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1.
Anal Chim Acta ; 1075: 137-143, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196419

RESUMO

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.


Assuntos
Sondas de DNA/química , DNA de Cadeia Simples/análise , Proteína BRCA2/genética , Sondas de DNA/genética , DNA de Cadeia Simples/genética , Desoxirribonuclease IV (Fago T4-Induzido)/química , Feminino , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Neoplasias Ovarianas/genética , Mutação Puntual , Telomerase/genética
2.
Anal Chim Acta ; 1075: 144-151, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196420

RESUMO

Salmonella is a widely distributed, extremely harmful bacteria, the presence of which requires confirmation via an on-site visual biosensor. In this study, we constructed a label-free, cascade amplification visualization biosensor for the sensitive and rapid detection of Salmonella enterica subsp. enterica serovar typhimurium based on the RDTG principle (recombinase polymerase amplification (RPA), duplex-specific enzyme (DSN) cleavage, terminal deoxynucleotidyl transferase (TdT) extension and G-quadruplexes output). Following DNA extraction of Salmonella spp., the first step in the construction involved the recognition and amplification of nucleic acids, carried out by RPA, to achieve the first signal amplification within 10 min. This RPA product was then specifically cleaved by DSN to produce a large number of small double-stranded DNA (dsDNA) products with 3'-OH within 15 min to achieve the second signal amplification. Thereafter, TdT was employed to empower these small 3'-OH dsDNA products to extend and produce a large number of long G-rich single-stranded DNAs (ssDNAs) within 20 min, thus realizing the third signal increase. These long G-rich ssDNA products displayed a color change that could be directly observed through the naked eye by adding H2O2/3,3',5,5'-tetramethylbenzidine (TMB). The RDTG biosensor for the detection of Salmonella spp. has several advantages, including a low limit of 6 cfu/mL. It is an isothermal-free instrument, simple to operate, with a rapid detection time of less than 1.5 h. Furthermore, it can be visually characterized and quantified by a microplate reader to detect Salmonella spp., in food and environmental samples, and it has broad application prospects.


Assuntos
Técnicas Biossensoriais/métodos , DNA Bacteriano/análise , Salmonella/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Benzidinas/química , DNA Nucleotidilexotransferase/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Quadruplex G , Peróxido de Hidrogênio/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/química , Salmonella/genética , Sensibilidade e Especificidade , Iogurte/microbiologia
3.
Chem Commun (Camb) ; 55(51): 7358-7361, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31172143

RESUMO

Here, we describe a simple mix-and-read method for the detection of specific bacterial strains that uses a DNAzyme and a molecular beacon to generate a signal. We have greatly improved upon the previously described DNAzyme-based bacteria detection method by eliminating a tedious preparation step while maintaining detection sensitivity.


Assuntos
Extratos Celulares/análise , DNA Bacteriano/análise , DNA Catalítico/metabolismo , DNA de Cadeia Simples/análise , Escherichia coli/classificação , Técnicas Biossensoriais , Corantes Fluorescentes/química , Limite de Detecção
4.
Talanta ; 199: 442-448, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952281

RESUMO

Numerous nanomaterials have been utilized for novel biosensors with sensitivity and selectivity in the last decades due to their intrinsic unique properties. Herein, a facile fluorescence method for nucleic acid detection was developed by employing TiO2 nanowires (NWs) as the sensing platform. The quenching effect of TiO2 NWs to fluorophore-labelled single-stranded DNA (ssDNA) was found to be more significant than that to fluorophore-labelled double-stranded DNA (dsDNA) or triplex DNA probes. More importantly, the whole quenching process was also fast since it just took about ten minutes to reach the equilibrium. Based on the different affinities of TiO2 NWs to ssDNA, dsDNA and triplex DNA probes, the sequence-specific nucleic acids were detected with sensitivity and specificity. Further investigation has demonstrated that the quenching efficiency of TiO2 NWs to long ssDNA was apparently superior than that to short ssDNA. Moreover, the fluorescence from various ssDNA probes labelled with a wide spectrum of fluorescent dyes could also be quenched by TiO2 NWs. These inspiring results reveal that TiO2 NWs could be an excellent universal nanoquencher used in the next-generation biosensors.


