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1.
Phys Chem Chem Phys ; 21(29): 16367-16380, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31309941

RESUMO

Electrochemical DNA biosensors utilizing self-assembled monolayers (SAMs) with inserted DNA probes are promising biosensor designs because of their ease of preparation, miniaturization, and tunability. However, much is still unknown about the interactions between biomolecules such as DNA and various surfaces. A fundamental question regarding these sensors concerns the nature of diffusion of target molecules taking place on sensor surfaces and whether it speeds up the molecular recognition process. Lack of understanding of molecular interaction and surface diffusion in addition to questions regarding the behavior of DNA probes immobilized on these surfaces currently limits the rational design of nucleic acid biosensors. Using all-atom unbiased molecular dynamics (MD) simulations we found that single-stranded DNA (ssDNA) behavior on SAMs is drastically altered by different surface chemistries, with ssDNA adopting very different orientations upon adsorption and surface diffusivity varying over an order of magnitude. Probe behavior varies equally broadly as probes are considerably more stable in certain SAMs than others, which affects the accessibility of probes to the target molecules and likely changes DNA hybridization kinetics in multiple ways. We also found that nearby probes can alter each other's orientations substantially, which highlights the importance of surface density control. Our results elucidate nucleic acid biosensor dynamics vital to rational design and offer insights that can aid in the design of surface properties and patterning for specific applications.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples/química , Simulação de Dinâmica Molecular , Ácidos Nucleicos/análise , Técnicas Eletroquímicas , Propriedades de Superfície
2.
Chem Commun (Camb) ; 55(61): 9019-9022, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290863

RESUMO

DNA nanohydrogel assembled AuNPs were proposed as a high-throughput multidimensional sensing strategy for small molecule reductant profiling in rat brain. The equilibrium among AuNPs, DNA nanohydrogel and targets produced a unique fingerprint-like pattern for differentiating the reducing capacity.


Assuntos
Encéfalo/metabolismo , DNA de Cadeia Simples/química , Ouro/química , Hidrogéis/química , Nanopartículas Metálicas/química , Substâncias Redutoras/análise , Animais , Colorimetria/métodos , Análise Discriminante , Glutationa/líquido cefalorraquidiano , Oxirredução , Tamanho da Partícula , Ratos
3.
Soft Matter ; 15(21): 4284-4293, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31094392

RESUMO

Despite their great promise as fluorescent biological probes and sensors, the structure and dynamics of Ag complexes derived from single stranded DNA (ssDNA) are less understood than their double stranded counterparts. In this work, we seek new insights into the structure of single AgNssDNA clusters using analytical ultracentrifugation (AUC), nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular dynamics simulations (MD) of a fluorescent (AgNssDNA)8+ nanocluster. The results suggest that the purified (AgNssDNA)8+ nanocluster is a mixture of predominantly Ag15 and Ag16 species that prefer two distinct long-lived conformational states: one extended, the other approaching spherical. However, the ssDNA strands within these clusters are highly mobile. Ag(i) interacts preferentially with the nucleobase rather than the phosphate backbone, causing a restructuring of the DNA strand relative to the bare DNA. Infrared spectroscopy and MD simulations of (AgNssDNA)8+ and model nucleic acid homopolymers suggest that Ag(i) has a higher affinity for cytosine over guanine bases, little interaction with adenine, and virtually none with thymine. Ag(i) shows a tendency to interact with cytosine N3 and O2 and guanine N7 and O6, opening the possibility for a Ag(i)-base bifurcated bond to act as a nanocluster nucleation and strand stabilizing site. This work provides valuable insight into nanocluster structure and dynamics which drive stability and optical properties, and additional studies using these types of characterization techniques are important for the rational design of single stranded AgDNA nanocluster sensors.


