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Biosensors & Bioelectronics May 2017A QDs-DNA nano-probe was made by combining Mn-doped ZnS room-temperature phosphorescence (RTP) quantum dots (QDs) and DNA. Then an RTP sensor for quantitative detection...
A QDs-DNA nano-probe was made by combining Mn-doped ZnS room-temperature phosphorescence (RTP) quantum dots (QDs) and DNA. Then an RTP sensor for quantitative detection of genetically-modified mark sequence cauliflower mosaic virus 35S promoter (Ca MV 35S) DNA was built on basis of phosphorescent resonance energy transfer (PRET). The underlying principles were that a QDs-DNA water-soluble nano-probe was built by connecting single-strand DNA to the surfaces of QDs via a ligand exchange method. This probe had good RTP performance and could well identify Ca MV 35S. Thereby, the simple, rapid and efficient detection of genetically-modified organisms was realized. With the increase of target DNA sequence, the phosphorescent intensity of QDs was gradually reduced due to the energy transfer between QDs and the organic quencher BHQ. This sensor had a detection limit of 4.03nM and a detection range of 12-300nM. Moreover, this sensor had high selectivity. This sensor could effectively detect the target DNA compared with mismatched and random sequences. Thus, this method is very promising for biological analysis.
Topics: Biosensing Techniques; Caulimovirus; DNA Probes; DNA, Single-Stranded; DNA, Viral; Energy Transfer; Luminescent Measurements; Promoter Regions, Genetic; Quantum Dots; Sulfides; Transgenes; Zinc Compounds
PubMed: 28088748
DOI: 10.1016/j.bios.2017.01.012 -
Journal of Nanoscience and... Mar 2016A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA...
A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA probe based hybridization detection methods are often called as 'bead based assays' and their success is substantially influenced by the dispensing and manipulation capability of the microfluidic technology. This study reports the detection of N-Ras cancer gene by fluorescence quenching of quantum dots immobilized on the surface of polystyrene beads. A microfluidic chip was constructed in which the quantum dots-bead-DNA probes were packed in the channel. The target DNA flowed across the beads and hybridized with immobilized probe sequences. The target DNA can be detected by the fluorescence quenching of the quantum dots due to their transfer of emission energy to intercalation dye after DNA hybridization. The mutated gene also induces fluorescence quenching but with less degree than the perfectly complementary target DNA.
Topics: DNA Probes; Fluorescence; Microfluidics; Quantum Dots
PubMed: 27455729
DOI: 10.1166/jnn.2016.11072 -
Analytical Chemistry Apr 2015Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the...
Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the studies about the protein-DNA binding are in an unconfined space. Here, we propose a new method, nanopore-based DNA-probe sequence-evolution (NDPSE), which includes up to 6 different DNA-probe systems successively designed in a nanoscale confined space which unveil the more realistic characteristics of protein-DNA binding phenomena. There are several features; for example, first, the edge-hindrance and core-hindrance contribute differently for the binding events, and second, there is an equilibrium between protein-DNA binding and DNA-DNA hybridization.
Topics: Base Sequence; Binding Sites; DNA; DNA Probes; Nanopores; Proteins
PubMed: 25751160
DOI: 10.1021/acs.analchem.5b00375 -
Jikken Dobutsu. Experimental Animals Jul 1989Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized...
Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.
Topics: DNA Probes; DNA, Bacterial; Mycoplasma pneumoniae; Nucleic Acid Hybridization
PubMed: 2507333
DOI: No ID Found -
Bioorganic & Medicinal Chemistry Jul 2008Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent...
Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent agent for use in a DNA probe. Thus, we have prepared 5'-terminal acridone-labeled DNAs by post-modification, and have examined their photophysical properties and their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with a 3'-terminal dabcyl-tagged DNA as an acceptor, which can detect the target DNA by emission-quenching caused by FRET. The FRET with an acridone and dabcyl pair has been found to complement that with fluorescence and dabcyl and other fluorescence-quencher pairs. Significant amounts of quenching of the acridone emissions by guanine in the DNA were observed when guanine was close to acridone, which can be applied as a quencher-free probe for the detection of special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could discriminate the opposite T-T base mismatch, although enhancement of discrimination ability is needed for the practical use of SNP typing.
