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1.
Biosens Bioelectron ; 151: 111970, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868609

RESUMO

Herein, a credible construction strategy to improve electrochemiluminescence (ECL) of luminol was developed based on Cu2O-Au heterostructures. Summarily, gold nanoparticles (AuNPs) were anchored on surface of Cu2O nanocube (Cu2O@AuNPs) by spontaneous reduction reaction. Then, luminol molecules were concentrated on Cu2O@AuNPs using L-Cysteine (Cys) as covalent linkage to build the composite emitter (Cu2O@AuNPs-Cys-luminol). The enhancement mechanism was realized by following aspects: (I) Cu2O@AuNPs worked as electrocatalyst for glucose to generate coreactant of H2O2 in situ, avoiding the instability of direct addition of H2O2. (II) luminol molecules were firmly attached on Cu2O@AuNPs to achieve centralized and strong luminescence at low consumption. (III) Cys acted as an intramolecular coreactant and directly linked to luminol to increase luminous efficiency. To validate the effectiveness, a sandwiched immunoassay was built using concanavalinA (ConA) as analyte. Electroreduced graphene film as substrate provided phenoxy-derivatized dextran (DexP) with abundant binding sites and improved conductivity. To improve the specificity, DexP was used to identify ConA via the specific carbohydrate-ConA interaction. Then, Cu2O@AuNPs-Cys-luminol was modified on electrode as ECL signal indicator. The ECL immunosensor achieved determination of ConA with low detection limit of 2.9 × 10-5 ng/mL and excellent stability of continuous potential scan for 8 cycles. Experimental results demonstrated that the proposed construction strategy made considerable progress in ECL efficiency and stability of luminol. The creational pattern of construction strategy achieves high detection capabilities to ConA and expands the applicability of luminol in ECL system. It is expected to have more potential application value in immunoassay with universality.


Assuntos
Cobre/química , Ouro/química , Luminol/química , Nanopartículas Metálicas/química , Técnicas Biossensoriais , Concanavalina A/análise , Cisteína/química , Dextranos/química , Técnicas Eletroquímicas , Eletrodos , Peróxido de Hidrogênio/química , Limite de Detecção , Medições Luminescentes , Oxirredução , Sensibilidade e Especificidade , Propriedades de Superfície
2.
Colloids Surf B Biointerfaces ; 173: 504-511, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30340178

RESUMO

Thermosensitive glucose-functionalized glycopolymers grafted gold nanoparticles (Glyco@GNPs) with good colloidal stability and thermosensitive in aqueous solution were fabricated by reversible addition-fragmentation chain transfer (RAFT) mediated one-pot synthesis. The formation of core-shell morphology with about a 60 nm gold core in diameter and a glycopolymer shell of about 80 nm in thickness was indicated by transmission electron microscopy (TEM). The recognition ability of the Glyco@GNPs toward lectin concannavalin A (Con A) was verified by ultraviolet-visible spectroscopy and dynamic light scattering (DLS). The good cytocompatibility of the glycopolymers and Glyco@GNPs was proven by MTT assay on L-929 cells. Glyco@GNPs could effectively inhibit hepatoma cells SMMC-7721 growth after recognizing Con A was also proved by MTT assay and flow cytometry assay.


Assuntos
Técnicas de Química Sintética , Concanavalina A/análise , Glucose/química , Glicoconjugados/química , Ouro/química , Nanopartículas Metálicas/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Concanavalina A/química , Fibroblastos/efeitos dos fármacos , Glicoconjugados/farmacologia , Hepatócitos/efeitos dos fármacos , Temperatura Alta , Humanos , Nanopartículas Metálicas/ultraestrutura , Metacrilatos/química , Camundongos , Polimerização , Soluções , Compostos de Vinila/química , Água/química
3.
Biosens Bioelectron ; 120: 40-46, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30144644

