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Angewandte Chemie (International Ed. in... Mar 2018A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a...
A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future.
Topics: Biosensing Techniques; DNA; DNA Probes; Fluorescent Dyes; Hemolysin Proteins; Limit of Detection; Nanoparticles; Nanopores; Nucleic Acid Hybridization; Particle Size; Protein Conformation; Sensitivity and Specificity; Spectrometry, Fluorescence; Surface Properties
PubMed: 29488675
DOI: 10.1002/anie.201711690 -
Biosensors & Bioelectronics Apr 2019Surface-enhanced Raman scattering (SERS) based DNA biosensors have considered as excellent, fast and ultrasensitive sensing technique which relies on the fingerprinting...
Surface-enhanced Raman scattering (SERS) based DNA biosensors have considered as excellent, fast and ultrasensitive sensing technique which relies on the fingerprinting ability to produce molecule specific distinct spectra. Unlike conventional fluorescence based strategies SERS provides narrow spectral bandwidths, fluorescence quenching and multiplexing ability, and fitting attribute with short length probe DNA sequences. Herein, we report a novel and PCR free SERS based DNA detection strategy involving dual platforms and short DNA probes for the detection of endangered species, Malayan box turtle (MBT) (Cuora amboinensis). In this biosensing feature, the detection is based on the covalent linking of the two platforms involving graphene oxide-gold nanoparticles (GO-AuNPs) functionalized with capture probe 1 and gold nanoparticles (AuNPs) modified with capture probe 2 and Raman dye (Cy3) via hybridization with the corresponding target sequences. Coupling of the two platforms generates locally enhanced electromagnetic field 'hot spot', formed at the junctions and interstitial crevices of the nanostructures and consequently provide significant amplification of the SERS signal. Therefore, employing the two SERS active substrates and short-length probe DNA sequences, we have managed to improve the sensitivity of the biosensors to achieve a lowest limit of detection (LOD) as low as 10 fM. Furthermore, the fabricated biosensor exhibited sensitivity even for single nucleotide base-mismatch in the target DNA as well as showed excellent performance to discriminate closely related six non-target DNA sequences. Although the developed SERS biosensor would be an attractive platform for the authentication of MBT from diverse samples including forensic and/or archaeological specimens, it could have universal application for detecting gene specific biomarkers for many diseases including cancer.
Topics: Biosensing Techniques; DNA; DNA Probes; Gold; Graphite; Limit of Detection; Metal Nanoparticles; Nanostructures; Spectrum Analysis, Raman
PubMed: 30844598
DOI: 10.1016/j.bios.2019.02.028 -
Analytical Chemistry Mar 2000Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured...
Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.
Topics: Base Sequence; DNA; DNA Probes; Electrochemistry; Electrodes; Ferrous Compounds; Imides; Kinetics; Ligands; Magnetic Resonance Spectroscopy
PubMed: 10740879
DOI: 10.1021/ac991031j -
Analytical Biochemistry Oct 1991A general procedure for the cross-linking of enzyme to DNA has been developed for use as a nonradioactive probe. In this method, DNA is transaminated with diaminopropane...
A general procedure for the cross-linking of enzyme to DNA has been developed for use as a nonradioactive probe. In this method, DNA is transaminated with diaminopropane to introduce primary amino groups into the cytosine residues. Then the amino groups are converted to thiol groups using a heterobifunctional cross-linker. The thiolated DNA is conjugated with the maleimide-introduced enzyme. With this method, alkaline phosphatase was cross-linked to a single-stranded DNA (sspUCRf1). The conjugate was able to detect 5 pg of target DNA (pUCf1 plasmid, 3.2 kbp) fixed onto the nitrocellulose membrane, using a colorimetric assay. The enzyme-conjugated DNA was applied to "the universal probe system," which consisted of two single-stranded DNA probes (a primary probe and a labeled secondary probe). Using alkaline phosphatase-conjugated sspUCRf1 DNA as the secondary probe, the c-myc gene and HBV DNA were detected effectively on Southern and dot-blot hybridization.
