A mixed film composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) brushes to enhance selectivity for detection of single nucleotide polymorphisms

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (ΔT_m) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1-0.6 M). Increases of ΔT_m when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.     

A spatially resolved nucleic acid biochip based on a gradient of density of immobilized probe oligonucleotide

The potential for a new biochip design based on a continuous gradient of density of immobilized single-stranded DNA oligonucleotide probes (ssDNA) is explored. This gradient resolved information platform (GRIP) can provide sequence identification based on the spatial location and extent of hybridization by a target sequence. Surfaces based on indium-tin oxide (ITO) on glass were first functionalized by 3-aminopropyltriethoxysilane (APTES) followed by attachment of glutaraldehyde, prior to immobilization of oligonucleotide probe that was terminated with amine. The use of Cy₃ and Cy₅ dye-labelled ssDNA probes and targets allowed estimation of density and correlation of the location of binding of labelled targets. Probe molecules of 20 mer lengths were loaded to produce density gradients in the range of 1.0-200 ng/mm². The biochips could resolve a mixture of fully complementary five base-pair mismatched targets by the location of binding on the surface. Thermal control provided additional selectivity. Thermal cycling and washing provided for regeneration of the surface, and the fluorescence intensities showed no deterioration in at least five cycles of hybridization reactions.     

Base sequence- and <Emphasis Type="Italic">T</Emphasis><Subscript>m</Subscript>-dependent DNA oligomer separation by open tubular capillary columns carrying complementary DNA oligomers as probes

DNA chips prepared on a flat glass surface have unavoidable drawbacks when used for quantitative analysis. In an attempt to overcome this problem, we constructed an HPLC-type system suitable for quantitative analysis that enables base sequence- and T _m-dependent DNA oligomer separation in a flow system. A small open tubular capillary column (300-mm × 100-μm I.D.) was used. The DNA oligomers used as probes had an amino group at the 5′-end and were immobilized on the inner silica surface of the capillary column which had been sequentially treated with 3-aminopropyltriethoxysilane, butyltrimethoxysilane, and disuccinimidylglutarate. Using the combination of probe-immobilized column placed in a column oven equipped with temperature gradient function, a nano-flow-controllable pump, a small sample-loading injector, and a capillary-fitted UV detector, we succeeded in separating complementary and non-complementary DNA oligomers in specific and quantitative modes. We also designed a temperature gradient strategy for efficient separation of target DNA oligomers in DNA mixture samples. Using a column carrying two different probes with similar T _m values, their complementary target DNA oligomers were also separated and detected. The developed DNA open tubular capillary column system investigated in the present study could be further improved as an alternative tool to DNA chips to be used for the quantitative analysis of DNA or mRNA samples. Kamakshaiah Charyulu Devarayapalli and Seung Pil Pack contributed equally to this paper.     

Molecular and crystalline structure of pyrocatechol and hydroquinone dimethacrylates and their reactivity in melts

X-ray diffraction analysis of pyrocatechol and hydroquinone dimethacrylates (T _m = 18 and 86–88°C, respectively) shows that the oligomer molecules within crystals are packed in stacks where the methacrylate fragments of neighboring molecules are parallel to each other. The minimum distances between the centers of double bonds of adjacent methacrylate fragments in crystals of pyrocatechol and hydroquinone dimethacrylates are 4.621(3) and 4.269(4) Å. The curves showing the reduced rate of photopolymerization of oligomer melts versus conversion (9,10-phenanthrenequinone used as the initiator) display a maximum at conversions of 1.5–3.0%. The limiting conversion in photopolymerization of molten pyrocatechol dimethacrylate at 25 and 40°C is 20%; for hydroquinone dimethacrylate at 95°C, it is approximately 10%. As the temperature rises from 25 to 40°C, the maximum reduced rate of photopolymerization of pyrocatechol dimethacrylate increases by a factor of 1.4.     

