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1.
Life Sci ; 251: 117609, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32272180

RESUMO

AIMS: To identify the target of an adipose specific aptamer adipo-8, predict the potential interaction between adipo-8 and its target, and investigate lipid-lowering effect of adipo-8 in vitro and in vivo. MAIN METHODS: Distinct membranous protein of 3T3-L1 adipocyte pulled-down by adipo-8 was mass-spectrometry analyzed as target candidate(s), and affinity of adipo-8 to target protein-silent adipocyte was detected to validate it. Interaction between adipo-8 and target was predicted by bioinformatic analysis, further confirmed by aptamer truncation and competitive binding assay. To investigate lipid-lowering effect of adipo-8 and mechanism behind, 250 nmol/L adipo-8 or library was incubated with 3T3-L1 adipocyte or target-protein-silent adipocyte for 24 h, and 0.01 µg/g/day adipo-8 or library was administrated to high-fat-fed male mice for 21 days. KEY FINDINGS: APMAP (Adipocyte Plasma Membrane Associated Protein) was identified as adipo-8 target, and adipo-8 affinity to adipocytes was in proportional to APMAP expression. Docking model between the stem-loop structure of adipo-8 and APMAP were predicted that adipo-8 was likely to interact with APMAP at its amino-acid 275-411 sequence. Moreover, adipo-8 could ameliorate fat deposition through interaction with APMAP in vitro, and administration of adipo-8 in high-fat-diet fed mice resulted in body weight loss and blood triglyceride decrease without liver or renal dysfunction. SIGNIFICANCE: Adipo-8 could recognize APMAP specifically and interact with its targets to ameliorate fat deposition in vitro and in vivo. Aptamer adipo-8 has potential to act as an effective and safe targeted drug for obesity and obesity related diseases.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Células 3T3-L1 , Animais , Dieta Hiperlipídica , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular
2.
Food Chem ; 317: 126459, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32113141

RESUMO

The widespread exposure of bisphenol A (BPA) presents a significant risk to human health. A rapid, ultra-sensitive and label-free colorimetric aptasensor using high affinity truncated aptamers was developed for BPA detection. Truncated 38-mer and 12-mer aptamers specific for BPA were obtained through rationally truncation from 63-mer BPA aptamer. The dissociation constants (Kd) of 38-mer and 12-mer aptamers were determined to be 13.17 nM and 27.05 nM. Then, truncated aptamers were used in label-free colorimetric detection assays based on gold nanoparticles (AuNPs). The limit of detections of aptasensors using 38-mer and 12-mer aptamers were 7.60 pM and 14.41 pM, which were 265-fold and 140-fold lower than that of the aptasensor using 63-mer aptamer, respectively. The recovery rates in milk, orange juice and mineralized water samples were 93.88% to 107.30%. Therefore, the developed BPA colorimetric aptasensor using truncated aptamers has great application prospects in food safety control and environmental monitoring.


Assuntos
Aptâmeros de Nucleotídeos/química , Compostos Benzidrílicos/análise , Colorimetria/métodos , Análise de Alimentos/métodos , Fenóis/análise , Animais , Aptâmeros de Nucleotídeos/metabolismo , Compostos Benzidrílicos/química , Compostos Benzidrílicos/metabolismo , Análise de Alimentos/instrumentação , Contaminação de Alimentos/análise , Sucos de Frutas e Vegetais/análise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Simulação de Acoplamento Molecular , Fenóis/química , Fenóis/metabolismo , Sensibilidade e Especificidade
3.
Nat Commun ; 11(1): 1283, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152311

RESUMO

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (ß-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Fibroblastos/citologia , RNA/metabolismo , Imagem Individual de Molécula , Actinas/genética , Actinas/metabolismo , Sobrevivência Celular , Fibroblastos/metabolismo , Fluorescência , Células HEK293 , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
4.
Cardiovasc Ther ; 2020: 6869856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32042311

