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Analytica Chimica Acta Oct 2023Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped...
Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.
Topics: DNA Probes; Fluorescence; Fluorescent Dyes; G-Quadruplexes; Ionophores; RNA, Messenger
PubMed: 37604619
DOI: 10.1016/j.aca.2023.341633 -
The Analyst Oct 2020We have developed a bifunctional probe based on triplex molecular beacons for the measurement of environmental pH and quantification of microRNA-10b using a nanopore....
We have developed a bifunctional probe based on triplex molecular beacons for the measurement of environmental pH and quantification of microRNA-10b using a nanopore. The probe responds sharply to solution pH changes in the range of 6.0-7.5. The limit of detection for microRNA-10b is as low as 5.0 pM.
Topics: Biosensing Techniques; DNA Probes; Hydrogen-Ion Concentration; MicroRNAs; Nanopores
PubMed: 33103667
DOI: 10.1039/d0an01208d -
Analytical Chemistry Oct 2019The introduction of nanotechnology can overcome some inherent drawbacks of traditional DNA probes, thus promoting their applications in living cells. Herein, a...
The introduction of nanotechnology can overcome some inherent drawbacks of traditional DNA probes, thus promoting their applications in living cells. Herein, a three-dimensional DNA nanostructure, a DNA nanolantern, was prepared via simple nucleotide hybridization of four short-stranded oligonucleotides and successfully applied to the construction of a novel DNA probe and signal amplifier. Compared to most reported DNA nanostructures, a DNA nanolantern shows the distinct advantages of low cost, easy design and preparation, more and arbitrary adjusted probe numbers, and high fluorescence resonance energy transfer (FRET) signal readout. Compared to traditional DNA probes, the constructed nanolantern-based one has improved cell internalization efficiency, enhanced biostability, accelerated reaction kinetics, excellent biocompatibility, and greatly reduced false-positive output and was demonstrated to work well for probing the expression level of tumor-related mRNA and microRNA in living cells. The DNA nanolantern can also be easily integrated with some reported signal amplification strategies, e.g., isothermal hybridization chain reaction (HCR), and the obtained signal amplifier combines the advantages of the DNA nanolantern and the HCR, enabling sensitive imaging detection of ultralow abundance targets in living cells. This work demonstrated that this simple DNA nanostructure can not only improve the performance of traditional DNA probes but can also be easily integrated with reported DNA-based strategy and technology, thus showing a broad application prospect.
Topics: Biomarkers, Tumor; Cell Line, Tumor; DNA; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Limit of Detection; MicroRNAs; Nanostructures; Nucleic Acid Hybridization; RNA, Messenger; Thymidine Kinase
PubMed: 31512479
DOI: 10.1021/acs.analchem.9b03453 -
Mikrochimica Acta Aug 2018A star-shaped fluorescent DNA probe (S-probe) is described that can recognize target DNA and discriminate it from interfering DNA via strand displacement including...
A star-shaped fluorescent DNA probe (S-probe) is described that can recognize target DNA and discriminate it from interfering DNA via strand displacement including branch migration and toehold exchange. The artificially designed S-probe does not harm the strand displacement while it allows the fluorescently labelled strand and the quencher-labelled strand to be shared among different S-probes targeting different genetic variations. Generally, multiplexed detection of different MT/WT pairs requires different fluorophore-labelled and quencher-labelled strands. The two labelled oligonucleotides of S-probe have sequences decoupled from the target/interfering DNA sequence, so the same fluorescent and quencher strands can be used for different S-probes that target different sequences. The sensitivity, specificity, and general applicability of the method toward BRCA 41293497 mutation, KRAS G13D mutation and two types of EGFR mutations (T790 M and L858R) were experimentally demonstrated. The limit of quantification of the MT concentration is 2 nM, and the detection limit of the low abundance of the target sequence is 5% (40 nM of MT strand in the background of 760 nM of WT strand). The fluorometric assay with excitation/emission wavelengths of 485/582 nm was successfully applied to clinical samples spiked with mutant-type and wild-type DNA. The unique structure of the S-probe provides a useful tool for the regulation of the strand displacement reaction. Conceivably, the star-shaped DNA probe can be widely adopted to multiplexed detection of genetic variations and provide novel insights into the regulation of strand displacement processes as utilized in DNA based nanomachines. Graphical abstract A star-shaped fluorescent DNA probe (S-probe) with a detection limit of 2 nM was adopted to multiplexed detection of genetic variations via strand displacement including branch migration and toehold exchange.