Assuntos
Sondas de DNA/análise , DNA de Cadeia Simples/análise , DNA/análise , Fluorescência , Nanofios/química , Titânio/química
5.
Analyst ; 144(8): 2773-2779, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30869659

RESUMO

With the use of a double-cycle system involving two catalytic reactions by RNase H and DNAzyme, the signal of oligoDNAs has been specifically amplified in an isothermal mode. The precursor of DNAzyme was introduced to the system as a ring-structured and inactivated form, which involves the 6-nt RNA portion being complementary to target oligoDNA. In the presence of target oligoDNA, the RNA portion forms a DNA/RNA hetero-duplex and is cut by RNase H. This scission converts the precursor to catalytically active DNAzyme, which in turn disconnects the molecular beacon to produce the amplified signal. Because the covalent bonds were disconnected to provide discrete structural changes in both cycles, high sensitivity and specificity are obtained, indicating the strong potential of this double catalytic cycle method for versatile applications.


Assuntos
DNA Catalítico/química , DNA de Cadeia Simples/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Oligonucleotídeos/análise , Ribonuclease H/química , Antraquinonas/química , DNA Catalítico/genética , DNA de Cadeia Simples/genética , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Perileno/química , Ribonuclease H/genética
6.
Nanoscale ; 11(8): 3633-3638, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30741288

RESUMO

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 µL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.


Assuntos
DNA Bacteriano/análise , Endonucleases Flap/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Chlamydia trachomatis/genética , DNA/análise , DNA/sangue , DNA/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/sangue , DNA de Cadeia Simples/metabolismo , Humanos , Limite de Detecção , Ácidos Nucleicos/sangue , Ácidos Nucleicos/metabolismo , Compostos Orgânicos/química
7.
Anal Chim Acta ; 1048: 42-49, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30598156

RESUMO

A novel enhanced photoelectrochemical (PEC) DNA biosensor, based on a compact heterojunction g-C3N4/MoS2 and co-sensitization effect with CdSe quantum dots (QDs), was first proposed for simple and accurate analysis of a short ssDNA. In this work, the g-C3N4/MoS2 was successfully synthesized and used as the electrode matrix material to construct PEC biosensor. 2D/2D heterojunction was formed between g-C3N4 and MoS2, which could promote the separation of photogenerated electron-hole pairs resulting in an enhanced photocurrent. In the presence of target DNA, CdSe QDs labeled reporter DNA was complementary pairing with target DNA which was specific recognized by capture DNA loading on self-assembled CdS QDs film, leading to close contact between CdSe QDs and g-C3N4/MoS2 modified electrode surface, thereby resulting in the enhanced photocurrent intensity due to the co-sensitization effect. Under the optimal operating conditions, the photoelectrochemical biosensor demonstrated favorable accuracy and could respond to 0.32 pM (S/N = 3) with a linear concentration range from 1.0 pM to 2.0 µM. Moreover, the proposed PEC DNA biosensor exhibits high sensitivity, excellent specificity, acceptable reproducibility and accuracy, showing a promising potential in DNA bioanalysis and other relative fields.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/análise , Técnicas Eletroquímicas/métodos , Fotoquímica/métodos , Pontos Quânticos/química , Compostos de Cádmio/química , Compostos de Cádmio/efeitos da radiação , DNA de Cadeia Simples/genética , Dissulfetos/química , Eletrodos , Luz , Limite de Detecção , Molibdênio/química , Nitrilos/química , Hibridização de Ácido Nucleico , Pontos Quânticos/efeitos da radiação , Compostos de Selênio/química , Compostos de Selênio/efeitos da radiação
8.
ACS Appl Mater Interfaces ; 11(7): 6759-6768, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30682241

