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1.
Int J Nanomedicine ; 14: 4833-4847, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308661

RESUMO

Background: The use of functionalized graphene oxide (fGO) has led to a new trend in the sensor field, owing to its high sensitivity with regards to sensing characteristics and easy synthesis procedures. Methods: In this study, we developed an ultra-sensitive carboxyl-graphene oxide (carboxyl-GO)-based surface plasmon resonance (SPR) aptasensor using peptides to detect human chorionic gonadotropin (hCG) in clinical serum samples. The carboxyl-GO based SPR aptasensor provided high affinity and stronger binding of peptides, which are great importance to allow for a non-immunological label-free mechanism. Also, it allows the detection of low concentrations of hCG, which are in turn considered to be important clinical parameters to diagnose ectopic pregnancies and paraneoplastic syndromes. Results: The high selectivity of the carboxyl-GO-based SPR aptasensor for hCG recombinant protein was verified by the addition of the interfering proteins bovine serum albumin (BSA) and human serum albumin (HSA), which did not affect the sensitivity of the sensor. The carboxyl-GO-based chip can enhance the assay efficacy of interactions between peptides and had a high affinity binding for a ka of 17×106 M-1S-1. The limit of detection for hCG in clinical serum samples was 1.15 pg/mL. Conclusion: The results of this study demonstrated that the carboxyl-GO-based SPR aptasensor had excellent sensitivity, affinity and selectivity, and thus the potential to be used as disease-related biomarker assay to allow for an early diagnosis, and possibly a new area in the field of biochemical sensing technology.


Assuntos
Técnicas Biossensoriais/instrumentação , Gonadotropina Coriônica/sangue , Grafite/química , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Bovinos , Eletroquímica , Humanos , Peptídeos/química , Espectroscopia Fotoeletrônica , Soroalbumina Bovina/química
2.
Nat Commun ; 10(1): 2385, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160589

RESUMO

Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.


Assuntos
Antineoplásicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/metabolismo , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Cristalização , Cristalografia por Raios X , Humanos , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Ressonância de Plasmônio de Superfície
3.
Anal Bioanal Chem ; 411(20): 5023-5031, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31177332

RESUMO

A simple method to determine hazardous silver nanoparticles (AgNPs) based on ionic liquid (IL) dispersive liquid-liquid microextraction and back-extraction is described. This approach involves AgNP stabilization using a cationic surfactant followed by extraction from the sample matrix by means of an IL as an extraction phase. Certain ILs have high affinity for metals, and preliminary experiments showed that those ILs consisting of imidazolium cation efficiently extracted AgNPs in the presence of a cationic surfactant and a chelating agent. Afterward, histamine was used as a dispersing agent to promote phase transfer of differently coated AgNPs from the IL in aqueous solution to be subsequently analyzed by UV-visible spectrometry. The analytical procedure allows AgNPs to be recovered from the sample matrix in an aqueous medium, the enrichment factor being up to 4, preserving both AgNP size and AgNP shape as demonstrated by transmission electron microscopy images and the localized surface plasmon resonance band characteristic of each AgNP. The present method exhibited a linear response for AgNPs in the range from 3 to 20 µg/mL, the limit of detection being 0.15 µg/mL. Method efficiency was assessed in spiked orange juice and face cream, yielding recoveries ranging from 75.7% to 96.6%. The method was evaluated in the presence of other nanointerferents (namely, gold nanoparticles). On the basis of diverse electrophoretic mobilities and surface plasmon resonance bands for metal nanoparticles, capillary electrophoresis was used to prove the lack of interaction of the target AgNPs with gold nanoparticles during the whole protocol; thus, interferents do not affect AgNP determination. As a consequence, the analytical approach described has great potential for the analysis of engineered nanosilver in consumer products. Graphical abstract Simple protocol for the determination of silver nanoparticles (AgNPs) based on dispersive liquid-liquid extraction with a specific short alkyl side chain ionic liquid and their quantitative detection with a UV-visible spectrometer. HMIM•PF6 1-hexyl-3-methylimidazolium hexafluorophosphate, NP nanoparticle, SPR surface plasmon resonance.


