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1.
PLoS One ; 15(9): e0239366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32991599

RESUMO

Platelet-derived growth factor-bb (PDGF-BB) is a potent chemokine and mitogen for fibroblasts, keratinocytes, and vascular endothelium in the injured area, believed to be effective in wound healing. However, the short half-life of PDGF-BB and its rapid release from the wound surface limited its efficacy in vivo and vitro. To evaluate the wound healing effects of dorsal skin in SD rats with polydopamine-assisted immobilized PDGF-BB on PLGA nanofibrous substrate. First, the effects of pDA-coating and PDGF-BB immobilization on the morphology, compositions, and hydrophilicity of substrates were evaluated in details. Second, the wound healing effect of pDA/PLGA/PDGF-BB substrate was assessed in the dorsal skin of SD rats. Last, the cytokine analysis by ELISA method was employed to evaluate the advantages of pDA/PLGA/PDGF-BB substrate on anti-inflammatory, angiogenesis, and cellular proliferation. This method significantly improved the immobilization amount and stability of PDGF-BB on the substrate (p<0.01), further improved the hydrophilicity of substrates (p<0.05). Furthermore, the wound closure process was much more accelerated in the pDA/PLGA/PDGF-BB group (p<0.05). H&E and CD31 staining informed that the wound treated by pDA/PLGA/PDGF-BB substrate showed a high degree of regeneration and angiogenesis. The cytokine analysis showed that pDA significantly reduced the high level of inflammatory cytokines such as TNF-α (p<0.05). And the immobilized PDGF-BB significantly elevated the level of TGF-ß and VEGF (p<0.05). The pDA/PLGA/PDGF-BB substrate showed great therapeutic effect on wound healing compared with other control groups via regulating anti-inflammatory, angiogenesis, and cellular proliferation. Absolutely, this report offered an available novel method for skin regeneration.


Assuntos
Becaplermina/química , Becaplermina/farmacologia , Citocinas/metabolismo , Indóis/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Polímeros/química , Cicatrização/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Cinética , Masculino , Ratos , Ratos Sprague-Dawley
2.
Anal Chim Acta ; 1113: 26-35, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32340666

RESUMO

Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipid-nanodisc systems (membrane-mimicking environment) and miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach was exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 µg of protein per column) were fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound (whose binding site is known) with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range can be detected. At last, the applicability of this method was demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.


Assuntos
Proteínas Imobilizadas/metabolismo , Nanopartículas/química , Compostos Orgânicos/análise , Receptor A2A de Adenosina/metabolismo , Cromatografia de Afinidade/métodos , Descoberta de Drogas , Humanos , Proteínas Imobilizadas/química , Ligantes , Membranas Artificiais , Compostos Orgânicos/metabolismo , Estudo de Prova de Conceito , Ligação Proteica , Receptor A2A de Adenosina/química
3.
Chem Commun (Camb) ; 56(40): 5393-5396, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32285901

RESUMO

A cytochrome c (Cyt c)-modified glass nanopore sensing platform was constructed to sensitively detect glucose in single cells based on changes in the ionic current rectification (ICR) of the system. A difference in glucose content between single H8 and HeLa cells in their satiety or starvation states was clearly able to be detected using this method.


Assuntos
Vidro/química , Glucose/análise , Nanoporos , Análise de Célula Única/métodos , Citocromos c/química , Técnicas Eletroquímicas/métodos , Glucose/química , Glucose Oxidase/química , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Proteínas Imobilizadas/química , Limite de Detecção , Oxirredução
4.
Langmuir ; 36(16): 4556-4562, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32239960

RESUMO

In biological systems, membrane proteins play major roles in energy conversion, transport, sensing, and signal transduction. Of special interest are the photosynthetic reaction centers involved in the initial process of light energy conversion to electrical and chemical energies. The oriented binding of membrane proteins to solid surfaces is important for biotechnological applications. In some cases, novel properties are generated as a result of the interaction between proteins and solid surfaces. We developed a novel approach for the oriented tagging of membrane proteins. In this unique process, bifunctional molecules are used to chemically tag the exposed surfaces of membrane proteins at selected sides of membrane vesicles. The isolated tagged membrane proteins were self-assembled on solid surfaces, leading to the fabrication of dens-oriented layers on metal and glass surfaces, as seen from the atomic force microscopy (AFM) images. In this work, we used chromatophores and membrane vesicles containing protein chlorophyll complexes for the isolation of the bacterial reaction center and photosystem I, from photosynthetic bacteria and cyanobacteria, respectively. The oriented layers, which were fabricated on metal surfaces, were functional and generated light-induced photovoltage that was measured by the Kalvin probe apparatus. The polarity of the photovoltage depended on the orientation of proteins in the layers. Other membrane proteins can be tagged by the same method. However, we preferred the use of reaction centers because their orientation can be easily detected by the polarity of their photovoltages.


