DNA Hybridization Efficiency Research Articles

Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3

Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4-4.5h. The parallel quantification was possible in a concentration range of 9.6 × 10(5)-1.4 × 10(10)genomic units (GU)/mL for bacteriophage MS2, 1.4 × 10(5)-3.7 × 10(8)GU/mL for bacteriophage ΦX174, and 6.5 × 10(3)-1.2 × 10(5) for hAdV2, respectively, by using a measuring temperature of 40°C. Detection limits could be calculated to 6.6 × 10(5)GU/mL for MS2, 5.3 × 10(3)GU/mL for ΦX174, and 1.5 × 10(2)GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.

DNA Hybridization Efficiency on Concave Surface Nano-Structure in Hemispherical Janus Nanocups

We examined the effect of a concave structure on DNA hybridization efficiency using an inner surface of hemispherical Janus nanocups in the range from 140 to 800 nm. Target DNA was specifically immobilized onto the inner cup surface, hybridized with complementary DNA-attached 20 nm Au probes, and the number of the hybridized probes was counted by scanning electron microscopy. The hybridization density of the attached Au probes on 800 nm nanocups was 255 μm(-2), which was 0.57 times that on a flat surface, 449 μm(-2), and increased to 394 μm(-2) on a 140 nm cup, 0.88 times of a flat surface, as the cup size decreased. The local density of attached Au probes within the central 25% at the bottom of the 800 nm nanocups was 444 μm(-2), which was closer to that on a flat surface, and the tendency was the same for all sizes of cups, indicating that the size dependency of DNA hybridization efficiency on the concave structures were mostly affected by the lower efficiency of side wall hybridization.

Effect of Ionic Strength on End-Tethered ssDNA Molecules on Gold Surfaces

DNA conformational and mechanical properties play a significant role in determining DNA protein interactions and hybridization efficiency. Counterions differently screen the electrostatic charge carried by the DNA backbone affecting its curvature and flexibility. In order to improve the understanding of DNA conformation in crowded cellular environments and for the realization of more efficient biosensors, we analyse the collective response of DNA brushes to changes of ionic strength. We report here about an atomic force microscopy (AFM) study of the ionic effects on single stranded (ssDNA) confined monolayers tethered on ultra flat gold surfaces. We realized via Nanografting, an AFM-based lithography, micrometer sized brushes of short ssDNAs with controlled, variable surface density, confined inside a biorepellent self-assembled monolayer. Varying concentration and salt species (NaCl, KCl, CaCl2) inside the AFM liquid cell we monitored with high precision the corresponding ssDNA brush height variations. We will show the measured height can be related to scaling law of salt concentration with an exponent α =-1/6, in agreement with the theory of polyelectrolyte brush.

3P291 ナノ粒子表面へのプローブオリゴヌクレオチドの修飾密度がDNAハイブリダイゼーション効率に与える影響(26. 計測,ポスター,第52回日本生物物理学会年会(2014年度))

3P291 ナノ粒子表面へのプローブオリゴヌクレオチドの修飾密度がDNAハイブリダイゼーション効率に与える影響(26. 計測,ポスター,第52回日本生物物理学会年会(2014年度))

A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform

Biosensor based on DNA hybridization holds great potential to get higher sensitivity as the optimal DNA hybridization efficiency can be achieved by controlling the distribution and orientation of probe strands on the transducer surface. In this work, an innovative strategy is reported to tap the sensitivity potential of current electrochemiluminescence (ECL) biosensing system by dispersedly anchoring the DNA beacons on the gold nanoparticles (GNPs) array which was electrodeposited on the glassy carbon electrode surface, rather than simply sprawling the coil-like strands onto planar gold surface. The strategy was developed by designing a “signal-on” ECL biosensing switch fabricated on the GNPs nanopatterned electrode surface for enhanced ultra-sensitivity detection of Hg2+. A 57-mer hairpin-DNA labeled with ferrocene as ECL quencher and a 13-mer DNA labeled with Ru(bpy)32+ as reporter were hybridized to construct the signal generator in off-state. A 31-mer thymine (T)-rich capture-DNA was introduced to form T–T mismatches with the loop sequence of the hairpin-DNA in the presence of Hg2+ and induce the stem-loop open, meanwhile the ECL “signal-on” was triggered. The peak sensitivity with the lowest detection limit of 0.1nM was achieved with the optimal GNPs number density while exorbitant GNPs deposition resulted in sensitivity deterioration for the biosensor. We expect the present strategy could lead the renovation of the existing probe-immobilized ECL genosensor design to get an even higher sensitivity in ultralow level of target detection such as the identification of genetic diseases and disorders in basic research and clinical application.

