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1.
Talanta ; 236: 122868, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635250

RESUMO

Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.


Assuntos
COVID-19 , Nanopartículas Metálicas , Sistemas CRISPR-Cas/genética , DNA , Ouro , Humanos , RNA Viral , RNA Polimerase Dependente de RNA , SARS-CoV-2
2.
Food Chem ; 367: 130617, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352696

RESUMO

The abuse application of glyphosate can result in a potential hazard for environment and human, however its ultrasensitive detection remains challenging. Herein, a Cu2+ modulated DNA-templated silver nanoclusters (DNA-AgNCs) sensor was constructed to sensitively determine glyphosate based on the turn-on fluorescence strategy. The fluorescence quenching of DNA-AgNCs occurred with the existence of Cu2+. Upon the presence of glyphosate, the functional groups on the surface of glyphosate could chelate with Cu2+, following the fluorescence recovery of DNA-AgNCs. Through the stoichiometric methods, we unveil that Cu2+-trigged fluorescence quenching mode is a combination of static and dynamic quenching with the static mode being predominant. In DNA-AgNCs/Cu2+ system, the carboxylate, amine, and phosphonate groups of glyphosate interact with Cu2+ through chelation, in which the carboxylate oxygen, the phosphonate oxygen atoms, and the monoprotonated secondary amine nitrogen atom and Cu2+ form chelate rings. This fluorescence sensor showed a desired linearity of glyphosate analysis under the optimum conditions, ranging from 15 to 100 µg/L with a low detection down to 5 µg/L. Moreover, the proposed sensor was successfully utilized to measure glyphosate in real samples, indicating a promising application in pesticide residues detection.


Assuntos
Nanopartículas Metálicas , Prata , DNA/genética , Glicina/análogos & derivados , Humanos
3.
Food Chem ; 367: 130754, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34384983

RESUMO

N-acetylneuraminic acid (Neu5Ac) is widely spread in many biologically significant glycans of mammals, commonly as a terminal α-glycoside. It is of great significance to develop analytical techniques for detection of Neu5Ac. Herein, a high-sensitive fluorescent biosensor for Neu5Ac has been developed based on FRET between CdSe/ZnS quantum dots (QDs) and BHQ2, as well as exonuclease III (Exo III)-assisted recycling amplification strategy. Employing the specially designed three-level FRET systems and fluorescent signal recovery mechanism, together with five-step recycling signal amplification chain reactions, an ultralow detection limit of 24 fM was achieved. Meanwhile, good linear response ranges within 0.2-12.5 pM and 12.5-1000 pM were founded. The assay has excellent performance in real sample detection, and thus offers great potential for detection of sialic acids modified glycans/lipids in the fields of medical diagnosis and food testing.


Assuntos
Compostos de Cádmio , Pontos Quânticos , Compostos de Selênio , Animais , DNA , Exodesoxirribonucleases , Transferência Ressonante de Energia de Fluorescência , Limite de Detecção , Ácidos Siálicos , Sulfetos , Compostos de Zinco
4.
Food Chem ; 366: 130648, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34325245

RESUMO

A novel label-free fluorescence aptasensor was established for chloramphenicol (CAP) detection by DNA four-arm junction-assisted target recycling and SYBR Green I dye-aided fluorescence-signal amplification. The CAP aptamer was hybridized to its complementary strand (primer) to form a double-stranded primer/aptamer complex. In the presence of CAP, aptamers can specifically bind with CAP to dissociate primers, which can trigger the self-assembly of four hairpins to continuously generate DNA four-arm junctions. After digesting the excess hairpins using T7 exonuclease, SYBR Green I was inserted into the base pair-rich DNA four-arm junctions, which led to a significant increase in fluorescence intensity. Under optimal conditions, the developed aptasensor can detect CAP in a linear range of 1.0 pg mL-1 to 10 ng mL-1 with a detection limit of 0.72 pg mL-1. The recovery rates in milk and honey ranged from 90.3% to 106.6%. Thus, the method shows substantial potential for CAP detection in food products.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Animais , Cloranfenicol/análise , DNA , Limite de Detecção , Leite/química
5.
Talanta ; 236: 122821, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635211