Assuntos
Proteínas de Bactérias/química , Proteínas Imobilizadas/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Bactérias/efeitos da radiação , Reagentes para Ligações Cruzadas/química , Eletroquímica , Ouro/química , Proteínas Imobilizadas/efeitos da radiação , Luz , Lipossomos/química , Complexo de Proteína do Fotossistema I/efeitos da radiação , Rhodobacter/enzimologia , Succinimidas/química , Synechocystis/enzimologia
5.
J Chromatogr A ; 1620: 461003, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32156458

RESUMO

The enormous growth in drug discovery paradigm has necessitated continuous exploration of new methods for drug-protein interaction analysis. To enhance the role of these methodologies in designing rational drugs, this work extended an immobilized angiotensin II type I receptor (AT1R) based affinity chromatography in antihypertensive compound identification. We fused haloalkane dehalogenase at C-terminus of AT1R and expressed the fusion receptor in E. coli. The expressed receptor was covalently immobilized onto 8.0 µm microspheres by mixing the cell lysate with 6-chlorocaproic acid-modified amino polystyrene microspheres. The immobilized AT1R was utilized for thermodynamic and kinetic interaction analysis between the receptor and four specific ligands. Following confirmation of these interactions by molecular docking, we identified puerarin and rosmarinic acid by determining their binding to the receptor. Azilsartan, candesartan, valsartan and olmesartan displayed two kinds of binding sites to AT1R by injection amount-dependent method. By molecular docking, we recognize the driving forces of the interaction as electrostatic interaction, hydrogen bonds and van der Waals force. The dissociation rate constants (kd) of azilsartan, candesartan, valsartan and olmesartan to AT1R were 0.01138 ± 0.003, 0.05142 ± 0.003, 0.07547 ± 0.004 and 0.01310 ± 0.005 min-1 by peak profiling assay. Comparing with these parameters, puerarin and rosmarinic acid presented lower affinity (KA: 0.12 × 104 and 1.5 × 104/M) and slower kinetics (kd: 0.6864 ± 0.03 and 0.3005 ± 0.01 min-1) to the receptor. These results, taking together, indicated that the immobilized AT1R has the capacity to probe antihypertensive compounds.


Assuntos
Anti-Hipertensivos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Receptor Tipo 1 de Angiotensina/metabolismo , Anti-Hipertensivos/química , Benzimidazóis/química , Benzimidazóis/metabolismo , Sítios de Ligação , Cromatografia de Afinidade , Cinamatos/metabolismo , Depsídeos/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Isoflavonas/metabolismo , Cinética , Ligantes , Simulação de Acoplamento Molecular , Oxidiazóis/química , Oxidiazóis/metabolismo , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Tetrazóis/química , Tetrazóis/metabolismo , Termodinâmica , Valsartana/química , Valsartana/metabolismo
6.
Talanta ; 213: 120845, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32200931

RESUMO

As important disease diagnostic markers, circulating miRNAs have been put on an urgent agenda in recent years with the focus on their highly sensitive and specific multiplex detection in one reaction. In this article, we proposed a unique miRNAs detection method based on tailored-designed stem-loop structure ligation strategy to realize ideal detection performance. The stem-loop ligation probe had a target miRNA mediated quick ligation, which the relative ligation efficiency of stem-loop structure was superior evidently to straight chain structure about 76% at 10 min on account of the preferentially formed partial dimer. Moreover, the streptavidin-coated magnetic microspheres combined with optimized ligation probes concentration at 10 nM were utilized to purify ligation products and completely eliminated nonspecific ligation substantially. Due to the stem-loop structure ligation products contained universal primer regions, let-7 family as model was used to evaluate the detection performance, demonstrating high sensitivity as the minimum detection limit of 2.5 fM with a five-orders of magnitude dynamic detection range. To some degree, the stem-loop structure-based ligation may inspire the flexible design strategy in the future application and provide a significant quick and efficient platform to miRNAs detection.