Sensitive sequence-specific molecular identification system comprising an aluminum micro-nanofluidic chip and associated real-time confocal detector

A method for the development of continuous density gradients of immobilized oligonucleotide probes (20mer) along the length of microfluidic channels is demonstrated. The development of continuous density gradients was achieved using variable electrokinetic transport of probes in hybrid glass-polydimethylsiloxane microfluidic chips. The probes were terminated with an amine functional group, and were delivered by electrokinetic pumping to the flat glass channel wall after it had been densely coated with covalently immobilized aldehyde groups. This method provided probe immobilization densities ranging from 4.5(±0.8)×10(13) to 2.5(±0.8)×10(11) molecules cm(-2), with longitudinal dilution and differential mass transport of the injected plug of probes being the primary factors responsible for the gradient of density. The utility of the resulting density gradient of immobilized probes to control the selectivity of hybridization was demonstrated at room temperature by discrimination between a fully complementary oligonucleotide target, and a target strand containing 3 base pair mismatches (3 BPM) based on the spatial pattern of hybridization for sub-picomole quantities of targets. Single nucleotide polymorphism (SNP) discrimination was possible when temperature control was implemented to improve resolution of the mismatch discrimination, allowing SNP discrimination at 35 °C with a contrast ratio of almost 5 to 1.     

Quantitative detection of well-based DNA array using switchable lanthanide luminescence

In this report a novel wash-free method for multiplexed DNA detection is demonstrated employing target specific probe pairs and switchable lanthanide luminescence technology on a solid-phase array. Four oligonucleotide capture probes, conjugated at 3′ to non-luminescent lanthanide ion carrier chelate, were immobilized as a small array on the bottom of a microtiter plate well onto which a mix of corresponding detection probes, conjugated at 5′ to a light absorbing antenna ligand, were added. In the presence of complementary target nucleic acid both the spotted capture probe and the liquid-phase detection probe hybridize adjacently on the target. Consequently the two non-luminescent label molecules self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide luminescence is thereafter measured without a wash step from the spots by scanning in time-resolved mode. The homogeneous solid-phase array-based method resulted in quantitative detection of synthetic target oligonucleotides with 0.32 nM and 0.60 nM detection limits in a single target and multiplexed assay, respectively, corresponding to 3× SD of the background. Also qualitative detection of PCR-amplified target from Escherichia coli is described.     

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO₂NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)₃]^2+/3+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)₃]^2+/3+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10⁻¹⁵ to 1 × 10⁻⁸ mol L⁻¹. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL⁻¹ with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)₃]^2+/3+ interaction with ssDNA before and after hybridization.     

Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system

Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T _m values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T _m values were separated and detected quantitatively by using a packed column carrying two different DNA probes.     

Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole

We report a new approach for detecting DNA hybridisation using non faradaic electrochemical impedance spectroscopy. The technique was applied to a system of DNA probes bearing amine groups that are immobilized by covalent grafting on a supporting polypyrrole matrix functionalised with activated ester groups.The kinetics of the attachment of the ss-DNA probe was monitored using the temporal evolution of the open circuit potential (OCP). This measurement allows the determination of the time necessary for the chemical reaction of ss-DNA probe into the polypyrrole backbone.The hybridisation reactions with the DNA complementary target and non complementary target were investigated by non faradaic electrochemical impedance spectroscopy. Results show a significant modification in the Nyquist plot upon addition of the complementary target whereas, in presence of the non complementary target, the Nyquist plot is not modified. The spectra, in the form of Nyquist plot, were analysed with the Randles circuit. The transfer charge resistance R₂ shows a linear variation versus the complementary target concentration. Sensitivity and detection limit (0.2 nM) were determined and detection limit was lower of one order of magnitude than that obtained with the same system and measuring variation of the oxidation current at constant potential.     

Thermodynamics for the Formation of Double‐Stranded DNA–Single‐Walled Carbon Nanotube Hybrids

For the first time, the thermodynamics are described for the formation of double‐stranded DNA (ds‐DNA)–single‐walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds‐DNAs d(A)₂₀–d(T)₂₀ and nuclear factor (NF)‐κB decoy. UV/Vis/near‐IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single‐stranded DNAs (ss‐DNAs), including d(A)₂₀ and d(T)₂₀, are also used for comparison. The d(A)₂₀–d(T)₂₀ shows a drastic change in its thermodynamic parameters around the melting temperature (T_m) of the DNA oligomer. No such T_m dependency was measured, owing to high T_m in the NF‐κB decoy DNA and no T_m in the ss‐DNA.     

