Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.192
Filtrar
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 ; 1621: 461051, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32268955

RESUMO

The strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.


Assuntos
Avidina , Biotina , Cromatografia de Afinidade/métodos , Cromatografia/métodos , Proteínas/isolamento & purificação , Biotinilação , DNA Ligase Dependente de ATP/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Endonucleases Flap/isolamento & purificação , Humanos , Plasmídeos , Antígeno Nuclear de Célula em Proliferação , Proteínas/genética , Proteína SUMO-1
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
14.
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
15.
Clin Lab ; 65(6)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31232038

RESUMO

BACKGROUND: GLP-1 as an incretin, has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Besides the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Thus, the action of GLP-1 is preserved in patients with type 2 diabetes and substantial pharmaceutical research has therefore been directed towards the development of GLP-1-based treatment. METHODS: In this work, we reported an electrochemical sense array based on the aptamer and biotin-avidin system for the detection of glucagon-like peptide-1 (GLP-1). The sense array employed a "stem-loop" conformation ap-tamer which was immobilized on the electrode of the 16-unit gold array via pre-labeled thiol group (-SH). Pre-labeled biotin serves as an affinity tag for the binding of avidin-horseradish peroxidase (avidin-HRP). The stem-loop structure of the aptamer kept the biotin from being approached by a bulky avidin-HRP by means of the steric hindrance. After the interaction of the target (GLP-1) and the aptamer, the aptamer would undergo a significant conformational change to force biotin away from the electrode, giving the avidin-HRP easy access to the labeled biotin. The HRP in the substrate could sensitively transduce the concentration of GLP-1 into the electrical signals, which were then measured by electrochemical technology of cyclic voltammetry and amperometric i-t curve. RESULTS: Under the optimal experimental conditions, the proposed sense array for GLP-1 had a good linear relationship from 0.1 pmol/L to 20 pmol/L with a detection limit of 0.05 pmol/L and can be used to accurately detect the GLP-1 in serum. CONCLUSIONS: The experimental results show that GLP-1 could be selectively detected by the electrochemical sense array, indicating that the proposed sense array based on the biotin-avidin system and the stem-loop aptamer has great potential in the detection of GLP-1.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Avidina/metabolismo , Técnicas Biossensoriais/métodos , Biotina/metabolismo , Técnicas Eletroquímicas/métodos , Peptídeo 1 Semelhante ao Glucagon/sangue , Aptâmeros de Nucleotídeos/química , Diabetes Mellitus Tipo 2/sangue , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Ligação Proteica , Reprodutibilidade dos Testes
16.
Chem Asian J ; 14(24): 4616-4624, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31210021

RESUMO

The dynamics of cell-cell adhesion are complicated due to complexities in cellular interactions and intra-membrane interactions. In the present work, we have reconstituted a liposome-based model system to mimic the cell-cell adhesion process. Our model liposome system consists of one fluorescein-tagged and one TRITC (tetramethyl-rhodamine isothiocyanate)-tagged liposome, adhered through biotin-neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin-neutravidin interaction. We have compared the dynamics in a cell-mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time-scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET-based physical chemistry approach.


Assuntos
Adesão Celular/fisiologia , Membrana Celular/metabolismo , Lipossomos/metabolismo , Animais , Avidina/química , Biotina/química , Células CHO , Cumarínicos/química , Cricetulus , Fluoresceína/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Maleimidas/química , Microscopia Confocal , Microscopia de Fluorescência , Oxazinas/química , Rodaminas/química
17.
Molecules ; 24(11)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31141958