RESUMO

DNA nanotechnology has a great potential in biosensor design including nanostructuring of the biosensor surface through DNA origami, target recognition by means of aptamers, and DNA-based signal amplification strategies. In this paper, we use DNA nanotechnology to describe for the first time the concept of real-time solid-phase monitoring of DNAzyme cleavage activity for the detection of specific single-stranded DNA (ssDNA) with a fiber optic surface plasmon resonance (FO-SPR) biosensor. Hereto, we first developed a robust ligation strategy for the functionalization of the FO-SPR biosensing surface with ssDNA-tethered gold nanoparticles, serving as the substrate for the DNAzyme. Next, we established a relation between the SPR signal change, due to the cleavage activity of the 10-23 DNAzyme, and the concentration of the DNAzyme, showing faster cleavage kinetics for higher DNAzyme concentrations. Finally, we implemented this generic concept for biosensing of ssDNA target in solution. Hereto, we designed a DNAzyme-inhibitor complex, consisting of an internal loop structure complementary to the ssDNA target, that releases active DNAzyme molecules in a controlled way as a function of the target concentration. We demonstrated reproducible target detection with a theoretical limit of detection of 1.4 nM, proving that the presented ligation strategy is key to a universal DNAzyme-based FO-SPR biosensing concept with promising applications in the medical and agrofood sector.


Assuntos
DNA Catalítico/química , DNA de Cadeia Simples/análise , Ouro/química , Nanopartículas Metálicas/química , Ressonância de Plasmônio de Superfície/métodos , DNA de Cadeia Simples/química
9.
J Photochem Photobiol B ; 188: 159-176, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30308399

RESUMO

Owing to the emerging applications of DNA-functionalized TiO2 nanocrystals towards DNA damage detection, it is inevitable to understand the better chemistry as well as in-depth molecular interaction phenomena. Fundamentally, energy difference underlies the layer-by-layer construction, resulted in the increase of the interaction energy and thus, altering the electrochemical behavior. Herein, Density functional theory (DFT) calculations were performed using DMol3 and DFTB+ codes successfully to elucidate the structural, electronics, and vibrational properties of the layer-by-layer components composing ss-DNA/dopamine/TiO2/FTO. The obtained results are in good agreement with the experimental findings. The band gaps of FTO and TiO2 were computationally obtained at 3.335 and 3.136 eV which are comparable with the experimental data (3.500 eV; FTO and 3.200 eV; TiO2). Frontier orbital analysis is also considered to elucidate their electron transfer phenomena. Further, a 100 ns MD simulations are carried out using canonical ensemble embedded with COMPASS-Universal Forcefields generating useful thermodynamics parameters. Binding energies indicate increasing interaction energies for the layer-by-layer nanosystem, in agreement with the increasing diameter of electrochemical impedance spectroscopy (EIS) semicircle. Our results reveal the fundamental understanding of the DNA-functionalized TiO2 nanocrystals down to molecular and electronic level and further, paving a way of its application towards nanoelectrochemical DNA biosensors.


Assuntos
Dano ao DNA , DNA/química , Luz , Nanopartículas/química , Titânio/química , Técnicas Biossensoriais , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/metabolismo , Espectroscopia Dielétrica , Dopamina/química , Flúor/química , Simulação de Dinâmica Molecular , Nanopartículas/toxicidade , Teoria Quântica , Compostos de Estanho/química
10.
World J Microbiol Biotechnol ; 34(10): 149, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30220026

RESUMO

Aptamers are short nucleotide sequences which can specifically bind to a variety of targets with high affinity. They are identified and selected via systematic evolution of ligands by exponential enrichment (SELEX). Compared to antibodies, aptamers offer several advantages including easy labeling, high stability and lower cost. Those advantages make it possible to be a potential for use as a recognition probe to replace antibody in the diagnostic field. This article is intended to provide a comprehensive review, which is focused on systemizing recent advancements concerning SELEX procedures, with special emphasis on the key steps in SELEX procedures. The principles of various aptamer-based detections of pathogenic bacteria and their application are discussed in detail, including colorimetric detection, fluorescence detection, electrochemical detection, lateral flow strip test, mass sensitive detection and PCR-based aptasensor. By discussing recent research and future trends based on many excellent publications and reviews, we attempt to give the readers a comprehensive view in the field of aptamer selection against pathogenic bacteria and their diagnostics application. Authors hope that this review will promote lively and valuable discussions in order to generate new ideas and approaches towards the development of aptamer-based methods for application in pathogenic bacteria diagnosis.