Electrochemical Detection of Avian Influenza Virus Genotype Using Amino‐ssDNA Probe Modified Gold Electrodes

A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH₂‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal.     

Langmuir probe measurements of electron temperature in an inductively coupled plasma

The feasibility of using double Langmuir probes to measure electron temperature (T_e) in an Ar inductively coupled plasma (ICP) was evaluated. Experimental methods for probing the plasma and for reducing rf interference were devised. Despite these measures, the probe signal was noisy and erratic if the ICP had the normal analytical configuration with a hole through its center, so measurements were restricted to an ICP without an axial channel. Theoretical criteria indicated that Langmuir probe measurements in an atmospheric pressure ICP were in a borderline regime in which the measured T_e values may have been depressed somewhat (relative to the actual T_e values in the ICP) due to cooling of electrons as they approached the probe. The T_e values obtained from the center of the ICP were 7500 K at a forward power of 1.0 kW and 10 000 K at 1.25 kW for a measurement position 8 mm above the load coil. Electron density (n_e) measurements by the Langmuir probe method were comparable to or higher than n_e values calculated from the Saha equation at the measured T_es. The T_e and n_e values were high enough to indicate that, if electron cooling and ion-electron recombination occurred near the probes, these effects were not extreme and/or the use of two probes compensated for them in some fashion. The probe measurements also indicated that T_e increased with the potential difference between the probes. This latter observation provided tentative evidence that the electron kinetic energy distribution was non-Maxwellian with an excess of higher energy electrons relative to lower energy electrons.     

Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids

Hybridization probes are often inefficient in the analysis of single‐stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real‐time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T_m>80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure‐folded analytes, whereas a MB‐based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.     

The structure of the triplet excimer in hexachlorobenzene crystals — an endor study

The lowest triplet state T₁ of hexacholorobenzene (HCB) crystals doped with trichlorobenzene (TCB) is excimeric. By an ESR and ENDOR (¹H and ³⁵Cl) analysis it is shown that the triplet excimers are dimeric and located in one of the two stacks constituting the crystals with misorientation of 20°. From H-ENDOR it is concluded that the dopant TCB is nearest neighbour and translationaily equivalent to the excimer. The same is true for the HCB molecule which is nearest neighbour on the other side of the excimer in the stack. From Cl-ENDOR orientation and size of the quadrupole tensor, spin density distribution, symmetry and orientation of the excimer are derived.     

A Sequence‐Selective Electrochemical DNA Biosensor Based on HRP‐Labeled Probe for Colorectal Cancer DNA Detection

Abstract

A highly‐sensitive sequence‐selective DNA sensor based on HRP‐labeled probe to detect specific K‐ras gene which is highly associated with colorectal cancer has been reported. Capture probe modified with–SH was first chemically adsorbed on the gold electrode through self‐assembly. Then, the hybridization of a complementary nucleic acid (target DNA:K‐ras gene) and HRP labeled oligonucleotide detection probe occurred in a sandwich way. Finally, H₂O₂ electroreduction current catalyzed by HRP was measured amperometrically in the presence of hydroquinon as mediator. The sequence selectivity is double guaranteed by the complementary hybridization of target DNA with capture and detection probes. The experimental conditions were optimized. The linear range is 1.17×10^?11～1.17×10^?7 mol l^?1 with a detection limit of 5.85×10^?12 mol l^?1. The electrode with capture probe can be reused after regeneration in boiling water.     

