitivity. With the use of blockers, such as DL-2-amino-5-phosphonopentanoic acid (APPA) or lanthanum chloride (LaCl3), we have demonstrated that the NMDA (N-methyl-d-aspartate) receptor plays a critical role in the perception of chemoeffectors, whereas the Ca2+ channel is related to the motility of the ciliate. These results demonstrate that our microfluidic chemotaxis assay system is useful not only for the study of ciliate chemotaxis but also for a better understanding of the signal transduction mechanism on their receptors.     

On the possibility of shear-driven chromatography: a theoretical performance analysis.

The use of shear forces for the generation of the mobile phase flow in chromatographic separations is proposed. This novel chromatographic operating principle, referred to as shear-driven chromatography (SDC), completely circumvents the pressure-drop limitation of conventional pressure-driven GC and LC without affecting the operational flexibility (choice of mobile and stationary phases, possibility of solvent and/or temperature programming, etc.). In the present paper, the expression for the height equivalent to a theoretical plate in SDC in a channel with a flat rectangular cross-section is established and is used to demonstrate the large gain in analysis speed under LC, GC and supercritical fluid chromatography conditions.     

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.     

Amplifying the electrical hybridization signals of DNA array by multilayer assembly of Au nanoparticle probes

This paper describes a versatile method for amplifying the signals of Au-nanoparticle-based DNA hybridization detecting systems. The Au nanoparticles usually serve as labels to enhance DNA hybridization signal. We further assembled several layers of nanoparticles to selectively increase the number of labelled nanoparticles. Through silver enhancement, the multilayer nanoparticles may produce significantly higher amounts of metal silver on the their surfaces than the monolayer nanoparticles did. This finally accounts for the greatly enhanced DNA hybridization signal. Particularly, the amplification of electrical detection system was demonstrated here. Electrical measuring results indicated that the current values were enhanced by approximately 3 orders of magnitude, and the single nucleotide mismatch discrimination ratio was enlarged to approximately 10(9):1.     

Shear-driven pumping and Fourier transform detection for on chip circular chromatography applications

A circular, shear-driven pumping system combined with Fourier Transform detection has been developed for the application of chip based cyclic chromatography. Using this system, it is possible to perform an injection of a sample plug into a circular micro-channel and then drive the sample through the channel, using shear flow induced by a rotation stage. High pumping rates at uniform speeds are attainable with this system with very little heat production. Typical pumping rates of up to 1.423 mm s(-1) +/- 2 microm s(-1) were used in experiments although much higher rates >14 mm s(-1) are also possible with this system. Fluorescence detection was used to detect a sample plug of Coumarin dyes, flowing around the channel. A coating of porous polymethacrylate was used to immobilise RP-HPLC beads onto the glass surface and was applied to the glass micro-channel by selective photopolymerisation. This coating acted as a stationary phase and differences in retention time were observed for an injection of Coumarin dyes for different methanol-water, mobile phase ratios. Full sample retention occurred for 30 : 70 (v/v) methanol-water whereas no retention occurred for 92 : 8 (v/v) methanol-water which can be expected for such a reverse phase, open tubular system. Fourier transform detection applied to the fully retained and non-retained cases showed frequency domain data from a single detection point corresponding to that which may be expected from shear flow theory.     

Rapid DNA hybridization analysis using a PDMS microfluidic sensor and a molecular beacon.

A rapid DNA analysis has been developed based on a fluorescence intensity change of a molecular beacon in a PDMS microfluidic channel. Recently, we reported a new analytical method of DNA hybridization involving a PDMS microfluidic sensor using fluorescence energy transfer (FRET). However, there are some limitations in its application to real DNA samples because the target DNA must be labelled with a suitable fluorescent dye. To resolve this problem, we have developed a new DNA microfluidic sensor using a molecular beacon. By monitoring the change in the restored fluorescence intensity along the channel length, it is possible to rapidly detect any hybridization of the molecular beacon to the target DNA. In this case, the target DNA does not need to be labelled. Our experimental results demonstrate that this microfluidic sensor using a molecular beacon is a promising diagnostic tool for rapid DNA hybridization analysis.     