RESUMO

A novel ratiometric electrochemiluminescent (ECL) biosensor was designed for the detection of concanavalin A (Con A) based on two ECL emitters competing for the dissolved oxygen (O2). In this strategy, CdTe quantum dots (QDs) were used as the cathodic emitter and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) was used as the anodic emitter. With the presence of dissolved O2 utilized as co-reactant, CdTe QDs showed an ECL emission at - 1.7 V, and ABEI showed an emission at + 0.6 V. Phenoxy-derivatized dextran (Dexp), as a recognition element, was immobilized by graphene oxide functionalized CdTe QDs (G-CdTe QDs) to recognize Con A, and further combine with Dexp, gold and platinum nanoparticles decorated ABEI (Dexp-Au-Pt-ABEI) to form a sandwich structure. With the increasing concentration of analyte Con A, the ECL signal of cathodic CdTe QDs decreased and the anodic response of ABEI increased, thus achieving a ratiometry detection of Con A with a wide linear range from 1.0 × 10-4 ng/mL to 10 ng/mL. The detection limit was low to 3.0 × 10-5 ng/mL. This proposed ratiometric ECL biosensor not only conquered the false errors caused by external interferences, but excluded the disability caused by exogenous co-reactant via the application of common dissolved O2. It also exhibited an excellent stability, selectivity and reproducibility.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Concanavalina A/análise , Oxigênio/química , Pontos Quânticos , Reprodutibilidade dos Testes
4.
Biosensors (Basel) ; 8(3)2018 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-29966294

RESUMO

Improvement upon, and expansion of, diagnostic tools for clinical infections have been increasing in recent years. The simplicity and rapidity of techniques are imperative for their adoption and widespread usage at point-of-care. The fabrication and evaluation of such a device is reported in this work. The use of a small bioreceptor array (based on lectin-carbohydrate binding) resulted in a unique response profile, which has the potential to be used for pathogen identification, as demonstrated by Principal Component Analysis (PCA). The performance of the chemiresistive device was tested with Escherichia coli K12, Enterococcus faecalis, Streptococcus mutans, and Salmonella typhi. The limits of detection, based on concanavalin A (conA) lectin as the bioreceptor, are 4.7 × 10³ cfu/mL, 25 cfu/mL, 7.4 × 104 cfu/mL, and 6.3 × 10² cfu/mL. This shows that the detection of pathogenic bacteria is achieved with clinically relevant concentrations. Importantly, responses measured in spiked artificial saliva showed minimal matrix interference. Furthermore, the exploitation of the distinctive outer composition of the bacteria and selectivity of lectin-carbohydrate interactions allowed for the discrimination of bacterial infections from viral infections, which is a current and urgent need for diagnosing common clinical infections.


Assuntos
Bactérias/isolamento & purificação , Técnicas Biossensoriais , Lectinas/análise , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Infecções Bacterianas/diagnóstico , Concanavalina A/análise , Concanavalina A/metabolismo , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Humanos , Lectinas/metabolismo , Limite de Detecção , Monossacarídeos/química , Monossacarídeos/metabolismo , Nanotubos de Carbono/química , Análise de Componente Principal , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/isolamento & purificação , Streptococcus mutans/metabolismo
5.
ACS Nano ; 12(3): 2455-2465, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29512997

RESUMO

Glycoproteins adhered on the cellular membrane play a pivotal role in a wide range of cellular functions. Their importance is particularly relevant in the recognition process between infectious pathogens (such as viruses, bacteria, toxins) and their host cells. Multivalent interactions at the pathogen-cell interfaces govern binding events and can result in a strong and specific interaction. Here we report an approach to mimic the cell surface presentation of carbohydrate ligands by the multivalent display of sugars on the surface of peptoid nanosheets. The constructs provide a highly organized 2D platform for recognition of carbohydrate-binding proteins. The sugars were displayed using different linker lengths or within loops containing 2-6 hydrophilic peptoid monomers. Both the linkers and the loops contained one alkyne-bearing monomer, to which different saccharides were attached by copper-catalyzed azide-alkyne cycloaddition reactions. Peptoid nanosheets functionalized with different saccharide groups were able to selectively bind multivalent lectins, Concanavalin A and Wheat Germ Agglutinin, as observed by fluorescence microscopy and a homogeneous Förster resonance energy transfer (FRET)-based binding assay. To evaluate the potential of this system as sensor for threat agents, the ability of functionalized peptoid nanosheets to bind Shiga toxin was also studied. Peptoid nanosheets were functionalized with globotriose, the natural ligand of Shiga toxin, and the effective binding of the nanomaterial was verified by the FRET-based binding assay. In all cases, evidence for multivalent binding was observed by systematic variation of the ligand display density on the nanosheet surface. These cell surface mimetic nanomaterials may find utility in the inactivation of pathogens or as selective molecular recognition elements.


