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Biosensors Jun 2021Owing to the lack of specific diagnostic methods, Scrub typhus can sometimes be difficult to diagnose in the Asia-Pacific region. Therefore, an efficient and rapid...
Owing to the lack of specific diagnostic methods, Scrub typhus can sometimes be difficult to diagnose in the Asia-Pacific region. Therefore, an efficient and rapid detection method urgently needs to be developed. Based on competitive single-stranded DNA over modified glassy carbon electrode (GCE), an electrochemical biosensor was established to detect the disease. The nano-flower NiFe layered double hydroxide (NiFe-LDH) modified GCE has a large specific surface area, which supported a large amount of gold nanoparticles, so that a wide linear detection range from 25 fM to 0.5 μM was obtained. The beacon DNA (B-DNA) with the same sequence as the Scrub typhus DNA (T-DNA), but labeled with methylene blue, was used to construct a competitive relationship. When T-DNA and B-DNA were present on the sensor simultaneously, they would hybridize with probe DNA in a strong competition, and the corresponding electrochemical response signal would be generated via testing. It contributed to reducing tedious experimental procedures and excessive response time, and achieved fast electrochemical detection of DNA. The strategy provides a worthy avenue and possesses promising applications in disease diagnosis.
Topics: Biosensing Techniques; Carbon; DNA; DNA Probes; DNA, Bacterial; Electrochemical Techniques; Electrochemistry; Electrodes; Gold; Metal Nanoparticles; Nucleic Acid Hybridization; Scrub Typhus
PubMed: 34202437
DOI: 10.3390/bios11070207 -
Sensors (Basel, Switzerland) Aug 2023A label-free-based fiber optic biosensor based on etched tilted Bragg fiber grating (TFBG) is proposed and practically demonstrated. Conventional phase mask technic has...
A label-free-based fiber optic biosensor based on etched tilted Bragg fiber grating (TFBG) is proposed and practically demonstrated. Conventional phase mask technic has been utilized to inscribe tilted fiber Bragg grating with a tilt angle of 10°, while the etching has been accomplished with hydrofluoric acid. A composite of polyethylenimine (PEI)/poly(acrylic acid) (PAA) has been thermally deposited on the etched TFBG, followed by immobilization of probe DNA (pDNA) on this deposited layer. The hybridization of pDNA with the complementary DNA (cDNA) has been monitored using wavelength-dependent interrogation. The reproducibility of the probes has been demonstrated by fabricating three identical probes and their response has been investigated for cDNA concentration ranging from 0 μM to 3 μM. The maximum sensitivity has been found to be 320 pm/μM, with the detection limit being 0.65 μM. Furthermore, the response of the probes towards non-cDNA has also been investigated in order to establish its specificity.
Topics: Reproducibility of Results; DNA; DNA, Complementary; Fiber Optic Technology
PubMed: 37631556
DOI: 10.3390/s23167019 -
Journal of the American Chemical Society Oct 2020Thienoguanosine (G) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for...
Thienoguanosine (G) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, 20 DNA duplexes, where the bases facing and neighboring G were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations, and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, G's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of G independently of its flanking nucleobases. G can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of G's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of G. Due to this exquisite sensitivity, G appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, G's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlight G as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes, and exquisite sensitivity to local structural changes.
Topics: DNA; DNA Probes; Fluorescent Dyes; Guanosine; Kinetics; Molecular Dynamics Simulation; Nucleic Acid Conformation; Solvents; Spectrometry, Fluorescence; Structure-Activity Relationship
PubMed: 32915558
DOI: 10.1021/jacs.0c06165 -
Nature Nanotechnology Dec 2023There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance...
There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance clinical diagnostics beyond the single biomarker model. Here we perform nanopore sequencing of DNA-barcoded molecular probes engineered to recognize a panel of analytes. This allows for highly multiplexed and simultaneous quantitative detection of at least 40 targets, such as microRNAs, proteins and neurotransmitters, on the basis of the translocation dynamics of each probe as it passes through a nanopore. Our workflow is built around a commercially available MinION sequencing device, offering a one-hour turnaround time from sample preparation to results. We also demonstrate that the strategy can directly detect cardiovascular disease-associated microRNA from human serum without extraction or amplification. Due to the modularity of barcoded probes, the number and type of targets detected can be significantly expanded.
Topics: Humans; MicroRNAs; Nanopore Sequencing; DNA; DNA Probes; Sequence Analysis, DNA; Biomarkers; Nanopores; High-Throughput Nucleotide Sequencing
PubMed: 37749222
DOI: 10.1038/s41565-023-01479-z -
Biosensors Jul 2020BRCA1 is the biomarker for the early diagnosis of breast cancer. Detection of BRCA1 has great significance for the genetic analysis, early diagnosis and clinical...
