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1.
Nat Commun ; 11(1): 3859, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737298

RESUMO

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins.


Assuntos
Anidrase Carbônica II/química , Preparações de Ação Retardada/síntese química , Hidrogéis/química , Ribonucleases/química , Sefarose/química , Animais , Anticorpos/química , Avidina/química , Biotina/química , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Bovinos , Ativação Enzimática , Transição de Fase , Reologia , Ribonucleases/antagonistas & inibidores , Sulfonamidas/química
2.
J Chromatogr A ; 1627: 461422, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823117

RESUMO

Sialylation, an important form of glycosylation, is involved in many biological processes and plays an important role in the development of diseases. However, due to the low abundance among various glycosylation and lack of efficient enrichment method with high specificity, the study of sialylation remains a challenge. Herein, multi-histidine modified microspheres (MHM) were synthesized to enrich sialylated glycopeptides. It was found that MHM could selectively enrich sialylated glycopeptides from over 100 times of non-sialylated glycopeptides, which indicated MHM possessed good enrichment specificity towards sialylated glycopeptides. Furthermore, MHM were utilized to the large-scale analysis of protein sialylation, and 510 intact glycopeptides were identified with over 94.5% sialylated glycopeptide specificity from 4 µL human serum. The good specificity could be attributed to the synergistic effect by the electrostatic interaction and hydrophilic interaction. Hence, MHM could provide an alternative approach for the analysis of site-specific sialylation at proteome level from complex biological samples.


Assuntos
Glicopeptídeos/análise , Histidina/química , Ácido N-Acetilneuramínico/química , Sequência de Aminoácidos , Avidina/química , Fetuínas/química , Glicopeptídeos/sangue , Glicosilação , Humanos , Microesferas , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Biomater Sci ; 8(8): 2120-2128, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32211644

RESUMO

The development of a universal coating strategy for the construction of functional surfaces and modulation of surface properties is of great research interest. Tannic acid (TA) could serve as a sole precursor for the deposition of colorless coatings on substrate surfaces. However, the deposition of TA requires a high salt concentration (0.6 M), which may limit its practical application. Herein, primary amine moieties were introduced on the gallic acid groups in TA. The resultant amine-containing TA derivative (TAA) can self-polymerize under mild conditions (10 mM, Tris buffer), and form uniform and colorless coatings in a material-independent manner. In comparison with the TA coating under the same preparation conditions, the TAA coating exhibits an increased thickness as measured by ellipsometry. The TAA coating is adapted for secondary surface functionalization. The hydrophilic mPEG brushes can be grafted on the TAA coating to inhibit non-specific protein adsorption. A biotin probe can be immobilized on the TAA coating to promote specific binding with avidin. In addition, the TAA coating can be utilized for in situ reduction of silver ions to AgNPs. The resulting AgNP-loaded TAA coating can inhibit bacterial adhesion and prevent biofilm formation.


Assuntos
Aminas/química , Taninos/química , Avidina/química , Aderência Bacteriana , Biofilmes , Biotina/química , Escherichia coli/fisiologia , Ácido Gálico/química , Nanopartículas Metálicas/química , Polietilenoglicóis/química , Prata/química , Staphylococcus aureus/fisiologia , Ressonância de Plasmônio de Superfície , Propriedades de Superfície , Titânio/química
4.
Biosens Bioelectron ; 154: 112050, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32056957