Assuntos
Lectinas/análise , Nanoestruturas/química , Peptoides/química , Toxina Shiga/análise , Sítios de Ligação , Biomimética , Técnicas Biossensoriais , Concanavalina A/análise , Concanavalina A/metabolismo , Transferência Ressonante de Energia de Fluorescência , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Lectinas/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Monossacarídeos/química , Monossacarídeos/metabolismo , Nanoestruturas/ultraestrutura , Peptoides/metabolismo , Ligação Proteica , Toxina Shiga/metabolismo , Trissacarídeos/química , Trissacarídeos/metabolismo , Aglutininas do Germe de Trigo/análise , Aglutininas do Germe de Trigo/metabolismo
6.
Colloids Surf B Biointerfaces ; 162: 60-68, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29149729

RESUMO

Glycan-lectin interactions are commonly observed in nature. Analytical methods, which are used to detect lectins that rely on the use of glycan ligand-modified nanoprobes as affinity probes, have been developed. Most of the existing methods are focused on the use of synthetic glycan ligands. Nevertheless, naturally available glycoproteins, such as ovalbumin in chicken egg whites, are good sources for fabricating glycan-immobilized nanoprobes. In this study, we generated functionalized gold nanoparticles (Au NPs) from a one-pot reaction by reacting chicken egg white (cew) proteins with aqueous tetrachloroaurate. The generated Au@cew NPs are mainly encapsulated by ovalbumin, in which the surface is decorated by abundant hybrid mannose and Galß(1→4)GlcNAc-terminated glycan ligands. Thus, the generated Au@cew NPs containing hybrid mannose and Galß(1→4)GlcNAc have the capability to selectively bind with their corresponding lectins. Lectins including concanavalin A, banana lectin, and ricin B that have binding moieties toward specific sugars were used as the model samples. Our results showed that the generated AuNPs can be used as multiplex affinity probes for these model lectins. Lectins can be selectively released from the Au@cew NP-lectin conjugates by using specific sugars, such as mannose, glucose, and ß-lactose, as the releasing agents to release specific lectins from the conjugates. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used as the tool to characterize the released species from the nanoprobes. The limit of detection of these model lectins using the current approach was low (in nM). The feasibility of using the Au@cew NP-based affinity MALDI-MS to selectively detect specific lectins from complex samples was also demonstrated.


Assuntos
Concanavalina A/análise , Ouro/química , Nanopartículas Metálicas/química , Sondas Moleculares/química , Ovalbumina/química , Ricina/análise , Animais , Galinhas , Clara de Ovo/química , Glucose/química , Lactose/química , Limite de Detecção , Manose/química , Sondas Moleculares/síntese química , Ovalbumina/isolamento & purificação , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
7.
Biosens Bioelectron ; 96: 113-120, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28475956

RESUMO

An electrochemiluminescence (ECL) biosensor was developed for detection of Concanavalin A (Con A). Chitosan/Ru(bpy)32+/silica/Fe3O4 nanomaterials (CRuSi-Fe3O4) were synthesized through W/O microemulsion route. The added Fe3O4 nanoparticles can simplify the prepared process and enhance the conductivity of nanomaterials which can increase the ECL intensity of luminophor CRuSi-Fe3O4. In addition, the layered structure of CRuSi-Fe3O4 can immobilize lots of Con A using glutaraldehyde as the coupling agent which can improve the sensitivity of the biosensor. Then the quenching probe glucose functionalized NiCo2S4 nanoparticles-grown on carboxylic graphene (NiCo2S4-COOH-rGO@Glu) was anchored on the modified-electrode via the specific carbohydrate-Con A interaction. Here, NiCo2S4 was used to quench the ECL of CRuSi-Fe3O4, graphene was used to grow NiCo2S4 nanoparticles as carrier materials and glucose was served as the recognition element for bounding Con A. Therefore, a desirable quenching ECL signal was measured with S2O82- as the coreactant of CRuSi-Fe3O4. Under the optimization of determination conditions, a linear response range for Con A from 0.5pgmL-1 to 100ngmL-1 was obtained, and the detection limit was calculated to be 0.18pgmL-1 (S/N=3).