RESUMO

Well-defined structures and compositions of nucleic acids afford oligonucleotide probes with unique chemical properties and biological functions for various biosensing applications. Herein, a unique and special oligonucleotide probe, named multifunction-integrated linear oligonucleotide probe (MI-LOP), was facile designed and reported for label-free and turn-on fluorescent detection of the codon component of genetically modified organisms (GMOs). The MI-LOP contains four different functional regions including recognition of target, serving as polymerization template, and creating polymerization primer-linked G-quadruplex (PP-G-quadruplex). Without the aid of any other oligonucleotides, the introduction of target DNA can make each function of the MI-LOP executed one-by-one, during which the species of target DNA, target analogue, and PP-G-quadruplex can be cyclically utilized and in turn induce a multiplex signal amplification responsible for substantial collection of the G-quadruplex moieties under isothermal conditions. The stable G-quadruplexes can combine with N-methyl mesoporphyrin IX (NMM) and function as efficient fluorescence light-up probes, rapidly leading to a dramatic increase in the fluorescence intensity for the amplified detection of the target codon component. Our results strongly demonstrate that the developed MI-LOP with multiplex amplification effect confers the sensing strategy a high sensitivity and specificity for quantitative and qualitative detection of the target codon. And it has also been successfully applied for analyzing target codon in the complex extractions of soybean. The achievements highlight the significance of using oligonucleotide probes as promising analytical tools to promote the basic biochemical research and help in food and environmental analysis.


Assuntos
Quadruplex G , DNA/genética , Fluorescência , Sondas de Oligonucleotídeos/genética , Plantas Geneticamente Modificadas
6.
Talanta ; 236: 122840, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635230

RESUMO

Ultrasensitive detection of biomarkers at an early stage is generally limited by external influence factors such as high reaction temperature, complex operations, and sophisticated instruments. Here, we circumvent these problems by using nicotinamide adenine dinucleotide (NAD+) to control electroinitiated reversible addition fragmentation chain transfer (electro-RAFT) polymerization for biosensing that enables the detection of a few molecules of target DNA. In this coenzyme-catalyzed electro-RAFT polymerization, numerous ferrocenylmethyl methacrylate (FCMMA) as monomer with electrochemistry signal were linked to the biomarker on Au electrode. Afterwards, a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA. The sensitivity of this methodology was presented by detecting DNA from 10-1 to 104 fM concentration and detection limit (LOD) being down to 4.39 aM in 10 µL samples. This is lower by factors than detection limits of most other ultra-sensitive electrochemical DNA assays.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Catálise , Coenzimas , DNA , Polimerização
7.
Talanta ; 236: 122846, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635236

RESUMO

Simultaneous detection of multiple microRNAs (miRNAs) with high sensitivity can give accurate and reliable information for clinical applications. By uniformly anchoring hairpin probes on the surface of DNA nanolantern, a three-dimensional DNA nanostructure contains abundant and adjustable modification sites, highly integrated DNA nanoprobes were designed and developed as catalytic hairpin assembly (CHA)-based signal amplifiers for enzyme-free signal amplification detection of target miRNAs. The nanolantern-based CHA (NLC) amplifiers, which were facilely prepared via a simple "one-pot" annealing method, showed enhanced biostability, improved cell internalization efficiency, accelerated CHA reaction kinetics, and increased signal amplification capability compared to the single-stranded DNA hairpin probes used in traditional CHA reaction. By co-assembling multiple hairpin probes on a DNA nanolantern surface, as-prepared NLC amplifiers were demonstrated to work well for highly sensitive and specific imaging, expression level fluctuation analysis of two miRNAs in living cells, and miRNAs-guided tumor imaging in living mice. The proposed DNA nanolantern-based nanoamplifier strategy might provide a feasible way to promote the cellular and in vivo applications of nucleic acid probes.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Animais , Catálise , DNA/genética , Camundongos , MicroRNAs/genética , Sondas de Ácido Nucleico
8.
Anal Chim Acta ; 1182: 338945, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602198

RESUMO

Conventionally, the photoelectrochemistry relies on freely diffusive signal molecules in solution to stimulate the photocurrent output, leading to limited sensing strategies. Herein, we showcase the methylene blue (MB) embedded duplex DNA for efficient signal stimuli and its application for ultrasensitive photoelectrochemical (PEC) bioassay. Specifically, the MB embedded duplex DNA scavenged the photogenerated holes of petal-like BiVO4 efficiently, and thus greatly augmented the anodic photocurrent output. Taking the miRNA-21 as a model target, whose biorecognition reaction was aided by the rolling circle amplification (RCA) reaction to finally produce an amplified amount of double-stranded DNA (dsDNA) with embedded MB on the photoelectrode's surface, a "label-free" and "signal-on" PEC biosensing platform was implemented with ultra-sensitivity and high selectivity. The proposed strategy could detect miRNA-21 in the concentration range of 5.0 fM to 10 nM, with the detection limit as low as 0.3 fM. This work opens up the utilization of redox substance intercalated duplex DNA for an efficient signal stimulator, which hints the prospect of other more intercalators embedded DNA for versatile biosensing purposes. Importantly, considering the large quantities of bioreactions that involve duplex DNA as reactants/products, the developed signal transduction strategy may further find wide applications in bioanalysis for targeting more analytes.


