Detection Of Single-base Mismatch Research Articles

Ultra-sensitive DNA detection with single-base mismatch resolution using a tapered thin-diameter photonic crystal fiber interferometer.

A tapered thin-diameter photonic crystal fiber interferometer (TTD-PCFI) biosensor is proposed and demonstrated for DNA hybridization detection with an ultra-low limit of detection (LoD) and high specificity. The TTD-PCFI is fabricated by arc-discharged melting and adiabatic tapering, with a lower loss and stronger evanescent optical field. The fabricated TTD-PCFI with a diameter of 8.63 µm achieves a high refractive index sensitivity of 3842.5 nm/RIU. DNA hybridization detection of concentration ranging from 1 fM to 1 pM is achieved, with a sensitivity of 632 pm/lg(fM). The measured LoD is as low as 410 aM. Single-base mismatch detection of 25-base target single-stranded DNA is also achieved. The proposed label-free DNA biosensor has the advantages of the ultra-low LoD, high specificity, and real-time detection, which has great application prospects in the fields of disease diagnosis, microbial detection, and environmental science.

Photoelectrochemical biosensor based on FTO modified with BiVO4 film and gold nanoparticles for detection of miRNA-25 biomarker and single-base mismatch

MicroRNAs (miRNAs) are of great important as non-invasive biomarkers for early diagnosis of cancer. Based on this, a photoelectrochemical (PEC) biosensor was developed to measure the concentration of microRNA-25 (miR-25), as a lung cancer biomarker. To fabricate the PEC biosensor, a suitable amino modified probe was immobilized on the surface of a florin tin oxide (FTO) electrode modified with bismuth vanadate (BiVO4) film and gold nanoparticles (AuNPs). The fabrication steps of the PEC biosensor were monitored by electrochemical impedance spectroscopy (EIS) and PEC measurements. The results indicate that the combination of BiVO4 film and AuNPs enhances the separation of photogenerated electrons and increase the photocurrent signal. Upon hybridization of the probe immobilized on the biosensor with miR-25, the photocurrent is significantly decreased. This current decrease is attributed to the fact that the hybridization processes blocks the electrode surface by creating a steric hindrance and by decrease the diffusion of AA to the electrode surface, it reduces the AA oxidation and subsequently decreases the capture photogenerated holes. Therefore, the amount of photocurrent decrease is dependent on the miR-25 concentration, and this PEC biosensor can be used to measure miR-25. The developed PEC biosensor shows good sensitivity for the determination of miR-25 in the linear concentration range of 1.0 × 10−14 M to 1.0 × 10−6 M with a detection limit of 3.4 fM, under optimal conditions. Using this biosensor, miR-25 can be detected from a non-complementary and one base mismatch sequence. It is also possible to measure miR-25 in human serum samples with satisfactory results using the PEC biosensor.

Ionic Signal Enhancement by the Space Charge Effect through the DNA Rolling Circle Amplification on the Outer Surface of Nanochannels.

DNA species are recognized as a powerful probe for nanochannel analyses to address the issues of specific target recognition and highly efficient signal conversion due to their programmable and predictable Watson-Crick bases. However, in the conventional view, abundant sophisticated DNA structures synthesized by DNA amplification strategies are unsuitable for use in nanochannel analyses owing to their low probability to enter a nanochannel restricted by the smaller opening of the nanochannel, as well as the faint ion signal produced by the steric effect. Here, we present an integrated strategy of nanochannel analyses that combines the target recognitions by encoded rolling circle amplification (RCA) in solution and the ionic signal enhancement by the space charge effect through the immobilization of highly negative-charged RCA amplicons on the outer surface of the nanochannels. Owing to the highly negative-charged RCA amplicons with 100 nm sizes, a sharp increase of ionic current up to 7454% has been achieved. The RCA amplicon triggered by mRNA-21 on the outer surface of the poly(ethylene terephthalate) membrane with a single nanochannel realized the single-base mismatch detection of mRNA-21 with a sensitivity of 6 fM. The DNA amplicon endows the nanochannel with high sensitivity and selectivity that could extend to other applications, such as DNA sequencing, desalination, sieving, and water-energy nexus.

