Detection Of Oligonucleotides Research Articles

Label-free oligonucleotide detection method based on a new L-cysteine-dihydroartemisinin complex electroactive indicator

A novel base-mismatched oligonucleotide assay method based on label-free electrochemical biosensor was developed, in which the L-cysteine (Cys)-dihydroartemisinin (DHA) complex was used as a new electroactive indicator. In DNA sensor, Cys-DHA complex was initially formed on electrode surface by cathodic scanning, and target oligonucleotide was conjugated with Cys-terminated DHA indicator through electrostatic interaction under optimal pH. The subsequent sequence assay was responsive to hybridization recognition, which target oligonucleotide was captured by the surface-anchored DNA/Cys-DHA probe. The electrochemical signals of biosensor before and after hybridization were compared basing the measurements of semi-derivative linear scan voltammetry (SDLSV) and electrochemical impedance spectroscopy (EIS). On the basis of signal amplification of electroactive indicator and specific recognition of DNA probe, five target oligonucleotides with different mismatched bases were assayed, and a detection limit reached 0.3 nM. Furthermore, atomic force microscopy (AFM) was used to visually characterize specific recognition spots of biosensor at nanoscale. This study demonstrated a new electroactive molecule-based, biomolecule-involved electroactive indicator and its application in recognition and detection of complementary and base-mismatched oligonucleotide.

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 x 10(-12) M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.

Optical DNA detection based on gold nanorods aggregation

Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Herein, the localized surface plasmon resonance properties of unmodified gold nanorods (GNRs) in 1 mM cetyltrimethyl-ammonium bromide solution were used for sensing DNA sequences, with good simplicity and sensitivity. The intensity of typical plasmon resonance absorption bands of the GNRs decreased with increasing cDNA concentration. The detection of a 30-mer single-stranded oligonucleotide as a model reached a detection limit of about 0.1 pM. This study will be significant for as-prepared GNRs for future application in biological systems.

Novel chitosan/gold-MPA nanocomposite for sequence-specific oligonucleotide detection

A 20-mer single-stranded oligodeoxyribonucleotide (ssODNs) was covalently probed onto the nanocomposite electrode, made up of an indium-tin oxide (ITO) glass surface coated with chitosan (CHIT), which is bonded with carboxyl functionalized thiol capped gold nanoparticles (gold-mercaptopropionic acid, Au-MPA NPs). The prepared CHIT/Au-MPA/ITO and the ssODNs/CHIT/Au-MPA/ITO electrodes were characterized with FTIR spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and cyclic voltammetry (CV). TEM micrograph of the composite electrode showed that Au-MPA NPs had the diameter ranging from 4 to 16 nm. The ssODNs/CHIT/Au-MPA/ITO electrode showed a linear increase in the amperometric current with an increasing concentration of the single-stranded target complementary ODNs (cODNs) within the range of 0.03–325 fM. The ssODNs/CHIT/Au-MPA/ITO electrode exhibited a sensitivity of 0.572 μA/fM with a response time of 12 s. The ssODNs/CHIT/Au-MPA/ITO electrode had a shelf life of ∼182 days, when stored at 4 °C.

Synthesis of Glass‐Coated SERS Nanoparticle Probes via SAMs with Terminal SiO2 Precursors

Surface-enhanced Raman scattering (SERS) combines the benefits of vibrational Raman scattering with highest sensitivity, using nanostructures that can support localized plasmon resonances. [1‐4] The main application of SERS is the label-free detection of analytes. [5‐8] An alternative and more recent approach uses SERS as a readout method in bioanalytical applications: the selective detection of proteins and oligonucleotides is achieved by employing target-specific SERS nanoparticle probes. [7‐11] The central motivation for this strategy is the option to detect numerous target molecules within a single measurement (multiplexing). The basis of spectral multiplexing in SERS arises from the small linewidth of vibrational Raman bands compared with the significantly broader emission profiles of molecular fluorophores. [12,13] Further important advantages are quantification of target concentration and the extreme sensitivity of SERS, in particular SERRS (surface-enhanced resonance Raman scattering). [14,15] Additionally, the simultaneous excitation of spectrally distinct SERS nanoparticle probes requires only a single laser wavelength. Different designs for nanoparticle-based SERS probes (SERSlabels,SERSnanotags)areavailable,whichdifferinthe plasmonic nanostructure, the Raman reporter molecule, and the optional protective shell. [9,16‐18] Using a self-assembled monolayer(SAM)ofRamanreportermoleculesonthesurface of the nanoparticle has several advantages. [18‐22] Firstly,

Electrochemical detection of oligonucleotide by attaching redox probes onto its backbone

An approach was demonstrated to detect oligonucleotide by attaching redox probes onto its backbone. First, peptide nucleic acid (PNA) with a neutral backbone was immobilized onto a gold (Au) electrode surface as a capture. Second, when the PNA capture hybridized with a target oligonucleotide (a short DNA), an assembly of Au–PNA–DNA formed and phosphate groups were thus brought into the assembly from the DNA's backbone. The linker ion of Zr 4+ exhibits a strong coordination interaction with the phosphate group and the carboxylic group. The hybridized target DNA provides the phosphate group while a derivatized redox probe of ferrocene (Fc) carboxyl acid offers the carboxylic group. Therefore, the redox probe can be attached to the phosphate group by the linker to form an assembly of Au–PNA–DNA–Zr–Fc. Its redox process was studied and the detection conditions of oligonucleotide were optimized. A limit of detection of 1.0 × 10 −12 M or ∼2 attomol was reached.