RESUMO

Objectives: To observe the effect of avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Background: Percutaneous transluminal coronary angioplasty (PTCA) is currently the preferred method for the treatment of coronary heart disease. However, vascular restenosis still occurs after PTCA treatment, severely affecting the clinical efficacy of PTCA. Integrin avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Methods: In this experiment, we used systematic evolution of ligands by exponential enrichment (SELEX) to screen out avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. Results: In the present study, we found that avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. P < 0.05). Avß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism. P < 0.05). AvP < 0.05). Av. Conclusions: The findings suggest that avß3 ssDNA inhibited the proliferation and migration of VSMCs by suppressing the activation of Ras-PI3K/MAPK signaling.ß3 single-stranded (ss) DNA on proliferation and migration of vascular smooth muscle cells (VSMCs) and its potential mechanism.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Movimento Celular , Proliferação de Células , DNA de Cadeia Simples/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Aptâmeros de Nucleotídeos/genética , Células Cultivadas , DNA de Cadeia Simples/genética , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Integrina alfaVbeta3/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Osteopontina/genética , Osteopontina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas ras/genética
5.
Nucleic Acids Res ; 48(7): 3975-3986, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32095808

RESUMO

Guanine-rich regions of the human genome can adopt non-canonical secondary structures. Their role in regulating gene expression has turned them into promising targets for therapeutic intervention. Ligands based on polyaromatic moieties are especially suitable for targeting G-quadruplexes utilizing their size complementarity to interact with the large exposed surface area of four guanine bases. A predictable way of (de)stabilizing specific G-quadruplex structures through efficient base stacking of polyaromatic functional groups could become a valuable tool in our therapeutic arsenal. We have investigated the effect of pyrene-modified uridine nucleotides incorporated at several positions of the thrombin binding aptamer (TBA) as a model system. Characterization using spectroscopic and biophysical methods provided important insights into modes of interaction between pyrene groups and the G-quadruplex core as well as (de)stabilization by enthalpic and entropic contributions. NMR data demonstrated that incorporation of pyrene group into G-rich oligonucleotide such as TBA may result in significant changes in 3D structure such as formation of novel dimeric topology. Site specific structural changes induced by stacking of the pyrene moiety on nearby nucleobases corelate with distinct thrombin binding affinities and increased resistance against nuclease degradation.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Pirenos/química , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/metabolismo , Desoxirribonucleases , Dimerização , Entropia , Humanos , Termodinâmica , Trombina/metabolismo , Nucleotídeos de Uracila/química
6.
Nucleic Acids Res ; 48(5): 2709-2722, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31943114

RESUMO

RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit RT in enzymatic and viral replication assays. Some aptamers inhibit RT from only a few viral clades, while others show broad-spectrum inhibition. Biophysical determinants of recognition specificity are poorly understood. We investigated the interface between HIV-1 RT and a broad-spectrum UCAA-family aptamer. SAR and hydroxyl radical probing identified aptamer structural elements critical for inhibition and established the role of signature UCAA bulge motif in RT-aptamer interaction. HDX footprinting on RT ± aptamer shows strong contacts with both subunits, especially near the C-terminus of p51. Alanine scanning revealed decreased inhibition by the aptamer for mutants P420A, L422A and K424A. 2D proton nuclear magnetic resonance and SAXS data provided constraints on the solution structure of the aptamer and enable computational modeling of the docked complex with RT. Surprisingly, the aptamer enhanced proteolytic cleavage of precursor p66/p66 by HIV-1 protease, suggesting that it stabilizes the productive conformation to allow maturation. These results illuminate features at the RT-aptamer interface that govern recognition specificity by a broad-spectrum antiviral aptamer, and they open new possibilities for accelerating RT maturation and interfering with viral replication.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , Aptâmeros de Nucleotídeos/química , Simulação de Acoplamento Molecular , Mutagênese/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Multimerização Proteica , Inibidores da Transcriptase Reversa/farmacologia
7.
FASEB J ; 34(1): 365-385, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914616

RESUMO

Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP90-231) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP90-231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP90-231 phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs.