RESUMO

In this work we discuss a new label-free biosensing device based on indium tin oxide (ITO) overlaid section of a multimode optical fiber fused silica core. The sensor has been used to optical measurements also simultaneously interrogated electrochemically (EC). Due to optimized thickness and optical properties of ITO film, a lossy-mode resonance (LMR) could be observed in the optical domain, where electrical properties of the film allowed for application of the sensor as a working electrode in an EC setup. It has been confirmed that the LMR response depends on optical properties of the external medium, as well as potential applied to the electrode during cyclic voltammetry. After the ITO surface functionalization with amine groups and covalently attached biotin, the device has been applied for label-free biosensing of avidin in both the domains simultaneously. On the example of biotin-avidin detection system it was demonstrated that when avidin concentration increases a decrease in current and increase in LMR wavelength shift were recorded in EC and optical domain, respectively. Both optical and EC responses follow the protein interaction process, and thus can be used as cross-verification of the readouts. Moreover, an extended information has been achieved comparing to solely EC interrogation, i.e., the grafting process of biotin and avidin was directly monitored optically displaying individual steps of an incubation procedure.


Assuntos
Avidina/isolamento & purificação , Técnicas Biossensoriais , Biotina/isolamento & purificação , Técnicas Eletroquímicas , Avidina/química , Biotina/química , Eletrodos , Óptica e Fotônica , Compostos de Estanho/química
5.
PLoS One ; 15(2): e0229000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092106

RESUMO

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.


Assuntos
Avidina/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi , Antígeno Nuclear de Célula em Proliferação , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/isolamento & purificação , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/isolamento & purificação , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Células Sf9 , Spodoptera , Ubiquitina/química , Ubiquitina/isolamento & purificação , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/isolamento & purificação , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/isolamento & purificação
6.
Food Chem ; 317: 126433, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092613

RESUMO

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm-1. With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL.


Assuntos
Biotina/análise , Análise de Alimentos/métodos , Prata/química , Análise Espectral Raman/métodos , Animais , Avidina/química , Carbono/química , Catálise , Citratos/química , Análise de Alimentos/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Sonda Molecular/instrumentação , Sondas Moleculares/química , Compostos Orgânicos/química , Pontos Quânticos/química , Nitrato de Prata/química , Análise Espectral Raman/instrumentação
7.
Nat Commun ; 11(1): 32, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896744

RESUMO

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Orthoreovirus Mamífero 3/fisiologia , Nanopartículas Metálicas/química , Actinas/metabolismo , Animais , Avidina/química , Biotina/química , Capsídeo/química , Células Cultivadas , Fibroblastos/virologia , Ouro , Células HeLa , Humanos , Integrinas/metabolismo , Cinética , Orthoreovirus Mamífero 3/química , Orthoreovirus Mamífero 3/patogenicidade , Nanopartículas Metálicas/virologia , Modelos Teóricos , Miosinas/metabolismo , Ratos , Vírion/patogenicidade , Vírion/fisiologia
8.
Biosens Bioelectron ; 148: 111764, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31707325

RESUMO

We are reporting an original supramolecular architecture based on a rationally designed new nanohybrid with enhanced peroxidase-like activity and site-specific biorecognition properties using avidin-functionalized multi-walled carbon nanotubes (MWCNTs-Av) and Ru nanoparticles (RuNPs). The nanohybrid-electrochemical interface was obtained by drop-coating of MWCNTs-Av dispersion at glassy carbon electrodes (GCE) followed by solvent evaporation and further electrodeposition of RuNPs (50 ppm RuCl2 for 15 s at -0.600 V). The simultaneous presence of MWCNTs and RuNPs produces a synergic effect on the non-enzymatic catatalytic reduction of H2O2 and allows the quantification of H2O2 in a wide linear range (from 5.0 × 10-7 M to 1.75 × 10-3 M) with a low limit of detection (65 nM). The avidin residues present in MWCNTs-Av/RuNPs hybrid nanomaterial allowed the anchoring by bioaffinity of biotinylated glucose oxidase (biot-GOx) as proof-of-concept of the analytical application of MWCNTs-Av platform for biosensors development. The resulting nanoarchitecture behaves as a bienzymatic-like glucose biosensor with a competitive analytical performance: linear range between 2.0 × 10-5 M and 1.23 × 10-3 M, sensitivity of (0.343 ±â€¯0.002) µA mM-1 or (2.60 ±â€¯0.02) µA mM-1 cm-2, detection limit of 3.3 µM, and reproducibility of 5.2% obtained with five different GCE/MWCNTs-Av/RuNPs/biot-GOx bioplatforms prepared the same day using the same MWCNTs-Av dispersion, and 9.1% obtained with nine biosensors prepared in different days with nine different MWCNTs-Av dispersions. The average concentrations of glucose in Gatorade®, Red bull® and Pepsi® with the biosensor demonstrated excellent agreement with those reported in the commercial beverages.