Angle-Resolved XPS Analysis and Characterization of Monolayer and Multilayer Silane Films for DNA Coupling to Silica

We measure silane density and Sulfo-EMCS cross-linker coupling efficiency on aminosilane films by high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) measurements. We then characterize DNA immobilization and hybridization on these films by (32)P-radiometry. We find that the silane film structure controls the efficiency of the subsequent steps toward DNA hybridization. A self-limited silane monolayer produced from 3-aminopropyldimethylethoxysilane (APDMES) provides a silane surface density of ~3 nm(-2). Thin (1 h deposition) and thick (19 h deposition) multilayer films are generated from 3-aminopropyltriethoxysilane (APTES), resulting in surfaces with increased roughness compared to the APDMES monolayer. Increased silane surface density is estimated for the 19 h APTES film, due to a ∼32% increase in surface area compared to the APDMES monolayer. High cross-linker coupling efficiencies are measured for all three silane films. DNA immobilization densities are similar for the APDMES monolayer and 1 h APTES. However, the DNA immobilization density is double for the 19 h APTES, suggesting that increased surface area allows for a higher probe attachment. The APDMES monolayer has the lowest DNA target density and hybridization efficiency. This is attributed to the steric hindrance as the random packing limit is approached for DNA double helices (dsDNA, diameter ≥ 2 nm) on a plane. The heterogeneity and roughness of the APTES films reduce this steric hindrance and allow for tighter packing of DNA double helices, resulting in higher hybridization densities and efficiencies. The low steric hindrance of the thin, one to two layer APTES film provides the highest hybridization efficiency of nearly 88%, with 0.21 dsDNA/nm(2). The XPS data also reveal water on the cross-linker-treated surface that is implicated in device aging.

Detection of DNA hybridization by field-effect DNA-based biosensors: mechanisms of signal generation and open questions

We model theoretically the electrostatic effects taking place upon DNA hybridization in dense DNA arrays immobilized on a layer of Au nano-particles deposited on the surface of a field-effect-based DNA capacitive biosensor. We consider the influence of separation of a charged analyte from the sensor surface and the salinity of electrolyte solution, in the framework of both linear and nonlinear Poisson–Boltzmann theories. The latter predicts a substantially weaker sensor signals due to electrostatic saturation effects that is the main conclusion of this paper. We analyze how different physical parameters of dense DNA brushes affect the magnitude of hybridization signals. The list includes the fraction of DNA charge neutralization, the length and spatial conformations of adsorbed DNA molecules, as well as the discreteness of DNA charges. We also examine the effect of Donnan ionic equilibrium in DNA lattices on the sensor response. The validity of theoretical models is contrasted against recent experimental observations on detection of DNA hybridization via its intrinsic electric charge. The sensitivity of such biochemical sensing devices, their detection limit, and DNA hybridization efficiency are briefly discussed in the end.

Modulation of molecular hybridization and charge screening in a carbon nanotube network channel using the electrical pulse method

In this paper, we investigate the effect of electrical pulse bias on DNA hybridization events in a biosensor platform, using a Carbon Nanotube Network (CNN) and Gold Nano Particles (GNP) as an electrical channel. The scheme provides both hybridization rate enhancement of bio molecules, and electrical measurement in a transient state to avoid the charge screening effect, thereby significantly improving the sensitivity. As an example, the probe DNA molecules oscillate with pulse trains, resulting in the enhancement of DNA hybridization efficiency, and accordingly of the sensor performances in Tris-EDTA (TE) buffer solution, by as much as over three times, compared to the non-biasing conditions. More importantly, a wide dynamic range of 10(6) (target-DNA concentration from 5 pM to 5 μM) is achieved in human serum. In addition, the pulse biasing method enables one to obtain the conductance change, before the ions within the Electrical Double Layer (EDL) are redistributed, to avoid the charge screening effect, leading to an additional sensitivity enhancement.