Assuntos
DNA de Cadeia Simples/química , Prata/química , Sequência de Bases , DNA de Cadeia Simples/genética , Conformação Molecular , Simulação de Dinâmica Molecular
4.
Anal Chim Acta ; 1070: 112-122, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31103164

RESUMO

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has proven to be an effective technique for aptamers selection. In this study, we present an online reaction based convenient single-step CE-SELEX (ssCE-SELEX) mode with human thrombin (H-Thr) as a model target. The selection progress was monitored through bulk Kd analysis, which showed more than a 1000-fold improvement over the initial library after two rounds of selection. Three selected candidate sequences presented high binding affinities against H-Thr with nanomolar (nM) Kd determined by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM, 56.4-177.1 nM) and CE based non-linear fitting (CE-NLF, 98.2-199.7 nM). They also exhibited high specificities towards H-Thr compared with bovine thrombin, IgG, lysozyme, and lactoferrin. Meanwhile, the Kd results by isothermal titration calorimetry (ITC) confirmed the effective CE in measuring the aptamer affinity. In addition, three candidates were applied as aptasensors in the AuNPs based colorimetric assay, which showed visible color change and good linear relationships (R2 > 0.93) with H-Thr concentration. Furthermore, molecular dynamics (MD) simulation was performed to validate the binding of the three candidates with H-Thr by binding sites and binding free energy. The ssCE-SELEX method avoids off-line incubation, saves time and sample, and may provide a universal and convenient method for aptamers selection.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Sistemas On-Line , Técnica de Seleção de Aptâmeros/métodos , Animais , Calorimetria , Bovinos , Eletroforese Capilar , Humanos , Imunoglobulina G/análise , Lactoferrina/análise , Ligantes , Muramidase/análise , Muramidase/metabolismo , Trombina/análise
5.
Chem Soc Rev ; 48(10): 2698-2737, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31080987

RESUMO

Calcium phosphates (CaPs) are ubiquitous in nature and vertebrate bones and teeth, and have high biocompatibility and promising applications in various biomedical fields. Nanostructured calcium phosphates (NCaPs) are recognized as promising nanocarriers for drug/gene/protein delivery owing to their high specific surface area, pH-responsive degradability, high drug/gene/protein loading capacity and sustained release performance. In order to control the structure and surface properties of NCaPs, various biomolecules with high biocompatibility such as nucleic acids, proteins, peptides, liposomes and phosphorus-containing biomolecules are used in the synthesis of NCaPs. Moreover, biomolecules play important roles in the synthesis processes, resulting in the formation of various NCaPs with different sizes and morphologies. At room temperature, biomolecules can play the following roles: (1) acting as a biocompatible organic phase to form biomolecule/CaP hybrid nanostructured materials; (2) serving as a biotemplate for the biomimetic mineralization of NCaPs; (3) acting as a biocompatible modifier to coat the surface of NCaPs, preventing their aggregation and increasing their colloidal stability. Under heating conditions, biomolecules can (1) control the crystallization process of NCaPs by forming biomolecule/CaP nanocomposites before heating; (2) prevent the rapid and disordered growth of NCaPs by chelating with Ca2+ ions to form precursors; (3) provide the phosphorus source for the controlled synthesis of NCaPs by using phosphorus-containing biomolecules. This review focuses on the important roles of biomolecules in the synthesis of NCaPs, which are expected to guide the design and controlled synthesis of NCaPs. Moreover, we will also summarize the biomedical applications of NCaPs in nanomedicine and tissue engineering, and discuss their current research trends and future prospects.


Assuntos
Fosfatos de Cálcio/química , Nanocompostos/química , Materiais Biocompatíveis/química , DNA de Cadeia Simples/química , Portadores de Fármacos/química , Química Verde , Humanos , Nanomedicina , Albumina Sérica/química , Engenharia Tecidual
6.
Nat Methods ; 16(6): 533-544, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110282

RESUMO

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.


Assuntos
DNA/análise , Corantes Fluorescentes/metabolismo , Hibridização in Situ Fluorescente/métodos , Oligonucleotídeos/química , Imagem Óptica/métodos , RNA/análise , Retina/metabolismo , Animais , Células Cultivadas , DNA/genética , DNA de Cadeia Simples/química , Humanos , Camundongos , RNA/genética , Retina/diagnóstico por imagem
7.
Talanta ; 199: 634-642, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952308