Topics: Acridines; Acridones; Base Pair Mismatch; DNA; DNA Mutational Analysis; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Guanine
PubMed: 18539465
DOI: 10.1016/j.bmc.2008.05.050 -
The Analyst Feb 2017Matrix metalloproteinases (MMPs) have been considered to contribute to the progression of tumorigenesis and tumor invasion; MMP-9 in particular, has been regarded as a...
Matrix metalloproteinases (MMPs) have been considered to contribute to the progression of tumorigenesis and tumor invasion; MMP-9 in particular, has been regarded as a priority target in cancer treatment due to its massive up-regulation in malignant tissues and its ability to degrade type IV collagen. In this work, we employed a DNA-directed covalent conjugation method to design a fluorescence probe for in vitro detection of functional matrix metalloproteinases, by which a nitrilotriacetic acid (NTA)-modified DNA probe can combine with the Zn in the active site of MMPs, and then a molecule beacon (MB) modified FITC and BHQ1 can open to bond with their complementary base, NTA-modified DNA. We can evaluate the amount of MMPs in the medium according to the fluorescence intensity. The detection procedure can be finished in 30 min with good selectivity, cheap reagents and easy preparation. All the results and the amount of secreted MMPs under three different cell culture conditions are in accordance with previous reports. Satisfactory results are obtained. Furthermore, owing to the importance of MMP-9, we designed an approach to achieve the desired selectivity and specificity of our work, using dual amplification for improving fluorescence intensity based on RCA to detect the amount of MMP-9.
Topics: Cell Line, Tumor; DNA Probes; Fluorescence; Humans; Matrix Metalloproteinase 9; Nitrilotriacetic Acid; Oligonucleotides
PubMed: 28112295
DOI: 10.1039/c6an02339h -
Bioorganic & Medicinal Chemistry Sep 2016Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we...
Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40°C at 100mM NaCl concentration.
Topics: Anthraquinones; Base Sequence; DNA; DNA Probes; Fluorescent Dyes; Nucleic Acid Conformation; Nucleic Acid Hybridization; Peptide Nucleic Acids; Perylene; Spectrometry, Fluorescence
PubMed: 27394693
DOI: 10.1016/j.bmc.2016.06.055 -
Analytical and Bioanalytical Chemistry Nov 2014In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA...
In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA have been used for the detection of DNA. The photoluminescence of C-dots@RGO is quenched by MB, which is restored in the presence of a target DNA. The combination of the C-dots@RGO, MB, and a DNA probe is selective for perfectly matched DNA over mismatched DNA, mainly because relative to single-stranded DNA, double-stranded DNA intercalates more strongly with MB, but interacts more weakly with RGO. In the presence of a target DNA, MB intercalates with the as-formed double-stranded DNA and is released from the surface of C-dots@RGO, leading to "turn-on" photoluminescence. The practicality of this assay has been validated by the determination of tumor suppressor gene BRCA1, with linearity over the concentration range from 25 to 250 nM and a limit of detection (LOD, at a signal-to-noise ratio of 3) of 14.6 nM. The C-dots@RGO probe provides higher specificity towards target DNA than towards common salts, carbohydrates, amino acids, and proteins found in real samples. Having the advantages of simplicity, cost-effectiveness, selectivity, and sensitivity, the DNA-P/C-dots@RGO-MB probe on microwells has been successfully employed for the detection of DNA, suggesting its potential for multiple analyses of DNA targets when various DNA probes are employed.
Topics: Base Sequence; DNA Probes; Luminescence; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Molecular Sequence Data
PubMed: 24553661
DOI: 10.1007/s00216-014-7658-2 -
Analytical Chemistry Feb 2021Flap endonuclease 1 (FEN1), an endogenous nuclease with the ability to cleave the 5' overhang of branched dsDNA, is of significance in DNA replication and repair. The...