Assuntos
Amiloide/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Extração Líquido-Líquido/métodos , Doenças Priônicas/patologia , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Animais , Camundongos , Conformação de Ácido Nucleico , Doenças Priônicas/metabolismo , Proteínas Priônicas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnica de Seleção de Aptâmeros
8.
Talanta ; 207: 120280, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594565

RESUMO

Thrombin and its aptamers have been well studied and widely used as models in aptamer based assays and sensors. Here we reported a thrombin-linked sandwich immunoassay for proteins to demonstrate new applications of thrombin and the aptamers, converting protein detection to analysis of thrombin label. In this assay, target protein was sandwiched by the capture antibody on a microplate and the biotinylated detection antibody. Thrombin bound to one biotinylated aptamer, and then the thrombin-labeled aptamer was attached on the sandwich complex through streptavidin-biotin interaction by using streptavidin as a linker. Thrombin catalyzed cleavage of fluorogenic peptide substrates, generating fluorescence signals for target detection. Among a few different anti-thrombin aptamers, the use of one nuclease resistant RNA aptamer having phosphorodithioate (PS2) modification on a specific backbone position enabled higher assay sensitivity due to its much higher affinity. This thrombin-linked sandwich immunoassay allowed detection of prostate-specific antigen (PSA) at 2 pM, an important protein related cancer disease, with high sensitivity and specificity. The strategy was general, and also enabled sensitive detection of botulinum neurotoxin type A (BoNTA) light chain, one toxin protein causing risk to human health. This assay combines advantages of antibody recognition, aptamer affinity labeling, high affinity of aptamers, and enzyme activity of thrombin. Labeling thrombin on the immunosandwich complex through simple affinity binding overcomes limitations of covalent conjugating enzyme on antibody in conventional immunoassay. This assay is promising in applications for protein detection.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Limite de Detecção , Fosfatos/química , Proteínas/análise , Estudos de Viabilidade , Humanos , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/metabolismo , Proteínas/metabolismo
9.
Talanta ; 207: 120321, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594568

RESUMO

An optical aptasensor-based sensing platform for rapid insulin detection was fabricated. Aminated porous silica microparticles (PSiMPs) were synthesized via a facile mini-emulsion method to provide large surface area for covalent immobilization of insulin-binding DNA aptamer (IGA3) by glutaraldehyde cross-linking protocol. A Nickel-salphen type complex with piperidine side chain [Ni(II)-SP] was synthesized with a simple one-pot reaction, and functionalized as an optical label due to strong π-π interaction between aromatic carbons of G-quadruplex DNA aptamer and planar aromatic groups of Ni(II)-SP to form the immobilized IGA3-Ni(II)-SP complex, i.e. the dye-labeled aptamer, thereby bringing yellow colouration to the immobilized G-quartet plane. Optical characterization of aptasensor towards insulin binding was carried out with a fiber optic reflectance spectrophotometer. The maximum reflectance intensity of the immobilized IGA3-Ni(II)-SP complex at 656 nm decreased upon binding with insulin as aptasensor changed to brownish orange colouration in the background. This allows optical detection of insulin as the colour change of aptasensor is dependent on the insulin concentration. The linear detection range of the aptasensor is obtained from 10 to 50 µIU mL-1 (R2 = 0.9757), which conformed to the normal fasting insulin levels in human with a limit of detection (LOD) at 3.71 µIU mL-1. The aptasensor showed fast response time of 40 min and long shelf life stability of >3 weeks. Insulin detection using healthy human serums with informed consent provided by participants suggests the DNA aptamer biosensor was in good agreement with ELISA standard method using BIOMATIK Human INS (Insulin) ELISA Kit.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/instrumentação , Insulina/análise , Dispositivos Ópticos , Compostos Organometálicos/química , Fenilenodiaminas/química , Aptâmeros de Nucleotídeos/química , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Insulina/sangue , Insulina/química , Insulina/metabolismo , Limite de Detecção , Porosidade , Bases de Schiff/química , Dióxido de Silício/química , Fatores de Tempo
10.
Talanta ; 207: 120300, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594586