Assuntos
Aptâmeros de Nucleotídeos , Bactérias/química , Técnicas de Diagnóstico Molecular/métodos , Técnica de Seleção de Aptâmeros/métodos , Bactérias/patogenicidade , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/análise , RNA/análise
11.
Chem Commun (Camb) ; 54(79): 11108-11111, 2018 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-30101270

RESUMO

We describe an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin functionalised ssDNA probes. The sensitivity at the attomolar concentration level arises from an increased density of redox labels on the electrode surface compared to sensors without NP modification. The electrode detects as few as 23 DNA molecules, approaching single molecule detection.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Ouro/química , Nanopartículas Metálicas/química , Metaloporfirinas/química , Adsorção , Cobalto/química , DNA de Cadeia Simples/genética , Técnicas Eletroquímicas/métodos , Eletrodos , Limite de Detecção , Hibridização de Ácido Nucleico , Oxirredução , Tamanho da Partícula
12.
Talanta ; 188: 325-331, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029383

RESUMO

Owing to their unique physical and chemical properties like stability, non-toxic, biocompatibility and feasible to modification with various biomolecules, gold nanoparticle has become a versatile nanomaterial in the field of therapeutic, diagnostic and analytical studies. Various surface plasmon resonance based pathogen detection systems, relying on change in colour, have been proposed. However, all the approaches developed so far were designed for the detection of a single pathogen. In the present study, we have designed a new colorimetric approach based on distant-dependent properties of gold nanoparticle for the detection of multiple targets. A modified multiplex asymmetric PCR in which a universal primer amplifies the multiple targets with the same efficiency was performed. The Limit of detection (LOD) of the designed visual assay is 10 pg of Brucella and Leptospira target DNA and 100 pg of Bovine herpes virus-1 (BoHV-1) target DNA. LOD of 0.5 pg, 0.7 pg and 3.8 pg for Brucella, Leptospira and BoHV-1 respectively was obtained spectrophotometrically. A study on dark field microscopy as a qualitative supporting detection system has also been presented in this study. The designed assay has advantages over earlier reports in terms of multiple organisms detection, specificity and sensitivity of the test.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/análise , Ouro/química , Nanopartículas Metálicas/química , Sondas Moleculares/genética , Brucella/genética , Colorimetria/métodos , DNA de Cadeia Simples/genética , Herpesvirus Bovino 1/genética , Leptospira/genética , Limite de Detecção , Microscopia/métodos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase
13.
Talanta ; 188: 58-65, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029416

RESUMO

In this study,we proposed a triangular silver nanosheets (Tri-SNSs)-layered, Chitosan (CS)-supported three-dimensional of reduced graphene oxide (3D-ERGO) electrochemiluminescence (ECL) biosensing platform using self-designed dual-Ru(bpy)32+ scDNA (Ru2-DNA) as capture probe for ECL biosensing of single-chain DNA (scDNA). Based on the different affinity with scDNA and double chain DNA (dcDNA), the biosensor is designed to recognize the target DNA (t-DNA), which leads to the desorption of a hybrid molecule from the surface of the biosensor, further removing the Ru2-DNA and inhibiting the ECL. Analytical results clearly showed that the electrochemical and ECL behaviors of proposed biosensing platform on the glassy carbon electrode (GCE) exhibited outstanding performance, which was due to large specific surface area, high carrier mobility and strong π-π non-covalent attraction toward single-chain DNA (scDNA) of the stable 3D platform, and ECL amplification of Tri-SNSs. Besides, based on such a system, this strategy can effectively identify full match and mismatched target DNA (M-DNA) with a wide concentration range beyond 7 orders of magnitude and detection limit down to 16.2 aM. Therefore, the 3D biosensing strategy shows potential for the application of bioassays.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/análise , Técnicas Eletroquímicas/métodos , Grafite/química , Nanoestruturas/química , Prata/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Quitosana/química , Complexos de Coordenação/química , DNA de Cadeia Simples/química , Eletrodos , Limite de Detecção , Luminescência , Medições Luminescentes/métodos , Hibridização de Ácido Nucleico , Óxidos/química , Reprodutibilidade dos Testes
14.
J Phys Chem Lett ; 9(15): 4379-4384, 2018 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30016106