Electrochemical biosensor microarray functionalized by means of biomolecule friendly photolithography

Microfabrication permits the incorporation of dense electrode arrays in microsystems and small volume diagnostic devices. However, the specific functionalization of arbitrary shape electrodes with different biomolecules remains a challenging issue. In the present work, the problem of fabricating closely spaced microelectrodes (20 μm sensor diameter and 20 μm-spaced interdigitated electrodes array) that can be modified selectively in order to create multi-analyte sensor arrays is addressed by employing a biomolecule friendly photolithographic procedure for the sequential immobilization of different biomolecules onto separated electrodes of the same array. The concept was demonstrated with selective detection of oligonucleotides for breast cancer gene mutation detection, the hormone T4 detected with specific antibodies and sarcosine and glucose detected with specific enzymes immobilized in two-analyte arrays in order to assure that the method is compatible with all the types of biorecognition molecules used in biosensors. Electrochemical techniques were used in this array, because of the low cost, high sensitivity and easy miniaturisation of these transducers. Although the array was composed of only two sets of electrodes, the results demonstrate that the method proposed is generic and could be used for patterning of electrochemical multi-analyte biosensors at even higher resolution.

A binary Cy3 aptamer probe composed of folded modules

Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on their high affinity to a target molecule. Dye-binding aptamers are promising tools for real-time detection of not only DNA or RNA sequences but also proteins of interest both in vitro and in vivo. In this study, we aimed to isolate an RNA aptamer to Cy3, a widely used, membrane-permeant, and nontoxic fluorescent cyanine dye. Extensive selection of affinity RNA molecules to Cy3 yielded a unique sequence aptamer named Cy3_apt. The selected Cy3_apt was 83 nucleotides long and successfully shortened to 49 nucleotides long with increased affinity to Cy3 by multiple base changes. The shortest Cy3_apt is composed of two separate hairpin modules that are required for the affinity to Cy3 as monitored by the surface plasmon resonance (SPR) assay. Also, the fluorescence of Cy3 increased on binding to Cy3_apt. The two modules of Cy3_apt, when detached from each other, functioned as a binary aptamer probe. We demonstrate that the binary Cy3_apt probe is applicable to the detection of target oligonucleotides or RNA–RNA interaction by tagging with target sequences. This binary probe consists of two folded modules, referred to as a folded binary probe.

Luminescent Probes for Ultrasensitive Detection of Nucleic Acids

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Separation and identification of oligonucleotides by hydrophilic interaction liquid chromatography (HILIC)-inductively coupled plasma mass spectrometry (ICPMS).

A method for the separation and detection of oligonucleotides utilizing hydrophilic interaction liquid chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICPMS) is described. Polythymidylic acids of various lengths (10, 15, 20 and 30 nucleotides) were separated under gradient HILIC conditions. Selective detection of oligonucleotides was possible through monitoring m/z 47, corresponding to (31)P(16)O(+), using ICPMS. Oxygen was used as a reaction gas in the collision/reaction cell to produce PO(+) by reacting with phosphorus in the gas phase, thereby effectively eliminating the interferences for phosphorus normally seen at m/z 31. Limits of detections (LODs) were determined to be 1.69 pmol, 1.21 pmol, 1.0 pmol and 0.55 pmol loaded on column for the 10, 15, 20 and 30 mer, respectively.

Reply to evolutionary flux of canonical microRNAs and mirtrons in Drosophila.

Reply to evolutionary flux of canonical microRNAs and mirtrons in Drosophila.

Detection of microarray-hybridized oligonucleotides with magnetic beads

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.