RESUMO

A "signal-on" chemiluminescence biosensor was established for detecting thrombin. The thrombin aptamer1-functionalized magnetic sodium alginate (Malg-Apt1) hydrogel was synthesized by physical interaction between sodium alginate and Ca2+, and it was used in the biosensor for separating and enriching thrombin. Ethylenediamine tetraacetic acid (EDTA) was used to chelate with Ca2+ to dissolve the hydrogel and release thrombin. A metalloporphyrinic metal-organic framework nanosheet, named as Cu-TCPP(Co) MOFs, was prepared as signal amplification strategy. Cu-TCPP(Co) MOFs/Au-ssDNA (ssDNA: single-strand DNA) was synthesized for controllable further amplification of chemiluminescent signal. The thrombin aptamer2-functionalized magnetic carbon nanotubes (MCNTs-Apt2) were used as a matrix, and Cu-TCPP(Co) MOFs/Au-ssDNA was adsorbed on the MCNTs by the complementary pairing of the partial bases between ssDNA and Apt2. Compared with ssDNA, Apt2 has a stronger interaction with thrombin. Therefore, thrombin can trigger the release of Cu-TCPP(Co) MOFs/Au-ssDNA to achieve signal amplification. Under the optimal conditions, the biosensor could detect thrombin as low as 2.178 × 10-13 mol/L with the range from 8.934 × 10-13 to 5.956 × 10-10 mol/L and exhibited excellent selectively. Moreover, the "signal-on" chemiluminescence biosensor showed potential application for the detection of thrombin in body fluids.


Assuntos
Alginatos/química , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/química , Hidrogéis/química , Estruturas Metalorgânicas/química , Nanotubos de Carbono/química , Trombina/análise , Adsorção , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , DNA de Cadeia Simples/genética , Medições Luminescentes , Imãs/química , Modelos Moleculares , Conformação Molecular , Porfirinas/química , Trombina/metabolismo
11.
Food Chem ; 302: 125359, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31442702

RESUMO

A simple and rapid sensing strategy was proposed for chloramphenicol (CAP) detection based on structure-switching signaling aptamers. In this protocol, the aptamer can bind to both the fluorophore (FAM)-labeled complementary strand and the quencher (BHQ1)-labeled complementary strand, thus leading to the effective quenching of FAM fluorescence by BHQ1. However, when CAP is present, the structure switch is reversed because the aptamer recognizes CAP, resulting in fluorescence recovery. Such a fluorescence-sensing platform can monitor CAP within a good linear range (1-100 ng/mL), with a detection limit of 0.70 ng/mL. Cross-reactivity with other common antibiotics is negligible, indicating the excellent selectivity of the strategy. Moreover, as the aptamers are not modified, this method is simple and low-cost. The present work reveals a new direction for detecting CAP or other target compounds without prior knowledge of the secondary or tertiary structures of the aptamer.


Assuntos
Aptâmeros de Nucleotídeos/química , Cloranfenicol/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Análise de Alimentos/métodos , Animais , Antibacterianos/análise , Aptâmeros de Nucleotídeos/metabolismo , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Limite de Detecção , Leite/química , Sensibilidade e Especificidade
12.
Talanta ; 208: 120369, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816724

RESUMO

In this study, we developed a simple and cost effective aptasensor based on TiO2 nanotubes-reduced graphene oxide (TiO2 nanotube-rGO) linked to MUC1 aptamers for ultrasensitive electrochemical detection of breast cancer cell (MCF-7). Moreover, the photothermal performance of nanohybrid TiO2-rGO was investigated for cancer treatment. In this regard, after synthesize of TiO2 nanotubes via anodization process, TiO2 nanotubes-rGO hybrid was synthesized by UV assisted reduction of GO and subsequent TiO2 nanotubes attachment to rGO sheets. The resultant hybrid could provide an excellent large surface area leading to improvement of suitable sites for MUC1 aptamer immobilization. Our results revealed that TiO2-rGO aptasensor exhibited superior analytical performance for MCF-7 cell detection with the detection limit of 40 cells.ml-1 within the detection range of 103-107 cells. ml-1. In addition, the designed aptasensor was effectively applied to detect MUC1 marker in a real sample. Moreover, the TiO2 nanotube-rGO hybrid nanoparticles revealed great photothermal performance exposed to NIR laser. It could be concluded that nanohybrid TiO2-rGO would be a useful and beneficial platform for detection and treatment of breast cancer.


