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1.
Anal Bioanal Chem ; 412(4): 915-922, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900531

RESUMO

A tetrahedral DNA probe can effectively overcome the steric effects of a single-stranded probe to obtain well-controlled density and minimize nonspecific adsorption. Herein, a highly sensitive electrochemical biosensor is fabricated for determination of protein using a tetrahedral DNA probe and rolling circle amplification (RCA). N- and P-co-doped graphene (NP-rGO) is prepared, and AuNPs are then electrodeposited on it for DNA probe immobilization. Benefitting from the synergistic effects of the excellent electrical conductivity of NP-rGO, the stability of the tetrahedral DNA probe and the signal amplification of RCA, the biosensor achieves a low limit of 3.53 × 10-14 M for thrombin and a wide linear range from 1 × 10-13 to 1 × 10-7 M. This study provides a sensitive and effective method for the detection of protein in peripheral biofluids, and paves the way for future clinical diagnostics and treatment of disease. Graphical abstract.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Grafite/química , Trombina/análise , Sondas de DNA/química , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico/métodos
2.
Chem Commun (Camb) ; 56(4): 647-650, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31840153

RESUMO

Here, we demonstrate use of a Mg2+-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.


Assuntos
DNA Catalítico/metabolismo , Ácidos Nucleicos/metabolismo , Resinas Acrílicas/química , Resinas Acrílicas/metabolismo , Géis/química , Géis/metabolismo , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/metabolismo , Magnésio/química , Magnésio/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/química , Tamanho da Partícula , Propriedades de Superfície
3.
Talanta ; 206: 120220, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514891

RESUMO

This work addresses a technological advance applied to the construction of a magnetogenoassay with electrochemical transduction for the maize taxon-specific (HMGA gene) detection using gold-coated magnetic nanoparticles as nanosized platform. Superparamagnetic core-shell Fe3O4@Au nanoparticles (10.4 ±â€¯1.7 nm) were used to assemble the genoassay through the covalent immobilization of HMGA DNA probes onto carboxylated self-assembled monolayers at the nanoparticles surface. A hybridization reaction using sandwich format was selected to prevent inefficient hybridization connected with stable secondary DNA structures using also fluorescein isothiocyanate as DNA signaling tag. The labelling of the hybridization reaction with enzymes allowed the chronoamperometric measurement of the peroxidase activity linked to the nanoplatform located on gold surface. Using this electrochemical magnetogenoassay a linear concentration range from 0.5 to 5 nM and a LOD of 90 pM with a RSD <1.2% was calculated. Certified maize was evaluated without further purification after PCR amplification. This work highlights the efficacy of the electrochemical magnetogenoassay for the HMGA detection, showing its potential as alternative procedure for the verification of the compliance of the legislation.


Assuntos
Técnicas Biossensoriais/métodos , Genes de Plantas , Ouro/química , Proteínas HMGA/genética , Nanopartículas de Magnetita/química , Zea mays/genética , Sequência de Bases , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Técnicas Eletroquímicas/métodos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Hibridização de Ácido Nucleico , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética
4.
Talanta ; 206: 120246, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514901

RESUMO

We develop a novel label-free liquid crystal (LC) aptasensor based on intrinsic properties of nematic LCs for ultra-sensitive detection of tetracycline. The aptasensor is assembled by immobilizing aptamers onto the glass slide modified with both homeotropic alignment and silane coupling agents. Designed aptasensor makes use of the target-induced aptamer conformational switching and disruption of the orientation of LCs which lead to an obvious change of the optical appearance from a dark to a bright response. We describe the optimized condition for maintaining the homeotropic orientation of LCs, which are suitable for the tetracycline detection. The average gray-scale intensities of polarizing optical microscopy images were calculated to quantitatively detect tetracycline concentrations. The aptasensor works especially at trace level of tetracycline as low as 0.5 pM. Moreover, the LC aptasensor was successfully used to detect tetracycline in the real milk sample. According to the results, the proposed LC aptasensor for tetracycline detection is simple, ultra-sensitive, label free and ease of preparation.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Cristais Líquidos/química , Tetraciclina/análise , Animais , Antibacterianos/análise , Compostos de Bifenilo/química , Contaminação de Alimentos/análise , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Microscopia de Polarização/métodos , Leite/química , Nitrilos/química
5.
Anal Bioanal Chem ; 411(25): 6745-6754, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31482291