Assuntos
Avidina/química , Técnicas Biossensoriais/métodos , Nanopartículas/química , Nanotubos de Carbono/química , Rutênio/química , Aspergillus niger/enzimologia , Bebidas/análise , Materiais Biomiméticos/química , Biotinilação , Catálise , Técnicas Eletroquímicas/métodos , Glucose/análise , Glucose Oxidase/química , Peróxido de Hidrogênio/análise , Limite de Detecção , Nanopartículas/ultraestrutura , Nanotubos de Carbono/ultraestrutura , Peroxidase/química
9.
J Mol Model ; 25(12): 361, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31773283

RESUMO

Biotin is well known to be bound with exceptional strength by the avidin class of proteins. This ability comes from a match between the biotin-binding pocket of the protein and the structural elements of biotin, including its ureido and thiolane rings. Here we investigate the solvation shell of biotin in water as revealed by classical force field molecular dynamics with GAFF force field. Snapshots from the classical molecular dynamics were then used to generate microsolvated structures. Details of hydrogen bonding patterns present in these microsolvated structures were studied by symmetry-adapted perturbation theory (SAPT). Interaction energy values for small models of biotin hydrated by 5 or 6 water molecules show that the cooperativity constitutes 15-22% of the total interaction energy and corresponds roughly to formation of one additional hydrogen bond to biotin. The SAPT analysis shows the differences underlying hydrogen bonds of similar strength (with oxygen or sulfur atoms as the hydrogen bond acceptors, and with nitrogen atom playing a dual role of the donor and acceptor).


Assuntos
Avidina/metabolismo , Biotina/metabolismo , Solventes/química , Água/química , Avidina/química , Sítios de Ligação , Biotina/química , Ligação de Hidrogênio , Ligantes , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade
10.
J Am Chem Soc ; 141(44): 17847-17853, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31642667

RESUMO

A molecular signal displayed on the external surface of one population of vesicles was used to trigger a catalytic process on the inside of a second population of vesicles. The key recognition event is the transfer of a protein (NeutrAvidin) bound to vesicles displaying desthiobiotin to vesicles displaying biotin. The desthiobiotin-protein complex was used to anchor a synthetic transducer in the outer leaflet of the vesicles, and when the protein was displaced, the transducer translocated across the bilayer to expose a catalytic headgroup to the internal vesicle solution. As a result, an ester substrate encapsulated on the inside of this second population of vesicles was hydrolyzed to give a fluorescence output signal. The protein has four binding sites, which leads to multivalent interactions with membrane-anchored ligands and very high binding affinities. Thus, biotin, which has a dissociation constant 3 orders of magnitude higher than desthiobiotin, did not displace the protein from the membrane-anchored transducer, and membrane-anchored biotin displayed on the surface of a second population of vesicles was required to generate an effective input signal.