Electrochemistry-assisted surface plasmon resonance detection of miRNA-145 at femtomolar level

In this work, we combined electrochemical techniques with SPR (eSPR) for the label-free detection of cancer biomarker miRNA-145. Detection was performed in a simple two-step assay. In the first step, the gold sensor surface, previously functionalized with a self-assembled monolayer (SAM) of thiolated RNA probes is incubated with the sample containing the target RNA biomarker. In this step, hybridization of RNA fragments with complementary immobilized probes was monitored in real-time by SPR. In the second step, eSPR measurements were performed to improve the sensitivity of the hybridization assay. Potential-induced deposition of a redox probe at the sensor surface resulted in enhanced SPR response promoted by the electrochemical process, thereby allowing the detection of miRNA-145 at femtomolar level (LOD = 0.56 fM), without sample derivatization or post-hybridization treatment for signal amplification. Good linearity was achieved (R2 = 0.984) over the concentration range from 1.0 fM and 10 nM. Furthermore, the developed eSPR biosensor showed high selectivity towards single-base and two-base mismatch sequences and detection of target miRNA-145 in synthetic human serum was successful achieved.

Molecular AND logic gate for multiple single-nucleotide mutations detection based on CRISPR/Cas9n system-trigged signal amplification

Precise detection of single-nucleotide mutations (SNMs) is extremely important in various biomedical applications, but the simultaneous detection of multiple SNMs remains a great challenge. Herein, we developed a new method based on CRISPR/Cas9 system for multiple SNMs detection. The CRISPR/Cas9 system transduces the nucleic acid into an intermediate trigger to initiate the isothermal amplification reaction and further form fluorescence signals. According to this strategy, we established nucleic acid bio-computing operations-molecule logic gate in simultaneous distinction of the genetic locus. We demonstrate that the fluorescence signals generated from different input combinations can be used to discriminate the multiple genetic locus, and the molecular logic gate has great potential in single-base mismatch detection. In addition, the successful assay of real samples indicates that the novel strategy could further adapt for the pathogenic monitoring and biomedical research.

Ratiometric Multicolor Analysis of Intracellular MicroRNA Using a Chain Hybrid Substitution-Triggered Self-Assembly of Silver Nanocluster-Based Label-Free Sensing Platform.

A simple and label-free sensing platform with low background based on the chain-displacement triggered self-assembly of Ag NCs was developed for ratiometric visual analysis of intracellular miRNA-21. Based on this sensitively ratiometric sensing approach, a picomole limit detection for miRNA-21 can be obtained. Most importantly, compared with the traditional single base mismatch detection method, our proposed method can realize single base mismatch detection according to the remarkable fluorescence color conversion, rather than simple fluorescence intensity change, which can obviously improve the accuracy and reliability. In addition, successful multicolor real-time monitoring of intracellular miRNA-21 makes the probe a potential candidate for miRNA-21 inhibiting drug screening. Furthermore, MCF-7, HeLa, and normal L02 cells can also be visually differentiated according to the fluorescence color by using the label-free sensing platform, showing its potential prospect in target visual analysis.

Aligner mediated cleavage of nucleic acids for site-specific detection of single base mismatch

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5′-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.

Fluorescent DNA Biosensor for Single-Base Mismatch Detection Assisted by Cationic Comb-Type Copolymer.

Simple and rapid detection of DNA single base mismatch or point mutation is of great significance for the diagnosis, treatment, and detection of single nucleotide polymorphism (SNP) in genetic diseases. Homogeneous mutation assays with fast hybridization kinetics and amplified discrimination signals facilitate the automatic detection. Herein we report a quick and cost-effective assay for SNP analysis with a fluorescent single-labeled DNA probe. This convenient strategy is based on the efficient quenching effect and the preferential binding of graphene oxide (GO) to ssDNA over dsDNA. Further, a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran (PLL-g-Dex), significantly accelerates DNA hybridization and strand-exchange reaction, amplifying the effective distinction of the kinetic barrier between a perfect matched DNA and a mismatched DNA. Moreover, in vitro experiments indicate that RAW 264.7 cells cultured on PLL-g-Dex exhibits excellent survival and proliferation ability, which makes this mismatch detection strategy highly sensitive and practical.

Amplified detection of single base mismatches with the competing-strand assay reveals complex kinetic and thermodynamic behavior of strand displacement at the electrode surface

Detection of single-base mismatches with respect to a probe strand has been a predominant pursuit in electrochemical biosensor efforts, due to links found between single nucleotide polymorphisms (SNPs) and the predisposition to various diseases. We report an osmium tetroxide bipyridine-based, thermally-controlled, competitive-strand electrochemical assay to allow amplified detection of single-base mismatches. Optimally designed competitive-strand displacement and hybridization temperature allows us to distinguish the single-mismatched-target from the fully complementary sequence with unambiguous, highly reproducible, robust signal differences of over 90%. Furthermore, we find a complex interplay between the position of the redox label, variations in strand displacement kinetics due to mismatches incorporated into the competitive strand, and alterations in the melting temperature of DNA duplexes tethered on the gold surface, when probed by square-wave voltammetry. These insights will apply to any surface-tethered DNA-based electrochemical biosensor, and can help with understanding complex phenomena involved in these types of assays.

Label-free detection of DNA single-base mismatches using a simple reflectance-based optical technique.

Rapid and quantitative detection of the binding of nucleic acids to surface-immobilized probes remains a challenge in many biomedical applications. We investigated the hybridization of a set of fully complementary and defected 12-base long DNA oligomers by using the Reflective Phantom Interface (RPI), a recently developed multiplexed label-free detection technique. Based on the simple measurement of reflected light intensity, this technology enables to quantify the hybridization directly as it occurs on the surface with a sensitivity of 10 pg mm(-2). We found a strong effect of single-base mismatches and of their location on hybridization kinetics and equilibrium binding. In line with previous studies, we found that DNA-DNA binding is weaker on a surface than in the bulk. Our data indicate that this effect is a consequence of weak nonspecific binding of the probes to the surface.

Electrochemical Biosensor for Detection of Target DNA Sequence and Single-Base Mismatch Related to Helicobacter Pylori Using Chlorogenic Acid as Hybridization Indicator

An electrochemical DNA hybridization biosensor was developed using a novel electroactive indicator. The DNA hybridization was detected based on the interaction of chlorogenic acid, CGA, with an 18-mer alkanethiol DNA probe (from Helicobacter pylori) self-assembled gold electrode (S1/MCH/AuE) and its hybridization form (S2-S1/MCH/AuE). The conditions were optimized for the immobilization of probe DNA, the exposure of MCH, the accumulation of indicator and the hybridization process. The differential pulse voltammetric response of the accumulated CGA, as an electroactive indicator, on different electrode surfaces was used to assess the DNA hybridization. The results indicate that there is a significant difference between the voltammetric responses of CGA accumulated on the probes hybridized with non-complementary, mismatch and complementary targets. The potential of the developed biosensor is, thus, confirmed in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. In optimum conditions, CGA electrochemical signals had a linear dependence on the concentration of the complementary DNA within the range of 20.0 to 410.0 nM. Finally, a detection limit of 7.2 nM was obtained for the target DNA.

Scanning Electrochemical Microscopy for Electrochemical Detection of Single‐base Mismatches by Tagging Ferrocenecarboxylic Acid as a Redox Probe to DNA

AbstractScanning electrochemical microscopy (SECM) was employed for sensitive detection of single base mismatches (SBMs) in a sandwiched dsDNA. Ferrocenecarboxylic acid (Fc), covalently conjugated to the dsDNA, was oxidized to Fc+ via the DNA‐mediated charge transfer from the underlying gold substrate, and reduced back to Fc by SECM tip generated ferrocyanide. The electrocatalytic oxidation of SECM tip‐generated ferrocyanide was sensitive to presence, as well as the type of SBMs. Apparent standard rate constants (k0app) values for different SBMs, both near the electrode surface and far from it, were evaluated by SECM. The method can detect SBMs independent of their position in dsDNA.

A biosensor for the detection of single base mismatches in microRNA.

Graphene oxide quenches fluorescence corresponding to only a mismatched target due to selective denaturing of the thermo-unstable duplex composed of probe peptide nucleic acid and single base mismatched target RNA and thus, the fluorescence signal only from perfectly matched target RNA is measured.

Detection of positional mismatch in oligonucleotide by electrochemical method

Detection of single base mismatch in DNA has been achieved by various techniques. However, the detection of position of that mismatch in the DNA duplex sequence has not been adequately addressed. In the present work, sensing of positional change of DNA base mismatch is achieved, with enhanced detection limit, using methylene blue as a redox indicator in electrochemical differential pulse voltammetric method (DPV). Interfacially synthesized Gold Polyaniline nanocomposites (Au-PAni) is coated on Pt electrode and attached with thiol modified single stranded DNA to construct the sensor electrode. Position sensitive detection of a single base mismatch in oligomeric duplexes is achieved here, with a detection limit for concentration as low as 10−8M for mixed oligomeric sequences and 10−6M for dA-dT duplexes. In this case, the sensitivity of the electrode is 0.12μApM−1cm−2. Reusable sensor electrode coated with Au-PAni nanocomposites was employed without compromising the sensitivity of the system. This is the first step in developing a highly sensitive method for detecting the effect of varying position of single base mismatch in the DNA.

A microfluidic chip‐based fluorescent biosensor for the sensitive and specific detection of label‐free single‐base mismatch via magnetic beads‐based “sandwich” hybridization strategy

A novel microfluidic chip-based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads-based "sandwich" hybridization strategy, was developed for the sensitive and ultra-specific detection of single-base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single-base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer-related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10(-11) M, a RSD (n = 5) < 5% and a discrimination factor of 3.58-4.54 for one-base mismatch.

Ultralocalized thermal reactions in subnanoliter droplets-in-air

Miniaturized laboratory-on-chip systems promise rapid, sensitive, and multiplexed detection of biological samples for medical diagnostics, drug discovery, and high-throughput screening. Within miniaturized laboratory-on-chips, static and dynamic droplets of fluids in different immiscible media have been used as individual vessels to perform biochemical reactions and confine the products. Approaches to perform localized heating of these individual subnanoliter droplets can allow for new applications that require parallel, time-, and space-multiplex reactions on a single integrated circuit. Our method positions droplets on an array of individual silicon microwave heaters on chip to precisely control the temperature of droplets-in-air, allowing us to perform biochemical reactions, including DNA melting and detection of single base mismatches. We also demonstrate that ssDNA probe molecules can be placed on heaters in solution, dried, and then rehydrated by ssDNA target molecules in droplets for hybridization and detection. This platform enables many applications in droplets including hybridization of low copy number DNA molecules, lysing of single cells, interrogation of ligand-receptor interactions, and rapid temperature cycling for amplification of DNA molecules.

An approach toward SNP detection by modulating the fluorescence of DNA-templated silver nanoclusters

A rapid, homogeneous, and in-situ labelable format designed to detect single nucleotide polymorphism (SNP) is presented. Fluorescent silver nanoclusters produced following hydride-mediated reduction of Ag+ bound to a partial double-stranded oligodeoxynucleotide were employed as probes for SNPs. The sensing mechanism is based on the fluorescence enhancement of silver nanoclusters through an irreversible cluster transfer upon hybridization. The key element of our design is modulating the “turn on” mechanism by introducing a competitor that can be displaced in response to a target DNA. In a controlled model system, the fluorescence intensity of silver nanoclusters is approximately 3-fold enhanced upon hybridization with a perfectly matched target DNA, and single-base mismatch detection is achieved within 5min, regardless of the position of mismatches. Human aldehyde dehydrogenase 2 (ALDH2), which is responsible for the oxidation of aldehydes to carboxylic acids, is then utilized as a target for SNP. Upon addition of a perfectly matched sequence, dramatically enhanced fluorescence intensity is observed (ca. 48-fold), and prompt SNP genotyping is accomplished.

