-
Scientific Reports Jul 2019Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and...
Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and label-free detection. In nucleic acid detection, GC-rich nucleic acid sequences form self- and cross-dimers and stem-loop structures, which can easily obtain data containing signals from nonspecific DNA binding. The features of GC-rich nucleic acid sequences cause inaccuracies in nucleic acid detection and hinder the development of precision medicine. To improve the inaccurate detection results, we used phosphate-methylated (neutral) nucleotides to synthesize the neutralized chimeric DNA oligomer probe. The probe fragment originated from a primer for the detection of hepatitis C virus (HCV) genotype 3b, and single-mismatched and perfect-matched targets were designed for single nucleotide polymorphisms (SNP) detection on the SiNW FET device. Experimental results revealed that the HCV-3b chimeric neutralized DNA (nDNA) probe exhibited better performance for SNP discrimination in 10 mM bis-tris propane buffer at 25 °C than a regular DNA probe. The SNP discrimination of the nDNA probe could be further improved at 40 °C on the FET device. Consequently, the neutralized chimeric DNA probe could successfully distinguish SNP in the detection of GC-rich target sequences under optimal operating conditions on the SiNW FET device.
Topics: Biosensing Techniques; DNA Probes; Genotyping Techniques; Nanowires; Polymorphism, Single Nucleotide; Sensitivity and Specificity; Silicon; Transistors, Electronic
PubMed: 31363139
DOI: 10.1038/s41598-019-47522-9 -
Biosensors & Bioelectronics May 2017We present a non-modification electrochemical DNA sensing strategy, which used Potential-Assisted Au-S Deposition and a clamp-like DNA probe. The dual-hairpin probe DNA...
We present a non-modification electrochemical DNA sensing strategy, which used Potential-Assisted Au-S Deposition and a clamp-like DNA probe. The dual-hairpin probe DNA was tagged with a methylene blue (MB) at the 3' terminal and a thiol at the 5' terminal., Without being hybridized with target DNA, the loop of probe prevented the thiol from reaching the bare gold electrode surface with an applied potential., After hybridization with the target DNA, the probe' s loop-stem structure opened through two distinct and sequential events, which led to the formation of a triplex DNA structure. Then the thiol easily contacted with electrode and resulted in potential-assisted Au-S self-assembly. Electrochemical signals of MB were measured by differential pulse voltammetry (DPV) and used for target quantitative detection. This strategy offered a detection limit down to 2.3pM. and an inherently high specificity for detecting even single mismatch.
Topics: Base Sequence; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Gold; Limit of Detection; Nucleic Acid Conformation; Nucleic Acid Hybridization; Sulfhydryl Compounds
PubMed: 28011414
DOI: 10.1016/j.bios.2016.10.008 -
Analytical and Bioanalytical Chemistry Jan 2012Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here...
Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.
Topics: Biosensing Techniques; DNA; DNA Probes; Electrochemistry; Kinetics; Proteins
PubMed: 21928081
DOI: 10.1007/s00216-011-5361-0 -
Biosensors & Bioelectronics Mar 2013Intensive efforts have been focused on the development of ultrasensitive DNA biosensors capable of quantitative gene expression analysis. Various neutralized nucleic... (Comparative Study)
Comparative Study
Intensive efforts have been focused on the development of ultrasensitive DNA biosensors capable of quantitative gene expression analysis. Various neutralized nucleic acids have been demonstrated as alternative and attractive probe for the design of a DNA chip. However, the mechanism of the improvements has not been clearly revealed. In this investigation, we used a newly developed neutral ethylated DNA (E-DNA), a DNA analog with the "RO-P-O" backbone (wherein R could be methyl, ethyl, aryl, or alkyl group) obtained from synthetic procedures, and a silicon nanowire (SiNW) field-effect transistor (FET) to evaluate the difference in DNA detection performance while using E-DNA and DNA as probes. It is demonstrated that using the E-DNA probe in the FET measurement could have a significantly enhanced effect upon the detection sensitivity. Surface plasmon resonance imaging (SPRi) was used to evidence the mechanism of the improved detection sensitivity. SPRi analysis showed the amounts of probe immobilization on the sensor surface and the hybridization efficiency were both enhanced with the use of E-DNA. Consequently, neutral ethylated DNA probe hold a great promise for DNA sensing, especially in the electrical-based sensor.