Assuntos
Técnicas Biossensoriais/métodos , Neoplasias da Mama/patologia , Grafite/química , Nanopartículas/química , Nanotubos/química , Temperatura , Titânio/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Neoplasias da Mama/sangue , Eletroquímica , Humanos , Células MCF-7 , Modelos Moleculares , Conformação Molecular , Oxirredução
13.
Mikrochim Acta ; 187(1): 25, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811449

RESUMO

The detection of thrombin by using CdS nanocrystals (CdS NCs), gold nanoparticles (AuNPs) and luminol is investigated in this work. Thrombin is detected by three methods. One is called the quenching method. It is based on the quenching effect of AuNPs on the yellow fluorescence of CdS NCs (with excitation/emission wavelengths of 355/550 nm) when placed adjacent to CdS NCs. The second method (called amplification method) is based on an amplification mechanism in which the plasmonics on the AuNPs enhance the emission of CdS NCs through distance related Förster resonance energy transfer (FRET). The third method is ratiometric and based on the emission by two luminophores, viz. CdS NCs and luminol. In this method, by increasing the concentration of thrombin, the intensity of CdS NCs decreases, while that of luminol increases. The results showed that ratiometric method was most sensitive (with an LOD of 500 fg.mL-1), followed by the amplification method (6.5 pg.mL-1) and the quenching method (92 pg.mL-1). Hence, the latter is less useful. Graphical abstract Schematic representation of three different methods (quenching, amplification and ratiometric) were applied for detection of thrombin via aptasensor. The CdS nanocrystals, streptavidin (Str) coated AuNPs and also Str-luminol coated AuNPs were used for the construction steps of the electrochemiluminescence (ECL)-based biosensor.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Compostos de Cádmio/química , Pontos Quânticos/química , Sulfetos/química , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Eletroquímica , Eletrodos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Medições Luminescentes , Trombina/metabolismo
14.
Mikrochim Acta ; 187(1): 13, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31802241

RESUMO

A proof-of-concept aptamer-based optical assay is described for the determination of the immuno signalling molecule interleukin-6 (IL-6), a key marker of acute inflammation. The optical assay is based on the aggregation of gold nanoparticles (AuNP) coated in two complimentary "sandwich-style" aptamers, each with different IL-6 target moieties. IL-6 will recognise the complimentary aptamer pair and bind to it, thereby causing the aggregation of the corresponding functionalised nanoparticles. The aggregation of the AuNPs after exposure to IL-6 induces a visible colour change from red to pink, with a corresponding change in the absorption maximum from 520 to 540 nm. The change in the absorption maximum can be monitored visually, or by using a spectrophotometer or a plate reader. The optimal size and functionalisation of aptamer-coated AuNPs, and the potential assay formats were investigated using UV-vis spectrophotometry, transmission electron microscopy, and dynamic light scattering. The optical assay was applied for detecting mouse IL-6 in a mixed protein solution as a representative biological sample. The assay works in the 3.3 to 125 µg·mL-1 IL-6 concentration range, and the detection limit (at S/N = 3) is 1.95 µg·mL-1. This study was performed as a proof-of-concept demonstration of this versatile assay design, with a view to developing a similar assay for use in clinical samples in future. Graphical abstractSchematic representation of the aggregation of aptamer-functionalised nanoparticles in the presence of interleukin-6 (IL-6). The presence of mouse IL-6 in a mixed protein solution leads to a visible colour change, and a change in the absorption spectrum of the nanoparticles.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Ouro/química , Interleucina-6/análise , Nanopartículas Metálicas/química , Aptâmeros de Nucleotídeos/metabolismo , Inflamação/diagnóstico , Interleucina-6/metabolismo , Cinética
15.
Mikrochim Acta ; 187(1): 5, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31797120