Topics: Alkaline Phosphatase; Amination; Blotting, Southern; Colorimetry; Cross-Linking Reagents; DNA Probes; Dithiothreitol; Humans; Maleimides; Nucleic Acid Hybridization; Succinimides
PubMed: 1789434
DOI: 10.1016/0003-2697(91)90508-q -
Biosensors & Bioelectronics Feb 2020A promising electrochemical system was explored for DNA methylation detection according to the construction of a signal-on biosensor. Based on the ingenious design of...
A promising electrochemical system was explored for DNA methylation detection according to the construction of a signal-on biosensor. Based on the ingenious design of probe DNA and auxiliary DNA, methylated target DNA triggered the exonuclease III (Exo III) digestion of auxiliary DNA from 3'-terminus, resulting in the conformational change of probe DNA with an electroactive methylene blue (MB) tag at 5'-terminus. Consequently, the MB tag in the probe DNA was close to the electrode surface for electron transfer, generating an increased current signal. Because of the target recycling of methylated DNA, significant signal amplification was obtained. Moreover, bisulfite conversion conferred an efficient approach for the universal analysis of any CpG sites without the restriction of specific DNA sequence. As a result, the target DNA with different methylation statuses were clearly recognized, and the fully methylated DNA was quantified in a wide range from 10 fM to 100 pM, with a detection limit of 4 fM. The present work realized the assay of methylated target DNA in serum samples with satisfactory results, illustrating the application performance of the system in complex sample matrix.
Topics: Aptamers, Nucleotide; Biosensing Techniques; DNA Methylation; DNA Probes; Electrochemical Techniques; Exodeoxyribonucleases; Gold; Limit of Detection; Nucleic Acid Conformation
PubMed: 31733487
DOI: 10.1016/j.bios.2019.111847 -
Analytical Methods : Advancing Methods... Dec 2022Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing...
Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing system was based on two DNA hybrid probes, each of which was constructed by annealing a reconfigurable DNA hairpin with single-stranded DNA. Upon target recognition by the first DNA hybrid probe, a reconfigurable DNA switch was liberated, triggering a toehold-mediated strand displacement reaction (TSDR) with the second DNA hybrid probe, which was accompanied by the release of another reconfigurable DNA switch. This released allosteric DNA switch could further interact with the first hybrid DNA probe the TSDR strategy to form a reciprocal strand displacement network between the two DNA hybrid probes. Theoretically, this reciprocal strand displacement reaction would continue till the complete consumption of the reaction substrates. Thus, it provides a new signal amplification method leading toward target recognition. More interestingly, it creates a ratiometric signal response mode for target recognition, which involves the fluorescence increment of one fluorophore (Cy5) and concurrent decrement of another fluorophore (Cy3) accompanied by the target-triggered reciprocal strand displacement reaction. This process could achieve a low detection limit of about 0.1 pM toward the target nucleic acid and selective discrimination toward different mismatched targets. It could also be applied for detection in a serum sample. Thus, the developed catalytic DNA circuit reaction strategy together with ratiometric signal readout provides a new avenue for programmable, reliable and sensitive detection of nucleic acids and might also pave the way for developing more advanced DNA circuits or biosensors.
Topics: DNA, Catalytic; Nucleic Acid Hybridization; Limit of Detection; DNA; DNA Probes
PubMed: 36504112
DOI: 10.1039/d2ay01751b -
Journal of Nanoscience and... Oct 2015The development of chip-based, quantum dot (QD)-bead-DNA conjugate probes for hybridization detection is a prime research focus in the field of microfluidics....