RESUMO

Systems for efficient and selective capture of micro-scale objects and structures have application in many areas and are of particular relevance for selective isolation of mammalian cells. Systems for the latter should also not interfere with the biology of the cells. This study demonstrates the capture of microspheres through orthogonal coupling using biotin (ligand) and (strept)avidin (receptor). Fibrous poly(ethylene terephthalate) (PET) meshes were hydrolyzed under controlled alkaline conditions to obtain activated surfaces with COOH groups allowing for the functionalization of the PET with biotin of various spacer length. The system capture efficiency was optimized by varying the length of spacer presenting the biotin against streptavidin. In a proof of concept experiment, avidin-functionalized microspheres were used as surrogates for cells, and their capture under dynamic conditions including virous mixing and high-flow rate perfusion is demonstrated. Functionalization of PET meshes with biotin conjugated to longest spacer yielded the most efficient capture of microspheres. These preliminary results lay the foundation for the development of biosystems for capture of specific cells under physiologically relevant conditions, using biorthogonal avidin-biotin interactions.


Assuntos
Avidina/química , Biotina/química , Microesferas , Polímeros/química , Álcalis/química , Reatores Biológicos , Hidrólise , Perfusão , Polietilenotereftalatos/química
18.
Nanoscale ; 11(19): 9436-9443, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31038504

RESUMO

Ionic concentration-polarization (CP)-based biomolecule preconcentration is an established method for enhancing the detection sensitivity of a target biomolecule immunoassay. However, its main drawback lies in its inability to directly control the spatial overlap between the preconcentrated plug of biomolecules and the surface immobilized antibodies. To overcome this, we simultaneously preconcentrated freely suspended, surface functionalized nanoparticles and target molecules along the edge of a depletion layer, thus, increasing the binding kinetics and avoiding the need to tune their relative locations to ensure their spatial overlap. After the desired incubation time, the nanoparticles were dielectrophoretically trapped for postprocessing analysis of the binding signal. This novel combination of CP-based preconcentration and dielectrophoresis (DEP) was demonstrated through binding of avidin and biotin-conjugated particles as a model bead-based immunoassay, wherein increased detection sensitivity was demonstrated compared to an immunoassay without CP-based preconcentration. The DEP trapping of the beads following binding is important not only for an enhanced detection signal due to the preconcentration of the beads at the electrode edges but also for controlling their location for future applications integrating localized sensors. In addition, DEP may be important also as a preprocessing step for controlling the number of beads participating in the immunoassay.


Assuntos
Imunoensaio/métodos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Nanopartículas/química , Imagem com Lapso de Tempo
19.
Colloids Surf B Biointerfaces ; 180: 371-375, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079030

RESUMO

Functional poly(ethylene glycol) diacrylate (PEGDA) hydrogel microparticles for the detection of bioactive macromolecules were fabricated via oxygen-inhibited photopolymerization in a droplet microfluidic device. Hydrogel network functionalization and architecture were characterized using a biotin-avidin binding assay, which revealed radial network inhomogeneities dependent on exposure conditions. Empirical results were corroborated using a reaction-diffusion model, describing the effects of exposure intensity on the spatial photopolymerization kinetics and resulting polymeric mesh network. The combination of finely controlled exposure conditions and predictive simulations enables the generation of tailored particles with microengineered interfaces and gradients in crosslinking density, which dictate solute diffusivity and elasticity, augmenting the utility of this approach in engineering multifunctional, size-excluding hydrogel particles for multiplexed biomolecular sensing.


Assuntos
Hidrogéis/química , Luz , Microesferas , Oxigênio/química , Polimerização , Engenharia Tecidual/métodos , Avidina/química , Biotina/química , Difusão , Fluorescência , Rodaminas/química
20.
Chem Commun (Camb) ; 55(35): 5159-5162, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30984931

RESUMO

We have reported a versatile nanopore method based on the combination of analyte-controlled liposome signal amplification and the nanopore detection of a reporter molecule, which largely extends the nanopore application range, and easily elevates the nanopore sensitivity to the fM level from the µM level.


Assuntos
Avidina/análise , Proteínas Hemolisinas/química , Nanoporos , Hormônio Liberador de Tireotropina/análise , Lipossomas Unilamelares/química , Biotina/química , Fosfatidilcolinas/química , Ácido Fítico/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA