Single-base Mismatch Sequences Research Articles

Measurement of microRNA-106b as a gastric cancer biomarker based on Zn-BTC MOF label-free genosensor

Due to the late detection of stomach cancer, this cancer usually causes high mortality. The development of an electrochemical genosensor to measure microRNA 106b (miR-106b), as a gastric cancer biomarker, is the aim of this effort. In this regard, first, 1,3,5-benzenetricarboxylate (BTC) metal-organic frameworks (Zn-BTC MOF) were self-assembled on the glassy carbon electrode and then the probe (ssDNA) was immobilized on it. The morphology Zn-BTC MOF was characterized by SEM, FT-IR, Raman and X-Ray techniques. Zn-BTC MOF as a biosensor substrate has strong interaction with ssDNA. Quantitative measurement of miR-106b was performed by electrochemical impedance spectroscopy (EIS). To perform this measurement, the difference of the charge transfer resistances (ΔRct) of Nyquist plots of the ssDNA probe modified electrode before and after hybridization with miR-106b was obtained and used as an analytical signal. Using the suggested genosensor, it is possible to measure miR-106b in the concentration range of 1.0 fM to 1.0 μM with a detection limit of 0.65 fM under optimal conditions. Moreover, at the genosensor surface, miR-106b can be detected from a non-complementary and a single base mismatch sequence. Also, the genosensor was used to assess miR-106b in a human serum sample and obtained satisfactory results.

A sensitive electrochemical genosensor for measuring gastric cancer biomarker, microRNA-106b, based on asparagine polymerization on carbon quantum dots modified electrode

The aim of this work is to develop an electrochemical genosensor to measure microRNA 106b (miR-106b) as a gastric cancer biomarker. To construct the miR-106b genosensor, asparagine (As) was electropolymerized on the surface of carbon quantum dots modified glassy carbon electrode (CQDs/GCE). In the next step, ssDNA was immobilized on the modified electrode surface (As/CQDs/GCE) and then hybridized with miR-106b. Finally, differential pulse voltammograms of the accumulated chlorogenic acid (CGA), as an electroactive label, on the surface of immobilized ssDNA was recorded before (ssDNA/As/CQDs/GCE) and after hybridization with miR-106b (miR-106b/BSA/ssDNA/As/CQDs/GCE). The difference in the peak current of these two voltammograms was used as an analytical signal to measure of miR-106b. Using this genosensor, a linear concentration range from 1.0 fM to 1.0 µM and a detection limit of 0.39 fM were obtained for the measurement of miR-106b. The obtained data indicate that it is possible to recognize miR-106 from a non-complementary and a single-base mismatch sequence using the genosensor. In addition, the measurement of miR-106b in a human serum sample was successfully performed using the genosensor.

An enzyme-free turn-on fluorescent strategy for nucleic acid detection based on hybridization chain reaction and transferable silver nanoclusters.

A fluorescence biosensor has been developed based on hybridisation chain reaction (HCR) amplification coupled with silver nanoclusters (AgNCs) for nucleic acid detection. The fluorescence was activated via end-to-end transfer of dark AgNCs caged within a DNA template to another DNA sequence that could enhance their red fluorescence emission at 611nm. Such cluster-transfer approach allows us to introduce fluorogenic AgNCs as external signal transducers, thereby enabling HCR to perform in a predictable manner. The resulted HCR-AgNC biosensor was able to detect target DNA with a detection limit of 3.35 fM, and distinguish the DNA target from single-base mismatch sequences. Moreover, the bright red fluorescence emission was detectable with the naked eye, with concentration of target DNA down to 1pM. The biosensor also performed well in human serum samples with good recovery. Overall, our cluster-transfer approach provides a good alternative to construct HCR-AgNC assay with less risk of circuit leakage and produce AgNCs in a controllable manner.

Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection.

Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.

Single-Step FRET-Based Detection of Femtomoles DNA.

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

A Ratiometric Fluorescent Bioprobe Based on Carbon Dots and Acridone Derivate for Signal Amplification Detection Exosomal microRNA.