Assuntos
Células Artificiais/química , Avidina/química , Lipossomos/química , Transdução de Sinais , Biotina/análogos & derivados , Biotina/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química
11.
Dalton Trans ; 48(43): 16233-16241, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31598614

RESUMO

Biotinylated pharmaceuticals are of great interest due to the strong interactions between biotinyl-functionality and streptavidin/avidin, which opens up avenues for efficient targeting and localisation. Three new carbon monoxide-releasing molecules (CO-RMs) have been synthesised and characterised using chemical and biological analysis. An alkyne-containing CO-RM 2 was found to be toxic to RAW 264.7 murine macrophages; and thus therapeutically viable CO-RM 1 was employed as the alkyne precursor for [3 + 2] cycloaddition chemistry enabling a new acid-containing CO-RM 4 and biotin-bioconugate-CO-RM (BiotinCORM 5) to be prepared. CO-RM 4 showed significantly improved solubility and BiotinCORM 5 acts as a photo-CO-RM. We have found that an avidin-CORM adduct of 5 is a CO-releasing protein, releasing CO on irradiation with light (400 nm). The avidin-biotinCORM adduct of 5 was found to have a binding energy of 10 kcal mol-1.


Assuntos
Avidina/química , Biotina/química , Monóxido de Carbono/química , Portadores de Fármacos/química , Estreptavidina/química , Alquinos/química , Animais , Reação de Cicloadição , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Camundongos , Estrutura Molecular , Processos Fotoquímicos , Células RAW 264.7
12.
Nat Commun ; 10(1): 4347, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554812

RESUMO

Spatiotemporal control over engineered tissues is highly desirable for various biomedical applications as it emulates the dynamic behavior of natural tissues. Current spatiotemporal biomaterial functionalization approaches are based on cytotoxic, technically challenging, or non-scalable chemistries, which has hampered their widespread usage. Here we report a strategy to spatiotemporally functionalize (bio)materials based on competitive supramolecular complexation of avidin and biotin analogs. Specifically, an injectable hydrogel is orthogonally post-functionalized with desthiobiotinylated moieties using multivalent neutravidin. In situ exchange of desthiobiotin by biotin enables spatiotemporal material functionalization as demonstrated by the formation of long-range, conformal, and contra-directional biochemical gradients within complex-shaped 3D hydrogels. Temporal control over engineered tissue biochemistry is further demonstrated by timed presentation and sequestration of growth factors using desthiobiotinylated antibodies. The method's universality is confirmed by modifying hydrogels with biotinylated fluorophores, peptides, nanoparticles, enzymes, and antibodies. Overall, this work provides a facile, cytocompatible, and universal strategy to spatiotemporally functionalize materials.


Assuntos
Avidina/química , Materiais Biocompatíveis/química , Biotina/química , Substâncias Macromoleculares/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Avidina/metabolismo , Materiais Biocompatíveis/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Biotinilação/métodos , Linhagem Celular , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Nanopartículas/química , Peptídeos/química , Peptídeos/metabolismo , Análise Espaço-Temporal , Engenharia Tecidual/métodos
13.
ACS Appl Mater Interfaces ; 11(40): 36435-36443, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525892

RESUMO

Self-assembled phototheranostic nanomaterials used for photodynamic therapy (PDT) have attracted increasing attention owing to their several advantages. Herein, we developed a novel strategy for size-tunable self-assembled nanophotosensitizers for PDT through a simple method. A series of switchable self-assembled nanophotosensitizers (NanoPc90, NanoPc40, NanoPc20, and NanoPc10) of different particle sizes were readily prepared based on an amphiphilic silicon(IV) phthalocyanine (SiPc)-biotin conjugate by regulating the amount of the Cremophor EL surfactant used. The photoactivities, including fluorescence and reactive oxygen species (ROS), of the self-assemblies could be regulated by the particle size. The self-assemblies could be specifically disassembled by tumor-overexpressing biotin receptors, leading to the recovery of quenched photoactivities. Demonstrated by the competitive assay, the self-assemblies were able to enter HepG2 cells through a biotin-receptor-mediated pathway, followed by biotin-receptor-triggered fluorescence recovery at the cellular level. Moreover, the particle size could also affect the in vitro and in vivo PDT effects and tumor targeting. The photocytotoxicity of NanoPc20 against HepG2 cells was more potent compared to that of NanoPc90 because of its strong intracellular fluorescence, higher intracellular ROS generation, and different subcellular localization. In addition, NanoPc20 showed higher in vivo tumor targeting and photodynamic therapeutic efficacy than NanoPc90. This work would provide a valuable reference for the development of self-assembled nanophotosensitizers for cancer diagnosis and therapy.