RESUMO

Gastric cancer (GC) is associated with high morbidity and mortality rates worldwide. Poorly differentiated GC predicts a poor prognosis and is related to patients' response to chemotherapy and targeted therapy. Therefore, it is very important to accurately evaluate the tumour differentiation status for the treatment of poorly differentiated GC. To develop a molecular probe to analyse poorly differentiated GC, we selected aptamers against poorly differentiated GC by subtractive Cell-SELEX using the poorly differentiated GC cell line BGC-823 as the target and the moderately differentiated GC cell line SGC-7901 as the negative control. After 15 rounds of selection, aptamer PDGC21 exhibited the highest affinity, and the Kd value of the truncated aptamer PDGC21-T was 35.2 ±â€¯1.1 nM. Aptamer PDGC21-T not only specifically bound to the target cells but also bound to other poorly differentiated GC cells. When combined with fluorescent nanoparticle quantum dots (QDs), the PDGC21-T-QD probe could distinguish poorly differentiated GC cells in mixed culture cells and clinical specimens. Furthermore, in a tissue microarray containing 15 cases from patients, there was a higher positive rate in GC tissues compared with adjacent normal tissues; in poorly differentiated tissues, in particular, the fluorescence signal was significantly higher than that in well/moderately differentiated tissues. Therefore, aptamer PDGC21-T holds great potential for use as a molecular imaging probe for the detection of poorly differentiated GC, which is of great significance for diagnosis and treatment.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Técnica de Seleção de Aptâmeros , Neoplasias Gástricas/diagnóstico por imagem , Diferenciação Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Neoplasias Gástricas/patologia , Análise Serial de Tecidos , Células Tumorais Cultivadas
8.
Chem Commun (Camb) ; 55(37): 5387-5390, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30997454

RESUMO

DNA-triggered reversible isolation and recovery of circulating tumor cells (CTCs) is presented based on a multivalent dual-specific aptamer-tethered rolling circle amplification (MA-RCA) network. The multivalent binding sites endow the MA-RCA network with a strong binding ability towards CTCs, and the repeated cell capture/release processes are also actualized in a noninvasive manner.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Células Neoplásicas Circulantes/patologia , Linhagem Celular Tumoral , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Células Neoplásicas Circulantes/química , Células Neoplásicas Circulantes/metabolismo , Técnicas de Amplificação de Ácido Nucleico
9.
Phys Rev E ; 99(3-1): 032404, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30999428

RESUMO

Overstretching of B-DNA is currently understood as force-induced melting. Depending on the geometry of the stretching experiment, the force threshold for the overstretching transition is around 65 or 110 pN. Although the mechanisms behind force-induced melting have been correctly described by Rouzina and Bloomfield [Biophys. J. 80, 882 (2001)BIOJAU0006-349510.1016/S0006-3495(01)76067-5], neither force threshold has been exactly calculated by theory. In this work, a detailed analysis of the force-extension curve is presented, based on a description of single-stranded (ss) DNA in terms of the discrete Kratky-Porod model, consistent with (i) the contour length expected from the crystallographically determined monomer distance and (ii) a high value of the elastic stretch modulus arising from covalent bonding. The value estimated for the ss-DNA persistence length, λ=1.0 nm, is at the low end of currently known estimates and reflects the intrinsic stiffness of the partially, or fully stretched state, where electrostatic repulsion effects are expected to be minimal. A detailed analysis of single- and double-stranded DNA free energies provides estimates of the overstretching force thresholds. In the unconstrained geometry, the predicted threshold is 64 pN. In the constrained geometry, after allowing for the entropic penalty of the plectonemic topology of the molten state, the predicted threshold is 111 pN.


Assuntos
DNA de Forma B , DNA de Cadeia Simples , Modelos Químicos , Modelos Moleculares , Algoritmos , Fenômenos Biofísicos , Simulação por Computador , DNA de Forma B/química , DNA de Cadeia Simples/química , Módulo de Elasticidade , Modelos Genéticos , Desnaturação de Ácido Nucleico , Termodinâmica
10.
PLoS Comput Biol ; 15(4): e1006768, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30933978