Recently, sensitive and selective detection of exosomal microRNAs (miRNAs) has been garnering significant attention, because it is related to many complex diseases, including cancer. Herein, we report a ratiometric fluorescent bioprobe based on DNA-labeled carbon dots (DNA-CDs) and 5,7-dinitro-2-sulfo-acridone (DSA) coupling with the target-catalyzing signal amplification for the detection of exosomal miRNA-21. There was high fluorescence resonance energy transfer (FRET) efficiency between carbon dots (CDs) and DSA when the bioprobe was assembled. However, in the presence of the target, with disassembling of the fluorescent bioprobe, the fluorescence intensities of CDs and DSA were changed simultaneously. Because of the ratio of dual fluorescence intensities, this ratiometric fluorescent bioprobe was able to cancel out environmental fluctuations by calculating emission intensity ratio at two different wavelengths, being robust and stable enough for detection of exosomal miRNA-21. In addition, we displayed that a single miRNA-21 can catalyze the disassembly of multiple CDs with DSA theoretically, yielding significant change in the fluorescence ratio for the detection of miRNA-21. With this signal amplification strategy, the limit of detection was as low as 3.0 fM. Furthermore, because of the introduction of lock nucleic acid to mediate the strand displacement reaction, the selectivity of this strategy was improved remarkably, even against single base mismatch sequence. More importantly, our strategy could monitor the dynamic change of exosomal miRNA-21, which maybe becomes a potential tool to distinguish cancer exosomes and nontumorigenic exosomes. In a short, this ratiometric fluorescence bioprobe possessed high stability, sensitivity and selectivity coupling with ease of operation and cost efficiency, leading to great potential for wide application.

Design and Characterization of a Singly Labeled Fluorescent Smart Probe for In Vitro Detection of miR-21.

A sensitive hairpin smart probe (SP) has been developed and tested for its sequence-specificity and sensitivity for detecting microRNAs (miRNAs). The loop sequence of this SP is perfectly complementary to microRNA-21 (miR-21) sequence. This miRNA regulates certain biological processes and has been implicated in certain forms of cancer. The stem of the new SP consists of a fluorophore on one end and multiple guanine bases on the opposing end are used as quenchers. The fluorescence of the SP is significantly quenched by the guanine bases at room temperature and in the absence of the miR-21 target. The presence of miR-21 switches on the fluorescence due to spontaneous hybridization of the SP with this target, which also forces the stem hybrid of the SP apart. This new SP successfully discriminated between the perfect miR-21 target and two closely similar single-base mismatch sequences. When the SP was incubated with the miR-21 at 37 ℃, the hybridization kinetics increased seven times, compared to room temperature hybridization. Overall, this new SP shows good detection sensitivity and gives a limit of detection and limit of quantitation of 14.0 nM and 46.7 nM, respectively. This detection platform represents a simple, fast, mix-and-read homogeneous assay for sequence-specific detection of miR-21, and it can be adapted for other related diagnostic applications.

Surface modifications to boost sensitivities of electrochemical biosensors using gold nanoparticles/silicon nanowires and response surface methodology approach

This work describes fabrication of a DNA electrochemical sensor utilized of gold nanoparticles/silicon nanowires/indium tin oxide (AuNPs/SiNWs/ITO) as a modified substrate for detection of dengue virus DNA oligomers using methylene blue (MB) as a redox indicator. The response surface methodology (RSM) was applied as one of the advanced optimization methods for fabrication of SiNWs/AuNPs/ITO electrode and immobilization of DNA probes to enhance the sensitivity of DNA detection. Several factors were successfully optimized using RSM, including volume of SiNWs, concentration of dithiopropionic acid (DTPA), volume of AuNPs, DNA probe concentration, and DNA probe immobilization time. RSM approach shows that AuNPs and DNA probe concentration were the prominent factors affecting on the MB current signal and immobilization of DNA probe on AuNPs/SiNWs surface. This new developed sensor was able to discriminate complementary target sequences, noncomplementary and single-base mismatch sequences, for DNA dengue virus detection.

A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM) detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

Ultrasensitive electrochemical detection of microRNA with star trigon structure and endonuclease mediated signal amplification