Topics: Blood Specimen Collection; DNA; DNA Probes; Female; Fluorescent Dyes; Fluorometry; Genetic Variation; Humans; Limit of Detection; Mutation; Ovarian Neoplasms
PubMed: 30105500
DOI: 10.1007/s00604-018-2941-0 -
Analytical Chemistry Dec 2019Multiblock DNA probe attracted a large amount of scientific attention, for the development of multitarget biosensor and improved specificity/sensitivity. However, the...
Multiblock DNA probe attracted a large amount of scientific attention, for the development of multitarget biosensor and improved specificity/sensitivity. However, the development of multiblock DNA probes highly relied on the chemical synthesis of organic linkers or nanomaterials, which limited their practicability and biological compatibility. In this work, we developed a label-free assembling strategy using a triblock DNA capture probe, which connects two DNA probes with its intrinsic polyA fragment (probe-PolyA-probe, PAP). The middle polyA segment has a high affinity to the gold electrode surface, leading to excellent reproducibility, stability, and regeneration of our biosensor. Two flanking capture probes were tandemly co-assembled on the electrode surface with consistent spatial relationship and exactly the same amount. When combined with the target DNA, the hybridization stability was improved, because of the strong base stacking effect of two capture probes. The sensitivity of our biosensor was proved to be 10 fM, with a wide analysis range between 10 fM to 1 nM. Our PAP-based biosensor showed excellent specificity when facing mismatched DNA sequences. Even single nucleotide polymorphisms can be distinguished by each probe. The excellent practicability of our biosensor was demonstrated by analyzing genomic DNA both with and without PCR amplification.
Topics: Base Pair Mismatch; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Escherichia coli; Gold; Limit of Detection; Nucleic Acid Hybridization; Poly A; Polymorphism, Single Nucleotide; Reproducibility of Results
PubMed: 31746200
DOI: 10.1021/acs.analchem.9b04757 -
Talanta Apr 2024Alkaline phosphatase (ALP) is a zinc-containing metalloprotein that shows very great significance in clinical diagnosis, which can catalyze the hydrolysis of... (Review)
Review
Alkaline phosphatase (ALP) is a zinc-containing metalloprotein that shows very great significance in clinical diagnosis, which can catalyze the hydrolysis of phosphorylated species. ALP has the potential to serve as a valuable biomarker for detecting liver dysfunction and bone diseases. On the other hand, ALP is an efficient biocatalyst to amplify detection signals in the enzyme-linked assay. It has always been a major research focus to develop novel biosensors that can detect ALP activity with high selectivity and sensitivity. There have been numerous reports on the development of biosensors to determine ALP activity using a phosphorylated DNA probe. Among them, various beneficial strategies, such as λ exonuclease-mediated cleavage reaction, terminal deoxynucleotidyl transferase-triggered DNA polymerization, and Klenow fragment polymerase-catalyzed elongation, are employed to generate amplified and more intuitive signal. This review discusses and summarizes the development and advances of biosensors for ALP activity detection that use a well-designed phosphorylated DNA probe, aiming to provide some guidelines for the design of more sophisticated sensing strategies that exhibit improved sensitivity, selectivity, and adaptability in detecting ALP activity.
Topics: Alkaline Phosphatase; DNA Probes; Hydrolysis; Biosensing Techniques; DNA; Limit of Detection
PubMed: 38215586
DOI: 10.1016/j.talanta.2024.125622 -
The Analyst Apr 2016A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA...
A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA tetrahedron and a hairpin DNA on one of the vertices of the DNA tetrahedron. It is composed of four modified DNA segments: S1-Au nanoparticle (NP) inserting a telomerase strand primer (TSP) and S2-S4, three Cy5 dye modified DNA segments. Fluorescence of Cy5 at three vertices of the DNA tetrahedron is quenched by the Au NP at the other vertex due to the effective fluorescence resonance energy transfer (FRET) ("off" state). When the probe meets telomerase, the hairpin structure changes to rod-like through complementary hybridization with the telomerase-triggered stem elongation product, resulting in a large distance between the Au NP and Cy5 and the recovery of Cy5 fluorescence ("on" state). The molar ratio of 3 : 1 between the reporter (Cy5) and the target related TSP makes the probe show high sensitivity and recovery efficiency of Cy5 in the presence of telomerase extracted from HeLa cells. Given the functional and compact nanostructure, the mechanically stable and noncytotoxic nature of the DNA tetrahedron, this FRET-based probe provides more opportunities for biosensing, molecular imaging and drug delivery.