RESUMO

In the literature, there are reports of the utilization of various hydrogels to create generic platforms for protein microarray applications. Here, a novel strategy was developed to obtain high-performance microarrays. In it, a dextran hydrogel is used to covalently immobilize oligonucleotides and proteins. This method employs aqueous solutions of dextran methacrylate (Dx-MA), which is a biocompatible photopolymerizable monomer. Capture probes are immobilized inside the hydrogel via a light-induced thiol-acrylate coupling reaction at the same time as the dextran polymer is formed. Hydrogel microarrays based on this technique were prepared on different surfaces, such as a Blu-ray Disk and polycarbonate or alkene-functionalized glass slides, and these systems showed high probe-loading capabilities and good biorecognition yields. This methodology presents advantages such as a low cost, a short analysis time, a low limit of detection, and multiplexing capabilities, among others. Confocal fluorescence microscopy analysis demonstrated that in these hydrogel-based microarrays, receptor immobilization and the biorecognition event occurred within the hydrogel and not merely on the surface.


Assuntos
Dextranos/química , Ácidos Nucleicos Imobilizados/química , Metacrilatos/química , Química Click/métodos , Hidrogéis/química , Ácidos Nucleicos/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cimento de Policarboxilato/química , Compostos de Sulfidrila/química
6.
Analyst ; 144(17): 5277-5283, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31369000

RESUMO

The faster a disease can be diagnosed, the sooner effective treatment can be initiated, motivating a drive to replace standard laboratory techniques with point-of-care technologies that return answers in minutes rather than hours. Thus motivated, we describe the development of an E-DNA scaffold sensor for the rapid and convenient measurement of antibodies diagnostic of syphilis. To achieve this (and in contrast to previous sensors of this class, which relied on single, linear epitopes for detection), we utilized a near full-length antigen as the sensor's recognition element, allowing us to simultaneously display multiple epitopes. The resultant sensor is able to detect antibodies against Treponema pallidum pallidum, the causative agent of syphilis, at clinically relevant concentrations in samples in less than 10 min. Preliminary results obtained using sero-positive and sero-negative human samples suggest the clinical sensitivity and specificity of the approach compare well to current gold-standard tests, while being simple and rapid enough to deploy at the point of care.


Assuntos
Anticorpos Antibacterianos/sangue , DNA/química , Sífilis/diagnóstico , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Sequência de Bases , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Escherichia coli/genética , Humanos , Ácidos Nucleicos Imobilizados/química , Azul de Metileno/química , Oxirredução , Treponema pallidum/química
7.
Chem Commun (Camb) ; 55(69): 10288-10291, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31396601

RESUMO

A simple nanopore modification and sensing strategy was developed for the detection of miRNAs. This preparation and sensing approach provides a quick, simple and facile tool for the detection of specific biomolecules with high sensitivity and selectivity, and may find a wide range of applications in bio-analysis.


Assuntos
Ouro/química , Ácidos Nucleicos Imobilizados/química , MicroRNAs/análise , Nanoporos/ultraestrutura , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Hibridização de Ácido Nucleico
8.
Biosens Bioelectron ; 142: 111569, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31404881