MicroRNAs play important roles in gene regulation. They can be used as effective biomarkers for diagnosis and prognosis of diseases like cancers. Due to their intrinsic properties of short length, low abundance and sequence homology among family members, it is difficult to realize sensitive and selective detection with economical use of time and cost. Herein, we report an ultrasensitive electrochemical method for microRNA analysis employing two oligonucleotides and one endonuclease. Generally, a glassy carbon electrode is first covered with gold nanoparticles (AuNPs) mediated by poly(diallyldimethylammonium chloride) (PDDA). Then, thiolated capture probe (CP) with methylene blue (MB) labeled at 5′ end is modified on the pretreated electrode. Hybridization occurs among target microRNA, CP and auxiliary probe (AP), forming a star trigon structure on the electrode surface. Subsequently, endonuclease recognizes and cleaves CP on CP/AP duplex, releasing microRNA and AP back to the solution. The two regenerated elements can then form another star trigon with other CP molecules, initiating cycles of CP cleavage and MB departure. Significant decrease of electrochemical signals is thus observed, which can be used to reflect the concentration of microRNA. This proposed method has a linear response to microRNA in a wide range from 100aM to 1nM and the sensitivity of attomolar level can be achieved. Moreover, it has high selectivity against single-base mismatch sequences and can be used directly in serum samples. Therefore, this method shows great feasibility for the detection of microRNA and may have potential applications in cancer diagnosis and prognosis.

Label-free and non-enzymatic detection of DNA based on hybridization chain reaction amplification and dsDNA-templated copper nanoparticles

A label-free and non-enzymatic amplification fluorescent method for detection of DNA has been developed by using hybridization chain reaction (HCR) and dsDNA-templated copper nanoparticles (CuNPs). First, the biotinylated capture DNA probes were immobilized on the streptavidin-modified beads through the interaction of biotin and streptavidin. Then, target DNA hybridized with the capture DNA probes, which formed a hybridized DNA with sticky end. The sticky end triggered the HCR process and formation of dsDNA polymers while two hairpin probes coexisted. Subsequently, the dsDNA polymers were employed as template for synthesis of CuNPs with excellent fluorescent properties, which provided a label-free, non-enzymatic signal response. Meanwhile, the fluorescence sensing depended on the target DNA triggered HCR, which render this method a high selectivity against single-base mismatch sequences. The concept and methodology developed in this work show great promise in the quantitative detection of DNA in biological and medical applications.

Label-free fluorescent biosensor based on the target recycling and Thioflavin T-induced quadruplex formation for short DNA species of c-erbB-2 detection

Non-invasive early diagnosis of breast cancer is the most effective way to improve the survival rate and increase more chances of breast-conserving. In this paper, we developed a label-free fluorescent biosensor based on nuclease assisted target recycling and Thioflavin T-induced quadruplex formation for short DNA species of c-erbB-2 detection in saliva. By employing the strategy, the sensor can detect as low as 20fM target DNA with high discrimination ability even against single-base mismatch sequence. To the best of our knowledge, the proposed sensor is the first attempt to apply Thioflavin T that possesses outstanding structural selectivity for G-quadruplex in DNA amplification techniques, which may represent a promising path toward direct breast cancer detection in saliva at the point of care.

A new hybrid signal amplification strategy for ultrasensitive electrochemical detection of DNA based on enzyme-assisted target recycling and DNA supersandwich assemblies

A highly sensitive electrochemical sequence-specific DNA detection strategy is demonstrated by coupling N.BstNB I (a nicking endonuclease)-assisted target recycling amplification with DNA supersandwich assembly signal enhancement. The proposed method avoids any extra chemical labeling steps and offers high selectivity against single-base mismatch sequences and a low detection limit down to 0.36 fM.

In Situ Hybridization Chain Reaction Amplification for Universal and Highly Sensitive Electrochemiluminescent Detection of DNA

In this work, we describe a new universal and highly sensitive strategy for electrochemiluminescent (ECL) detection of sequence specific DNA at the femtomolar level via in situ hybridization chain reaction (HCR) signal amplification. The DNA capture probes are self-assembled on a gold electrode. The presence of the target DNA and two hairpin helper DNAs leads to the formation of extended dsDNA polymers through HCR on the electrode surface. The in situ, HCR-generated dsDNA polymers cause the intercalation of numerous ECL indicators (Ru(phen)(3)(2+)) into the dsDNA grooves, resulting in significantly amplified ECL signal output. The proposed strategy combines the amplification power of the DNA HCR and the inherent high sensitivity of the ECL technique and enables low femtomolar detection of sequence specific DNA. The developed strategy also shows high selectivity against single-base mismatch sequences, which makes our new universal and highly sensitive HCR-based method a useful addition to the amplified DNA detection arena.