Topics: Biosensing Techniques; Conductometry; DNA; DNA Probes; Equipment Design; Equipment Failure Analysis; Molecular Probe Techniques; Reproducibility of Results; Sensitivity and Specificity; Static Electricity; Surface Plasmon Resonance; Transistors, Electronic
PubMed: 23116544
DOI: 10.1016/j.bios.2012.10.010 -
Mikrochimica Acta Jan 2018A fluorescent method is described for simultaneous recognition of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). It is based on the quenching of the...
A fluorescent method is described for simultaneous recognition of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). It is based on the quenching of the fluorescence of fluorophore labeled DNA probes by gold nanoparticles (AuNPs). To demonstrate feasibility, two DNA probes labeled with spectrally different fluorophores were designed. The first DNA probe (P) was modified with 6-carboxyfluorescein (FAM; with green fluorescence, peaking at 518 nm), while the second (P) was modified with carboxy-X-rhodamine (ROX; with yellow fluorescence, 610 nm). The fluorescence signals of the labels are quenched if P or P are adsorbed on AuNPs. Upon addition of ssDNA and dsDNA, hybridization occurs between P and ssDNA to form a dsDNA. In contrast, P hybridizes with dsDNA such that a triplex DNA is formed. As a result, the dsDNA and the triplex DNA, respectively, are desorbed from the surface of the AuNPs so that quenching no longer can occur and strong fluorescence can be observed. Under the optimal conditions, ssDNA and dsDNA can be detected simultaneously via the green and yellow fluorescence, respectively. The detection limits can be as low as 330 pM. In particular, the method has excellent selectivity for the target DNAs over control DNAs. Graphical abstract A gold nanoparticle based fluorescent probe for simultaneous recognition of single-stranded DNA and double-stranded DNA is developed based on the fluorescence quenching of gold nanoparticles to different fluorophore labeled DNA probes.
Topics: DNA; DNA Probes; DNA, Single-Stranded; Fluorescence; Fluorescent Dyes; Gold; Limit of Detection; Metal Nanoparticles; Nucleic Acid Hybridization
PubMed: 29594738
DOI: 10.1007/s00604-017-2633-1 -
ACS Applied Bio Materials Jan 2024Cancers remain the leading cause of mortality worldwide. It is crucial to detect cancer at an early stage for improving survival rates. Biomarkers have precise...
Cancers remain the leading cause of mortality worldwide. It is crucial to detect cancer at an early stage for improving survival rates. Biomarkers have precise implications for cancer progression. Here, we built a straightforward DNA probe system that could be activated by near-infrared light to detect dual miRNAs with a high specificity. This probe is built on the basis of upconversion nanoparticles, which could emit ultraviolet light and activate DNA probes adsorbed on the outer layer. The DNA probe system is remotely controlled through manipulation of the near-infrared (NIR) light, enabling simultaneous detection of dual miRNAs. The DNA nanosystem could be effectively endocytosed by cancer cells and reflect expression levels of dual miRNAs. Overall, this study demonstrates a promising remote-controlled DNA nanoplatform for the simultaneous detection of dual miRNAs, which has tremendous potential for precise cancer diagnostics and therapies.
Topics: Humans; MicroRNAs; Ultraviolet Rays; DNA; DNA Probes; Nanoparticles; Neoplasms
PubMed: 38151236
DOI: 10.1021/acsabm.3c01079 -
Dalton Transactions (Cambridge, England... Dec 2023Sensitively monitoring metallothionein (MT), a heavy metal-binding protein with substantial cysteine content, is of significance for evaluating heavy metal poisoning in...
Sensitively monitoring metallothionein (MT), a heavy metal-binding protein with substantial cysteine content, is of significance for evaluating heavy metal poisoning in both humans and animals. Based on a new metal ion-coordinated DNA probe and the heavy metal ion binding capability of MT, as well as the substantial signal enhancement of the hybridization chain reaction (HCR) and rolling circle amplification (RCA), we demonstrate a highly sensitive fluorescence MT detection assay. MT binds the metal ions in the hairpin structured, metal ion-coordinated DNA probe to switch its hairpin structure into ssDNA, which triggers subsequent RCA reactions and HCRs to open plenty of fluorescently quenched signal hairpins to exhibit drastically amplified fluorescence recovery for assaying MT down to 0.58 nM within a dynamic range of 1-320 nM. In addition, the investigation of low contents of MT in diluted human serum by such an assay has also been verified, indicating its promising application potential for diagnosing heavy metal poisoning.