RESUMO

Recognition of single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) is important for many fundamental cellular functions. A variety of single-stranded DNA-binding proteins (ssDBPs) and single-stranded RNA-binding proteins (ssRBPs) have evolved that bind ssDNA and ssRNA, respectively, with varying degree of affinities and specificities to form complexes. Structural studies of these complexes provide key insights into their recognition mechanism. However, computational modeling of the specific recognition process and to predict the structure of the complex is challenging, primarily due to the heterogeneity of their binding energy landscape and the greater flexibility of ssDNA or ssRNA compared with double-stranded nucleic acids. Consequently, considerably fewer computational studies have explored interactions between proteins and single-stranded nucleic acids compared with protein interactions with double-stranded nucleic acids. Here, we report a newly developed energy-based coarse-grained model to predict the structure of ssDNA-ssDBP and ssRNA-ssRBP complexes and to assess their sequence-specific interactions and stabilities. We tuned two factors that can modulate specific recognition: base-aromatic stacking strength and the flexibility of the single-stranded nucleic acid. The model was successfully applied to predict the binding conformations of 12 distinct ssDBP and ssRBP structures with their cognate ssDNA and ssRNA partners having various sequences. Estimated binding energies agreed well with the corresponding experimental binding affinities. Bound conformations from the simulation showed a funnel-shaped binding energy distribution where the native-like conformations corresponded to the energy minima. The various ssDNA-protein and ssRNA-protein complexes differed in the balance of electrostatic and aromatic energies. The lower affinity of the ssRNA-ssRBP complexes compared with the ssDNA-ssDBP complexes stems from lower flexibility of ssRNA compared to ssDNA, which results in higher rate constants for the dissociation of the complex (koff) for complexes involving the former.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Animais , Sequência de Bases , Biologia Computacional , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Termodinâmica
11.
IET Nanobiotechnol ; 13(1): 77-83, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30964042

RESUMO

Molecular logic gate has been proposed using single-strand DNA (ssDNA) consisting of basic four nucleobases. In this study, density functional theory and non-equilibrium Green's function based first principle approach is applied to investigate the electronic transmission characteristics of ssDNA chain. The heavily hydrogen-doped-ssDNA (H-ssDNA) chain is connected with gold electrode to achieve enhanced quantum-ballistic transmission along 〈1 1 1〉 direction. Logic gates OR, Ex-OR, NXOR have been implemented using this analytical model of H-ssDNA device. Enhanced logic properties have been observed for ssDNA after H adsorption due to improved electronic transmission. Dense electron cloud is considered as logic 'high' (1) output in presence of hydrogen molecule and on the contrary sparse cloud indicate logic 'low' (0) in the absence of hydrogen molecule. Device current is significantly increased from 0.2 nA to 2.4 µA (approx.) when ssDNA chain is heavily doped with hydrogen molecule. The current-voltage characteristics confirm the formation of various Boolean logic gate operations.


Assuntos
Computadores Moleculares , DNA de Cadeia Simples , Hidrogênio/química , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Eletrodos , Ouro/química , Lógica
12.
Sensors (Basel) ; 19(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934691

RESUMO

In this research, we developed a miRNA sensor using an electrical double layer (EDL) gated field-effect transistor (FET)-based biosensor with enhanced sensitivity and stability. We conducted an in-depth investigation of the mechanisms that give rise to fluctuations in the electrical signal, affecting the stability and sensitivity of the miRNA sensor. Firstly, surface characteristics were studied by examining the metal electrodes deposited using different metal deposition techniques. The lower surface roughness of the gold electrode improved the electrical current stability. The temperature and viscosity of the sample solution were proven to affect the electrical stability, which was attributed to reducing the effect of Brownian motion. Therefore, by controlling the test conditions, such as temperature and sample viscosity, and the surface characteristics of the metal electrodes, we can enhance the stability of the sensor. Metal electrodes deposited via sputtering and e-beam evaporator yielded the lowest signal fluctuation. When ambient temperature was reduced to 3 °C, the sensor had better noise characteristics compared to room temperature testing. Higher viscosity of samples resulted in lower signal fluctuations. Lastly, surface functionalization was demonstrated to be a critical factor in enhancing the stability and sensitivity. MiRNA sensors with higher surface ratios of immobilized DNA probes performed with higher sensitivity and stability. This study reveals methods to improve the characteristics of EDL FET biosensors to facilitate practical implementation in clinical applications.