Assuntos
Técnicas Biossensoriais , Azul de Metileno , Bioensaio , DNA , Difusão , Técnicas Eletroquímicas , Substâncias Intercalantes , Limite de Detecção
9.
BMC Genomics ; 22(1): 726, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620074

RESUMO

BACKGROUND: The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. RESULTS: Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3' side of several coding regions. CONCLUSIONS: Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.


Assuntos
Genoma Mitocondrial , Leontopithecus , Nanoporos , Animais , DNA , Epigenoma , Fezes
10.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3071-3087, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622618

RESUMO

In recent years, the genome editing technologies based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) have developed rapidly. The system can use homologous directed recombination (HDR) to achieve precise editing that it medicated, but the efficiency is extremely low, which limits its application in agriculture and biomedical fields. As an emerging genome editing technology, the CRISPR/Cas-mediated DNA base editing technologies can achieve targeted mutations of bases without generating double-strand breaks, and has higher editing efficiency and specificity compared with CRISPR/Cas-mediated HDR editing. At present, cytidine base editors (CBEs) that can mutate C to T, adenine base editors (ABEs) that can mutate A to G, and prime editors (PEs) that enable arbitrary base conversion and precise insertion and deletion of small fragments, have been developed. In addition, glycosylase base editors (GBEs) capable of transitioning from C to G and double base editors capable of editing both A and C simultaneously, have been developed. This review summarizes the development, advances, advantages and limitations of several DNA base editors. The successful applications of DNA base editing technology in biomedicine and agriculture, together with the prospects for further optimization and selection of DNA base editors, are discussed.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Agricultura , Sistemas CRISPR-Cas/genética , DNA/genética , Tecnologia
11.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3162-3178, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622625

RESUMO

Deoxyribonucleic acid (DNA) not only serves as the material basis of biological inheritance, but also shows great potential in the development of novel biological materials due to its programmability, functional diversity, biocompatibility and biodegradability. DNA hydrogel is a three-dimensional mesh polymer material mainly formed by DNA. It has become one of the most interesting emerging functional polymer materials in recent years because of the perfect combination of the DNA biological properties that it retained and the mechanical properties of its own skeleton. At present, single- or multi-component DNA hydrogels developed based on various functional nucleic acid sequences or by combining different functional materials have been widely used in the field of biomedicine, molecular detection, and environmental protection. In this paper, the development of preparation methods and classification strategies of DNA hydrogels are summarized, and the applications of DNA hydrogels in drug delivery, biosensing and cell culture are also reviewed. Finally, the future development direction and potential challenges of DNA hydrogels are prospected.


Assuntos
Hidrogéis , Polímeros , DNA/genética , Sistemas de Liberação de Medicamentos
12.
13.
Nanoscale ; 13(37): 15552-15559, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34596187

RESUMO

Dynamic DNA origami nanostructures that respond to external stimuli are promising platforms for cargo delivery and nanoscale sensing. However, the low stability of such nanostructures under physiological conditions presents a major obstacle for their use in biomedical applications. This article describes a stable tetrahedral DNA nanorobot (TDN) programmed to undergo a controlled conformational change in response to epithelial cell adhesion molecule (EpCAM), a molecular biomarker specifically expressed on the circulating tumor cells. Multiresolution molecular dynamics simulations verified the overall stability of the folded TDN design and characterized local distortions in the folded structure. Atomic force microscopy and gel electrophoresis results showed that tetragonal structures are more stable than unfolded DNA origami sheets. Live cell experiments demonstrated the low cytotoxicity and target specificity of TDN. In summary, the proposed TDN can not only effectively resist nuclease catalysis but also has the potential to monitor EpCAM-positive cells precisely.