RESUMO

Uracil-DNA glycosylase (UDG) is a typical initiator for base excision repair (BER) process. Since aberrant expression of UDG is relevant to a variety of cancers, analysis of UDG activity with high sensitivity and accuracy is of great importance. We reported herein a sensitive and reliable dual-signal bioassay for UDG activity by coupling photoelectrochemical (PEC) and electrochemical (EC) strategies in one probe electrode. The Au/TiO2 hybrid was used as a matrix to immobilize substrate DNA (sDNA), which modified with AgInS2 quantum dot (AIS QD) on the terminal. When UDG exist, the base of uracil was eliminated from the sDNA, and the produced apyrimidinic (AP) site could be cleaved by endonuclease IV (Endo. IV) immediately. Under this situation, the PEC labels of AIS QDs were detached from the electrode, resulting in a "signal-off" trend for PEC signal. After assistant DNA (aDNA) was then assembled, the hybridization chain reaction (HCR) was triggered, and EC labels of ferrocene molecules were introduced, producing a "signal-on" trend for EC signal. Besides, as the produced long double-stranded DNA by the HCR had evident steric hindrance, the PEC signal further decreased. Based on this meticulous design, the dual-signal bioassay for UDG activity showed low detection limits of 4.3 × 10-5 and 1.9 × 10-4 U/mL with PEC and EC detection, and accurate analysis of UDG activity in living cells was realized. By just changing the recognition site, this sensitive and reliable dual-signal strategy can be extended to diagnose other DNA repair-related enzymes in the real samples.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Uracila-DNA Glicosidase/análise , Linhagem Celular Tumoral , Eletrodos , Ensaios Enzimáticos/instrumentação , Desenho de Equipamento , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Pontos Quânticos/química , Titânio/química , Uracila/química
9.
Biosens Bioelectron ; 142: 111578, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31422223

RESUMO

The sensitive and accurate detection of cardiac troponin I (cTnI) is critical for myocardial infarction diagnosis. In this work, a dual-aptamer-based electrochemical (EC) biosensor was designed for cTnI detection based on the DNA nanotetrahedron (NTH) capture probes and multifunctional hybrid nanoprobes. First, the NTH-based Tro4 aptamer probes were anchored on a screen printed gold electrode (SPGE) surface through the Au-S bond, providing an enhanced spatial dimension and accessibility for capturing cTnI. Then, the hybrid nanoprobes were fabricated by using magnetic Fe3O4 nanoparticles as nanocarriers to load a large amount of cTnI-specific Tro6 aptamer, natural horseradish peroxidase (HRP), HRP-mimicking Au@Pt nanozymes and G-quadruplex/hemin DNAzyme. This signaling nanoprobes are capable of specifically recognizing the target cTnI based on the Tro6 aptamer and amplifying the signals to improve the detection sensitivity via enzymatic processes. We found the remarkable enhanced effect of EC signal to be attributed to the co-catalysis effect of hybrid nanozymes, HRP and DNAzyme. The target cTnI was sandwiched between the two types of aptamers (Tro4 and Tro6) on the electrode interface. Finally, this EC aptasensing platform exhibited great analytical performance with a wide dynamic range of 0.01-100 ng mL-1 and a low detection limit of 7.5 pg mL-1 for cTnI. The high selectivity, sensitivity and reliability of EC aptasensor can provide great potential in the clinic disease diagnostics.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Ácidos Nucleicos Imobilizados/química , Troponina I/sangue , Catálise , Sondas de DNA/química , DNA Catalítico/química , Técnicas Eletroquímicas/métodos , Quadruplex G , Ouro/química , Hemina/química , Peroxidase do Rábano Silvestre/química , Humanos , Limite de Detecção , Platina/química , Reprodutibilidade dos Testes , Troponina I/análise
10.
Biosens Bioelectron ; 142: 111544, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376717

