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Journal of Materials Chemistry. B May 2020Homogeneous electrochemical DNA biosensors' unique qualities have been of great interest to researchers, mainly due to their high recognition efficiency in solutions....
Homogeneous electrochemical DNA biosensors' unique qualities have been of great interest to researchers, mainly due to their high recognition efficiency in solutions. However, the processes of introducing additional markers and extra operations to obtain a signal are tedious and time consuming, which limits their overall potential applications. Herein, a novel tetraferrocene was synthesized and used as a homogeneous electrochemical DNA biosensor probe label. It contains four ferrocene units, which provide greater signaling potential compared to monoferrocene. Furthermore, the target DNA triggers the digestion of the double hairpin DNA probe with the aid of exonuclease III, promoting short single stranded DNA probe formation. With the combination of the incorporated tetraferrocene labeled short DNA probe strands and graphene's ability to adsorb single stranded DNA, the hybridization process can produce an electrode signal provided by tetraferrocene. A low detection limit of 8.2 fM toward target DNA with excellent selectivity was achieved. The proposed sensing system avoids tedious and time-consuming steps of DNA modification, making the experimental processes simpler and convenient. The advantages of high sensitivity, selectivity and simple operation make this strategy applicable to DNA detection.
Topics: Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Exodeoxyribonucleases; Ferrous Compounds; Humans; Metallocenes; Molecular Structure; Nucleic Acid Amplification Techniques; Particle Size; Surface Properties
PubMed: 32270173
DOI: 10.1039/d0tb00204f -
Analytical Chemistry Sep 2011MicroRNAs (miRNAs) are regulatory small RNAs that have important roles in numerous developmental, metabolic, and disease processes of plants and animals. The individual...
MicroRNAs (miRNAs) are regulatory small RNAs that have important roles in numerous developmental, metabolic, and disease processes of plants and animals. The individual levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis. Thus, innovative new tools for rapid, specific, and sensitive detection of miRNAs are an important field of research. Using the fluorescence properties of DNA-nanosilver clusters (DNA/AgNC), we have designed a DNA/AgNC probe that can detect the presence of target miRNA. Here, we show that the red fluorescence of the DNA/AgNC probe is diminished upon the presence of target miRNA without pre- or postmodification, addition of extra enhancer molecules, and labeling. The DNA/AgNC probe emission was lowest when the complementary miRNA target was present and was significantly higher for four other control miRNA sequences. Also, when adding whole plant endogenous RNA to the DNA/AgNC probe, the emission was significantly higher for the mutant where miRNA was deficient. On the basis of these findings, we suggest that these DNA/AgNC probes could be developed into a new, simple, inexpensive, and instant technique for miRNAs detection.
Topics: DNA; DNA Probes; Fluorometry; Metal Nanoparticles; MicroRNAs; Silver
PubMed: 21859161
DOI: 10.1021/ac201903n -
DNA probe for beta-hemolytic group B Streptococcus. Diagnostic accuracy in threatened preterm labor.The Journal of Reproductive Medicine Jul 1999To determine the diagnostic accuracy of a DNA probe for beta-hemolytic group B Streptococcus (GBS) in women with threatened preterm labor.
OBJECTIVE
To determine the diagnostic accuracy of a DNA probe for beta-hemolytic group B Streptococcus (GBS) in women with threatened preterm labor.
STUDY DESIGN
Two identical vaginal/perianal samples were collected from 75 pregnant women who were being evaluated for threatened preterm labor. One sample was managed in the traditional manner, with direct plating onto blood agar followed by plating after 8 and 24 hours of LIM broth enhancement. The "gold standard" was 24 hours of LIM broth enhancement followed by blood agar plating. The second sample was placed in LIM broth, and DNA probe testing was performed after incubation for 8 and 24 hours.
RESULTS
The prevalence of GBS colonization by the gold standard culture was 32%. After 8 hours of incubation in LIM broth, the DNA probe had poor sensitivity (79%); however, after 24 hours of incubation in LIM broth the DNA probe sensitivity rose to 96%. The DNA probe demonstrated only one false negative result after 24 hours of LIM broth enhancement. All DNA probe results were known 25 hours after sample collection.
CONCLUSION
This DNA probe gave results nearly identical to those of standard cultures and allowed a substantial saving of time.
Topics: Adult; DNA Probes; Female; Humans; Infant, Newborn; Infant, Premature, Diseases; Obstetric Labor, Premature; Predictive Value of Tests; Pregnancy; Prospective Studies; Sensitivity and Specificity; Streptococcal Infections; Streptococcus agalactiae; Vagina
PubMed: 10442319
DOI: No ID Found -
Current Protocols in Molecular Biology May 2001The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the "probe") can base-pair to a second DNA or RNA molecule...
The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the "probe") can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target") that has been immobilized on a nitrocellulose and nylon (uncharged and charged) membrane support. The approach taken in this unit is to present as the basic protocol an unsophisticated procedure for hybridization analysis with a radiolabeled DNA probe. The alternate protocol describes a similar method for probing DNA blots with a radiolabeled RNA probe. A support protocol for stripping blots for reprobing is also provided. The commentary describes modifications, including changes to prehybridization, hybridization, and wash solution formulations, and alterations to incubation times and conditions, the latter including a discussion of the wash conditions compatible with different degrees of stringency.
Topics: DNA; DNA Probes; Humans; Nucleic Acid Hybridization; RNA Probes
PubMed: 18265178
DOI: 10.1002/0471142727.mb0210s21 -
The Analyst Jun 2019A rapid and label-free fluorescence biosensing strategy for highly sensitive detection of microRNA-122 (miR-122) has been developed by the combination of DNA three-way...