Assuntos
DNA , Nanoestruturas , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico
14.
Anal Chim Acta ; 1184: 339018, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34625260

RESUMO

DNA methylation is an epigenetic modification that plays a vital role in X chromosome inactivation, genome imprinting, and gene expression. DNA methyltransferase establishes and maintains a stable methylation state in genomic DNA. Efficient and specific DNA methyltransferase testing is essential for the early diagnosis and treatment of cancer. In this study, we designed an ultra-sensitive fluorescent biosensor, based on a 3D tetrahedral fluorescent scaffold assisted by symmetrical double-ring dumbbells, for the detection of DNA-[N 6-adenine]-methyltransferase (Dam MTase). Double-stranded DNA was methylated by Dam MTase and then digested by DpnI to form two identical dumbbell rings. The 3D tetrahedral fluorescent scaffold was synthesized from four oligonucleotide chains containing hairpins. When the sheared dumbbells reacted with the 3D tetrahedral fluorescent scaffold, the hairpins opened and a fluorescence signal could be detected. The strategy was successful over a wide detection range, from 0.002 to 100 U mL-1 Dam MTase, and the lowest detection limit was 0.00036 U mL-1. Control experiments with M.SssI methyltransferase and HpaII methylation restriction endonuclease confirmed the specificity of the method. Experiments with spiked human serum and the 5-fluorouracil inhibitor proved the suitability of the method for early cancer diagnosis.


Assuntos
Metilação de DNA , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Adenina , DNA/genética , DNA/metabolismo , Humanos , Metiltransferases , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
15.
BMC Bioinformatics ; 22(1): 487, 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34627154

RESUMO

BACKGROUND: Alignment-free methods are a popular approach for comparing biological sequences, including complete genomes. The methods range from probability distributions of sequence composition to first and higher-order Markov chains, where a k-th order Markov chain over DNA has [Formula: see text] formal parameters. To circumvent this exponential growth in parameters, variable-length Markov chains (VLMCs) have gained popularity for applications in molecular biology and other areas. VLMCs adapt the depth depending on sequence context and thus curtail excesses in the number of parameters. The scarcity of available fast, or even parallel software tools, prompted the development of a parallel implementation using lazy suffix trees and a hash-based alternative. RESULTS: An extensive evaluation was performed on genomes ranging from 12Mbp to 22Gbp. Relevant learning parameters were chosen guided by the Bayesian Information Criterion (BIC) to avoid over-fitting. Our implementation greatly improves upon the state-of-the-art even in serial execution. It exhibits very good parallel scaling with speed-ups for long sequences close to the optimum indicated by Amdahl's law of 3 for 4 threads and about 6 for 16 threads, respectively. CONCLUSIONS: Our parallel implementation released as open-source under the GPLv3 license provides a practically useful alternative to the state-of-the-art which allows the construction of VLMCs even for very large genomes significantly faster than previously possible. Additionally, our parameter selection based on BIC gives guidance to end-users comparing genomes.


Assuntos
Genoma , Software , Teorema de Bayes , DNA , Cadeias de Markov
16.
BMC Genomics ; 22(1): 732, 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34627155

RESUMO

BACKGROUND: Enzyme-based host depletion significantly improves the sensitivity of clinical metagenomics. Recent studies found that real-time adaptive sequencing of DNA molecules was achieved using a nanopore sequencing machine, which enabled effective enrichment of microbial sequences. However, few studies have compared the enzyme-based host depletion and nanopore adaptive sequencing for microbial enrichment efficiency. RESULTS: To compare the host depletion and microbial enrichment efficiency of enzyme-based and adaptive sequencing methods, the present study collected clinical samples from eight children with respiratory tract infections. The same respiratory samples were subjected to standard methods, adaptive sequencing methods, enzyme-based host depletion methods, and the combination of adaptive sequencing and enzyme-based host depletion methods. We compared the host depletion efficiency, microbial enrichment efficiency, and pathogenic microorganisms detected between the four methods. We found that adaptive sequencing, enzyme-based host depletion and the combined methods significantly enriched the microbial sequences and significantly increased the diversity of microorganisms (p value < 0.001 for each method compared to standard). The highest microbial enrichment efficiency was achieved using the combined method. Compared to the standard method, the combined method increased the microbial reads by a median of 113.41-fold (interquartile range 23.32-327.72, maximum 1812), and the number of genera by a median of 70-fold (interquartile range 56.75-86.75, maximum 164). The combined method detected 6 pathogens in 4 samples with a median read of 547, compared to 5 pathogens in 4 samples with a median read of 4 using the standard method. CONCLUSION: The combined method is an effective, easy-to-run method for enriching microbial sequences in clinical metagenomics from sputum and bronchoalveolar lavage fluid samples and may improve the sensitivity of clinical metagenomics for other host-derived clinical samples.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Criança , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metagenômica
17.
Anal Chim Acta ; 1183: 338966, 2021 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-34627513