Topics: Humans; DNA; DNA Probes; Nucleic Acid Hybridization; Heavy Metal Poisoning; Metals, Heavy; Biosensing Techniques; Limit of Detection
PubMed: 38014455
DOI: 10.1039/d3dt03346e -
Analytical Chemistry Feb 2023The accurate discrimination of single-nucleotide variants is of great interest for disease diagnosis and clinical treatments. In this work, a unique DNA probe with...
The accurate discrimination of single-nucleotide variants is of great interest for disease diagnosis and clinical treatments. In this work, a unique DNA probe with "Hill-type" cooperativity was first developed based on toehold-mediated strand displacement processes. Under simulation, this probe owns great thermodynamics advantage for specificity due to two mismatch bubbles formed in the presence of single-nucleotide variants. Besides, the strategies of Δ' = 0 and more competitive strands are also beneficial to discriminate single-nucleotide variants. The feasibility of this probe was successfully demonstrated in consistent with simulation results. Due to "Hill-type" cooperativity, the probe allows a steeper dynamic range compared with previous probes. With simulation-guided rational design, the resulting probe can accurately discriminate single-nucleotide variants including nucleotide insertions, mutation, and deletions, which are arbitrarily distributed in target sequence. Two specificity parameters were calculated to quantitatively evaluate its good discrimination ability. Hence, "Hill-type" cooperativity can serve as a novel strategy in DNA probe's design for accurate discrimination of single-nucleotide variants.
Topics: Nucleic Acid Hybridization; DNA Probes; DNA; Mutation; Nucleotides
PubMed: 36695821
DOI: 10.1021/acs.analchem.2c04446 -
Analytical Chemistry Dec 2021Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor...
Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor development. The thermal stability of low-coverage DNA SAMs was studied for surfaces prepared using potential-assisted thiol exchange (E) and compared to DNA SAMs prepared without control over the substrate potential (OCP). Both surface preparation methods were studied using in situ fluorescence microscopy and electrochemistry with fluorophore or redox-modified DNA SAMs on a single-crystal gold bead electrode. Fluorescence microscopy showed that the influence of the underlying surface crystallography was important in both cases. The highest thermal stability was realized for square or rectangular surface atomic structure (e.g., surfaces from 110 to 100). The 111 and related surfaces were the least thermally stable. The low DNA coverage surfaces prepared by E had better thermal stability and higher DNA probe mobility as compared to OCP-prepared surfaces with the similar coverage. These results were correlated with methylene blue redox-tagged DNA probes, which directly measured the average DNA coverage. Both methods indicated that E DNA SAMs were more uniformly distributed across the electrode surface, while the surfaces prepared via OCP assembled into clusters with reduced mobility. The potential-assisted thiol-exchange approach to preparing low-coverage DNA SAMs was shown to quickly create modified surfaces that were consistent, had mobility characteristics which should yield superior DNA hybridization efficiencies, and having greater thermal stability which will translate into a longer shelf-life.
Topics: DNA; DNA Probes; Gold; Sulfhydryl Compounds; Surface Properties
PubMed: 34813297
DOI: 10.1021/acs.analchem.1c03353 -
Talanta Sep 2019A rapid, cost-effective and quencher-free fluorescence-based analytical method for sensitive detection of l-cysteine (Cys) based on 2-aminopurine (2-AP) labeled DNA...
A rapid, cost-effective and quencher-free fluorescence-based analytical method for sensitive detection of l-cysteine (Cys) based on 2-aminopurine (2-AP) labeled DNA probe and exonuclease I (Exo I) activity was developed. 2-AP labeled DNA probe includes two thymine (T)-T mismatches, which can bind with Hg to form T-Hg-T pairing bases, resulting in stable hairpin with five base pairs in its stem. The target Cys can remove Hg from the stem of the hairpin probe based on the high affinity of Cys with Hg, leading to the unfolding of the hairpin probe. At last, by adding Exo I, the resulted single-stranded DNA (ssDNA) will be digested to release free 2-AP with strong fluorescence. Under the optimal conditions, the sensing system exhibited a good and wider linear range from 0.4 to 400 nM (R = 0.997) and a detection limit as low as 0.16 nM for Cys. Furthermore, other amino acids without reductive sulfur group did not generate obvious change in fluorescence signals. Finally, the sensor can be used in diluted real samples with a good recovery rate, showing promising application in food, environmental and medical analysis.
Topics: 2-Aminopurine; Cysteine; DNA Probes; Fluorescence; Nucleic Acid Conformation
PubMed: 31171216
DOI: 10.1016/j.talanta.2019.05.007