Assuntos
Cosméticos/química , Sucos de Frutas e Vegetais/análise , Líquidos Iônicos/química , Microextração em Fase Líquida/métodos , Nanopartículas Metálicas/análise , Prata/química , Artefatos , Estudos de Viabilidade , Limite de Detecção , Nanopartículas Metálicas/química , Espectrofotometria Ultravioleta/métodos , Ressonância de Plasmônio de Superfície
4.
Opt Express ; 27(11): 15996-16011, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31163787

RESUMO

In this paper, the dynamics of the spontaneous emission rate of Rhodamine 6G dye molecules, coupled into disorder-induced optical cavities in a scattering medium, is investigated by a time-resolved spectroscopic technique. The system is a wedge-type wave-guiding system formed by a polymer with randomly positioned air inclusions. The scattering of light in the medium induces transverse Anderson localization, which gives rise to quasi-optical modes or Anderson-localized cavities. The presence of these modes strongly enhances the decay emission of the emitters. The waveguide is fabricated by a conventional fiber drawing technique inside a fused silica micro-rod. Localized optical modes are observed to appear in the form of sharp spectral resonance peaks at various frequencies throughout the photoluminescence spectrum of the dye molecules. The spontaneous emission rate of the molecules on resonance with the localized modes is measured to enhance by a factor of up to 6.8, which elucidates that the transverse Anderson localization enables an efficient way to alter the spontaneous emission rate of quantum emitters in an optically asymmetric simple wedge-type photonic waveguide, offering a moderate alternative to highly engineered sophisticated light-wave devices.


Assuntos
Corantes Fluorescentes/química , Aumento da Imagem/métodos , Rodaminas/química , Espalhamento de Radiação , Ressonância de Plasmônio de Superfície/instrumentação
5.
J Photochem Photobiol B ; 197: 111519, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31228688

RESUMO

Gold nanoparticles of comparable size were synthetized using honey mediated green method (AuNPs@honey) and citrate mediated Turkevich method (AuNPs@citrate). Their colloidal behavior in two cell media DMEM and RPMI, both supplemented with 10% FBS, was systematically investigated with different characterization techniques in order to evidence how the composition of the media influences their stability and the development of protein/NP complex. We revealed the formation of the protein corona which individually covers the nanoparticles in RPMI media, like a dielectric spacer according to UV-Vis spectroscopy, while DMEM promotes more abundant agglomerations, clustering together the nanoparticles, according to TEM investigations. In order to evaluate the biological impact of nanoparticles, B16 melanoma and L929 mouse fibroblasts cells were used to carry out the viability assays. Generally, the L929 cells were more sensitive than B16 cells to the presence of gold nanoparticles. Measurements of cell viability, proliferation and apoptotic activities of B16 cells indicated that the effects induced by AuNPs@honey were slightly similar to those induced by AuNPs@citrate, however, the toxic response improved in the L929 fibroblast cells following the treatment with AuNPs@honey within the same concentration range from 1 µg/ml to 15 µg/ml for 48 h. Results showed that honey mediated synthesis generates nanoparticles with reduced toxicity trends depending on the cell type, concentration of nanoparticles and exposure time toward various biomedical applications.


Assuntos
Citratos/química , Ouro/química , Mel/análise , Nanopartículas Metálicas/química , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Camundongos , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier , Ressonância de Plasmônio de Superfície
6.
Food Chem ; 297: 125022, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253281

RESUMO

Lactoferrin (LF) is a glycoprotein that serves as a potential vehicle for small bioactive molecules in food. In an effort to improve this functionality, the kinetic and thermodynamic interaction of LF with naringin (NR) was studied by surface plasmon resonance (SPR). The results demonstrated that the association rate constant between LF and NR was 5.00 × 104 M-1 s-1, while the dissociation rate of the complex was 0.36 s-1, at 25 °C. The stable complex predominated over free molecules (ΔG25°C0=-29.35 kJ mol-1), and the binding constant was 1.39 × 105 M-1, at 25 °C. The association of LF and NR to form an intermediate complex occurred in multi-steps. Nevertheless, the intermediate complex formation from the dissociation of the stable complex occurred in a single step with the activation energy independent of temperature. This study provides an important basis to explore LF as a vehicle for bioactive molecules.


