-
Biosensors & Bioelectronics Dec 2016A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially...
A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.
Topics: Complex Mixtures; Conductometry; DNA Probes; DNA, Bacterial; Equipment Design; Equipment Failure Analysis; Food Analysis; Food Contamination; Food Microbiology; Gold; Metal Nanoparticles; Molecular Probe Techniques; Oligonucleotide Array Sequence Analysis; Polyesters; Reproducibility of Results; Sensitivity and Specificity; Vibrio parahaemolyticus
PubMed: 27414245
DOI: 10.1016/j.bios.2016.06.077 -
Chemical Communications (Cambridge,... Sep 2012We report a fully covalent, dual-signalling electrochemical DNA sensor that exploits competitive binding and target hybridization-induced change in probe flexibility for...
We report a fully covalent, dual-signalling electrochemical DNA sensor that exploits competitive binding and target hybridization-induced change in probe flexibility for simple and robust detection of target DNA.
Topics: Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Nucleic Acid Conformation; Nucleic Acid Hybridization; Sensitivity and Specificity
PubMed: 22825042
DOI: 10.1039/c2cc34312f -
Analytical and Bioanalytical Chemistry Feb 2020A tetrahedral DNA probe can effectively overcome the steric effects of a single-stranded probe to obtain well-controlled density and minimize nonspecific adsorption....
A tetrahedral DNA probe can effectively overcome the steric effects of a single-stranded probe to obtain well-controlled density and minimize nonspecific adsorption. Herein, a highly sensitive electrochemical biosensor is fabricated for determination of protein using a tetrahedral DNA probe and rolling circle amplification (RCA). N- and P-co-doped graphene (NP-rGO) is prepared, and AuNPs are then electrodeposited on it for DNA probe immobilization. Benefitting from the synergistic effects of the excellent electrical conductivity of NP-rGO, the stability of the tetrahedral DNA probe and the signal amplification of RCA, the biosensor achieves a low limit of 3.53 × 10 M for thrombin and a wide linear range from 1 × 10 to 1 × 10 M. This study provides a sensitive and effective method for the detection of protein in peripheral biofluids, and paves the way for future clinical diagnostics and treatment of disease. Graphical abstract.
Topics: Aptamers, Nucleotide; Biosensing Techniques; DNA Probes; Electrochemical Techniques; Gold; Graphite; Humans; Immobilized Nucleic Acids; Limit of Detection; Metal Nanoparticles; Nucleic Acid Amplification Techniques; Thrombin
PubMed: 31900531
DOI: 10.1007/s00216-019-02314-y -
Medicinal Chemistry (Shariqah (United... 2016The ability of DNA to capture oligonucleotide molecules in solution is of great importance in genetics, medical diagnostics, and drug discovery. The DNA hybridization...
The ability of DNA to capture oligonucleotide molecules in solution is of great importance in genetics, medical diagnostics, and drug discovery. The DNA hybridization event in which the probe, which is usually a single-stranded DNA (ssDNA) covalently immobilized on a functionalized surface, recognizes the complementary target and forms a stable duplex structure that is the basis of highly specific bio recognizing devices. In this computational study, molecular modeling and Quantitative Structure Activity Relationship (QSAR) calculations were utilized at PM3 level in order to evaluate the interaction of aldehyde ssDNA on chitosan-functionalized silicon substrate and the biological activity of the proposed compounds. Molecular modeling of ssDNA 5'-(TTCA) attached on chitosan- functionalized silicon dioxide substrate was carried out. Molecular modeling and QSAR calculations were utilized at MM3 level in order to evaluate the interaction of target DNA on DNA probe on chitosan-functionalized silicon substrate through hydrogen bonding and the biological activity of the proposed compounds.
Topics: Chitosan; DNA; DNA Probes; Hydrogen Bonding; Models, Chemical; Models, Molecular; Nucleic Acid Hybridization; Quantitative Structure-Activity Relationship; Silicon Dioxide
PubMed: 26558377
DOI: 10.2174/1573406412666151112124836 -
The Analyst Oct 2016An ultrasensitive fluorescent platform for sequence-specific recognition of double-stranded DNA (dsDNA) based on the quenching of gold nanoparticles (AuNPs) to a...
An ultrasensitive fluorescent platform for sequence-specific recognition of double-stranded DNA (dsDNA) based on the quenching of gold nanoparticles (AuNPs) to a fluorophore labeled DNA probe was developed. The target dsDNA could hybridize with the loop portion of the molecular beacon (MB) to form a triplex DNA structure and opened the "stem-loop" structure of the MB; such triplex DNA was used as an assistant probe (AP). Meanwhile, a fluorophore labeled DNA-AuNP probe that contained a specific enzyme cleavage site was introduced and its fluorescence signal was efficiently quenched due to the vicinity of the fluorophore to the AuNP surface. Such a DNA-AuNP probe could hybridize with the 5' stem portion of the MB in the AP to form duplex DNA strands that contained a specific enzyme cleavage site for the nicking enzyme assisted cleavage reaction, and resulted in the release of the fluorophore from the AuNP surface and the recovery of the fluorescence signal. Because the AP remains intact during such a cleavage process, it could be reused to hybridize with the next DNA-AuNP probe and trigger the nicking nuclease assisted signal amplification. Under optimal conditions, a low detection limit of 3.8 pM was obtained for dsDNA detection, and the assay has high sequence specificity for dsDNA detection.