Dual amplified, sensitive electrochemical detection of pathogenic sequences based on biobarcode labels and functional graphene modified electrode

A dual signal amplified strategy for sensitive EC detection of pathogenic DNA sequences based on poly[G]20-conjugated biobarcode nanoparticles and carboxyl functionalized graphene is described. The capture probes are covalently linked to the graphene modified electrode. The addition of the target sequences and the biobarcode amplification labels leads to the formation of sandwich structures on the electrode surface. The presence of Ru(bpy)32+ in the detection buffer catalyzes the electro-oxidation of the numerous guanine bases captured on the electrode surface and generates significantly amplified current response for quantitation. Moreover, graphene can offer excellent heterogeneous electron transfer during electro-catalytic oxidation of guanine bases. This dual signal amplification strategy thus results in the determination of pathogenic DNA sequences down to the low picomolar level (1pM). Our approach also exhibits excellent discrimination between the target and single-base mismatch sequences, which provides great potential for highly sensitive and selective detection of different nucleic acid sequences in general.

Molecular beacon-based fluorescence biosensor for the detection of gene fragment and PCR amplification products related to chronic myelogenous leukemia

A novel fluorescence method has been established for the determination of gene fragment and PCR amplification products related to chronic myelogenous leukemia (CML). A molecular beacon (MB) which comprises a DNA loop section, a pair of fluorophore (tetramethoxyl rhodamine, TAMRA), and a quencher (4-(2-methyl-on-amino-azobenzene) benzoate, DABCYL) was designed. The loop sequence of MB was designed according to the DNA sequence relating to CML (type b3a2) which contained a single-stranded oligonucleotide. Before hybridization, the fluorescence from the TAMRA had been quenched by the DABCYL. After hybridization with the complementary DNA, the quencher will become far away from the TAMRA, and the fluorescence intensity detected will increase. Changes in the fluorescence intensity have a linear relationship with the concentration of complementary DNA (C) in the range of 4.0 × 10(-9)-3.2 × 10(-8) mol/L, with a correlation coefficient of 0.9973; the detection limit was 6.0 × 10(-10) mol/L (S/N = 3). The developed method has high selectivity, which can be used to discriminate single-base mismatch sequence. The method has been applied to detect the short-stranded CML DNA fragment (278 bp) with high sensitivity. This approach is a promising method for the detection of CML in real samples for medical diagnostics.

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.

Electrochemical DNA sensor by the assembly of graphene and DNA-conjugated gold nanoparticles with silver enhancement strategy

Sensitive and selective detection of DNA is in urgent need due to its important role in human bodies. Many disorders, such as Alzheimer's disease and various cancers, are closely related with DNA damage. In this work, a novel electrochemical DNA biosensor was constructed on a DNA-assembling graphene platform which provided a robust, simple and biocompatible platform with large surface area for DNA immobilization. The as-designed DNA sensor was fabricated by directly assembling captured ssDNA on a graphene-modified electrode through the π-π stacking interaction between graphene and ssDNA bases. Then, the target DNA sequence and oligonucleotide probes-labeled AuNPs were able to hybridize in a sandwich assay format, following the AuNPs-catalyzed silver deposition. The deposited silver was further detected by differential pulse voltammetry. Owing to the high DNA loading ability of graphene and the distinct signal amplification by AuNPs-catalyzed silver staining, the resulting biosensor exhibited a good analytical performance with a wide detection linear range from 200 pM to 500 nM, and a low detection limit of 72 pM. Additionally, the biosensor was proved to be able to discriminate the complementary sequence from the single-base mismatch sequence. The simple biosensor is promising in developing electronic, on-chip assays in clinical diagnosis, environmental control, and drug discovery.

Voltammetric Detection Of Microcystis Genus Specific-sequence With Disposable Screenprinted Electrode Modified With Gold Nanoparticles

A disposable electrochemical biosensor for detection of DNA sequence related to Microcystis was described. After depositing gold nanoparticles (AuNPs) onto a screen-printed carbon electrode (SPCE) surface by electrodeposition, the DNA probe which is complementary to a specific gene sequence related to Microcystis was immobilized onto the AuNPs modified SPCE via a thiol linker at the 5’ end. On the electrode surface the immobilized single-stranded DNA could undergo hybridization in the solution containing DNA specific-sequence of Microcystis. Using methylene blue as the indicator, DNA immobilization and hybridization were characterized by cyclic voltammetry and differential pulse voltammetry. Parameters, such as the deposition conditions of gold nanopariticle and the preconcentration time of MB, were optimized. In addition, the control experiments with non-complementary and single base mismatch sequences were investigated and results demonstrated the high selectivity of the biosensor. Copyright © 2010 VBRI press.

Chemically induced hairpin formation in DNA monolayers.

A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.