Assuntos
Flavanonas/química , Lactoferrina/química , Ressonância de Plasmônio de Superfície , Animais , Bovinos , Flavanonas/metabolismo , Cinética , Lactoferrina/metabolismo , Ligação Proteica , Temperatura Ambiente , Termodinâmica
7.
Anal Bioanal Chem ; 411(17): 3979-3988, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089787

RESUMO

Herein, a universal and multifunctional fluorescence sensor platform is designed by the interaction of aggregation/dispersion gold nanoparticles (AuNPs) with Tb-metal-organic frameworks (Tb-MOFs). It is found that the dispersed AuNPs rather than the aggregated ones can quench effectively the fluorescence of Tb-MOFs, and the quenching process presumably involves the mechanism of inner filter effect (IFE), dynamic quenching effect (DQE), and fluorescence resonance energy transfer (FRET). The different affinities of aptamer and aptamer-target complex toward AuNPs are employed to modulate the fluorescence signal change of Tb-MOFs. As the proof of concept, prostate-specific antigen (PSA), an efficient tumor indicator for prostate cancer, is selected as the target. At first, the PSA aptamer can protect AuNPs against salt-induced aggregation, leading to the fluorescence of Tb-MOFs quenching. Subsequently, upon PSA introduction, the rigid aptamer-PSA complex is formed and cannot stabilize AuNPs in high salt conditions, so the AuNPs aggregate significantly and the fluorescence of Tb-MOFs is restored. The linear range of PSA is achieved from 1 to 100 ng/mL with a detection limit of 0.36 ng/mL. Finally, this method has been validated to be sensitive and specific for PSA in human urine samples. Graphical abstract.


Assuntos
Técnicas Biossensoriais , Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Estruturas Metalorgânicas/química , Antígeno Prostático Específico/análise , Térbio/química , Fluorescência , Humanos , Limite de Detecção , Masculino , Microscopia Eletrônica de Transmissão , Antígeno Prostático Específico/urina , Espectroscopia de Infravermelho com Transformada de Fourier , Ressonância de Plasmônio de Superfície
9.
Talanta ; 201: 465-473, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122452

RESUMO

We report results of the studies relating to the fabrication of a surface plasmon resonance (SPR) based label-free immunosensor for real-time monitoring of endothelin-1 (ET-1), a colon cancer biomarker. A gold disk modified with a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) was functionalised via covalent immobilization of monoclonal anti-ET-1 antibodies using EDC-NHS (1-(3-(dimethylamine)-propyl)-3-ethylcarbodiimide hydrochloride, N-hydroxy succinimide) chemistry. This immunosensing platform (ethanolamine/anti-ET-1/11-MUA/Au) was characterized via atomic force microscopy (AFM), contact angle (CA) and Fourier transform infrared (FT-IR) spectroscopic techniques. The fabricated SPR electrode was further used to detect ET-1 in the broad concentration range 2-100 pg mL-1, with a detection limit of 0.30 pg mL-1 and remarkable sensitivity of 2.18 mo pg-1mL. The adsorption mechanism was studied using monophasic model and the values of association (ka) and dissociation (kd) constants for anti-ET-1 and ET-1 binding were calculated to be 4.4 ±â€¯0.4 × 105 M-1 s-1 and 2.04 ±â€¯0.0003 × 10-3 s-1, respectively. The results obtained via analysis of serum samples of colorectal cancer patients were found to be in good agreement with those obtained from enzyme-linked immunosorbent assay (ELISA) technique. Further, electrochemical studies were performed to prove the efficacy of the fabricated platform as a point of care device for the detection of ET-1.