Optimization of DNA Hybridization Efficiency by pH-Driven Nanomechanical Bending

The accessibility and binding affinity of DNA are two key parameters affecting the hybridization efficiency in surface-based biosensor technologies. Better accessibility will result in a higher hybridization efficiency. Often, mixed ssDNA and mercaptohexanol monolayers are used to increase the hybridization efficiency and accessibility of surface-bound oligonucleotides to complementary target DNA. Here, no mercaptohexanol monolayer was used. We demonstrate by differential microcantilever deflection measurements at different pH that the hybridization efficiency peaks between pH 7.5 and 8.5. At low pH 4.5, hydration and electrostatic forces led to tensile surface stress, implying the reduced accessibility of the bound ssDNA probe for hybridization. In contrast, at high pH 8.5, the steric interaction between neighboring ssDNA strands was decreased by higher electrostatic repulsive forces, bending the microcantilever away from the gold surface to provide more space for the target DNA. Cantilever deflection scales with pH-dependent surface hybridization efficiency because of high target DNA accessibility. Hence, by changing the pH, the hybridization efficiency is adjusted.

Enhancement of DNA hybridization under acoustic streaming with three-piezoelectric-transducer system

Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol® Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.

Preparation of Photoresponsive DNA Tethering Ortho‐Methylated Azobenzene as a Supra‐Photoswitch

This unit describes synthetic procedures of photoresponsive DNA via a phosphoramidite monomer composed of D-threoninol as a scaffold and 4-carboxy-2',6'-dimethylazobenzene or 4-carboxy-2'-methylazobenzene that works as a photoswitch more efficiently than previous nonmodified azobenzene (4-phenylazobenzoic acid). With these newly modified-azobenzenes, photoregulatory efficiency of DNA hybridization can be greatly improved. Furthermore, thermal stability of cis-azobenzene of 4-carboxy-2',6'-dimethylazobenzene remarkably increases compared with the previous non-modified azobenzene.

Study of Single-Stranded DNA Binding Protein–Nucleic Acids Interactions using Unmodified Gold Nanoparticles and Its Application for Detection of Single Nucleotide Polymorphisms

We have developed a label-free homogeneous phase bioassay to characterize the DNA binding properties of single-stranded DNA binding (SSB) protein, a key protein involved in various DNA processes such as DNA replication and repair. This assay uses gold nanoparticles (AuNPs) as sensing probe and is based on the phenomenon that preformed SSB-single-stranded DNA (ssDNA) complexes can protect AuNPs against salt-induced aggregation better than SSB or ssDNA alone. With the controlled particle aggregation/dispersion as measure, this assay can be used to detect the formation of SSB complexes with ssDNA of different length and nucleotide composition and to assess their binding properties without tedious and complicated assay procedures. On the basis of the inverse relationship between DNA hybridization efficiency and the tendency of SSB to form protection complex with unhybridized ssDNA to AuNPs, this assay is further developed to detect DNA hybridization with single nucleotide polymorphism selectivity. Owing to the high affinity between SSB and dissociated ssDNA, single-base mismatch discrimination in a long sequence of 30-mer DNA was achieved for both the end- and center-base mismatch. Unlike the conventional techniques for DNA and protein analysis, current AuNPs-based sensing strategy is simple in design, fast in detection, and economical for operation without the need of sophisticated equipment.

Fast DNA hybridization on a microfluidic mixing device based on pneumatic driving

A novel fluid mixing strategy was developed which significantly enhanced the efficiency of DNA hybridization. A pneumatic micro-mixing device consisting of two pneumatic chambers and an underneath DNA microarray chamber was built up. The fluid in the array chamber was pneumatically pumped alternately by the two pneumatic chambers. The chaotic oscillatory flow caused by the pumping greatly intensified the fluidic mixing. A homogeneous distribution of the tracer dye solution in the microarray chamber was observed after 2s mixing with a pumping frequency of 24Hz. Microarray DNA hybridization was substantially accelerated using this device, and the fluorescence intensity showed a plateau after oscillating 30s at room temperature. The corresponding signal level of the dynamic hybridization was 12.5-fold higher than that of the static hybridization performed at 42°C. A signal-to-noise ratio of 117 was achieved and the nonspecific adsorption of the targets to the sample array was minimized, which might be attributed to the strong shearing force generated during the pneumatic mixing process.

DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ∼33% and ∼24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.