A rapid and label-free fluorescence biosensing strategy for highly sensitive detection of microRNA-122 (miR-122) has been developed by the combination of DNA three-way junction (TWJ)-actuated strand displacement and a fluorescence light-up Ag nanocluster (AgNC) probe. In the presence of target miR-122, the attachment of miR-122 to its complementary DNA results in the unblocking of the toehold and branch migration domains in the TWJ, activating the strand displacement reaction (SDR) accompanied by the proximity between the G-rich DNA probe and DNA-AgNC probe; thus a remarkably enhanced fluorescence signal of AgNCs can be obtained owing to the G-rich fluorescence enhancement mechanism. The results reveal that this biosensor exhibits superb specificity and high sensitivity toward miR-122 with a detection limit of 0.030 nM. In addition, the practicality of the biosensor is demonstrated by analyzing miR-122 in three cell lines with satisfactory results. Furthermore, by the utilization of the toehold-mediated SDR and DNA-AgNC conjugates, this proposed strategy offers the advantages of rapidness, convenience, low cost, and simplified operation without the need for biological labeling and the addition of enzymes. Thus, the constructed biosensor might provide a valuable and practical tool for detecting miRNA and the related clinical diagnosis and fundamental biomedicine research.
Topics: Base Sequence; Biosensing Techniques; Cell Line, Tumor; DNA; DNA Probes; Fluorescence; Humans; Limit of Detection; Metal Nanoparticles; MicroRNAs; Nucleic Acid Hybridization; Silver; Spectrometry, Fluorescence
PubMed: 31095133
DOI: 10.1039/c9an00508k -
Chembiochem : a European Journal of... Jun 2009
Topics: Alleles; DNA Probes; Nucleic Acid Conformation; Nucleic Acid Hybridization; Polymorphism, Single Nucleotide; Temperature
PubMed: 19444831
DOI: 10.1002/cbic.200900264 -
Langmuir : the ACS Journal of Surfaces... Jan 2015Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically...
Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3' terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxythymidylate [(dT)n] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM-4 μM) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein-DNA binding mechanisms.
Topics: Biosensing Techniques; DNA; DNA Probes; DNA-Binding Proteins; Electrochemistry
PubMed: 25517392
DOI: 10.1021/la504083c -
ACS Applied Materials & Interfaces Oct 2023Signal amplification methods based on DNA molecular interactions are promising tools for detecting various biomarkers in low abundance. The entropy-driven circuit (EDC),...
Signal amplification methods based on DNA molecular interactions are promising tools for detecting various biomarkers in low abundance. The entropy-driven circuit (EDC), as an enzyme-free signal amplification method, has been used in detecting and imaging a variety of biomarkers. The localization strategy can effectively increase the local concentration of the DNA reaction modules to improve the signal amplification effect. However, the localization strategy may also amplify the leak reaction of the EDC, and effective signal amplification can be limited by the unclear structure-function relationship. Herein, we utilized locked nucleic acid (LNA) modification to enhance the stability of the localized entropy-driven circuit (LEDC), which suppressed a 94.6% leak signal. The coarse-grained model molecular simulation was used to guide the structure design of the LEDC, and the influence of critical factors such as the localized distance and spacer length was analyzed at the molecular level to obtain the best reaction performance. The sensitivities of miR-21 and miR-141 detected by a simulation-guided optimal LEDC probe were 17.45 and 65 pM, 1345 and 521 times higher than free-EDC, respectively. The LEDC was further employed for the fluorescence imaging of miRNA in cancer cells, showing excellent specificity and sensitivity. This work utilizes LNA and molecular simulations to comprehensively improve the performance of a localized DNA signal amplification circuit, providing an advanced DNA probe design strategy for biosensing and imaging as well as valuable information for the designers of DNA-based probes.
Topics: Entropy; DNA; DNA Probes; MicroRNAs; Biomarkers; Nucleic Acid Amplification Techniques; Biosensing Techniques
PubMed: 37773989
DOI: 10.1021/acsami.3c11189 -
Analytical Sciences : the International... 2015In vitro selection methods allow for isolation of DNAzymes (catalytic DNAs) from random DNA pools. Here we describe a fluorogenic DNAzyme, LYF5, isolated using a...
In vitro selection methods allow for isolation of DNAzymes (catalytic DNAs) from random DNA pools. Here we describe a fluorogenic DNAzyme, LYF5, isolated using a double-random selection approach: a random DNA pool was selected against a complex molecular mixture derived from a breast cancer cell line, T47D. LYF5 specifically indicates the T47D breast cancer cell line with high sensitivity. After sequence optimization, the second-generation DNAzyme, 2G-LYF5, exhibited an approximately 2-fold higher cleavage percentage. Finally, we have determined that the intramolecular stem-loop motif plays a crucial role in 2G-LYF5 activity. Our findings underscore the capability of single-stranded DNA molecules to perform highly sophisticated functions that are amenable to the development of diagnostic tests for early identification of breast cancer.
Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; DNA Probes; DNA, Catalytic; Humans; Inverted Repeat Sequences; Molecular Sequence Data
PubMed: 26256606
DOI: 10.2116/analsci.31.815 -
Biosensors & Bioelectronics Dec 2016In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene...
In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM.
Topics: Conductometry; DNA Probes; Equipment Design; Equipment Failure Analysis; Mercury; Molecular Probe Techniques; Nucleic Acid Amplification Techniques; Reproducibility of Results; Sensitivity and Specificity; Water Pollutants, Chemical
PubMed: 27497197
DOI: 10.1016/j.bios.2016.07.098