RESUMO

Rapid and efficient detection of microRNA (miRNA) of breast cancer 1 gene mutation (BRCA1) at their earliest stages is one of the crucial challenges in cancer diagnostics. In this study, a highly-sensitive electrochemical DNA biosensor was fabricated by double signal amplification (DSA) strategy for the detection of ultra-trace miRNA of BRCA1. In the presence of target miRNA of BRCA1, the well-matched RNA-DNA duplexes were specifically recognized by double-strand specific nuclease (DSN), and the DNA part of the duplexes were then cleaved and miRNAs were released to trigger another following cycle, which produced a primarily amplified signal by such a cyclic enzymatic signal amplification (CESA). Then triple-CdTe quantum dot labelled DNA nanocomposites (3-QD@DNA NC) was selectively hybridized with the cleaved DNA probe on the electrode and produced multiply amplified signals. The biosensor exhibited a high sensitivity for the detection of miRNA of BRCA1 in concentrations ranging from 5 aM to 5 fM, and its detection limit of 1.2 aM was obtained, which is two or three orders of magnitude lower than those by single signal amplification strategy such as CESA or QD-labeled DNA probes. The as-prepared biosensor was successfully used to detect the miRNA of BRCA1 in human serum samples with acceptable stability, good reproducibility, and good recovery. The proposed DNA biosensor based on double signal amplification strategy provided a feasible, rapid, and sensitive platform for early clinical diagnosis and practical applications.


Assuntos
Técnicas Biossensoriais/métodos , Genes BRCA1 , Ácidos Nucleicos Imobilizados/química , MicroRNAs/genética , Nanocompostos/química , Compostos de Cádmio/química , Desoxirribonucleases/química , Técnicas Eletroquímicas/métodos , Humanos , Ácidos Nucleicos Imobilizados/genética , MicroRNAs/sangue , Mutação , Pontos Quânticos/química , Telúrio/química
11.
Anal Bioanal Chem ; 411(26): 6813-6823, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432237

RESUMO

We demonstrate a DNA-based optical fiber device that uses an in-fiber grating, a light absorbing coating with surface anchored DNA, and a built-in optical thermometer. This device is used for precisely thermal cycling surface DNA spots bound by a simple UV cross-linking technique. Near-infrared light of wavelengths near 1550 nm and guided power near 300 mW is coupled out of the fiber core by a tilted fiber Bragg grating inscribed in the fiber and absorbed by the coating to increase its temperature to more than 95 °C. A co-propagating broadband light signal (also in the near-infrared region) is used to measure the reflection spectrum of the grating and thus the temperature from the wavelength shifts of the reflection peaks. The device is capable of sensitive DNA melt analysis and can be used for DNA amplification. Graphical abstract.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA/química , Tecnologia de Fibra Óptica/instrumentação , Hibridização de Ácido Nucleico , DNA/genética , Desenho de Equipamento , Calefação , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Desnaturação de Ácido Nucleico , Fibras Ópticas
12.
Analyst ; 144(16): 4865-4870, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31297492

RESUMO

Proteins play a key role in disease diagnosis, and protein discrimination is an important but difficult issue. Here, we report a novel strategy for improving protein discrimination through a facile colorimetric sensor array, which is based on DNA-gold nanoparticle (AuNP) conjugates manipulated by exonuclease I (Exo I). Different proteins exhibit diverse affinities toward the three DNAs, and the DNA-protein binding is resistant to the digestion of Exo I and protects the AuNPs from aggregation in high concentrations of NaCl media, forming distinct response patterns of the array. These response patterns as "fingerprints" can be acquired on the sensor array and then identified by linear discriminant analysis (LDA). The sensor array achieved the correct discrimination of 15 proteins at a 10 nM level in buffer solution and real serum samples. Also, the sensor array had the capability to discriminate individual proteins and the mixtures of them. Remarkably, the practicability of the sensor array was further confirmed by the identification of 35 unknown protein samples with 100% accuracy.