RESUMO

CircRNA is a type of covalently closed circular RNA molecule that serves as a potential biomarker for the disease early diagnosis and clinical researches. To achieve living cell imaging of specific circRNA, we developed a novel graphene oxide (GO)-based catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) signal dual amplification system (GO-CHA-HCR, abbreviated GO-AR) for circ-Foxo3 imaging in living cells. The developed system consists of four types of designed hairpin DNA HP1, HP2, H1, and fluorophore-labeled H2, which are absorbed on the GO nanosheets surface leading to fluorescence quenching. In the presence of circ-Foxo3, the CHA cycle was initiated to form a hybrid chain with split fragments, which triggered the HCR cycle to generate dsDNA nanowires that were then released from GO. This process recovered the quenched fluorescence, realizing two-stage signal amplification. The GO-AR system effectively improved the signal-to-noise ratio compared to the traditional GO-CHA and GO-HCR detection system. The detection limit of circ-Foxo3 was as low as 15 pM with excellent sensitivity and selectivity. In addition, the enzyme-free sensing system was successfully applied in living cell circRNA imaging and serum circRNA detection, indicating its high potential in clinical diagnostics.


Assuntos
Grafite , RNA Circular , DNA/genética , Hibridização de Ácido Nucleico
18.
Anal Chim Acta ; 1183: 338988, 2021 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-34627518

RESUMO

An electrochemical biosensor for determination of DNA is developed based on T7 exonuclease-assisted regulatory strand displacement dual recycling signal amplification strategy. First, the hairpin probe recognized and bound the target DNA to form a double strand nucleotide structure, and then the T7 exonuclease was introduced. After be digested by T7 exonuclease, the target DNA was released and entered the next cycle of T7 exonuclease-assisted recycle amplification, while accompanied by a large number of mimic targets (output DNAs) into another cycle. Second, the mimic target reacted with double-chain substrated DNA (CP) by a regulated toehold exchange mechanism, yielding the product complex of detection probes with the help of assisted DNA (S). Finally, after many cycles, a large number of detection probes were produced for binding numerous streptavidin-alkaline phosphatases. The electrochemical biosensor showed very high sensitivity and selectivity with a dynamic response ranged from 0.1 fM to 10 pM with a detection limit of 31.6 aM. Furthermore, this proposed biosensor was successfully applied to the detection of target DNA in 20% diluted serum. The developed strategy has been demonstrated to have the potential for application in molecular diagnostics.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , DNA/genética , Exodesoxirribonucleases
19.
Nanoscale ; 13(29): 12687-12696, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34477619

RESUMO

Measuring the electrophoretic mobility of molecules is a powerful experimental approach for investigating biomolecular processes. A frequent challenge in the context of single-particle measurements is throughput, limiting the obtainable statistics. Here, we present a molecular force sensor and charge detector based on parallelised imaging and tracking of tethered double-stranded DNA functionalised with charged nanoparticles interacting with an externally applied electric field. Tracking the position of the tethered particle with simultaneous nanometre precision and microsecond temporal resolution allows us to detect and quantify the electrophoretic force down to the sub-piconewton scale. Furthermore, we demonstrate that this approach is suitable for detecting changes to the particle charge state, as induced by the addition of charged biomolecules or changes to pH. Our approach provides an alternative route to studying structural and charge dynamics at the single molecule level.


Assuntos
Nanopartículas , Nanotecnologia , DNA , Eletroforese
20.
Nanoscale ; 13(32): 13746-13757, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34477649

RESUMO

Manipulation of temperature can be used to actuate DNA origami nano-hinges containing gold nanoparticles. We develop a physical model of this system that uses partition function analysis of the interaction between the nano-hinge and nanoparticle to predict the probability that the nano-hinge is open at a given temperature. The model agrees well with experimental data and predicts experimental conditions that allow the actuation temperature of the nano-hinge to be tuned over a range of temperatures from 30 °C to 45 °C. Additionally, the model identifies microscopic interactions that are important to the macroscopic behavior of the system, revealing surprising features of the system. This combination of physical insight and predictive potential is likely to inform future designs that integrate nanoparticles into dynamic DNA origami structures or use strand binding interactions to control dynamic DNA origami behavior. Furthermore, our modeling approach could be expanded to consider the incorporation, stability, and actuation of other types of functional elements or actuation mechanisms integrated into nucleic acid devices.


Assuntos
Ouro , Nanopartículas Metálicas , DNA , Conformação de Ácido Nucleico , Temperatura
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