Assuntos
Concanavalina A/análise , Técnicas Eletroquímicas/métodos , Glucose/química , Grafite/química , Medições Luminescentes/métodos , Nanopartículas/química , Técnicas Biossensoriais/métodos , Quitosana/química , Eletrodos , Óxido Ferroso-Férrico/química , Humanos , Limite de Detecção , Compostos Organometálicos/química , Tamanho da Partícula , Dióxido de Silício/química , Propriedades de Superfície
8.
Anal Biochem ; 528: 53-56, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28416394

RESUMO

Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1-2 days, making it unsuitable for rapid diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol. Collectively, the findings suggest the development of ELISA-based rapid diagnostics.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/análise , Concanavalina A/análise , Humanos , Imunoglobulina E/análise , Indicadores e Reagentes
9.
Biosens Bioelectron ; 87: 802-806, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27657841

RESUMO

A sandwich-configuration electrochemiluminescence (ECL) biosensor was constructed for detecting concanavalin A (ConA) based on peroxydisulfate/oxygen (S2O82-/O2) system. In this work, the gold nanoflower modified Zn-doped SnO2 was used as a substrate to adsorb recognition element horseradish peroxidase (HRP) for binding ConA. Then, Au nanoparticles-thiosemicarbazide functionalized PtNi nanocubes (AuNPs-TSC-PtNi NCs), as a novel ECL signal tracer, were incubated onto the electrode through a specific carbohydrate-ConA interaction, thus achieving a sandwiched structure. The integration of amplifying effect of both TSC and PtNi NCs on the ECL of S2O82-/O2 system endowed the biosensor a high sensitivity. The linear range for ConA detection was from 0.0010ng/mL to 10ng/mL with a detection limit of 0.0002ng/mL (S/N=3).


Assuntos
Concanavalina A/sangue , Técnicas Eletroquímicas/métodos , Ouro/química , Medições Luminescentes/métodos , Nanopartículas Metálicas/química , Semicarbazidas/química , Técnicas Biossensoriais/métodos , Concanavalina A/análise , Peroxidase do Rábano Silvestre/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Níquel/química , Platina/química
10.
Anal Chim Acta ; 937: 119-26, 2016 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-27590553

RESUMO

Proteins are responsible for most biochemical events in human body. It is essential to develop sensitive and selective methods for the detection of proteins. In this study, liquid crystal (LC)-based sensor for highly selective and sensitive detection of lysozyme, concanavalin A (Con A), and bovine serum albumin (BSA) was constructed by utilizing the LC interface decorated with a nonionic surfactant, dodecyl ß-d-glucopyranoside. A change of the LC optical images from bright to dark appearance was observed after transferring dodecyl ß-d-glucopyranoside onto the aqueous/LC interface due to the formation of stable self-assembled surfactant monolayer, regardless of pH and ion concentrations studied in a wide range. The optical images turned back from dark to bright appearance after addition of lysozyme, Con A and BSA, respectively. Noteworthy is that these proteins can be further distinguished by adding enzyme inhibitors and controlling incubation temperature of the protein solutions based on three different interaction mechanisms between proteins and dodecyl ß-d-glucopyranoside, viz. enzymatic hydrolysis, specific saccharide binding, and physical absorption. The LC-based sensor decorated with dodecyl ß-d-glucopyranoside shows high sensitivity for protein detection. The limit of detection (LOD) for lysozyme, Con A and BSA reaches around 0.1 µg/mL, 0.01 µg/mL and 0.001 µg/mL, respectively. These results might provide new insights into increasing selectivity and sensitivity of LC-based sensors for the detection of proteins.


Assuntos
Técnicas Biossensoriais , Concanavalina A/análise , Cristais Líquidos/química , Muramidase/análise , Soroalbumina Bovina/análise , Tensoativos/química , Animais , Bovinos , Glucosídeos/análise , Ouro/química
11.
Anal Chim Acta ; 935: 97-103, 2016 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-27543018

RESUMO

In an attempt to develop a label- and reagent-free electrochemical method for the detection of lectin-glycoprotein interactions, we tested lectin-concanavalin A (ConA), glycoprotein-ovalbumin (Ova) and their complex using chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode. Incubation of ConA with Ova resulted in an increase of the CPS peak H of the complex as compared to the CPS peaks of individual Ova and ConA proteins. Qualitatively similar results were obtained with other glycoprotein-lectin couples (ConA-RNase B and lectin from Sambucus nigra-fetuin). Using the CPS method, we were able to follow the course of complex formation in solution. Comparable responses of Ova, ConA and ConA-Ova complex were obtained not only at the mercury electrode but also with solid amalgam electrodes, which are more suitable for parallel analysis. It can be anticipated that electrochemical methods, namely CPS, will find application in glycomics and proteomics.