Quantitative detection of E. coli O157:H7 eaeA gene using quantum dots and magnetic particles

A quantitative gene detection technique targeting the pathogenic E. coli O157:H7 eaeA gene was developed using magnetic bead (MB)-quantum dots (QDs) nanoparticle complexes. MBs allowed for the separation of DNA-conjugated QD nanoparticles via magnetic field manipulation. QDs provided internal fluorescence calibration to account for the intrinsically different numbers of nanoparticles interrogated in each assay. Based on the measurement of normalized fluorescence (Cy3/QD655), the linear quantification ranges of ssDNA and dsDNA targets were determined to be 10 through 103 fM (R2 = 0.992) and 2 × 102 through 6 × 107 gene copies (R2 = 0.972), with detection limits of 9.72 fM and 104 gene copies, respectively. The kinetic results indicate that adjustment of hybridization temperature in accordance to the amount of target DNA was required to maximize the efficiency of DNA hybridization. We were able to discriminate perfectly matched target DNA, 1-, 2-, and 41-base pair mismatched target DNAs in our approach and therefore demonstrated excellent selectivity. Our technique was also used on pure bacterial culture to showcase its ability to analyze environmental samples.

DNA Hybridization in Thermoresponsive Polymer Nanoparticles

We achieve very high hybridization efficiencies by using a new method to immobilize DNA strands on the surface of thermoresponsive polymer nanoparticles. Hybridization efficiencies of about 70 % are obtained between the DNA immobilized in the particles and a complementary strand in solution, even at very low ionic strengths (1 mM). The polymer nanoparticles have a glassy poly(methylmethacrylate) (PMMA) core and a thermoresponsive shell of poly(N-isopropylacrylamide) (PNIPAM) containing positive charges. After a DNA strand labeled with a fluorescence probe is loaded onto the particles at room temperature, the temperature is increased above the volume phase transition temperature of the PNIPAM shell, TVPT approximately 28 degrees C. The collapse of the particle shell immobilizes the DNA while maintaining its availability for hybridization with a complementary strand. Förster resonance energy transfer (FRET) is used to detect the hybridization with a complementary DNA strand labeled with a FRET acceptor probe.

Effect of probe characteristics on the subtractive hybridization efficiency of human genomic DNA

BackgroundThe detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics.FindingsIn this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA.ConclusionsThe characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase.

Effect of the ortho Modification of Azobenzene on the Photoregulatory Efficiency of DNA Hybridization and the Thermal Stability of its cis Form

We synthesized various azobenzenes methylated at their ortho positions with respect to the azo bond for more effective photoregulation of DNA hybridization. Photoregulatory efficiency, evaluated from the change of T(m) (DeltaT(m)) induced by trans-cis isomerization, was significantly improved for all ortho-modified azobenzenes compared with non-modified azobenzene due to the more stabilized trans form and the more destabilized cis form. Among the synthesized azobenzenes, 4-carboxy-2',6'-dimethylazobenzene (2',6'-Me-Azo), in which two ortho positions of the distal benzene ring with respect to carboxyl group were methylated, exhibited the largest DeltaT(m), whereas the newly synthesized 2,6-Me-Azo (4-carboxy-2,6-dimethylazobenzene), which possesses two methyl groups on the two ortho positions of the other benzene ring, showed moderate improvement of DeltaT(m). Both NMR spectroscopic analysis and computer modeling revealed that the two methyl groups on 2',6'-Me-Azo were located near the imino protons of adjacent base pairs; these stabilized the DNA duplex by stacking interactions in the trans form and destabilized the DNA duplex by steric hindrance in the cis form. In addition, the thermal stability of cis-2',6'-Me-Azo was also greatly improved, but not that of cis-2,6-Me-Azo. Solvent effects on the half-life of the cis form demonstrated that cis-to-trans isomerization of all the modified azobenzenes proceeded through an inversion route. Improved thermal stability of 2',6'-Me-Azo but not 2,6-Me-Azo in the cis form was attributed to the retardation of the inversion process due to steric hindrance between lone pair electrons of the pi orbital of the nitrogen atom and the methyl group on the distal benzene ring.

Trinity DNA Detection Platform by Ultrasmooth and Functionalized PEDOT Biointerfaces

An oligonucleotide-grafted poly(3,4-ethylenedioxythiophene) (PEDOT) thin film is developed for three DNA biosensor detection methods, including fluorescence, quartz crystal microbalance, and electrochemical methods. By electrocopolymerization of hydroxyl-functionalized EDOT and carboxylic-functionalized EDOT in microemulsion solutions, ultrasmooth films with a controlled surface density of carboxylic groups are created. The probe oligonucleotides are immobilized on PEDOT thin films by using a N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride coupling method. By monitoring the DNA hybridization efficiency on thin films with different oligonucleotide densities, the optimized density for DNA hybridization is obtained. The feasibility and limitation of using this platform for electrochemical detection are also discussed.

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI–TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 °C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 μM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.