Assuntos
Biotina/química , Indóis/química , Nanoestruturas/química , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Animais , Avidina/química , Proliferação de Células/efeitos dos fármacos , Fluorescência , Células Hep G2 , Humanos , Concentração Inibidora 50 , Camundongos , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo
14.
Bioorg Med Chem Lett ; 29(20): 126663, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31521477

RESUMO

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity to detect small tumor nodules during surgery or endoscopic surgery. Here, we introduce an avidin-conjugated fluorescence probe, Avidin-Leu-HMRG, which consists of a cancer-targeting macromolecule (avidin) and a protease-activatable probe. The conjugate has a high affinity for lectin on cancer cells and undergoes endocytosis, followed by irreversible fluorescence activation due to cleavage by lysosomal leucine aminopeptidase. In a mouse model of peritoneal ovarian metastases, the probe could detect submillimeter-sized tumor nodules with a high S/N ratio at 1 h after intraperitoneal injection.


Assuntos
Avidina/química , Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Peptídeo Hidrolases/química , Rodaminas/química , Animais , Linhagem Celular Tumoral , Humanos , Lectinas/química , Lectinas/metabolismo , Leucil Aminopeptidase/metabolismo , Camundongos , Microscopia de Fluorescência , Neoplasias Experimentais , Imagem Óptica
15.
Biomacromolecules ; 20(9): 3408-3424, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31389692

RESUMO

This study describes new mechanistic insights in the sequential polyassociation of streptavidin with biotinylated poly(ethyleneimine) glycopolymers and biotinylated PEGylated folic acid components for the preparation of biohybrid structures (BHS) for controlled targeting experiments. Characterization of the BHS revealed that during the formation and postfunctionalization of BHS, reversible dissociation and reassociation processes occur. The BHS are stable over weeks after finalizing the equilibrium-driven polyassociation process. Cellular uptake studies showed that this sequential polyassociation involving biotinylated PEGylated folic acid components does not lead to enhanced cellular uptake of the resulting BHS. In contrast, polyplexes, containing small interfering RNA and bioconjugates (1:1 molar ratio between biotinylated glycopolymer and monomeric streptavidin-lectin fusion protein), enabled us to control the targeting of tumor cells as revealed by knockdown of the tumor-associated protein survivin. Overall, this study demonstrates the high potential of (networklike) streptavidin-biotin interactions with a dynamic character in the formation of complex BHS and extracellular matrix materials.


Assuntos
Ácido Fólico/química , Nanopartículas/química , Polietilenoimina/química , RNA Interferente Pequeno/química , Avidina/química , Biotina/química , Biotinilação , Ácido Fólico/síntese química , Humanos , Polietilenoimina/síntese química , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/efeitos dos fármacos , Estreptavidina/química
16.
PLoS One ; 14(8): e0221931, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31469884

RESUMO

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types.


Assuntos
Avidina/química , Hidrogéis/química , Polissacarídeos Bacterianos/química , Adesivos/química , Biotinilação , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Fenômenos Químicos , Fibroblastos , Humanos , Ligantes , Tecidos Suporte/química
17.
Acta Biomater ; 99: 33-52, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31425893