Molecular dynamics simulation of local motion of polystyrene chain end—comparison with the fluorescence depolarization study

Molecular dynamics (MD) simulation of the local motion of a polystyrene (PS) chain with anthryl group at the chain end surrounded by benzene molecules was performed and the results were compared with those obtained experimentally by the fluorescence depolarization method. The molecular weight dependence of the relaxation time of the probe obtained by the MD simulation was qualitatively in agreement with the results obtained by the fluorescence depolarization method. We also estimated the molecular weight dependence of the relaxation time for the end-to-end vector. Below the degree of polymerization (DP)≤3, the mean relaxation time T_m for the end-to-end vector was similar to that for the vector corresponding to the transition moment of the probe. With the increase of DP, the T_m for the probe tended to reach an asymptotic value unlike that for the end-to-end vector, which monotonically increased with DP. This indicates that the entire motion of a polymer coil contributes to the local motion to a lesser extent as the molecular weight increases. The MD simulations using artificial restraints showed that the rotational relaxation of the probe at the chain end for a dynamically stiff PS chain is realized by the cooperative rotation of the main chain bonds. The internal modes which takes place below 5 monomer units mainly led to the rotational relaxation of the probe at the PS chain end. Finally, the change of T_m with the position along the PS main chain was examined.     

Nanostructured zirconium oxide based genosensor for Escherichia coli detection

A deoxyribonucleic acid (DNA) biosensor has been fabricated via immobilization of 17 base terminal single stranded DNA (ssDNA) identified from the 16s rRNA coding region of Escherichia coli onto sol–gel derived nanostructured zirconium oxide (NanoZrO₂) film. An oligonucleotide probe with a terminal 5′-phosphate group has been attached to the surface of the electrode via affinity of NanoZrO₂ for phosphate. The results of hybridization studies carried out with the complementary, non-complementary and genomic DNA reveal that ssDNA/NanoZrO₂/ITO bioelectrode has a high selectivity and sensitivity towards hybridization detection with limits of 10^?6–10⁶ pM of complementary DNA.     

Time-resolved fluorescence based DNA detection using novel europium ternary complex doped silica nanoparticles

A two-probe tandem DNA hybridization assay including capture DNA₁, probe DNA₂, and target DNA₃ was prepared. The long-lived luminescent europium complex doped nanoparticles (NPs) were used as the biomarker. The complex included in the particle was Eu(TTA)₃(5-NH₂-phen)-IgG (ETN-IgG), the europium complex Eu(TTA)₃(5-NH₂-phen) linking an IgG molecule. Silica NPs containing ETN-IgG were prepared by the reverse microemulsion method, and were easy to label oligonucleotide for time-resolved fluorescence assays. The luminophores were well-protected from the environmental interference when they were doped inside the silica network. The sequences of Staphylococcus aureus and Escherichia coli genes were designed using software Primer Premier 5.0. Amino-modified capture DNA₁ was covalently immobilized on the common glass slides surface. The detection was done by monitoring the fluorescence intensity from the glass surface after the hybridization reaction with the NPs labeled probe DNA₂ and complementary target DNA₃. The sensing system presented short hybridization time, satisfactory stability, sensitivity, and selectivity. This approach was successfully employed for preliminary application in the detection of pure cultured E. coli, it might be an effective tool for pathogen DNA monitoring.     

Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors

Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.     

A competitive displacement assay with quantum dots as fluorescence resonance energy transfer donors

The unique optoelectronic properties of semiconductor quantum dots (QDs) make them well-suited as fluorescent bioprobes for use in various biological applications. Modification of CdSe/ZnS QDs with biologically relevant molecules provides for multipotent probes that can be used for cellular labeling, bioassays, and localized optical interrogation by means of fluorescence resonance energy transfer (FRET). Herein, we demonstrate the use of red-emitting streptavidin-coated QDs (QD₆₀₅) as donors in FRET to introduce a competitive displacement-based assay for the detection of oligonucleotides. Various QD–DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23 were prepared. The single-stranded oligonucleotide probes were hybridized to dye-labeled (Alexa Fluor 647) reporter sequences, which were provided for a FRET-sensitized emission signal due to proximity of the QD and dye. The dye-labeled sequence was designed to be partially complementary and include base-pair mismatches to facilitate displacement by a more energetically favorable, fully complementary recognition motif embedded within a 98-mer displacer sequence. Overall, this study demonstrates proof-of-concept at the nM level for competitive displacement hybridization assays in vitro by reduction of fluorescence intensity that directly correlates to the presence of oligonucleotides of interest. This work demonstrates an analytical method that could potentially be implemented for monitoring of intracellular gene expression in the future.