Assuntos
Técnicas Biossensoriais/métodos , MicroRNAs/análise , Transistores Eletrônicos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/instrumentação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Condutividade Elétrica , Eletrodos , Ouro/química , MicroRNAs/metabolismo , Hibridização de Ácido Nucleico , Polímeros/química , Propriedades de Superfície , Temperatura Ambiente
13.
Int J Pharm ; 563: 208-216, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30953763

RESUMO

Biopharmaceutical development is progressing rapidly. It is imperative that novel drug delivery systems are designed to protect the integrity of the biopharmaceutical, and, at the same time, transport and distribute the drug efficaciously to the target site. Administration of highly specific and sensitive molecules, like therapeutic proteins or nucleic acid-based drugs, present distinct challenges. In this study, we investigate the topical drug delivery of 10-23 DNAzymes; short single-stranded oligonucleotides with RNA-cleaving properties. We developed different hydrogel formulations based on chitosan. These natural-based polymers are particularly suitable for biopharmaceuticals due to their high biocompatibility and biodegradability. We tested these hydrogels for penetration enhancement and for protective efficacy against DNAzymes degradation. Additionally, we examined the physicochemical characteristics and the storage stability of several hydrogel preparations. The formulations developed in this study demonstrate adequate antimicrobial activity, even without the addition of preservatives. A DNAse II degradation assay confirmed their ability to prevent enzymatic degradation of the oligonucleotide. The recovery of intact oligonucleotides in full thickness porcine skin samples indicated that hydrogel formulations composed of DNA/chitosan polyplexes provided satisfactory skin penetration.


Assuntos
Quitosana/administração & dosagem , DNA Catalítico/administração & dosagem , DNA de Cadeia Simples/administração & dosagem , Hidrogéis/administração & dosagem , Administração Cutânea , Animais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/química , Quitosana/química , DNA Catalítico/química , DNA de Cadeia Simples/química , Hidrogéis/química , Pele/metabolismo , Absorção Cutânea , Suínos
14.
Analyst ; 144(9): 3111-3118, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30924836

RESUMO

In this work, we fabricated a metal-organic framework derived magnetic porous carbon (MPC) composite using a one-pot solid state template method. The formation of the synthesized composite was confirmed with various spectroscopic techniques, and it was proved that the composite can effectively quench the fluorescence of ssDNA. This property was utilized in the specific and efficient recognition of harmful arsenate ions. FAM-labelled single strand DNA (FAM-ssDNA) was adsorbed on the surface of the MPC composite and immobilized viaπ-π stacking interactions, which resulted in the fluorescence emission being quenched. A fluorescence quenching efficiency of 96% was achieved, due to the huge surface area of the MPC composite. Upon the addition of As(v) ions into our sensing system, the fluorescence emission dramatically increased, due to the strong affinity for As(v) of the surface of the MPC composite. Consequently, the adsorbed FAM-ssDNA was spontaneously displaced from the surface of the MPC composite, and so the fluorescence intensity was regained. Based on this mechanism, the fabricated biosensor exhibited a highly sensitive fluorescence response to As(v) in the range from 0 to 15 nM, with a detection limit as low as 630 pM. Furthermore, the sensing system is suitable for diverse biological and environmental samples.


Assuntos
Arseniatos/análise , Carbono/química , DNA de Cadeia Simples/química , Estruturas Metalorgânicas/química , Adsorção , Técnicas Biossensoriais/métodos , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Limite de Detecção , Fenômenos Magnéticos , Estruturas Metalorgânicas/síntese química , Porosidade , Eletricidade Estática
15.
J Fish Dis ; 42(6): 851-858, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30859598

RESUMO

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single-stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem-loop structures, which could form the basis of aptamers' specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Vibrioses/veterinária , Vibrio alginolyticus/genética , Animais , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Citometria de Fluxo , Ligantes , Sensibilidade e Especificidade , Vibrio alginolyticus/patogenicidade
16.
Biosens Bioelectron ; 133: 24-31, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30903938

