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1.
PLoS One ; 15(9): e0238467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32877464

RESUMO

Resolving the genetic architecture of painful neuropathy will lead to better disease management strategies. We aimed to develop a reliable method to re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. In this study, we compared sensitivity, specificity, targeting efficiency, performance and cost effectiveness of Molecular Inversion Probes-Next generation sequencing (MIPs-NGS) and TruSeq® Custom Amplicon-Next generation sequencing (TSCA-NGS). Capture probes were designed to target nine sodium channel genes (SCN3A, SCN8A-SCN11A, and SCN1B-SCN4B). One hundred sixty-six patients with diabetic and idiopathic neuropathy were tested by both methods, 70 patients were validated by Sanger sequencing. Sensitivity, specificity and performance of both techniques were comparable, and in agreement with Sanger sequencing. The average targeted regions coverage for MIPs-NGS was 97.3% versus 93.9% for TSCA-NGS. MIPs-NGS has a more versatile assay design and is more flexible than TSCA-NGS. The cost of MIPs-NGS is >5 times cheaper than TSCA-NGS when 500 or more samples are tested. In conclusion, MIPs-NGS is a reliable, flexible, and relatively inexpensive method to detect genetic variations in a large cohort of patients. In our centers, MIPs-NGS is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sondas Moleculares/genética , Análise de Sequência de DNA/métodos , Inversão Cromossômica/genética , Sondas de DNA/genética , Testes Genéticos/métodos , Humanos , Mutação , Neuralgia/genética , Sensibilidade e Especificidade
2.
Chem Commun (Camb) ; 56(40): 5409-5412, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32286590

RESUMO

A simple multiplexed digital microRNA detection strategy with fluorescence flow cytometry was proposed. By isothermal ligation-rolling circle amplification, multiplexed microRNAs could be simultaneously converted to a series of nanoflower balls (NFBs) and counted by flow cytometry directly.


Assuntos
Citometria de Fluxo/métodos , MicroRNAs/análise , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/química , MicroRNAs/genética , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Estudo de Prova de Conceito
3.
J Infect Chemother ; 26(5): 523-526, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32147375

RESUMO

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect.


Assuntos
Sondas de DNA/química , Gastroenterite Suína Transmissível/diagnóstico , Nanopartículas/química , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Vírus da Gastroenterite Transmissível/isolamento & purificação , Animais , Sondas de DNA/genética , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Gastroenterite Suína Transmissível/virologia , Limite de Detecção , Imãs , Vírus da Diarreia Epidêmica Suína/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Vírus da Gastroenterite Transmissível/genética
4.
Sci Rep ; 10(1): 4018, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132554

RESUMO

The characteristic features of stem-loop structured probes make them robust tools to detect targets with high sensitivity and selectivity. The basis of the hairpin based sensors operation is a conformational change that occurs upon hybridization of target with stem-loop probe. The design of the stem-loop probe has an important role in target recognition. Therefore, we designed a label-free stem loop probe for targeting miR-21 as a cancer biomarker investigated by web-based tools; its thermodynamic parameters obtained by thermal UV spectroscopy. The efficiency of stem-loop structure opening in the presence of target and non-target sequences was evaluated by fluorescence spectroscopy and circular dichroism spectro-polarimetry. The results showed that the target sequence opens the structure of hairpin efficiently in comparison to non-target sequences. To optimize the stem-loop hybridization to its target, the buffer ionic strength was changed by adding different concentrations of NaCl, KCl and MgCl2. It was shown that buffering conditions have a significant role in loop structure opening and its optimization, led to an increase in sensitivity detection and have improved LOD from 60 pM to 45 pM.


Assuntos
Técnicas Biossensoriais , Sondas de DNA , MicroRNAs , Dicroísmo Circular , Sondas de DNA/química , Sondas de DNA/genética , Humanos , MicroRNAs/análise , MicroRNAs/química , MicroRNAs/genética , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
5.
Talanta ; 212: 120735, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113527

RESUMO

Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Técnicas Eletroquímicas/métodos , Glucuronidase/genética , Compostos de Sulfidrila/química , Fosfatase Alcalina/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Naftalenos/química , Hibridização de Ácido Nucleico , Compostos Organofosforados/química
6.
Talanta ; 212: 120754, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113533

RESUMO

Robust, reliable, and sensitively quantitative detection of genetic biomarkers at single-base resolution has the potential to revolutionize medical diagnostics, especially for precision medicine. Here, taking the advantages of the high specificity of ligase reaction and the powerful amplification features of the isothermally exponential amplification, we have demonstrated a novel methodology to sensitively quantify genetic biomarkers at one-base resolution. The methodology is based on the ligase reaction of two stem-loop DNA probes templated by the nucleic acid targets to form a double stem-loop DNA, which subsequently initiates the isothermally exponential amplification reaction with high amplification efficiency. With the proposed method, high sensitivity to determine as low as 0.01 fM DNA or 0.1 fM RNA targets and high specificity to detect single-base changes can be achieved. The new methodology is robust to be performed by using a pair of universal primers under isothermal conditions, which should be employed to quantitatively detect any genetic biomarkers because all DNA/RNA targets can be directly used as the templates to ligate the stem-loop DNA probes with single-base resolution.