Assuntos
Proteínas Sanguíneas/análise , DNA/química , Exodesoxirribonucleases/química , Nanopartículas Metálicas/química , Animais , Proteínas Sanguíneas/química , Bovinos , Colorimetria/métodos , Análise Discriminante , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Oligodesoxirribonucleotídeos/química
13.
Biosens Bioelectron ; 142: 111528, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31362202

RESUMO

DNA polymerase catalyzes the replication of DNA, one of the key steps in cell division. The control and understanding of this reaction owns great potential for the fundamental study of DNA-enzyme interactions. In this context, we developed a label-free microfluidic biosensor platform based on the principle of localized surface plasmon resonance (LSPR) to detect the DNA-polymerase reaction in real-time. Our microfluidic LSPR chip integrates a polydimethylsiloxane (PDMS) channel bonded with a nanoplasmonic substrate, which consists of densely packed mushroom-like nanostructures with silicon dioxide stems (~40 nm) and gold caps (~22 nm), with an average spacing of 19 nm. The LSPR chip was functionalized with single-stranded DNA (ssDNA) template (T30), spaced with hexanedithiol (HDT) in a molar ratio of 1:1. The DNA primer (P8) was then attached to T30, and the second strand was subsequently elongated by DNA polymerase assembling nucleotides from the surrounding fluid. All reaction steps were detected in-situ inside the microfluidic LSPR chip, at room temperature, in real-time, and label-free. In addition, the sensor response was successfully correlated with the amount of DNA and HDT molecules immobilized on the LSPR sensor surface. Our platform represents a benchmark in developing microfluidic LSPR chips for DNA-enzyme interactions, further driving innovations in biosensing technologies.


Assuntos
DNA Polimerase I/análise , Escherichia coli/enzimologia , Ácidos Nucleicos Imobilizados/química , Técnicas Analíticas Microfluídicas/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , DNA de Cadeia Simples/química , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Nanoestruturas/química , Nanoestruturas/ultraestrutura
14.
Prep Biochem Biotechnol ; 49(9): 900-907, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31271333

RESUMO

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola's blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100 nM and limit of detection was calculated as 8.7 nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.


Assuntos
Técnicas Biossensoriais/instrumentação , Hepacivirus/genética , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Peptídeos/química , Sondas de DNA/química , Sondas de DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Oligonucleotídeos/análise
15.
Biosens Bioelectron ; 141: 111351, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176113

RESUMO

Zika virus (ZIKV) has recently become a global health challenge due to its rapid geographical expansion, since it is associated with serious neurological anomalies such as Guillain-Barré syndrome and microcephaly. Currently, the techniques for ZIKV diagnosis require labor-intensive, expensive and lengthy tests using sophisticated equipment. Moreover, false-positive or false-negative results can occur. In the present work, a DNA biosensor to detect ZIKV in real human serum samples was developed using an oxidized glassy carbon electrode (ox-GCE) modified with silsesquioxane-functionalized gold nanoparticles (AuNPs-SiPy). This nanohybrid was characterized by UV-Vis, FTIR and Raman spectroscopies, DLS, and XRD. The conditions for the immobilization of a ZIKV ssDNA probe on the electrode surface (ox-GCE-[AuNPs-SiPy]) were optimized by univariate and multivariate analysis. The optimized biosensor was characterized by CV, EIS and AFM experiments. The ZIKV target recognition was based on the variation of the charge transfer resistance (ΔRct) of the redox marker ([Fe(CN)6]3-/4-) used and the roughness (Rq) of the electrode surface. The proposed biosensor presented a LOD of 0.82 pmol L-1, with a linear range of 1.0 x10-12 - 1.0 x10-6 mol L-1. Moreover, the reported device showed a suitable stability and satisfactory sensitivity and selectivity to quantify ZIKV in human serum samples, which suggests its promising clinical applications for the early diagnosis of ZIKV-associated pathologies.


Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Ácidos Nucleicos Imobilizados/química , Nanopartículas Metálicas/química , Infecção por Zika virus/sangue , Zika virus/isolamento & purificação , DNA de Cadeia Simples/química , Eletrodos , Humanos , Limite de Detecção , Compostos de Organossilício/química , Infecção por Zika virus/virologia
16.
Biosens Bioelectron ; 141: 111402, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185418

RESUMO

The development of ultrasensitive and specific methods for facile detection of trace nucleic acids is of great significance to human health and safety. In the present work, an ultrasensitive SERS-based strategy for detecting nucleic acids was proposed by integrating the SERS-active AgNRs array with double signal amplifications, i.e. the primary target-triggered enzyme-free amplification recycling and the secondary signal enhancement of multiple-reporter. By comparing two SERS sensing routes, i.e. solid interface recycling (Route A) and solution recycling (Route B), the superior solution recycling was determined first, and then the sensing strategy was optimized by investigating the immobilization time, surface blocking, and number of reporters utilized in the SERS sensing. The experimental results indicate that an ultrasensitive SERS strategy can be achieved via the primary amplification of target-triggered enzyme-free recycling and additional enhancement by the usage of multiple reporters. Under the optimal conditions, the SERS sensing showed good specificity and uniformity, and a linear calibration curve of DNAs in human serum solution, ranging from 1 µM to 1 fM, was obtained with LOD as low as 40.4 aM, and the following recovery rate measurements confirmed that the proposed SERS sensing had good repeatability and reliability, which shows great potential for facile detecting trace DNAs, especially disease-related nucleic acids in the liquid biopsy of early-stage cancer detection.


Assuntos
Técnicas Biossensoriais/métodos , DNA/sangue , Análise Espectral Raman/métodos , DNA/análise , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Nanotubos/química , Reprodutibilidade dos Testes
17.
Talanta ; 203: 49-57, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31202349

RESUMO

In this work, an electrochemical enzyme-linked oligonucleotide array to achieve simple and rapid multidetection of aflatoxin B1 (AFB1) is presented. The assay is based on a competitive format and disposable screen-printed cells (SPCs). Firstly, the electrodeposition of poly(aniline-anthranilic acid) copolymer (PANI-PAA) on graphite screen-printed working electrodes was performed by means of cyclic voltammetry (CV). Aflatoxin B1 conjugated with bovine serum albumin (AFB1-BSA) was then immobilized by covalent binding on PANI-PAA copolymer. After performing the affinity reaction between AFB1 and the biotinylated DNA-aptamer (apt-BIO), the solution was dropped on the modified SPCs and the competition was carried out. The biotinylated complexes formed onto the sensor surface were coupled with a streptavidin-alkaline phosphatase conjugate. 1-naphthyl phosphate was used as enzymatic substrate; the electroactive product was detected by differential pulse voltammetry (DPV). The response of the enzyme-linked oligonucleotide assay was signal-off, according to the competitive format. A dose-response curve was obtained between 0.1 ng mL-1 and 10 ng mL-1 and a limit of detection of 0.086 ng mL-1 was achieved. Finally, preliminary experiments in maize flour samples spiked with AFB1 were also performed.


Assuntos
Aflatoxina B1/análise , Aptâmeros de Nucleotídeos/química , Ácidos Nucleicos Imobilizados/química , Oligodesoxirribonucleotídeos/química , Aflatoxina B1/química , Fosfatase Alcalina/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Farinha/análise , Contaminação de Alimentos/análise , Grafite/química , Limite de Detecção , Naftalenos/química , Compostos Organofosforados/química , Zea mays
18.
Methods Appl Fluoresc ; 7(3): 035006, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31042679

RESUMO

Due to the concern over food safety, it is important to detect the pesticides residues in agricultural products. Here, a highly sensitive and low background fluorescent strategy for the detection of pesticides residues has been developed. The fluorescence intensity of N-methyl mesoporphyrin IX (NMM) binding G-quadruplex could be turn off because of inhibiting effect of the pesticides on the acetylcholinesterase (AChE) activity. For that, four single-stranded DNAs (named linker, trigger, H1 and H2, respectively) are rational designed and T-Hg-T mismatches duplex DNAs as a recognizer combined with the separation of magnetic beads. The design of hybridization chain reaction (HCR) amplification strategy assisted by magnetic separation has been adopted to improve the detection sensitivity. In the presence of pesticides, the amount of the thiol group generated by hydrolysis reaction of acetylcholine (ACh) is reduced, lead to release of less trigger DNA. Therefor subsequent HCR process is retarded with decreased fluorescence intensity. The reduced fluorescence intensity has a quantitative relationship with the pesticide concentration. The limit of detection of chlorpyrifos was estimated to be 2.0 ng ml-1. It has been applied to detect the pesticides residues in real samples.