BRCA1 is the biomarker for the early diagnosis of breast cancer. Detection of BRCA1 has great significance for the genetic analysis, early diagnosis and clinical treatment of breast cancer. In this work, we developed a simple electrochemical DNA sensor based on a DNA tetrahedral-structured probe (TSP) and poly-adenine (polyA) mediated gold nanoparticles (AuNPs) for the sensitive detection of BRCA1. A thiol-modified TSP was used as the scaffold on the surface of the screen-printed AuNPs electrode. The capture DNA (TSP) and reporter DNA were hybridized to the target DNA (BRCA1), respectively, to form the typical sandwich system. The nanocomposites of reporter DNA (polyA at the 5' end) combined with AuNPs were employed for signal amplification which can capture multiple enzymes by the specificity between biotin and streptavidin. Measurements were completed in the electrochemical workstation by cyclic voltammetry and amperometry and we obtained the low limit of detection of 0.1 fM with the linear range from 1 fM to 1 nM. High sensitivity and good specificity of the proposed electrochemical DNA sensor showed potential applications in clinical early diagnosis for breast cancer.
Topics: BRCA1 Protein; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Gold; Humans; Metal Nanoparticles; Poly A
PubMed: 32698331
DOI: 10.3390/bios10070078 -
Chemistry (Weinheim An Der Bergstrasse,... Jan 2022We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs...
We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100-1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.
Topics: Binding Sites; DNA; DNA Probes; Nucleic Acid Conformation; Nucleic Acid Hybridization; Nucleic Acids; Nucleotides; RNA
PubMed: 34693570
DOI: 10.1002/chem.202103616 -
Sensors (Basel, Switzerland) Jul 2018Taking advantage of the high selectivity of aptamers and enzyme-free catalyzed hairpin assembly (CHA) amplification strategy, we herein describe a label-free and...
Taking advantage of the high selectivity of aptamers and enzyme-free catalyzed hairpin assembly (CHA) amplification strategy, we herein describe a label-free and enzyme-free sensitive fluorescent and colorimetric strategy for thrombin detection in this paper. In the presence of target, the corresponding aptamer of the partial dsDNA probes will bind to the target and liberate the initiation strand, which is artfully designed as the “on” switch for hairpin assembly. Moreover, the displaced initiation strand partakes in a multi-cycle process and produces numerous G-quadruplexes, which have a remarkable enhancement in fluorescent/colorimetric signal from NMM (-methyl-mesoporphyrin IX) and TMB (3,3′,5,5′-tetramethylbenzidine), respectively. The proposed amplification strategy for thrombin detection is of high sensitivity, down to 2.4 pM, and also achieves colorimetric signals that are able to be distinguished by naked eye. More importantly, the thermodynamics of interacting DNA strands used in our work, and the process of toehold strand displacement-driven assembly are simulated before biological testing, verifying the feasibility theoretically, and simplifying the subsequent actual experiments. Therefore, our approach and simulation have a certain potential application in biomarker detection and quantitatively monitor for disease diagnosis.
Topics: Aptamers, Nucleotide; Biosensing Techniques; DNA; DNA Probes; G-Quadruplexes; Limit of Detection; Thrombin
PubMed: 29986462
DOI: 10.3390/s18072179 -
Biosensors Jan 2023In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper...
In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.
Topics: Copper; Streptavidin; Metal Nanoparticles; DNA; DNA Probes; Biosensing Techniques; Fluorescent Dyes; Spectrometry, Fluorescence; Limit of Detection
PubMed: 36831934
DOI: 10.3390/bios13020168 -
The Analyst Jun 2021Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety,...
Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.
Topics: COVID-19; DNA Probes; Humans; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; SARS-CoV-2
PubMed: 34106115
DOI: 10.1039/d1an00399b -
RSC Advances Mar 2022Nucleic acid-based biosensors, where the capture probe is a nucleic acid, , DNA or its synthetic analogue xeno nucleic acid (XNA), offer interesting ways of eliciting...
Nucleic acid-based biosensors, where the capture probe is a nucleic acid, , DNA or its synthetic analogue xeno nucleic acid (XNA), offer interesting ways of eliciting clinically relevant information from hybridization/dehybridization signals. In this respect, the application of XNA probes is attractive since the drawbacks of DNA probes might be overcome. Within the XNA probe repertoire, peptide nucleic acid (PNA) and morpholino (MO) are promising since their backbones are non-ionic. Therefore, in the absence of electrostatic charge repulsion between the capture probe and the target nucleic acid, a stable duplex can be formed. In addition, these are nuclease-resistant probes. Herein, we have tested the molecularly resolved nucleic acid sensing capacity of PNA and MO capture probes using a fluorescent label-free single molecule force spectroscopy approach. As far as single nucleobase mismatch discrimination is concerned, both PNA and MO performed better than DNA, while the performance of the MO probe was the best. We propose that the conformationally more rigid backbone of MO, compared to the conformationally flexible PNA, is an advantage for MO, since the probe orientation can be made more upright on the surface and therefore MO can be more effectively accessed by the target sequences. The performance of the XNA probes has been compared to that of the DNA probe, using fixed nucleobase sequences, so that the effect of backbone variation could be investigated. To our knowledge, this is the first report on molecularly resolved nucleic acid sensing by non-ionic capture probes, here, MO and PNA.
PubMed: 35424880
DOI: 10.1039/d2ra00386d