Assuntos
DNA/análise , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Bacteriófago T4/enzimologia , Biomarcadores/análise , DNA/química , DNA/genética , DNA Ligases/química , Metilação de DNA , Sondas de DNA/química , Sondas de DNA/genética , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Hibridização de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , RNA Ligase (ATP)/química , Proteínas Virais/química
7.
Talanta ; 212: 120764, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113537

RESUMO

A magnetic-separation-dual-targets fluorescent biosensor was fabricated to detect terminator nopaline synthase (TNOS) and promoter of cauliflower mosaic virus 35s (P35S) in transgenic soybean based on incorporation of bicolor CdTe quantum dots carried by silica nanospheres. In this protocol, the fixed probes for TNOS or P35S were magnetized firstly with Fe3O4@Au magnetic nanosphere by Au-S covalent bonding to achieve magnetized probes. Meanwhile, the capture probes for TNOS or P35S were functionalized with green or red fluorescent microspheres respectively to obtain fluorescently-labeled probes, which could emit relative strong green or red fluorescent signal. Two terminals of TNOS or P35S were recognized by magnetized probes and fluorescently-labeled probes respectively to form the sandwiched structures in the process of biosensor development subsequently, and it was separated by a magnet instantly. The fluorescence intensities of remnant supernatant were measured and analyzed accordingly to achieve simultaneous detection of TNOS and P35S. This biosensor exhibited a good dynamic range, low limit of detection and excellent selectivity in detecting transgenic soybean.


Assuntos
Aminoácido Oxirredutases/genética , Técnicas Biossensoriais/métodos , DNA Viral/análise , Corantes Fluorescentes/química , Nanosferas/química , Proteínas Virais/genética , Compostos de Cádmio/química , Caulimovirus/química , Caulimovirus/enzimologia , Sondas de DNA/química , Sondas de DNA/genética , DNA Viral/genética , Óxido Ferroso-Férrico/química , Ouro/química , Limite de Detecção , Hibridização de Ácido Nucleico , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Pontos Quânticos/química , Reprodutibilidade dos Testes , Soja , Telúrio/química
8.
Talanta ; 213: 120816, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32200921

RESUMO

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.


Assuntos
Técnicas Biossensoriais/métodos , Carbono/química , Desoxirribonuclease I/química , MicroRNAs/análise , Nanopartículas/química , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sondas de DNA/química , Sondas de DNA/genética , Feminino , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Espectrometria de Fluorescência/métodos
9.
Mikrochim Acta ; 187(2): 119, 2020 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-31927667

RESUMO

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.µL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.


Assuntos
Colorimetria/métodos , DNA Bacteriano/análise , Staphylococcus aureus/isolamento & purificação , Animais , Armoracia/enzimologia , Benzidinas/química , Galinhas , Corantes/química , Sondas de DNA/química , Sondas de DNA/genética , DNA Bacteriano/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Limite de Detecção , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Aves Domésticas/microbiologia , Staphylococcus aureus/química
10.
Talanta ; 209: 120505, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31891997

RESUMO

Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H2O2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10-15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research.


Assuntos
Eletroforese em Microchip/métodos , MicroRNAs/análise , Armoracia/enzimologia , Linhagem Celular Tumoral , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Exodesoxirribonucleases/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Luminescência , Medições Luminescentes , Luminol/química , MicroRNAs/genética , Neoplasias/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico
11.
Talanta ; 209: 120511, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892041

RESUMO

An electrochemical immuno-nanogenosensor is developed based on noble-metal-free nickel phosphate nanostructure (NiPNs) as an excellent biocompatible material for miRNA detection in blood serum and urine samples without using indicators for the first time. The pompon flower-like morphology of NiPNs is synthesized, and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction pattern (XRD), fourier transform-infrared spectroscopy (FT-IR), and electrochemical impedance methods. The novel NiPNs nanostructured interface was constructed by coordinate covalent bonding between Ni and phosphate group of probe DNA. The constructed NiPNs-p-DNA surface served as the amplified hybridization platform enabling efficient access to numerous target microRNA sequences. As a result, the developed NiPFNs biosensing platform displayed excellent sensitivity, selectivity, and ultralow experimental limit-of-detection (LOD) of 0.034 pM (S/N = 3) as compared with other Ni phosphide nanostructures. This simple and efficient approach is highly suitable for the development of point-of-care detection systems. To the extent of our knowledge, this is the first report on trace level detection of miRNAs employing non-noble Ni metal nanostructures based biosensing platform.