Assuntos
Contaminação de Alimentos/análise , Resíduos de Praguicidas/análise , Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Clorpirifos/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Quadruplex G , Gengibre/química , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Fenômenos Magnéticos , Malus/química , Mercúrio/química , Mesoporfirinas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
19.
Analyst ; 144(12): 3817-3825, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31086898

RESUMO

Herein, an ultrasensitive and label-free electrochemical biosensor was developed for microRNA (miRNA) based on rolling circle amplification (RCA)-mediated palladium nanoparticles (PdNPs). The sensor was fabricated by immobilizing dual-functionalized hairpin probes onto an electrode. The specific recognition of target miRNA-21 by the hairpin probes could trigger the RCA reaction, which produced numerous guanine (G)-rich long single-stranded DNAs (ssDNAs). Based on the interaction of the PdII species with the nitrogen atoms of the G bases, these G-rich long ssDNAs served as specific templates in the in situ synthesis of massive PdNPs as electrochemical indicators. The formation of PdNPs was demonstrated to be exactly along the RCA products by high-resolution transmission electron microscopy. Using this cascade signal amplification strategy, the developed biosensor achieved a linear range of 50 aM-100 fM with an ultralow detection limit of 8.6 aM miRNA-21. Furthermore, the developed biosensor exhibited good selectivity, reproducibility, stability and satisfactory feasibility for miRNA-21 detection in human serum samples; this ensured significant potential of this biosensor in disease diagnosis and prognosis applications.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , Ácidos Nucleicos Imobilizados/química , Nanopartículas Metálicas/química , MicroRNAs/sangue , Platina/química , Calibragem , Sondas de DNA/genética , Técnicas Eletroquímicas/métodos , Humanos , Ácidos Nucleicos Imobilizados/genética , Sequências Repetidas Invertidas , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes
20.
Mikrochim Acta ; 186(4): 264, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30929090

RESUMO

The presented voltammetric mercury(II) sensor is based on the specific interaction between Hg(II) ion and thymine-thymine base pairs. Reduced graphene oxide is functionalized with gold nanorods and then loaded with thionine and streptavidin (RGO@AuNR-TH-SA). A T-rich thiolated DNA (S1) is firstly immobilized on a gold electrode. In the presence of Hg (II), the T-rich biotin-DNA (biotin-S2) binds to S1 via T-Hg(II)-T interaction. Then, the RGO@AuNR-TH-SA is linked to the gold electrode by specific binding between SA and biotin-S2. This produces an electrochemical signal (at -0.208 V vs. Ag/AgCl) of TH that depends on the concentration of Hg (II). The peak current increases linearly in the 1 to 200 nM Hg (II) concentration range, and the detection limit is 0.24 nM. The sensor is highly selective for Hg (II) over other environmentally relevant metal ions, even at concentration ratios of >1000. Graphical abstract Schematic representation of a voltammetric biosensor for mercury(II) using reduced graphene oxide@gold nanorods (RGO@AuNRs) and thymine-Hg(II)-thymine interaction. It is based on the fact that RGO@AuNR can strongly adsorb thionine (TH) and streptavidin to realize the signal amplification.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Grafite/química , Mercúrio/análise , Nanotubos/química , Timina/química , Pareamento de Bases , DNA/genética , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Ácidos Nucleicos Imobilizados/química , Limite de Detecção , Fenotiazinas/química , Reprodutibilidade dos Testes , Estreptavidina/química , Poluentes Químicos da Água/análise
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