Topics: Carbocyanines; Cell Line, Tumor; DNA Probes; Fluorescence Resonance Energy Transfer; Humans; Intracellular Space; Inverted Repeat Sequences; Optical Imaging; Telomerase
PubMed: 26979920
DOI: 10.1039/c6an00241b -
Biosensors & Bioelectronics Feb 2018A novel competitive aptasensor for thrombin detection is developed by using a tetrahedral DNA (T-DNA) probe and hybridization chain reaction (HCR) signal amplification....
A novel competitive aptasensor for thrombin detection is developed by using a tetrahedral DNA (T-DNA) probe and hybridization chain reaction (HCR) signal amplification. Sulfur and nitrogen co-doped reduced graphene oxide (SN-rGO) is firstly prepared by a simple reflux method and used for supporting substrate of biosensor. Then, T-DNA probe is modified on the electrode by Au-S bond and a competition is happened between target thrombin and the complementary DNA (cDNA) of aptamer. The aptamer binding to thrombin forms an aptamer-target conjugate and make the cDNA remained, and subsequently hybridizes with the vertical domain of T-DNA. Finally, the cDNAs trigger HCR, which results in a great current response by the catalysis of horseradish peroxidase to the hydrogen peroxide + hydroquinone system. For thrombin detection, the proposed biosensor shows a wide linearity range of 10-10M and a low detection limit of 11.6fM (S/N = 3), which is hopeful to apply in biotechnology and clinical diagnosis.
Topics: Aptamers, Nucleotide; Biosensing Techniques; DNA Probes; DNA, Bacterial; Graphite; Humans; Limit of Detection; Nanostructures; Nucleic Acid Hybridization; Oxidation-Reduction; Oxides; Thrombin
PubMed: 28942209
DOI: 10.1016/j.bios.2017.09.022 -
Analytical Chemistry Jan 2020Determination of the methylation levels of genes of interest is fundamental for biological and medical research that involves DNA methylation. Using the average...
Determination of the methylation levels of genes of interest is fundamental for biological and medical research that involves DNA methylation. Using the average methylation levels of multiple CpG sites to represent the methylation levels of the whole gene is much more accurate than using that of only one CpG site. However, current methods that can provide the average methylation levels of several CpG sites are all expensive, time-consuming (several weeks), and labor-intensive. Herein, guided by the unique thermodynamics of the DNA strand-displacement process, we constructed a DNA fluorescent probe for determination of the average methylation levels of multiple CpG sites. Theoretical calculations of the reaction process and proof-of-concepts experiments on two to three CpG sites of synthesized DNA validated the basic principles of our probe. Taking two CpG sites in the promotor regions of () gene and () gene as the targets, we successfully measured their average methylation levels in nine endometrial cancer patients and two healthy persons. We believe our probe will be a very useful tool in the field, and we anticipate it being widely adopted by biological and medical investigators.
Topics: CpG Islands; DNA; DNA Methylation; DNA Probes; Endometrial Neoplasms; Female; Fluorescent Dyes; Humans; Promoter Regions, Genetic; Receptors, Calcitriol; Spectrometry, Fluorescence; Steroidogenic Factor 1; Thermodynamics
PubMed: 31763817
DOI: 10.1021/acs.analchem.9b03198 -
ACS Applied Materials & Interfaces Nov 2017A "sandwich-like" biosensor was developed on the basis of the magnetic bead platform for sensitive detection of breast cancer 1 (BRCA1) DNA. In the present study, a...
A "sandwich-like" biosensor was developed on the basis of the magnetic bead platform for sensitive detection of breast cancer 1 (BRCA1) DNA. In the present study, a tetrahedron-structured reporter probe (TSRP) was designed, in which 3 vertices of the tetrahedron were labeled with digoxin (Dig), and the other one was labeled with a detection probe. TSRP here provided accurate enzyme loading and well-organized spatial arrangement for optimized signal amplification. The detection limit of this biosensor was as low as 10 fM, which is at least 4 orders of magnitude lower than that of the single DNA probe (100 pM), and the signal gain was 2 times higher than the analysis using three one-dimensional (1D) reporter probes. We could distinguish DNA sequences with only 1 base mismatch, and the performance of our TSRP biosensor was proven to be equally good in both PCR products and real fetal calf serum (FCS) sample as in buffer. We believe this work provided a novel avenue for the development of signal amplification strategies.
Topics: Biosensing Techniques; Colorimetry; DNA Probes; Nanostructures; Nucleic Acid Hybridization; Polymerase Chain Reaction
PubMed: 29022698
DOI: 10.1021/acsami.7b11994