Topics: DNA; DNA Probes; Fluorescent Dyes; Gold; Metal Nanoparticles
PubMed: 27508282
DOI: 10.1039/c6an01145d -
The Analyst Aug 1999The acquisition of sequence, expression and other information concerning genetic material constitutes a crucial component of the modern revolution in molecular biology.... (Review)
Review
The acquisition of sequence, expression and other information concerning genetic material constitutes a crucial component of the modern revolution in molecular biology. One important advance in this area is the development of high density oligonucleotide/DNA microarrays which allows the rapid sequence analysis of genomic target samples in addition to diagnostic possibilities with respect to genetic and infectious disease. In the present article we review protocols for the design of such microarrays and their principles of operation. Together with a look at some recent applications we include brief remarks as to the possibilities for the future.
Topics: Animals; DNA Probes; Gene Targeting; In Situ Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes
PubMed: 10736845
DOI: 10.1039/a904581c -
Chemical Communications (Cambridge,... Aug 2020Herein, we develop a novel tripartite DNA probe to transport phosphorothioated substrate hairpins and an aptamer for the intramolecular CHA circuit, which achieves...
Herein, we develop a novel tripartite DNA probe to transport phosphorothioated substrate hairpins and an aptamer for the intramolecular CHA circuit, which achieves detection of a low amount of specific mRNA in living cells and mice. Our study provides an improved strategy to promote in vivo fluorescence imaging applications in early-stage clinical diagnosis.
Topics: Animals; Biocatalysis; DNA Probes; Inverted Repeat Sequences; Mice; Optical Imaging; RNA, Messenger
PubMed: 32618290
DOI: 10.1039/d0cc03596c -
Biosensors & Bioelectronics Nov 2013DNA methylation is an important epigenetic modification of genomes and is associated with various human diseases. Here, we develop a sensitive approach for DNA...
DNA methylation is an important epigenetic modification of genomes and is associated with various human diseases. Here, we develop a sensitive approach for DNA methylation assay using the short linear quencher-fluorophore DNA probe (QF probe) and two-stage isothermal amplification. With bisulfite treatment, the methylated DNA target is able to hybridize with the template to initiate the strand displacement amplification (SDA), generating abundant triggers which can further hybridize with the QF probes to form the DNA duplexes. The subsequent recognition of DNA duplexes and the cleavage of QF probes by the nicking enzyme can initiate the nicking enzyme signal amplification (NESA), inducing a significant fluorescence enhancement. While in the absence of methylated DNA, neither SDA nor NESA reaction can be initiated and no fluorescence enhancement is observed. This method exhibits high sensitivity with a detection limit of 0.78 pM, and can distinguish as low as 0.1% methylation level from the mixture of methylated and unmethylated DNA. Notably, the introduction of SDA into NESA can improve the detection sensitivity by up to 2 orders of magnitude as compared with the NESA assay, and it can even discriminate single-base mismatched methylated DNA.
Topics: Base Sequence; DNA; DNA Methylation; DNA Probes; Fluorescent Dyes; Humans; Limit of Detection; Nucleic Acid Amplification Techniques; Spectrometry, Fluorescence
PubMed: 23743329
DOI: 10.1016/j.bios.2013.05.009 -
Analytical Chemistry Oct 2009The paper described a label-free assay for the detection of single-nucleotide mismatches in which an unlabeled hairpin DNA probe and a MutS protein conjugate...
The paper described a label-free assay for the detection of single-nucleotide mismatches in which an unlabeled hairpin DNA probe and a MutS protein conjugate (His6-MutS-linker peptide-streptavidin binding peptide (HMLS)) are exploited for the detection of mismatches by electrochemical impedance spectroscopy (EIS). We demonstrate this method for eight single-nucleotide mismatches. Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened forming a duplex DNA. In duplexes containing a single nucleotide mismatch, the mismatch is present at the solvent exposed side, enabling more effective HMLS recognition and binding. The binding event is evaluated by EIS and analyzed with the help of Randles' equivalent circuits. The differences in the charge transfer resistance DeltaR(CT) before and after protein binding to the duplex DNA allows the unequivocal detection of all eight single-nucleotide mismatches. DeltaR(CT) allows the discrimination of a C-A mismatch with the concentration of the target strand as low as 100 pM.
Topics: Base Pair Mismatch; Base Sequence; DNA; DNA Probes; Electric Impedance; Electrochemistry; Escherichia coli Proteins; Inverted Repeat Sequences; Molecular Sequence Data; MutS DNA Mismatch-Binding Protein; Peptides; Streptavidin
PubMed: 19769379
DOI: 10.1021/ac901371n -
RNA (New York, N.Y.) Dec 2018Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes....
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Topics: Blotting, Northern; DNA Probes; Fluorescent Dyes; Nucleic Acid Hybridization; RNA; RNA Probes
PubMed: 30201850
DOI: 10.1261/rna.068213.118