RESUMO

Chemiresistive platforms are best suited for developing DNA hybridization detection systems, owing to their ease of fabrication, simple detection methodology and amenability towards electronics. In this work, we report development of a generic, robust, electrospun nanofiber based interdigitated chemiresistive platform for DNA hybridization detection. The platform comprises of interdigitated metal electrodes decorated with electrospun nanofibers on the top. Two approaches viz., drop casting of graphene doped Mn2O3 nanofibers (GMnO) and direct electrospinning of polyaniline/polyethylene oxide (PANi/PEO) composite nanofibers, have been utilized to decorate these electrodes. In both approaches, inter-device variability, a key challenge for converting this proof-of-concept into a tangible prototype/product, has been addressed using a shadow masking technique. Consequently, the relative standard deviation for multiple PANi/PEO nanofiber based chemiresistors has been brought down from 17.82% (without shadow masking) to 4.41% (with shadow masking). The nanofibers are further modified with single-stranded probe DNAs, to capture a desired hybridization event. To establish the generic nature of the platform, detection of multiple target DNAs has been successfully demonstrated. These targets include dengue virus specific consensus primer (DENVCP) and four DNAs corresponding to Staphylococcus aureus specific genes, namely nuc, mecA, vanA and protein A. The chemiresistive detection of DENVCP has been performed in the concentration range of 10 fM - 1 µM, whereas the detection of the other targets has been carried out in the range of 1 pM - 1 µM. Using a 3σ method, we have estimated the limit of detection for the chemiresistive detection of DENVCP to be 1.9 fM.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples/química , DNA/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/isolamento & purificação , DNA/química , DNA de Cadeia Simples/genética , Grafite/química , Humanos , Nuclease do Micrococo/genética , Nuclease do Micrococo/isolamento & purificação , Nanofibras/química , Hibridização de Ácido Nucleico , Proteínas de Ligação às Penicilinas/isolamento & purificação , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/isolamento & purificação , Staphylococcus aureus/genética
17.
Biosens Bioelectron ; 133: 48-54, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30909012

RESUMO

Carbon dots have been extensively used for the development of fluorescent based molecular affinity sensors. However, label free DNA sensing by electrochemical method is not reported so far. Herein, we report carbon dots stabilized silver nanoparticles (CD-AgNPs) lipid nano hybrids as a sensitive and selective platform for label free electrochemical DNA sensing. The CD-AgNPs were synthesized by wet chemical method and then characterized by UV-visible, Fourier-transform Infra-red (FT-IR), dynamic light scattering (DLS) and high resolution transmission electron microscopy (HR-TEM) techniques. These CD-AgNPs were used for decorating the binary lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) surface (named as lipid) and tethered on self-assembled monolayer of 3-mercaptopropionic acid (MPA) (MPA-lipid-CD-AgNPs). The formation of array of MPA-lipid-CD-AgNPs on Au electrode was confirmed by atomic force microscopy (AFM). Electrochemical behavior of MPA- lipid-CD-AgNPs was monitored in the presence of 1 mM potassium ferri/ferrocyanide (K3/K4 [Fe(CN)6]). The formation of layer-by-layer MPA-lipid-CD-AgNPs is indicated by increased anodic and cathodic peak (ΔEp) separation with decreased redox peak current of K3/K4 [Fe(CN)6]. Short chain DNA (30 mer oligonucleotide, representing the lung cancer) was used as a model system for label free DNA sensing. Un-hybridized (single stranded DNA), hybridized (complementary hybridized), single, double and triple base mismatched target DNA hybridized surfaces were efficiently discriminated at 1 µM target DNA concentration at the Au/MPA-lipid-CD-AgNPs electrode by change in the charge transfer resistance from impedance technique. Further, the modified electrode was successfully used to determine target DNA in a wide linear range from 10-16 to 10-11 M. The present work open doors for the utilization of CDs in molecular affinity based electrochemical sensor design and development.