Assuntos
Imãs/química , MicroRNAs/análise , Reação em Cadeia da Polimerase/métodos , Estreptavidina/química , Humanos , Proteínas Imobilizadas/química , Limite de Detecção
7.
Sci Rep ; 10(1): 3376, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32099058

RESUMO

Dye-sensitized solar cells (DSSCs) have been highlighted as the promising alternative to generate clean energy based on low pay-back time materials. These devices have been designed to mimic solar energy conversion processes from photosynthetic organisms (the most efficient energy transduction phenomenon observed in nature) with the aid of low-cost materials. Recently, light-harvesting complexes (LHC) have been proposed as potential dyes in DSSCs based on their higher light-absorption efficiencies as compared to synthetic dyes. In this work, photo-electrochemical hybrid devices were rationally designed by adding for the first time Leu and Lys tags to heterologously expressed light-harvesting proteins from Chlamydomonas reinhardtii, thus allowing their proper orientation and immobilization on graphene electrodes. The light-harvesting complex 4 from C. reinhardtii (LHC4) was initially expressed in Escherichia coli, purified via affinity chromatography and subsequently immobilized on plasma-treated thin-film graphene electrodes. A photocurrent density of 40.30 ± 9.26 µA/cm2 was measured on devices using liquid electrolytes supplemented with a phosphonated viologen to facilitate charge transfer. Our results suggest that a new family of graphene-based thin-film photovoltaic devices can be manufactured from rationally tagged LHC proteins and opens the possibility to further explore fundamental processes of energy transfer for biological components interfaced with synthetic materials.


Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Técnicas Eletroquímicas/métodos , Grafite/química , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Algas/genética , Corantes/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Energia Solar
8.
Mater Sci Eng C Mater Biol Appl ; 107: 110335, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761211

RESUMO

In order to stimulate the cellular response to implant materials, extracellular matrix (ECM) proteins, such as collagen and fibronectin (FN), are immobilized on the implant surface. Amongst all ECM proteins used for biomimetic materials for medical applications, FN is one of the most investigated proteins thanks to its ability to promote cell adhesion and its contribution to important physiological processes. However, its conformation and hence its bioactivity strongly depend on the hydrophilic/hydrophobic nature of the surface as well as on immobilization strategies. This work investigates the effect of these two parameters, as well as the effect of the crosslinker length. FN was grafted onto silicon wafers using eights different linking arms presenting different lengths, hydrophilic/hydrophobic characters and binding sites. The protein was linked through either its amino groups (lysine amino acids) or sulfhydryl functionalities (cysteine amino acids). The grafting of each crosslinker and subsequent FN conjugation onto the surfaces was evidenced by X-ray photoelectron spectroscopy, while the surface hydrophilicity was determined by contact angle measurements. Moreover, atomic force microscopy images revealed that the conformation of surface conjugated FN only depends on the hydrophilicity of the linking arm. The FN conformation was also probed by enzyme-linked immunosorbent assays (ELISA). ELISA data demonstrated that all of the three investigated parameters linking arm parameter (length, hydrophobic/hydrophilic character, and terminal end-group) somewhat influence the RGD accessibility.


Assuntos
Fibronectinas/química , Proteínas Imobilizadas/química , Oligopeptídeos/química , Sítios de Ligação , Reagentes para Ligações Cruzadas/química , Ensaio de Imunoadsorção Enzimática , Fibronectinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Imobilizadas/metabolismo , Microscopia de Força Atômica , Espectroscopia Fotoeletrônica , Propriedades de Superfície
9.
Talanta ; 208: 120445, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816708