Amplified label-free electrical detection of DNA hybridization

A new protocol is described for amplifying label-free electrochemical measurements of DNA hybridization based on the enhanced accumulation of purine nucleobases in the presence of copper ions . Such electrical DNA assays involve hybridization of the target to inosine-substituted oligonucleotide probes (captured on magnetic beads), acidic dipurinization of the hybrid DNA, and adsorptive chronopotentiometric stripping measurements of the free nucleobases in the presence of copper ions. Both amplified adenine and guanine peaks can be used for detecting the DNA hybridization. The dramatic signal amplification advantage of this type of detection has been combined with efficient magnetic removal of non-complementary DNA, use of microliter sample volumes and disposable transducers. Factors influencing the signal enhancement were assessed and optimized. A detection limit of 40 fmol (250 pg) was obtained with 10 min hybridization and 5 min adsorptive-accumulation times. The advantages of this procedure were demonstrated by its application in the detection of DNA segments related to the BRCA1 breast cancer gene. The copper enhancement holds great promise not only for the detection of DNA hybridization, but also for trace measurement of nucleic acids.     

Photoactivated capture molecule immobilization in plasmonic nanoapertures in the ultraviolet

We demonstrate a photoactivated surface coupling scheme for achieving spatial overlap between biomolecules of interest and optical near field excitation. Using aluminium nanoapertures, we obtained increased coupling efficiency of biotinylated capture probe oligos to the photoactivated surface due to ~3× nanoaperture enhancement of UV light. We further validate DNA sensor functionality via the hybridization of Cy-5 labeled target oligos, with up to 8× fluorescence enhancement obtained from a commercial microarray scanner. This generic photoimmobilization strategy is an essential step to realizing miniaturized plasmon enhanced detection arrays by virtue of localizing capture molecules to the region of plasmonic enhancement.     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 μL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides. Received: 26 September 2000 / Revised: 24 November 2000 / Accepted: 30 November 2000     

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     

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.     

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.     

Monitoring molecular beacon DNA probe hybridization at the single-molecule level

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.     

旋转玻碳电极上二茂铁的电化学阻抗行为及其与DNA的相互作用

应用旋转圆盘电极和电化学阻抗法研究了二茂铁在Tris-NaC l(pH=7.2)缓冲溶液中于旋转玻碳电极上的电化学阻抗行为及其与DNA的相互作用.结果表明,二茂铁于旋转电极的伏安曲线呈现明显的极限电流平阶,而其交流阻抗谱则出现两个电容弧.二茂铁与DNA的作用,若受扩散过程控制则其极限扩散电流随DNA浓度增大而减小,而在电化学控制过程中则表现为电化学反应电阻随DNA浓度增大而增大.根据旋转圆盘电极和电化学阻抗谱测试,表明由这两种方法数据拟合求得的二茂铁条件电位速率常数能够很好地相互吻合,但如存在DNA时,则其条件电位速率常数有一定程度的减小.     

Nanoparticle based enhancement of electrochemical DNA hybridization signal using nanoporous electrodes

A novel nanoparticle-based enhanced methodology for the detection of ssDNA using nanoporous alumina filter membranes, containing pores of 200 nm in diameter, is reported. The blockage of the pores due to the hybridization is detected by measuring the decrease in the differential pulse voltammetric response of the [Fe(CN)(6)](4-/3-) redox indicator and using screen-printed carbon electrodes as transducing platform. Furthermore, 20 nm gold nanoparticle (AuNPs) tags are used in order to increase the sensitivity of the assay. The enhancement mechanism of DNA detection is due to an additional blocking effect induced by hybridization reaction by bringing AuNPs inside the pores. The developed methodology can be extended to other biosensing systems with interest not only for DNA but also for proteins and cells. The developed nanochannel/nanoparticle biosensing system would have enormous potential in future miniaturized designs adapted to mass production technologies such as screen-printing technology.     

Enzyme-catalyzed signal amplification for electrochemical DNA detection with a PNA-modified electrode

The signal amplification technique of peptide nucleic acid (PNA)-based electrochemical DNA sensor was developed in a label-free and one-step method utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. The negatively charged mediator, ferrocenedicarboxylic acid, cannot diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of the breast cancer susceptibility gene BRCA1 at a sample concentration lower than 10(-9) M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.