RESUMO

Electrical properties, such as charge propagation, dielectrics, surface potentials, conductivity, and piezoelectricity, play crucial roles in biomolecules, biomembranes, cells, tissues, and other biological samples. However, characterizing these electrical properties in delicate biosamples is challenging. Atomic Force Microscopy (AFM), the so called "Lab on a Tip" is a powerful and multifunctional approach to quantitatively study the electrical properties of biological samples at the nanometer level. Herein, the principles, theories, and achievements of various modes of AFM in this area have been reviewed and summarized. STATEMENT OF SIGNIFICANCE: Electrical properties such as dielectric and piezoelectric forces, charge propagation behaviors play important structural and functional roles in biosystems from the single molecule level, to cells and tissues. Atomic force microscopy (AFM) has emerged as an ideal toolkit to study electrical property of biology. Herein, the basic principles of AFM are described. We then discuss the multiple modes of AFM to study the electrical properties of biological samples, including Electrostatic Force Microscopy (EFM), Kelvin Probe Force Microscopy (KPFM), Conductive Atomic Force Microscopy (CAFM), Piezoresponse Force Microscopy (PFM) and Scanning ElectroChemical Microscopy (SECM). Finally, the outlook, prospects, and challenges of the various AFM modes when studying the electrical behaviour of the samples are discussed.


Assuntos
Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Animais , Avidina/química , Biotina/química , Sistemas Computacionais , Eletrônica , Desenho de Equipamento , Geobacter , Glucose 1-Desidrogenase/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Íons , Camundongos , Células PC12 , Fotossíntese , Ratos , Silício , Eletricidade Estática , Células-Tronco/citologia
18.
Mikrochim Acta ; 186(8): 488, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267252

RESUMO

A nanocomposite was prepared from ß-cyclodextrin (ß-CD) functionalized graphene oxide and magnetic nanoparticles (GO/Fe3O4/ß-CD). In parallel, a polyamidoamine dendrimer conjugated to avidinylated alkaline phosphatase (PAMAM-avidin-ALP) was prepared and exploited as a signal amplification unit in a voltammetric immunoassay for 5-methylcytosine (5mC) in genomic DNA. The GO/Fe3O4/ß-CD as a substrate material exhibited good solubility, electrical conductivity and large surface. This is beneficial for the further modification of antibodies (Ab) by host-guest interaction and amide bonds. By taking advantage of three-dimensional structure to capture avidin-ALP by amide linkages, PAMAM was used as a catalytic signal amplification element in this assay. Under the optimized condition and at a typical working potential of 0.94 V, the response to 5mC is linear in the 0.01-50 nM concentration range with a detection limit of 3.2 pM (at S/N = 3). The method is stable, selective and reproducible. It was applied to the determination of 5mC in genomic DNA of human tissue. Graphical abstract An electrochemical immunoassay was constructed for 5-methylcytosine detection based on nanocomposite of graphene oxide, magnetite nanoparticles and ß-cyclodextrin, and enzymatic signal amplification.


Assuntos
5-Metilcitosina/análise , Técnicas Biossensoriais , 5-Metilcitosina/química , Fosfatase Alcalina/química , Avidina/química , Mama , Neoplasias da Mama/genética , DNA/química , Dendrímeros/química , Técnicas Eletroquímicas , Feminino , Grafite/química , Humanos , Imunoensaio , Nanopartículas de Magnetita/química , Nanocompostos/química , Estômago , Neoplasias Gástricas/genética , beta-Ciclodextrinas/química
19.
Chem Asian J ; 14(17): 2953-2957, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31321878

RESUMO

This paper describes the synthesis of protein microtube motors having a urease interior surface and highlights their nonbubble-propelled behavior driven by enzymatic reaction (urea→NH3 and CO2 ). The precursor microtubes were prepared by layer-by-layer assembly using a track-etched microporous polycarbonate membrane. Immobilization of a urease on the internal wall was accomplished using avidin-biotin interaction. The tubules swam smoothly in an aqueous media containing a physiological concentration of urea. Each tubule was rotating laterally while moving forward. It is remarkable that the microtubes were digested completely by proteases, demonstrating perfect biodegradability.


Assuntos
Avidina/química , Biotina/química , Urease/metabolismo , Avidina/metabolismo , Biotina/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cimento de Policarboxilato/química , Porosidade , Ureia/química , Ureia/metabolismo , Urease/química
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