RESUMO

Multi-channel capillaries (MC) formed from thousands individual microcapillaries with diameters ranging 10-100 µm are of a great interest for their use as platforms for molecular imprinting due to their relatively large surface area, high mechanical stability and possibility of facile integration in sensor systems. The manuscript proposes a new format of immunoassay based on imprinted protein immobilized on a MC inner surface modified with poly-l-lysine. The combination of the environmentally friendly, easy-to-produce and cheap recognition element with the carrier allowing to increase the assay sensitivity makes the described technique a perspective alternative for the existing screening tests. Two bioimprinting approaches were described. The imprinted protein (ovalbumin, OVA) primarily prepared separately and later immobilized on a MC structure was compared to the imprinted OVA directly prepared on the MC surface. Detection of a food contaminant zearalenone was chosen as a proof-of-concept. In a case of the immobilization of the primarily prepared imprinted OVA the reached limit of detection (LOD) was 0.8 ng/mL, and for the in-situ imprinted OVA the LOD was 0.12 ng/mL. The sensitivity of the developed bioimprinted assay was comparable to the commercially available ELISA kits for ZEN detection. The OVA in-situ imprinted on the MC surface was tested for the detection of ZEN in artificially spiked wheat samples. The high recovery values (88-112%) and good repeatability (RSD of 8.5-9.6%) were demonstrated allowing to conclude that the IPs-based MC-ELISA is a promising tool for analysis of the mycotoxin in complex matrices.


Assuntos
Contaminação de Alimentos/análise , Imunoensaio/métodos , Impressão Molecular , Ovalbumina/química , Triticum/química , Zearalenona/análise , Vidro , Peroxidase do Rábano Silvestre/química , Proteínas Imobilizadas/química , Polilisina/química , Soroalbumina Bovina/química , Zearalenona/química
10.
Biosens Bioelectron ; 151: 111950, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868605

RESUMO

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec 15) is a novel immunomodulatory target and was identified as an immune suppressor in the tumor microenvironment. Accurate assessment of Siglec 15 expression levels is critical for cancer prognosis and treatment. In this work, a natural receptor-based immunoelectrochemical sensor is designed to mimic the interaction between Siglec 15 and DNAX-activation protein (DAP 12) in the cellular signal pathway. DAP 12 labeled with the electrochemical signal molecule Fc is recognized by Siglec 15 through specific interaction on the electrode surface and used as the signal reporter. Anti-Siglec 15 modified MNPs (MNPs-Ab) were used as the extraction agent for the magnetic extraction of target analytes in complex matrices. Free Anti-Siglec 15 will "squeeze out" the DAP 12-Fc to bind the Siglec 15 on the electrode surface, resulting a sensitive electrochemical signal change according to the Siglec 15 concentration in sample. Natural receptor-based competitive assay ensure the efficient binding between antibody and Siglec 15 and decrease the nonspecific interaction. Therefore, this simple natural receptor-based competitive assay with sensitivity and selectivity has potential for practical clinical application.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Biomarcadores Tumorais/sangue , Imunoglobulinas/sangue , Nanopartículas de Magnetita/química , Proteínas de Membrana/sangue , Proteínas de Membrana/química , Neoplasias/diagnóstico , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Ligação Competitiva , Biomarcadores Tumorais/imunologia , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Eletrodos , Humanos , Proteínas Imobilizadas/química , Imunoglobulinas/química , Imunoglobulinas/imunologia , Proteínas de Membrana/imunologia , Sensibilidade e Especificidade , Transdução de Sinais , Propriedades de Superfície
11.
Food Chem ; 307: 125514, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639576

RESUMO

The thermodynamics and kinetics of binding between human serum albumin (HSA) and resveratrol (RES) or its analog (RESAn1) were investigated by surface plasmon resonance (SPR). The binding constant and the kinetic constants of association and dissociation indicated that RESAn1 has higher affinity toward HSA than does RES. The formation of these complexes was entropically driven ( [Formula: see text] , [Formula: see text]  KJ mol-1). However, for both polyphenols, the activation energy (Eact) of association (a) of free molecules was higher than that for dissociation (d) of the stable complex ( [Formula: see text]  KJ mol-1), and the rate of association was faster than that of dissociation since the activation Gibbs free energy (ΔG‡) was lower for the former (ΔGaHSA-RES‡â‰…54.73,ΔGdHSA-RES‡â‰…73.83,ΔGaHSA-RESAn1‡â‰…54.14,ΔGdHSA-RESAn1‡â‰…73.97 KJ mol-1). This study showed that small differences in the structure of polyphenols such as RES and RESAn1 influenced the thermodynamics and kinetics of the complex formation with HSA.