RESUMO

An impedimetric single-shot assay is described for the determination of the proteinic breast cancer marker MUC1. The surface of a glassy carbon electrode was modified with core-shell nanofibers, multi-walled carbon nanotubes and gold nanoparticles that were covalently modified with the MUC1-binding aptamer. Detection is based on the change of the resistance of the electrode surface as measured by electrochemical impedance spectroscopy using hexacyanoferrate(II/III) as an electrochemical probe in working potential is 0.25 V. Scanning electron microscopy and cyclic voltammetry were also applied to characterize the electrode. The analytical response ranges from 5 to 115 nM of MUC1, with a detection limit of 2.7 nM. The assay was successfully applied to MUC1 determination in spiked serum samples where it gave satisfactory results. Graphical abstractAn impedimetric nanoprobe for the tumor marker MUC1 is proposed. It is based on use of electrospun honey core-shell nanofibers. The nanoprobe exhibits excellent sensitivity, good stability and a low detection limit.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/análise , Eletricidade , Mucina-1/análise , Nanofibras , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Impedância Elétrica , Eletrodos , Ferrocianetos/química , Humanos , Imunoensaio , Limite de Detecção , Mucina-1/sangue , Mucina-1/metabolismo , Nanotubos de Carbono/química
16.
Nat Commun ; 10(1): 5476, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792209

RESUMO

There are disease-causing biohazards in the blood that cannot be treated with modern medicines. Here we show that an intelligently designed safe biomaterial can precisely identify, tow and dump a targeted biohazard from the blood into the small intestine. Positively charged mesoporous silica nanoparticles (MSNs) functionalized with EGFR-targeting aptamers (MSN-AP) specifically recognize and bind blood-borne negatively charged oncogenic exosomes (A-Exo), and tow A-Exo across hepatobiliary layers and Oddi's sphincter into the small intestine. MSN-AP specifically distinguish and bind A-Exo from interfering exosomes in cell culture and rat and patient blood to form MSN-AP and A-Exo conjugates (MSN-Exo) that transverse hepatocytes, cholangiocytes, and endothelial monolayers via endocytosis and exocytosis mechanisms, although Kupffer cells have been shown to engulf some MSN-Exo. Blood MSN-AP significantly decreased circulating A-Exo levels, sequentially increased intestinal A-Exo and attenuated A-Exo-induced lung metastasis in mice. This study opens an innovative avenue to relocate blood-borne life-threatening biohazards to the intestine.


Assuntos
Sangue/metabolismo , Exossomos/metabolismo , Intestino Delgado/metabolismo , Nanopartículas/metabolismo , Células A549 , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Análise Química do Sangue , Endocitose , Receptores ErbB/genética , Receptores ErbB/metabolismo , Exossomos/química , Hepatócitos/metabolismo , Humanos , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Ratos , Dióxido de Silício/química , Dióxido de Silício/metabolismo
18.
Mikrochim Acta ; 187(1): 20, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31807965

RESUMO

A multi-channel localized surface plasmon resonance system is described for absorptiometric determination of abscisic acid (ABA). The system is making use of gold nanoparticles and consists of a broadband light source, a multi-channel alignment device, and a fiber spectrometer. The method is based on the specific interaction between an ABA-binding aptamer and ABA. This induces the growth of gold nanoparticles (AuNPs) functionalized with a polyadenine-tailed aptamer that act as optical probes. Different concentrations of ABA give rise to varied morphologies of grown AuNPs. This causes a change of absorption spectra which is recorded by the system. ABA can be quantified by measurement of the peak wavelength shifts of grown AuNPs. Under optimized conditions, this method shows a linear relationship in the 1 nM to 10 µM ABA concentration range. The detection limit is 0.51 nM. The sensitivity of the ABA assay is strongly improved compared to the method based on salt-induced AuNP aggregation. This is attributed to the use of a poly-A-tailed aptamer and the catalytic ability of AuNPs. In the actual application, the ABA concentration of ABA in fresh leaves of rice is measured with the maximum relative error of 8.03% in comparison with the ELISA method. Graphical abstractSchematic representation of an absorptiometric approach for determination of abscisic acid based on the growth of polyA-tailed aptamer-AuNPs probes and a multi-channel localized surface plasmon resonance system.