Assuntos
MicroRNAs/sangue , MicroRNAs/urina , Nanoestruturas/química , Níquel/química , Fosfatos/química , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Espectroscopia Dielétrica , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico
12.
Chem Commun (Camb) ; 56(8): 1175-1178, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31919487

RESUMO

We developed a bright carbon nanodot-based miRNA detection method with signal amplification by concatenated hybridization chain reaction (CHCR). In the presence of target miRNA, CHCR was triggered and a frond-like DNA product was formed, which recovered remarkable fluorescence. The location and level of the target miRNA were then indicated.


Assuntos
Carbono/química , MicroRNAs/análise , Pontos Quânticos/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Humanos , Limite de Detecção , Células MCF-7 , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
14.
Chem Commun (Camb) ; 56(11): 1681-1684, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31939961

RESUMO

A functionalized dumbbell probe (FDP) based amplification method, termed as a cascading exponential amplification DNA machine (CEA-DNA machine), has been developed to autonomously accumulate single G-quadruplexes (SGQs) and twin-G-quadruplexes (TGQs) for robust fluorescence signal-on probing of miRNA-21.


Assuntos
DNA/química , MicroRNAs/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Espectrometria de Fluorescência/métodos , Benzotiazóis/química , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/química , Quadruplex G , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico
15.
J Pharm Biomed Anal ; 180: 113050, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31881396

RESUMO

A sensitive and specific approach was developed for the determination of Haemophilus influenza using DNA based bio-assay. In this study, citrate capped silver nanoparticle was synthesized and employed for bioconjugation with pDNA toward target sequences detection. In this study, synthesized probe (SH-5'-AAT TTT CCA ACT TTT TCA CCT GCA T-3') of Haemophilus influenza was detected with great sensitivity and selectivity after hybridization with cDNA (5'-ATG CAG GTG AAA AAG TTG GAA AAT T-3'). Regarding to the obtained results, the low limit of quantification (LLOQ) of DNA sample was 1 ZM using 15 µL of probe and 200 µL of Cit/AgNPs. According to ultra-sensitivity of the fabricated optical DNA-based bio-assay, it has potential for bacterial determination both in clinical and environmental specimens. To evaluate the selectivity of developed DNA based biosensor, three mismatch sequences were applied. Finally, the designed genosensor is a significant diagnostic strategy for detection of Haemophilus influenza with great selectivity.


Assuntos
Técnicas Biossensoriais/métodos , Ácido Cítrico/química , Sondas de DNA/química , DNA Bacteriano/análise , Haemophilus influenzae/isolamento & purificação , Nanopartículas Metálicas/química , Prata/química , Bioensaio , Técnicas Biossensoriais/instrumentação , Sondas de DNA/genética , DNA Bacteriano/genética , Haemophilus influenzae/genética , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Sensibilidade e Especificidade
16.
Biochemistry ; 59(4): 400-406, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31887028

RESUMO

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.


Assuntos
DNA Ligase Dependente de ATP/metabolismo , Sondas de DNA/química , Thermus thermophilus/enzimologia , Animais , Clonagem Molecular , DNA/química , DNA/metabolismo , DNA Ligase Dependente de ATP/fisiologia , DNA Ligases/metabolismo , DNA Ligases/fisiologia , Sondas de DNA/genética , Eletroforese em Gel de Poliacrilamida/métodos , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Oligonucleotídeos/química , Oligonucleotídeos/genética , Temperatura , Thermus thermophilus/metabolismo
17.
Anal Chim Acta ; 1095: 179-184, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31864620

RESUMO

Abnormal expression of specific microRNAs (miRNAs) is associated with the occurrence, development and prognosis of many diseases. In this study, a miRNA detection method based on exponential amplification reaction (EXPAR) and triplex DNA mediated aggregation of gold nanoparticles (AuNPs) was established. Specifically, one class of AuNPs is conjugated with an EXPAR probe, on which there is a complementary sequence of the target miRNA. The EXPAR reaction is triggered and duplex DNA is formed on the surface of AuNPs when the target miRNA exists. Then, single DNA probe on another class of AuNPs interacts with the duplex DNA to form triplex DNA, leading to the aggregation of the two classes of AuNPs, which could be quantified by UV-vis. The proposed method is highly selective and can afford a detection limit of 0.23 fM. Notably, all the ingredients needed for the analysis can pre-add to a tube and only 30 min is needed for the whole detection process. The method is simple, fast and with considerable selectivity and accuracy, so a great potential for this method is expected to meet the need of point-of-care testing of miRNA.