RESUMO

Photoluminescence (PL) multiplexing usually relies on spectral or temporal separation. A combination into higher-order multiplexing for biosensing is extremely challenging because the PL intensity is required for target quantification at very low concentrations and the interplay of color, lifetime, and intensity must be carefully adapted. Here, we demonstrate time-gated Förster resonance energy transfer (TG-FRET) from a long-lifetime Tb complex to Cy3.5 and Cy5.5 dyes for spectrotemporal multiplexing of four different DNA targets in the same sample by single-color excitation and two-color detection. We used rolling circle amplification (RCA) for high specificity and sensitivity and for placing Tb donors and dye acceptors at controlled distances within the amplified DNA concatemers. This precise distance tuning led to target-specific PL decays of the FRET pairs and simple, separation-free, and higher-order multiplexed quantification of DNA. The RCA-FRET DNA assay could distinguish very homologous target sequences and provided limits of detection down to 40 zeptomoles (300 aM).


Assuntos
DNA de Cadeia Simples/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carbocianinas/química , Complexos de Coordenação/química , Limite de Detecção , Sensibilidade e Especificidade , Térbio/química
15.
Anticancer Res ; 38(5): 2939-2943, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29715120

RESUMO

BACKGROUND/AIM: The main objective of the present study was to evaluate the significance of apoptosis in the Fletcher's risk classification for gastrointestinal stromal tumors (GISTs). MATERIALS AND METHODS: Apoptotic cells were identified by immunostaining for single-stranded DNA (ssDNA). We assigned each GIST to one of four risk groups: very low risk, n=32; low risk, n=53; intermediate risk, n=15; high risk, n=6). RESULTS: The mean ssDNA labeling index for each group was 8.0±44.2, 20.1±86.5, 18.7±38.6 and 5.7±5.7, respectively. Fletcher's risk classification for GISTs correlated significantly with the ssDNA labeling index (p=0.002). CONCLUSION: The ssDNA labeling index and Ki-67 labeling index were the most significant factors corresponding to the risk grade of GISTs. These findings suggest that the ssDNA labeling index might be useful for predicting aggressive biological behavior of GISTs.


Assuntos
Biomarcadores Tumorais/análise , DNA de Cadeia Simples/análise , Tumores do Estroma Gastrointestinal/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
16.
Acta Biochim Biophys Sin (Shanghai) ; 50(5): 507-515, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635339

RESUMO

Oligonucleotides were screened for strongly silver-stained repetitive sequences. An 'AG'-clustered purine sequence showed strong staining, and the staining density can be compromised by disrupting the continuity of the 'AG'-clustered sequence. The staining-favored sequence was then employed in rolling circle amplification (RCA) for its product detection. A tube-staining method was developed for convenient and visual RCA assay. Moreover, by introducing GalR into RCA, d-galactose was detected by RCA tube-staining with naked eyes without any equipment. About 10 mM d-galactose can be easily identified, and the detection of d-galactose was specific in comparison with that of several other monosaccharides.