Assuntos
Ácido Abscísico/análise , Aptâmeros de Nucleotídeos/química , Ouro/química , Nanopartículas Metálicas/química , Poli A/química , Ressonância de Plasmônio de Superfície/métodos , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Absorção Fisico-Química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Oryza/química
19.
Mikrochim Acta ; 187(1): 9, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31797061

RESUMO

The target-triggered DNA assembling probe is presented for highly selective protein detection. Target-triggered DNA assembling is used in an amplification strategy based on affinity binding for identification and determination of proteins in general. Specifically, it was applied to the platelet derived growth factor-BB (PDGF-BB). A hairpin DNA (H-DNA) probe was designed containing (a) an aptamer domain for protein recognition and (b) a blocked DNAzyme domain for DNAzyme cleavage. An assistant DNA (A-DNA) probe containing aptamer and complementary domains was also employed to recognize protein and to induce DNA assembly. Once H-DNA and A-DNA recognize the same protein, H-DNA and A-DNA are in close proximity to each other. This induces DNA assembling for protein-triggered complex (Protein-Complex) with free DNAzyme domains. The free DNAzymes trigger the circular cleavage of molecular beacons for amplified signals. The assay is performed by fluorometry at an excitation wavelength of 980 nm and by collecting fluorescence at 545 nm. The platelet derived growth factor-BB (PDGF-BB) was accurately identified and selectively determined by this assay with a 22 pM detection limit (using the 3σ criterion). The responses for PDGF-BB is nearly 6-fold higher than for PDGF-AB, and 16-fold higher than PDGF-AA. This upconversion assay avoids any interference by the autofluorescence of biological fluids. Graphical abstractSchematic representation of the principle of the target-triggered DNA assembling probes mediated amplification strategy based on affinity binding for PDGF-BB. The UCNP probe is used for the quantitation of PDGF-BB with high selectivity.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Becaplermina/análise , Técnicas Biossensoriais/métodos , Sondas de DNA/metabolismo , Nanopartículas/química , Aptâmeros de Nucleotídeos/química , Becaplermina/sangue , Becaplermina/metabolismo , Becaplermina/urina , Sondas de DNA/química , Ensaio de Imunoadsorção Enzimática , Estudos de Viabilidade , Fluorometria , Humanos
20.
Sensors (Basel) ; 20(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861555

RESUMO

Among prevalent food allergies, cow milk allergy (CMA) is most common and may persist throughout the life. The allergic individuals are exposed to a constant threat due to milk proteins' presence in uncounted food products like yogurt, cheese, and bakery items. The problem can be more severe due to cross-reactivity of the milk allergens in the food products due to homologous milk proteins of diverse species. This problem can be overcome by proper and reliable food labeling in order to ensure the life quality of allergic persons. Therefore, highly sensitive and accurate analytical techniques should be developed to detect the food allergens. Here, significant research advances in biosensors (specifically immunosensors and aptasensors) are reviewed for detection of the milk allergens. Different allergic proteins of cow milk are described here along with the analytical standard methods for their detection. Additionally, the commercial status of biosensors is also discussed in comparison to conventional techniques like enzyme-linked immunosorbent assay (ELISA). The development of novel biosensing mechanisms/kits for milk allergens detection is imperative from the perspective of enforcement of labeling regulations and directives keeping in view the sensitive individuals.


Assuntos
Alérgenos/análise , Técnicas Biossensoriais/métodos , Leite/metabolismo , Nanotecnologia/métodos , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Bovinos , Técnicas Eletroquímicas , Ensaio de Imunoadsorção Enzimática , Limite de Detecção , Magnetismo
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