Preparation of a new electrochemical biosensor for single base mismatch detection in DNA

In this study, a sandwich-type electrochemical DNA hybridization biosensor for the detection of single base mismatches (SBMs) has been described. 2,5-Dihydroxybenzoic acid (DHBA) is employed as a new redox probe in this study. For construction of the biosensor, thiolated capture DNA is self-assembled on a gold electrode surface and hybridized with one region of a target DNA sequence. Subsequently, a signaling amino-labeled probe DNA is hybridized to a second region of the target DNA to complete the DNA double-helix self-assembled monolayer (SAM). Finally DHBA is covalently attached to the signaling probe DNA using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) cross-linking reaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) are employed to characterize the modification steps. The electrochemical signal of DHBA was followed for studying different types of mismatches (GA, GT, TT, CA), both near the electrode surface (N-type) and far from it (F-type). The presence of a SBM leads to a considerable decrease in the electrochemical signal of DHBA compared to that of complementary DNA (cDNA), which makes the detection of mismatches (including thermodynamically stable GA mismatches) possible. The redox signal of DHBA was compared with that of methylene blue (MB) as a well-known and standard redox reporter in DNA biosensors. The results showed that for an N-type SBM, which is located between the electrode and the site of intercalation, the redox signal of MB switches off, whereas for an F-type SBM, which is located above the site of intercalation, electrons flow from the Au electrode to the intercalated MB, and hence the biosensor could not distinguish the presence and absence of an SBM. The proposed biosensor is straightforward with no need for target labeling, and is sensitive enough to detect SBMs independent of their position in DNA double helix.

A novel micro-fluidic biosensor for the rapid and sequence-specific detection of DNA with electrophoretic driving mode and laser-induced fluorescence detector

A novel DNA biosensor, which combines the merits of micro-fluidic chips, the electrophoretic driving mode, paramagnetic beads amplification, and laser-induced fluorescence detection was developed for the rapid and sequence-specific detection of DNA in this study. The proposed DNA biosensor has much higher discrimination ability for the detection of single-base mismatch and much stronger resistibility to the complex matrixes of real samples in comparison with previous biosensors. These features, as well as its ease of fabrication (the fabrication of the sensor takes only 10 min except the fabrication of micro-fluidic chip), operation convenience, stability, better re-usability (micro-fluidic chip can be reused without any extra treatment) and short analysis time (one determination only takes 15 min), make it a promising alternative to rapid detection of DNA in clinical diagnosis. With the help of the biosensor, we successfully determined DNA, which related to oral cancer, in a saliva sample without any pre-separation or dilution with a detection limit of 4.2 × 10−11 M and a relative standard deviation (n = 5) <5 %. The success in the present biosensor served as a significant step toward the practical application of the biosensor in clinical diagnosis.

Nanoporous gold electrode as a platform for the construction of an electrochemical DNA hybridization biosensor

The application of a nanoporous gold electrode (NPGE) in the fabrication of an electrochemical sensing system for the detection of single base mismatches (SBMs) using ferrocene-modified DNA probe has been investigated in the present manuscript. Ferrocene carboxylic acid is covalently attached to the amino-modified probe using EDC/NHS chemistry. By covalent attachment of the redox reporter molecules on the top of DNA, the direct oxidation of the ferrocene on the electrode surface is avoided. On the other hand, the electrochemical signals are amplified by anodizing the electrode surface and converting it to nanoporous form. By improving the sensitivity of the biosensor, the different SBMs including the thermodynamically stable G–A and G–T mismatches, can be easily distinguished. In this research, NPGE was prepared by anodization and chemical reduction of Au surface and used for signal amplification. Nanoporous electrode enhances the sensitivity of DNA biosensor and makes it capable to detect complementary target DNA in sub-nanomole scales.