The development of chip-based, quantum dot (QD)-bead-DNA conjugate probes for hybridization detection is a prime research focus in the field of microfluidics. QD-Bead-DNA probe-based hybridization detection methods are often called "bead-based assays," and their success is substantially influenced by the dispensing and manipulation capabilities of microfluidic technology. Met was identified as a prognostic marker in different cancers including lung, renal, liver, head and neck, stomach, and breast. In this report, the cancer causing Met gene was detected with QDs attached to polystyrene microbeads. We constructed a microfluidic platform using a flexible PDMS polymer. The chip consists of two channels, with two inlets and two outlets. The two channels were integrated with QD-bead-DNA probes for simultaneous detection of wild type target DNA and mutant DNA, containing three nucleotide changes compared to the wild type sequence. The fluorescence quenching ability of QDs within the channels of microfluidic chips were compared for both DNAs.
Topics: DNA Probes; Fluorescence; Humans; Lab-On-A-Chip Devices; Polystyrenes; Proto-Oncogene Proteins c-met; Quantum Dots
PubMed: 26726440
DOI: 10.1166/jnn.2015.11218 -
Journal of Materials Chemistry. B Dec 2022The real-time imaging of low-abundance tumor-related microRNAs (miRNAs) in living cells holds great potential for early clinical diagnosis of cancers. However, the...
The real-time imaging of low-abundance tumor-related microRNAs (miRNAs) in living cells holds great potential for early clinical diagnosis of cancers. However, the relatively low detection sensitivity and possible false-positive signals of a probe in complex cellular matrices remain critical challenges for accurate RNA detection. Herein, we developed a novel aptamer-functionalized cruciate DNA probe that enabled amplified multiple miRNA imaging in living cells catalytic hairpin assembly (CHA). The cross-shaped design of the cruciate DNA probe improved the stability against nucleases and acted as a modular scaffold for CHA circuits for efficient delivery into tumor cells. The cruciate DNA probe allowed self-assembly through thermal annealing and displayed excellent performance for sensitive miRNA detection . The cruciate DNA probe could be internalized into nucleolin-overexpressed cells specifically cell-targeting of the AS1411 aptamer, achieving amplified fluorescence imaging and quantitative evaluation of the expression of miRNAs in living cells. Through the simultaneous detection of intracellular multiple miRNAs, the developed cruciate DNA probe could provide more accurate information and reduce the chances of false positive signals for cancer diagnosis. This approach offers a new opportunity for promoting the development of miRNA-related biomedical research and tumor diagnostic applications.
Topics: MicroRNAs; DNA; Cell Line, Tumor; DNA Probes; Optical Imaging
PubMed: 36504047
DOI: 10.1039/d2tb02027k -
Biosensors & Bioelectronics Jan 2007In this paper, a label-free, rapid and simple method was proposed to study the hybridization specificity of hairpin DNA probe using methylene blue (MB) as a...
In this paper, a label-free, rapid and simple method was proposed to study the hybridization specificity of hairpin DNA probe using methylene blue (MB) as a hybridization indicator. Thiolated hairpin DNA probe was immobilized on the gold electrode by self-assembly. The voltammetric signals of MB were investigated at these modified electrodes by means of cyclic voltammetry (CV) detection. Single-base mutation oligonucleotide and random oligonucleotide can be easily discriminated from complementary target DNA. The effect of mismatch position in target DNA was investigated. Experimental results showed that mutation in the center of target DNA had greatest effect on the hybridization with hairpin DNA probe. The relationship between electrochemical responses and DNA target concentration was also studied. The reduction current of MB intercalation decreased with increasing the concentration of target DNA. Taken together, these experiments demonstrate that the hybridization indicator MB provides great promise for rapid and specific measurement of target DNA.
Topics: Biosensing Techniques; DNA Probes; Electrochemistry; In Situ Hybridization; Methylene Blue; Oligonucleotide Array Sequence Analysis
PubMed: 16730971
DOI: 10.1016/j.bios.2006.04.011 -
Biotechnic & Histochemistry : Official... Jan 1998We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA... (Review)
Review
We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.
Topics: Animals; DNA Probes; Humans; In Situ Hybridization, Fluorescence
PubMed: 9554580
DOI: 10.3109/10520299809140502