Combining Two Technologies for Full Genome Sequencing of Human

At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiDTM) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

Combining Two Technologies for Full Genome Sequencing of Human

At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

Construction of Oligonucleotide Microarrays (Biochips) via Thioether Linkage for the Detection of Bacterial Meningitis

Oligonucleotide-based arrays are increasingly becoming useful tools for the analysis of gene expression and single-nucleotide polymorphism. Here, we report a method that allows the direct immobilization of thiolated oligonucleotides onto an epoxy-activated glass surface via a stable thioether linkage under microwaves. The described chemistry efficiently immobilizes the probes via terminal thiol groups with uniform spot morphology. The thioether linkage could endure repeated PCR-like heat cycling with only 2.5% loss after 20 cycles, indicating that the chemistry can be used in integrated PCR/microarray devices. The highlighting feature of the proposed method is that the detection limit for the probe concentration can be reduced to 0.25 microM with 20-mer oligonucleotides. The efficiency of the projected method (approximately 33%) indicates its advantage over the existing standard methods, viz., NTMTA (approximately 9.8%), epoxide-amine (approximately 9.8%) and disulfide (approximately 1.7%). The constructed microarrays were validated through the detection of base mismatches and bacterial meningitis. These features make the projected strategy ideal for manufacturing oligonucleotide arrays and detection of mismatches and bacterial diseases.

Surface-enhanced Raman scattering biosensor for DNA detection on nanoparticle island substrates

We present a study on the surface-enhanced Raman scattering (SERS) properties of Ag nanoparticle island substrates (NIS) and their applications for target oligonucleotide (OND) detection. It has been found that the surface nanostructure of NIS samples can be controlled with a good degree of reproducibility, and a high SERS enhancement can be achieved when the peak extinction wavelength of NIS is tuned to a spectral window (approximately 60 nm) between the excitation wavelength and the scattered Raman wavelength. The highest SERS enhancement was obtained from the NIS substrates with a nominal thickness of 50 A. Detection of target OND was performed with a sandwich format in which the target OND was hybridized both to a capture OND immobilized on the NIS substrate, and a detection OND conjugated with a Raman-active dye for SERS signal generation. We compare the detection performance of two strategies based on the use of the detection OND with or without the gold nanoparticle (Au-NP). Our results confirm that, when the detection OND is coupled to the Au-NP, a better sensitivity for the target OND detection, in terms of a wider dynamic range and a lower detection limit (0.4 fM versus 1 nM without Au-NP), would be achieved.

A DNA biosensor based on the detection of doxorubicin-conjugated Ag nanoparticle labels using solid-state voltammetry

This report describes an electrochemical biosensor for the detection of short DNA oligonucleotide of the avian flu virus H5N1 with sequence 5′-CCA AGC AAC AGA CTC AAA-3′. To fabricate this DNA biosensor, a gold (Au) electrode surface was modified with thiolated DNA probes with a sequence complementary to the target DNA. This modified Au electrode was incubated in a buffer solution containing the target DNA to form double-stranded DNA (ds-DNA) through hybridization. The ds-DNA on the electrode surface was then labeled with silver nanoparticles conjugated with a well-known DNA intercalator, doxorubicin. By performing cyclic voltammetry in an aqueous KCl solution (0.3M), the silver nanoparticle labels were detected as a result of the highly characteristic solid-state Ag/AgCl redox process. The signal obtained was subsequently used to quantify the amount of DNA. A detection limit of 1pM has been achieved with this new DNA biosensor.

Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides

A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self-quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluorophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies.

Synthesis of DNA oligonucleotides in mesoporous silicon

AbstractIn‐situ oligonucleotide synthesis through an 8‐base sequence is demonstrated within 30 nm silicon mesopores utilizing sequential phosphoramidite reactions. Stepwise attachment of each base is confirmed by monitoring the porous silicon effective refractive index via a waveguide resonance angle. A linear increase in the effective refractive index is observed after the addition of each base, and the detection of oligonucleotides containing only a single thymine is possible. It is estimated that 50% of the available internal pore surface is occupied by the 8‐base oligonucleotides, which is a substantially greater coverage than obtained by using traditional methods of direct attachment of presynthesized DNA oligonucleotides. The greater surface coverage by DNA probes will enable more effective biosensing devices. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Development of an Oligonucleotide Functionalized Hydrogel Integrated on a High Resolution Interferometric Readout Platform as a Label-Free Macromolecule Sensing Device

Development of an oligonucleotide functionalized hydrogel integrated on a high resolution interferometric readout platform capable of determining changes in optical length of the hydrogel with 2 nm resolution is described. The hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network making up a network junction point in addition to the covalent cross-links. The hybridized dioligonucleotide network junctions were made with a 10 basepair complementary region flanked by additional basepairs that could aid in destabilizing the junction points in competitive displacement hybridization by the added probe oligonucleotides. The probe oligonucleotide destabilizing the junction point thus induces swelling of the functionalized hydrogel that is sensitive to the concentration of the probe, the sequence, and matching length between the probe and sensing oligonucleotide. This design yields a molecular amplification of the change in the optical length of the gel at least 5-fold compared to a hydrogel where sensing functionality is based on hybridization with a grafted oligonucleotide that is not a part of a network junction. Concentration sensitivity applied for specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. Applications of the resulting oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequences designed with aptamer functionalities for determination of other types of molecules are discussed.