Assuntos
Colorimetria/métodos , DNA/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Linhagem Celular Tumoral , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Ouro/química , Humanos , Limite de Detecção , MicroRNAs/genética , Mutação , Técnicas de Amplificação de Ácido Nucleico , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico
18.
Anal Chim Acta ; 1095: 212-218, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31864625

RESUMO

Sensitive and selective detection of miRNA is of great significance for the early diagnosis of human diseases, especially for cancers. Quartz crystal microbalance (QCM) is an effective tool for detecting biological molecules; however, the application of QCM for miRNA detection is still very limited. One of the great needs for QCM detection is to further improve the QCM signal. Herein, for the first time, we promote a new signal enhancement strategy for the detection of miRNA by QCM. First, a hairpin biotin-modified DNA was used as a probe DNA, which exposes the biotin site when interacting with target miRNA. Then, a streptavidin@metal-organic framework (SA@MOF) complex formed by electrostatic attractions between SA and a MOF was introduced into the QCM detection system. The SA@MOF complexes serve as both a signal amplifier and a specific recognition element via specific biotin-SA interactions. The strategy was applied to the detection of a colorectal cancer marker, miR-221, by using a stable Zr(IV)-MOF, UiO-66-NH2. The detection linear range was 10 fM-1 nM, the detection limit was 6.9 fM, and the relative standard deviation (RSD) (n = 5) was lower than 10% in both simulated conditions and the real serum environment. Furthermore, the detection limit reached 0.79 aM when coupled with the isothermal exponential amplification reaction (EXPAR).


Assuntos
Estruturas Metalorgânicas/química , MicroRNAs/análise , Estreptavidina/química , Animais , Técnicas Biossensoriais/métodos , Biotina/química , Bovinos , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Técnicas de Microbalança de Cristal de Quartzo/métodos
19.
Zool Res ; 41(1): 94-96, 2020 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-31840950

RESUMO

Many functional elements associated with traits and diseases are located in non-coding regions and act on distant target genes via chromatin looping and folding, making it difficult for scientists to reveal the genetic regulatory mechanisms. Capture Hi-C is a newly developed chromosome conformation capture technology based on hybridization capture between probes and target genomic regions. It can identify interactions among target loci and all other loci in a genome with low cost and high resolution. Here, we developed CaptureProbe, a user-friendly, graphical Java tool for the design of capture probes across a range of target sites or regions. Numerous parameters helped to achieve and optimize the designed probes. Design testing of CaptureProbe showed high efficiency in the design success ratio of target loci and probe specificity. Hence, this program will help scientists conduct genome spatial interaction research. CaptureProbe and source code are available at https://sourceforge.net/projects/captureprobe/.


Assuntos
Cromossomos/genética , Sondas de DNA/genética , Genômica/métodos , Software , Animais , Humanos
20.
Anal Chim Acta ; 1093: 52-60, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31735215

RESUMO

A high performance miRNA biosensor based on effective click chemistry assembly of a Ru(bpy)32+ labeled DNA probe and efficient electrochemiluminescence (ECL) quenching of the Ru(bpy)32+/BDEA (BDEA = N-butyldiethanolamine) system by surface-confined electroactive methylene blue (MB) dye is reported. When the target miRNA was present, the ECL signal instantly changed from "light off" to "light on" status. Using the specific miRNA let-7d as the target analyte, this biosensor provided sensitive detection over approximately six orders of magnitude (10 fM-10 nM), with a limit of detection of 10 fM (S/N = 3). Detailed study of the ECL quenching behavior of the Ru(bpy)32+/BDEA system by MB in solution suggested that the ECL quenching involves a combination of photoluminescence dynamic quenching and quenching processes directly associated with the redox reactions, as well as resonance energy transfer. A large binding constant of 4.7 × 1011 M-1 between let-7d and the DNA hairpin was estimated using an ECL-based extended Langmuir isotherm model, suggesting remarkably strong binding of the target to the probe. Furthermore, our biosensor exhibited excellent specificity and reproducibility. Using the developed system, the concentration of the target miRNA extracted from the A549 cell line could be obtained, demonstrating the potential application of the developed biosensor to practical biological sample analysis.


Assuntos
Técnicas Biossensoriais/métodos , Complexos de Coordenação/química , Técnicas Eletroquímicas/métodos , Medições Luminescentes/métodos , Azul de Metileno/química , MicroRNAs/análise , Células A549 , Sondas de DNA/química , Sondas de DNA/genética , Humanos , Sequências Repetidas Invertidas , MicroRNAs/genética , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes
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