Assuntos
Técnicas Biossensoriais , DNA/isolamento & purificação , Neoplasias Pulmonares/genética , Nanopartículas Metálicas/química , Carbono/química , DNA/química , DNA de Cadeia Simples/química , Ácidos Graxos Monoinsaturados/química , Ouro/química , Humanos , Limite de Detecção , Lipídeos/química , Lipossomos/química , Neoplasias Pulmonares/patologia , Microscopia de Força Atômica , Hibridização de Ácido Nucleico , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , Prata/química , Espectroscopia de Infravermelho com Transformada de Fourier
18.
RNA ; 25(6): 737-746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926754

RESUMO

Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.


Assuntos
Proteínas 14-3-3/química , Biomarcadores Tumorais/química , DNA de Cadeia Simples/química , Exorribonucleases/química , Magnésio/química , Proteínas Mitocondriais/química , Oligorribonucleotídeos/química , RNA/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sítios de Ligação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Magnésio/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA/genética , RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Analyst ; 144(8): 2755-2764, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30869681

RESUMO

A ratiometric and sensitive microfluidic chip based aptasensor was developed for antibiotic detection with kanamycin (Kana) as a model analyte. A novel stir bar assisted sorptive extraction and rolling circle amplification strategy was designed to largely amplify the signal and overcome complex matrix interference in food samples. The detection mechanism was as follows: firstly, many duplex DNA probes (a single-stranded DNA as a primer hybrid with an aptamer sequence) were modified on a stir bar. In the presence of Kana, the probes on the bar could specifically capture Kana and release the primer to trigger RCA in the presence of a circular DNA template (CDT). As the reaction proceeds, the amount of CDT decreased and the number of RCA products increased. It is worth mentioning that they can be efficiently separated and detected using a microfluidic chip. The signal ratio of RCA products and CDT (IR/IC) can be employed to qualify Kana in a wide linear range from 0.8 pg mL-1 to 10 ng mL-1 with a low detection limit of 0.3 pg mL-1. This method exhibited excellent sensitivity and selectivity and can obviously reduce the matrix interference through a ratiometric strategy combined with stir bar extraction. The aptasensor was successfully tested in milk and fish samples, confirming that it can be applied for on-site quantitation of antibiotic residues in foods.


Assuntos
Antibacterianos/análise , Técnicas Biossensoriais/métodos , Contaminação de Alimentos/análise , Canamicina/análise , Técnicas Analíticas Microfluídicas/métodos , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/instrumentação , Sondas de DNA/química , Sondas de DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Peixes , Ouro/química , Dispositivos Lab-On-A-Chip , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Leite/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Alimentos Marinhos/análise
20.
Biosens Bioelectron ; 132: 186-195, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30875630

RESUMO

In this study, a signal-on nanobiosensor based on bivalent aptamer-Cu nanocluster was designed and optimized for specific and sensitive detection of VEGF165. The VEGF165 is known as a promising biomarker in different diseases such as cancer in the angiogenic stage. Detection and quantification of VEGF165 is a crucial step in diagnosis and monitoring the treatment plan. The represented nanostructure consists of multimerized VEGF165 aptamer joint with ssDNA based linker in the middle and poly thymine sequences on both 3' and 5' ends as a template for Cu-nanocluster supraparticle formation. This self-assembled structure leads to accurate controlling of aggregation in the presence of VEGF165. This study is the first report for Cu nanocluster nucleation on ploy thymine tails of ssDNA which performed in two reduction steps to form stable CuNC supraparticle. The sensing strategy was designed based on the target-induced structure switching mode of the aptamer. In the presence of VEGF165, due to self-assembly induced emission and aggregation-induced emission phenomena this nanostructure depicted the visible wavelength shift and enhancement in the fluorescence emission intensity. Also, the results of the analytical performance of this nanobiosensor indicated the LOD of 12 pM which revealed high rate sensitivity. This aptasensor exhibited stability and decent response linearity range (10-800 pM, R2 = 0.9943). The selectivity and specificity assessment showed high discriminant capability in the real serum sample. In conclusion, this signal-on nanobiosensor provides a facile, sensitive and reliable assay for clinical monitoring of the VEGF165 concentration in serum without further sample preparation.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Cobre/química , Nanopartículas Metálicas/química , Fator A de Crescimento do Endotélio Vascular/sangue , DNA de Cadeia Simples/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Timina/química , Fator A de Crescimento do Endotélio Vascular/análise
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