Assuntos
Galactose/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Oligonucleotídeos/análise , Coloração pela Prata/métodos , Sequência de Bases , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Eletroforese em Gel de Poliacrilamida , Galactose/química , Galactose/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/genética , Reprodutibilidade dos Testes
17.
Anal Chim Acta ; 1013: 79-86, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-29501095

RESUMO

A novel dual-sensing fluorescence probe L was designed and synthesized for highly selective and sensitive detection of Zn2+ and DNA. The probe L achieved a detection limit of 3.8 nM for Zn2+, which is lower than the acceptable level of Zn2+ in living cells. The probe L displayed high selectivity toward Zn2+ over other interference metal ions and amino acids. Moreover, the probe L displayed low cytotoxicity and good cell permeability, indicating its potential for detecting and bio-imaging of Zn2+. In addition, the probe L-Zn2+ exhibited enhanced fluorescence signal for DNA detection through the metal-coordination interaction between Zn2+ and DNA. The enhanced signal is higher than that of the classical ethidium bromide probe. The experiments in aqueous media verified the feasibility of applying probe L in real samples.


Assuntos
DNA de Cadeia Simples/análise , Corantes Fluorescentes/química , Zinco/análise , Corantes Fluorescentes/síntese química , Células HeLa , Humanos
18.
Talanta ; 182: 259-266, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29501150

RESUMO

Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples/análise , DNA/análise , Nanopartículas de Magnetita/química , Nanoestruturas/química , Análise Espectral Raman/normas , Cloretos/química , Sondas de DNA/síntese química , Sondas de DNA/química , Compostos Férricos/química , Ouro/química , Humanos , Sequências Repetidas Invertidas , Nanopartículas de Magnetita/ultraestrutura , Nanoestruturas/ultraestrutura , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Sensibilidade e Especificidade , Dióxido de Silício/química , Soluções
19.
Talanta ; 181: 65-72, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29426543

RESUMO

Poly(acrylic acid) (PAA) brushes coated onto silica nanoparticles have been widely utilized in bioassays due to their abilities of providing favorable microenvironments and ensuring good biological activities for biomolecules. However, traditional PAA brushes are synthesized by reversible addition-fragmentation chain transfer polymerization. Hence, it is generally difficult to control and characterize the molecular weight of the PAA brushes, which may depress the reproducibility and bring more uncertain results. Herein, atom transfer radical polymerization method is employed to synthesize ß-cyclodextrin-cored PAA with uniform and controllable molecular weight. After loading on the surfaces of adamantane-functionalized silica nanoparticles via host-guest interactions, glucose oxidase and probe single strand DNA (ssDNA) are further immobilized on the as-prepared nanoparticles. Meanwhile, capture ssDNA is functionalized on amino modified magnetic beads. In the presence of ssDNA sequence of Hepatitis B Virus (HBV) containing completely matched sequence of both probe and capture ssDNA, a bioconjugate is formed and can be separated by an external magnet. The isolated glucose oxidase can further catalyze glucose into gluconic acid and H2O2, and then reduce HAuCl4 on Au seeds. By monitoring the absorption intensity change of the Au NPs at 530nm, the proposed biosensor with novel signal amplification probes can be used to detect DNA sequence of HBV with high sensitivity and selectivity in both buffer and serum samples. This developed strategy has presented a new way to construct silica nanoparticles coated by PAA brushes for the fields of clinical diagnosis and other life sciences.


Assuntos
Resinas Acrílicas/química , DNA Viral/análise , Nanopartículas Metálicas/química , Dióxido de Silício/química , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , DNA Viral/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Reprodutibilidade dos Testes , beta-Ciclodextrinas/química
20.
Bioelectrochemistry ; 118: 106-113, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28780443

RESUMO

Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH3)6,2Cl-] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10-21 to 1×10-9M with a lower detection limit of 1.55×10-21M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Peixes , Ouro/química , Nanopartículas Metálicas/química , Nanotubos de Carbono/química , Análise para Determinação do Sexo/métodos , Animais , Técnicas Biossensoriais/instrumentação , Ácidos Carboxílicos/química , DNA/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Eletrodos , Feminino , Concentração de Íons de Hidrogênio , Masculino , Hibridização de Ácido Nucleico , Sonicação , Fatores de Tempo , Raios Ultravioleta
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