Assuntos
Fenóis/química , Resveratrol/metabolismo , Albumina Sérica Humana/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Cinética , Ligação Proteica , Resveratrol/química , Albumina Sérica Humana/química , Ressonância de Plasmônio de Superfície , Temperatura , Termodinâmica
12.
Mater Sci Eng C Mater Biol Appl ; 107: 110209, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761232

RESUMO

In this paper cobalt oxide (Co3O4) nanoparticles were mixed with polyacrylonitrile to prepare Co3O4 doped carbon nanofiber (CNF) composite by electrospinning and carbonization, which was further used to modify on carbon ionic liquid electrode (CILE). Hemoglobin (Hb) was immobilized on Co3O4-CNF/CILE surface with Nafion acted as the protective film to fabricate an electrochemical biosensor (Nafion/Hb/Co3O4-CNF/CILE). Electrochemical behavior of Hb on the electrode was investigated with a pair of quasi-reversible redox peak appeared on cyclic voltammogram and electrochemical parameters were calculated. Moreover, this biosensor had good analytical capabilities for electrocatalytic reduction of different substrates including trichloroacetic acid, potassium bromate and sodium nitrite with wider detection range from 40.0 to 260.0 mmol L-1, 0.1 to 48.0 mmol L-1 and 1.0 to 12.0 mmol L-1 by cyclic voltammetry, respectively. The proposed method showed excellent anti-interferences ability with good selectivity and was successful used for quantitative detection of real samples, which displayed the potential applications to develop into a new analytical device.


Assuntos
Carbono/química , Cobalto/química , Técnicas Eletroquímicas/métodos , Hemoglobinas/química , Nanofibras/química , Óxidos/química , Técnicas Biossensoriais/métodos , Bromatos/análise , Catálise , Proteínas Imobilizadas/química , Líquidos Iônicos/química , Reprodutibilidade dos Testes , Nitrito de Sódio/análise , Propriedades de Superfície , Ácido Tricloroacético/análise
13.
Scanning ; 2019: 8175413, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819781

RESUMO

To investigate the stability and dynamic characteristics of monolayer adsorbed on unsaturated lipid dioleoylphosphatidylcholine (DOPC) with varying concentrations of myelin basic protein (MBP), the system is studied by applying Langmuir technique and making atomic force microscope (AFM) observation, which is based on the mass conservation equation analysis method referred to in the thermodynamics theory. As indicated by surface pressure-mean molecular area (π - A) and surface pressure-adsorption time (π - T) isotherms, the physical properties of monolayer derived from the interaction of varying concentrations of MBP with liquid crystalline unsaturated lipid DOPC molecules were qualitatively studied. As revealed by surface morphology analysis with AFM, the micro region was expanded as the concentration of MBP in the subphase was on the increase, suggesting that hydrophobic interactions led to the MBP insertion, thus causing accumulation of the MBP on the surface of the monolayer. Experimental results have demonstrated that the partition coefficient of the interaction between MBP and unsaturated phospholipid DOPC and the molecular area of MBP adsorbed on the monolayer film was calculated using the mass conservation equation. In addition, not only does the varying concentration of MBP in the subphase exerts significant effects on the arrangement and conformation of DOPC monolayer, it also has certain guiding significance to exploring the structural changes to biofilm supramolecular aggregates as well as the pathogenesis and treatment of related diseases.


Assuntos
Fenômenos Químicos , Proteínas Imobilizadas/química , Proteína Básica da Mielina/química , Fosfatidilcolinas/química , Adsorção , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Estabilidade Proteica , Termodinâmica
14.
Cells ; 8(12)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31847477

RESUMO

We report on the covalent immobilization of bone morphogenetic protein 6 (BMP-6) and its co-presentation with integrin ligands on a nanopatterned platform to study cell adhesion and signaling responses which regulate the transdifferentiation of myoblasts into osteogenic cells. To immobilize BMP-6, the heterobifunctional linker MU-NHS is coupled to amine residues of the growth factor; this prevents its internalization while ensuring that its biological activity is maintained. Additionally, to allow cells to adhere to such platform and study signaling events arising from the contact to the surface, we used click-chemistry to immobilize cyclic-RGD carrying an azido group reacting with PEG-alkyne spacers via copper-catalyzed 1,3-dipolar cycloaddition. We show that the copresentation of BMP-6 and RGD favors focal adhesion formation and promotes Smad 1/5/8 phosphorylation. When presented in low amounts, BMP-6 added to culture media of cells adhering to the RGD ligands is less effective than BMP-6 immobilized on the surfaces in inducing Smad complex activation and in inhibiting myotube formation. Our results suggest that a local control of ligand density and cell signaling is crucial for modulating cell response.