Flap endonuclease 1 (FEN1), an endogenous nuclease with the ability to cleave the 5' overhang of branched dsDNA, is of significance in DNA replication and repair. The overexpression of FEN1 is common in cancer because of the ubiquitous upregulation of DNA replication; thus, FEN1 has been recognized as a potential biomarker in oncological investigations. However, few analytical methods targeting FEN1 with high sensitivity and simplicity have been developed. This work developed a signal-amplified detection of FEN1 based on the cleavage-induced ligation of a dumbbell DNA probe and rolling circle amplification (RCA). A flapped dumbbell DNA probe (FDP) was rationally designed with a FEN1 cleavable flap at the 5' end. The cleavage generated a nick site with juxtaposed 5' phosphate and 3' hydroxyl ends, which were linkable by T4 DNA ligase to form a closed dumbbell DNA probe (CDP) with a circular conformation. The CDP functioned as a template for RCA, which produced abundant DNA that could be probed using SYBR Green I. The highly sensitive detection of FEN1 with a limit of detection of 15 fM was achieved, and this method showed high specificity, which enabled the quantification of FEN1 in real samples. The inhibitory effects of chemicals on FEN1 were also evaluated. This study represents the first attempt to develop an FEN1 assay that involves signal amplification, and the novel biosensor method enriches the tools for FEN1-based diagnostics.
Topics: Biomarkers, Tumor; Biosensing Techniques; DNA; DNA Probes; Flap Endonucleases; Nucleic Acid Amplification Techniques
PubMed: 33529005
DOI: 10.1021/acs.analchem.0c05275 -
Analytica Chimica Acta Jan 2024Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode...
A universal dual-mode hydrogel array based on phage-DNA probe for simultaneous rapid screening and precisely quantitative detection of Escherichia coli O157:H7 in foods by the fluorescent/microfluidic chip electrophoresis methods.
Rapid and specific detection of virulent bacterial strains is a great challenge for food safety regarding large amounts of contaminated samples. Herein, a dual-mode hydrogel array biosensor was constructed to simultaneously rapidly screen and precisely quantitatively detect virulent Escherichia coli O157:H7 (E. coli O157:H7) based on a novel DNA-modified phage probe. First, E. coli O157:H7 was incubated with alginate to form the E. coli O157:H7/hydrogel premix complex. Subsequently, hydrogel formation by cross-linking upon the addition of calcium ions and phages for E. coli O157:H7 modified with a DNA primer (phage-DNA) was added to the alginate hydrogel. The DNA on the complex could trigger rolling circle amplification (RCA) to form a phage probe containing a long-chain DNA skeleton (phage@RCA-DNA). The RCA-DNA was then hybridized with the complementary DNA (cDNA) to form double-stranded DNA fragments (phage@RCA-dsDNA), which could be stained by the SYBR Green dye to emit visual green fluorescence (FL) and determined by a smartphone for rapid screening. Meanwhile, the unreacted cDNA in the supernatant could be quantitatively detected by microfluidic chip electrophoresis (MCE). The signal decrement was also proportional to the bacterial concentration. The detection limit values of E. coli O157:H7 were 50 CFU mL by the FL signal and 6 CFU mL by the MCE signal. The two results could be mutually corrected to decrease the false-positive results. This assay was also employed to detect virulent Salmonella Typhimurium (S. Typhimurium) using the corresponding S. Typhimurium phage@RCA-DNA probe. All these results demonstrated that the universal bioassay was suitable for simultaneous rapid screening and precisely quantitative detection of virulent bacterial strains.
Topics: DNA, Complementary; Hydrogels; Escherichia coli O157; Microfluidics; DNA Probes; Alginates; Bacteriophages; Coloring Agents; Electrophoresis
PubMed: 38182365
DOI: 10.1016/j.aca.2023.342053