Assuntos
Concanavalina A/análise , Técnicas Eletroquímicas , Ovalbumina/análise , Animais , Canavalia/química , Galinhas , Modelos Moleculares , Soluções
12.
Anal Chim Acta ; 932: 88-97, 2016 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-27286773

RESUMO

In this work, we report a novel label-free fluorescence "turn off-on" biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS2 quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS2 QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS2 QDs surface were interacted with the amino groups (NH2), carboxyl groups (COOH) and hydroxyl groups (OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively "turned on". Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I0 (I and I0 were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2-192.5 nmol L(-1), And the detection limit could be down to 0.08 nmol L(-1). Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results.


Assuntos
Técnicas Biossensoriais/métodos , Quitosana/análogos & derivados , Concanavalina A/análise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Pontos Quânticos/química , Animais , Bovinos , Quitosana/química , Concanavalina A/química , Transporte de Elétrons , Humanos , Espectrometria de Fluorescência
13.
Analyst ; 141(7): 2250-8, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26934683

RESUMO

The multivalent display of carbohydrates on the cell surface provides cooperative binding to improve the specific biological events. In addition to multivalency, the spatial arrangement and orientation of sugars with respect to external stimuli also trigger carbohydrate-protein interactions. Herein, we report a non-covalent host-guest strategy to immobilize heptavalent glyco-ß-cyclodextrin on gold-coated glass slides to study multivalent carbohydrate-protein interactions. We have found that the localization of sugar entities on surfaces using ß-cyclodextrin (ß-CD) chemistry increased the avidity of carbohydrate-protein and carbohydrate-macrophage interactions compared to monovalent-ß-CD sugar coated surfaces. This platform is expected to be a promising tool to amplify the avidity of sugar-mediated interactions on surfaces and contribute to the development of next generation bio-medical products.


Assuntos
Concanavalina A/análise , Ouro/química , Macrófagos/citologia , beta-Ciclodextrinas/química , Adesão Celular , Linhagem Celular , Humanos , Propriedades de Superfície
14.
Chem Rec ; 16(2): 768-82, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26915980

RESUMO

This article describes recent developments in C3 -symmetric tris-urea low-molecular-weight gelators and their applications. The C3 -symmetric tris-ureas are excellent frameworks to form supramolecular polymers through noncovalent interactions. In organic solvents, hydrophobic tris-ureas form supramolecular gels. Amphiphilic tris-ureas form supramolecular gels in aqueous media. Functional supramolecular gels were prepared by introducing appropriate functional groups into the outer sphere of tris-ureas. Supramolecular hydrogels obtained from amphiphilic tris-ureas were used in the electrophoresis of proteins. These electrophoreses results showed several unique characteristics compared to typical electrophoreses results obtained using polyacrylamide matrices.


Assuntos
Hidrogéis/química , Compostos de Fenilureia/química , Acetona/química , Animais , Galinhas , Concanavalina A/análise , Concanavalina A/química , Dimetil Sulfóxido/química , Eletroforese em Gel de Poliacrilamida , Glucosídeos/química , Hidrocarbonetos Clorados/química , Hidrogéis/síntese química , L-Lactato Desidrogenase/análise , Compostos Organometálicos/química , Ovalbumina/análise , Transição de Fase , Dodecilsulfato de Sódio/química , Estereoisomerismo , beta-Galactosidase/análise
15.
Anal Chem ; 88(3): 1821-6, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26750769

RESUMO

A ratiometric, versatile, and selective fluorescent pattern to sense and distinguish proteins on the basis of dissociation of aqueous polymer-pyrene/γ-cyclodextrin (γ-CD) inclusion complexes was developed. First, two kinds of aqueous polymer-pyrene were prepared via atom transfer radical polymerization using pyrene functionalized initiator. Then, the pyrene molecules could be accumulated into γ-CD cavity and form polymer-pyrene/γ-CD complexes, resulting in appearance of excimer emissions. The resultant complexes responded to proteins in two ways: nonmetalloproteins binding to polymer component triggered dissociation of the inclusion complexes, accompanied by alteration of pyrene excimer/monomer emission and ratiometric fluorescent intensity changes; the presence of metalloproteins could quench pyrene excimer/monomer emission because of energy transfer. Moreover, the fluorescent responses of the inclusion complexes to different proteins could be modulated by changing polymer type and chain length, resulting in a tunable selectivity and sensitivity. The proposed fluorescent inclusion complexes could provide a promising platform for sensing proteins.