Assuntos
Proteína Morfogenética Óssea 6/química , Proteína Morfogenética Óssea 6/metabolismo , Adesão Celular/fisiologia , Ouro/química , Nanopartículas Metálicas/química , Mioblastos/metabolismo , Oligopeptídeos/química , Animais , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proteínas Imobilizadas/química , Integrinas/metabolismo , Ligantes , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosforilação , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo
15.
Nat Nanotechnol ; 14(12): 1143-1149, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31712665

RESUMO

Affinity-based electrochemical detection in complex biological fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 month of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy preparation, stability and simplicity of this nanocomposite allow the generation of electrochemical biosensors that can operate in complex biological fluids such as blood plasma or serum.


Assuntos
Técnicas Biossensoriais/instrumentação , Ouro/química , Nanopartículas Metálicas/química , Nanotubos de Carbono/química , Soroalbumina Bovina/química , Anticorpos/sangue , Incrustação Biológica , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Humanos , Proteínas Imobilizadas/química , Modelos Moleculares , Nanotubos de Carbono/ultraestrutura , Plasma/química
16.
ACS Appl Mater Interfaces ; 11(49): 46350-46360, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31722179

RESUMO

Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and high-throughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10-7 mol/m2 is obtained, when chemisorbed on EDC-NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC-NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas Imobilizadas/química , Dispositivos Lab-On-A-Chip , Polimetil Metacrilato/química , Ar , Proteínas de Fluorescência Verde/química , Análise em Microsséries/métodos , Gases em Plasma/química , Ondas de Rádio , Propriedades de Superfície
17.
Nat Nanotechnol ; 14(12): 1135-1142, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740795

RESUMO

Chemists have long sought the ability to modify molecules precisely when presented with several sites of similar reactivity. We reasoned that the confinement of substrates within nanostructures might permit site-selective reactions unachievable in bulk solution, even with sophisticated reagents. In particular, the stretching and alignment of polymers within nanotubes might allow site-specific cleavage or modification. To explore this proposition, macromolecular disulfide substrates were elongated within members of a collection of tubular protein nanoreactors, which contained cysteine residues positioned at different locations along the length of each tube. For each nanoreactor, we defined the reactive location by using a set of polymer substrates (site-selectivity) and which of the two sulfur atoms was attacked (regioselectivity), and found that disulfide interchange occurs with atomic precision. Our strategy has potential for the selective processing of a wide variety of biomacromolecules, and the chemistry and substrates might be generalized yet further by using alternative nanotubes.


Assuntos
Nanotecnologia , Nanotubos/química , Domínio Catalítico , Dissulfetos/química , Proteínas Hemolisinas/química , Proteínas Imobilizadas/química , Cinética , Modelos Moleculares , Nanotecnologia/métodos , Estereoisomerismo , Especificidade por Substrato
18.
Mikrochim Acta ; 186(11): 726, 2019 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-31655909

RESUMO

A reusable fiber optic chemiluminescent aptasensor (FOCA) is reported for the rapid and sensitive on-site detection of 17ß-estradiol (E2), an endocrine-disrupting compound frequently found in water samples. The E2-ovalbumin conjugate (E2-OVA) was covalently immobilized onto the optical fiber as a biorecognition element as well as a transducer. The affinity constant of the E2/aptamer complex was determined to be 1.35 × 106 M-1 using the FOCA. An indirect competitive assay was then developed for E2 detection. A certain concentration of HRP-E2 aptamers pre-reacted with samples containing E2 in various concentrations. Part of HRP-E2 aptamers specially bound to the sensor surface after introduction of the mixture. This catalyzed the chemiluminescece reaction of a chemiluminescent system composed of luminol and H2O2. A higher concentration of E2 led to less HRP-E2 aptamer bound to the biosensor surface, thus resulting in less chemiluminescence. Highly sensitive detection of E2 was achieved under optimal conditions, and the limit of detection is 48 ng ·L-1 (0.18 nM). The whole analytical process, including measurement and regeneration, can be performed in <15 min. The robustness of the biosensor allows its application to multiple assays with little activity loss. The selectivity, recovery, and accuracy of the sensor was demonstrated by evaluating its response to potentially interfering endocrine disruptors in spiked water samples. Graphical abstract Schematic diagram of the fiber optic chemiluminescent aptasensor system (A), detection mechanism of 17ß-estradiol (B), and its application for detection of 17ß-estradiol with rapidity and sensitivity (C and D).


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Disruptores Endócrinos/análise , Estradiol/análise , Poluentes Químicos da Água/análise , Sequência de Bases , Técnicas Biossensoriais/instrumentação , Água Potável/análise , Tecnologia de Fibra Óptica/instrumentação , Peróxido de Hidrogênio/química , Proteínas Imobilizadas/química , Limite de Detecção , Luminol/química , Ovalbumina/química , Águas Residuárias/análise
19.
PLoS One ; 14(9): e0222144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553730

RESUMO

Dengue virus (DENV) nonstructural 1 (NS1) protein is a specific and sensitive biomarker for the diagnosis of dengue. In this study, an efficient electrochemical biosensor that uses chemically modified affinity peptides was developed for the detection of dengue virus NS1. A series of amino acid-substituted synthetic peptides was rationally designed, chemically synthesized and covalently immobilized to a gold sensor surface. The sensor performance was monitored via square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). Potential affinity peptides specific for NS1 were chosen according to the dynamic current decrease in SWV experiments. Using circular dichroism, the molar ellipticity of peptides (DGV BP1-BP5) was determined, indicating that they had a mostly similar in random coil structure, not totally identical. Using SWV, DGV BP1 was selected as a promising recognition peptide and limit of detection for NS1 was found to be 1.49 µg/mL by the 3-sigma rule. DGV BP1 showed good specificity and stability for NS1, with low signal interference. The validation of the sensor to detect NS1 proteins was confirmed with four dengue virus culture broth (from serotype 1 to 4) as proof-of-concept. The detection performance of our sensor incorporating DGV BP1 peptides showed a statistically significant difference. These results indicate that this strategy can potentially be used to detect the dengue virus antigen, NS1, and to diagnosis dengue fever within a miniaturized portable device in point-of-care testing.


Assuntos
Técnicas Biossensoriais/métodos , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Dengue/virologia , Proteínas não Estruturais Virais/análise , Substituição de Aminoácidos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/estatística & dados numéricos , Vírus da Dengue/química , Espectroscopia Dielétrica , Glicoproteínas/análise , Humanos , Proteínas Imobilizadas/síntese química , Proteínas Imobilizadas/química , Limite de Detecção , Peptídeos/síntese química , Peptídeos/química
20.
ACS Appl Mater Interfaces ; 11(39): 35604-35612, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31495166

RESUMO

As an oxygen-transporting protein, free hemoglobin (Hb) often suffers from the disadvantage of undesirable stability and short blood circulation, which severely impairs the potential clinical applications as the blood substitute. In this work, Hb was facilely encapsulated into a kind of metal-organic frameworks (MOFs) (ZIF-8) inspired by the natural biomineralization process. The obtained ZIF-8 encapsulating Hb (ZIF-8@Hb) showed the small hydrodynamic size of 180.8 nm and neutral zeta potential of -2.1 mV by adjusting the ratio of Hb in ZIF-8 frameworks. Intriguingly, Hb encapsulated by ZIF-8 exhibited significantly enhanced stability in alkaline, oxidation, high temperature, or enzymatic environment compared with free Hb because of the excellent protective MOF coatings. More importantly, the negative charge of Hb neutralized the original positive charge of ZIF-8, which led to the better biocompatibility, lower protein adsorption, and macrophage uptake of ZIF-8@Hb than bare ZIF-8 nanoparticles. Furthermore, ZIF-8@Hb displayed extended blood circulation with the elimination half-life of 13.9 h as well as reduced nonspecific distribution in normal organs compared with free Hb or ZIF-8 nanoparticles. With the above advantages, ZIF-8@Hb showed significantly extended survival time of mice in a disease model of hemorrhagic shock compared with free Hb or bare ZIF-8 nanoparticles. Overall, this work offers a high-stable and long-circulating oxygen carrier platform, which may find wide applications as a blood substitute to treat various oxygen-relevant diseases.


Assuntos
Substitutos Sanguíneos , Hemoglobinas , Proteínas Imobilizadas , Estruturas Metalorgânicas , Choque Hemorrágico/tratamento farmacológico , Animais , Substitutos Sanguíneos/química , Substitutos Sanguíneos/farmacologia , Hemoglobinas/química , Hemoglobinas/farmacologia , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Camundongos , Oxigênio/sangue , Células RAW 264.7 , Choque Hemorrágico/sangue
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