Assuntos
Concanavalina A/análise , Fluorescência , Mioglobina/análise , Pepsina A/análise , Albumina Sérica/análise , Humanos , Estrutura Molecular , Polímeros/química , Pirenos/química , Água/química , gama-Ciclodextrinas/química
16.
J Colloid Interface Sci ; 462: 19-28, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26433474

RESUMO

Poly(methyl methacrylate) polymer based Localized Surface Plasmon Resonance biosensor chips were successfully fabricated using glycopolymer brushes carrying glucose moieties for the detection of concanavalin A. Poly(pentafluorostyrene), with pre-determined polymer chain lengths, were synthesized via a reversible addition-fragmentation chain transfer polymerization technique. The synthesized poly(pentafluorostyrene), was subsequently converted into glycopolymers via a para-fluoro-thiol "click" reaction and grafted onto the surface of sensor chips. The "glycocluster effect" induced by pendent carbohydrate moieties enabled a stronger affinity for concanavalin A binding, which resulted in a dramatic expansion of the sensors' response range. It was discovered that the longer polymer brushes did not guarantee additional enhancements for the sensor chips. Instead, they could lead to higher detection limits. In this study, the limit of detection for the sensor chips was discovered to be 1.3nmolL(-1) with a saturated response at 1054.2nmolL(-1). In addition to the superior performance, the capabilities of the reported sensor chips can be easily manipulated to detect a diverse range of analytes by "clicking" various sensing elements onto the polymer brushes.


Assuntos
Técnicas Biossensoriais , Concanavalina A/análise , Glucose/química , Polimetil Metacrilato/química , Ressonância de Plasmônio de Superfície , Modelos Moleculares , Estrutura Molecular , Polimetil Metacrilato/síntese química
17.
Cytometry A ; 87(9): 843-54, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26033928

RESUMO

Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.


Assuntos
Parede Celular/química , Parede Celular/metabolismo , Concanavalina A/análise , Citometria de Fluxo/métodos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fusão Celular/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos
19.
J Agric Food Chem ; 63(16): 4104-11, 2015 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-25865304

RESUMO

Herein, a novel aptamer that targets concanavalin A (Con A), a plant lectin, is isolated using systematic evolution of ligands by an exponential enrichment (SELEX) technique. Nine rounds of SELEX screening over an agarose spin column have resulted in enrichment of eight sequences having high affinity to Con A. The highest affinity sequence was selected as a potent aptamer and characterized it in detail. The evolved Con A aptamer (Con A-aptabody) is a 41 nt ssDNA that binds the Con A specifically with a dissociation constant of 172.7 ± 29.7 nM. In silico analyses predict the Con A-aptabody to form G-quadruplex due to its G-rich sequence (GGAAGGCGGAGGG). A detection method developed using Con A-aptabody is found to have a detection range of 10-750 ng/mL with limits of detection and quantification being 13.22 and 44.09 ng/mL, respectively. The utility of the method is demonstrated by analyzing jack bean (Canavalia ensiformis), kidney bean (Phaseolus vulgaris), wheat (Triticum spp.), mung bean (Vigna radiata), and lentil (Lens culinaris) for their Con A contents. Hence, the developed Con A-aptabody provides a useful substitute to Con A-antibody for food analysis and related applications.


Assuntos
Concanavalina A/análise , Fabaceae/química , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química
20.
Biosens Bioelectron ; 70: 89-97, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25796041

RESUMO

A novel signal-on electrochemiluminescence (ECL) biosensor for detecting concanavalin A (Con A) was fabricated with phenoxy dextran-graphite-like carbon nitride (DexP-g-C3N4) as signal probe. In this construction strategy, the nanocomposites of three-dimensional graphene and gold nanoparticles (3D-GR-AuNPs) were used as matrix for high loading of glucose oxidase (GOx), which served as recognition element for bounding Con A. Con A further interacted with DexP-g-C3N4 through a specific carbohydrate-Con A interaction to achieve a sandwiched scheme. With the increase of Con A incubated onto the electrode, the ECL signal resulted from DexP-g-C3N4 would enhance, thus achieving a signal-on ECL biosensor for Con A detection. Due to the integration of the virtues of 3D-GR-AuNPs and the excellent ECL performance of DexP-g-C3N4, the prepared biosensor exhibits a wide linear response range from 0.05 ng/mL to 100 ng/mL and a low detection limit of 17 pg/mL (S/N=3).


Assuntos
Concanavalina A/análise , Condutometria/instrumentação , Dextranos/química , Grafite/química , Medições Luminescentes/instrumentação , Nitrilos/química , Desenho de Equipamento , Análise de Falha de Equipamento , Ouro , Nanopartículas Metálicas/química , Técnicas de Sonda Molecular , Fenóis , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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