Assuntos
Técnicas Biossensoriais/métodos , Neoplasias do Colo/diagnóstico , Técnicas Eletroquímicas/métodos , Endotelina-1/sangue , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Neoplasias do Colo/sangue , Técnicas Eletroquímicas/instrumentação , Eletrodos , Endotelina-1/imunologia , Ácidos Graxos/química , Ouro/química , Humanos , Cinética , Limite de Detecção , Compostos de Sulfidrila/química
10.
Chem Commun (Camb) ; 55(49): 6964-6996, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31140997

RESUMO

Noble metals comprise any of several metallic chemical elements that are outstandingly resistant to corrosion and oxidation, even at elevated temperatures. This group is not strictly defined, but the tentative list includes ruthenium, rhodium, palladium, silver, osmium, iridium, platinum and gold, in order of atomic number. The emerging properties of noble metal nanoparticles are attracting huge interest from the translational scientific community and have led to an unprecedented expansion of research and exploration of applications in biotechnology and biomedicine. Noble metal nanomaterials can be synthesised both by top-down and bottom up approaches, as well as via organism-assisted routes, and subsequently modified appropriately for the field of use. Nanoscale analogues of gold, silver, platinum, and palladium in particular, have gained primary importance owing to their excellent intrinsic properties and diversity of applications; they offer unique functional attributes, which are quite unlike the bulk material. Modulation of noble metal nanoparticles in terms of size, shape and surface functionalisation has endowed them with unusual capabilities and manipulation at the chemical level, which can lead to changes in their electrical, chemical, optical, spectral and other intrinsic properties. Such flexibility in multi-functionalisation delivers 'Ockham's razor' to applied biomedical science. In this feature article, we highlight recent advances in the adaptation of noble metal nanomaterials and their biomedical applications in therapeutics, diagnostics and sensing.


Assuntos
Antibacterianos/química , Antifúngicos/química , Pesquisa Biomédica , Nanopartículas Metálicas/química , Metais Pesados/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Humanos , Metais Pesados/farmacologia , Ressonância de Plasmônio de Superfície
11.
Biosci Biotechnol Biochem ; 83(9): 1683-1696, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31094670

RESUMO

The fully synthetic humanized phage antibody library has the advantages including the minimized immunogenicity, which frequently happened in hybridoma cell-based antibody production. In this paper, using the constructed diverse complementarity determining region gene library and the germline gene as the backbone, we constructed eight single-chain antibody libraries and a combinatorial antibody library with a big capacity of 1.41 × 1010. M13EEA helper phage that was engineered from M13KO7 was applied to prepare phage antibody library. The eukaryotic expression of T-cell immune receptor with Ig and ITIM domain (TIGIT) antigen was used as a target antigen for screening. The screening of antigen-specific single-chain Fc-fused protein was performed through evaluation of binding affinity based on ELISA analysis. The IgG antibody was prepared with the screened single-chain protein. Finally, the CB3 antibody was screened out which exhibits the highest binding affinity with TIGIT with the Kd value of 8.155 × 10-10 M.


Assuntos
Bacteriófagos/genética , Imunoterapia/métodos , Neoplasias/terapia , Ensaio de Imunoadsorção Enzimática , Humanos , Neoplasias/imunologia , Ressonância de Plasmônio de Superfície
12.
Opt Express ; 27(8): 11348-11360, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31052980

RESUMO

The fiber geometry, fiber parameters and mode-guiding properties are crucial for realizing high-performance fiber-based sensors. In this work, we propose and demonstrate a few-mode fiber (FMF)-based surface plasmon resonance (SPR) biosensor. The FMF-SPR sensor was fabricated via side-polishing a few-mode fiber and coating a thin layer of gold film, on the basis of the optimization of fiber geometry, thickness of the gold film and mode selection, which were performed with the finite element method. The refractive index (RI) sensing performance of three such sensors with different residual fiber thicknesses were investigated. In the RI range from 1.333 to 1.404, the highest sensitivity up to 4903 nm/RIU and a figure of merit of 46.1 RIU-1 are achieved. For testing the bovine serum albumin (BSA) solution, an averaged BSA RI sensitivity of 6328 nm/RIU and an averaged BSA concentration sensitivity of 1.17 nm/(mg/ml) are realized. Benefiting from only a few modes supported in the FMF, a smaller line-width of the SPR spectrum is obtained, which further results in a higher figure of merit (FOM). Moreover, when combined with the superiority of the mode-multiplexing technology brought by the FMF, the FMF-SPR sensors may find applications in biochemical analysis with high performance and high throughputs.


Assuntos
Técnicas Biossensoriais/instrumentação , Tecnologia de Fibra Óptica/métodos , Ressonância de Plasmônio de Superfície/instrumentação , Desenho de Equipamento , Refratometria , Ressonância de Plasmônio de Superfície/métodos
13.
Anal Bioanal Chem ; 411(16): 3621-3629, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31098745

RESUMO

In this paper, we present a new colorimetric technique as a novel assay for the easy and direct detection of α-amylase activity. This detection system utilizes the interaction of α-amylase with starch that is supporting copper/gold (Cu/Au) nanoclusters. The Cu/Au nanoclusters are synthesized using starch as a stabilizing agent at room temperature. These nanoclusters show robust peroxidase-like activity and are able to catalyze the oxidation of TMB (3,3,5,5-tetramethylbenzidine) in the presence of hydrogen peroxide (H2O2), leading to the generation of a blue-colored solution. The α-amylase detection mechanism is based on the digestion of the starch by α-amylase, which results in nanocluster aggregation, leading to increased nanoparticle size and thus decreased peroxidase-like activity of the Cu/Au NCs. Experiments showed that the gradual addition of α-amylase causes the peroxidase activity to decrease step by step in a linear fashion. Using this method, colorimetric sensing of α-amylase was achieved with a detection limit (LOD) of 0.04 U/mL and a linear range of 0.1-10 U/mL. This method is significantly selective for α-amylase and could be affordably and conveniently applied to the detection of α-amylase in blood serum. Graphical Abstract.


Assuntos
Amilases/análise , Colorimetria/métodos , Cobre/química , Ouro/química , Nanopartículas Metálicas/química , Peroxidases/química , Amido/química , Benzidinas/química , Catálise , Peróxido de Hidrogênio/química , Limite de Detecção , Microscopia Eletrônica de Transmissão , Oxirredução , Ressonância de Plasmônio de Superfície
14.
Anal Bioanal Chem ; 411(17): 3721-3729, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139859

RESUMO

Combining mass spectrometry (MS) with surface plasmon resonance (SPR) makes it possible to identify the chemical structures of the interacting molecules studied by SPR. Different approaches for coupling surface plasmon resonance sensors to mass spectrometry were developed. This article aims to summarize the established approaches and their applications to study biomolecular interactions. Three representative interactions were reviewed: protein-protein interactions, enzyme-substrate/inhibitor interactions, and protein-small molecule interactions.


Assuntos
Espectrometria de Massas/métodos , Ressonância de Plasmônio de Superfície/métodos , Enzimas/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Especificidade por Substrato
15.
Int J Mol Sci ; 20(9)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31052424

RESUMO

The purpose of this study was to develop a new method for a determination of the cathepsin L-biosensor based on the Surface Plasmon Resonance Imaging technique. The cathepsin L is an endopeptidase, which degrades proteins and plays an important role in various processes occurring in the human body. The detection technique, Surface Plasmon Resonance Imaging, is an optical, label-free technique, which can be used for quantitative determination of the different proteins. In order to bind the enzyme, the cathepsin L inhibitor-RKLLW-NH2 was used. The validation process showed that parameters: precision, accuracy, and selectivity of the method were acceptable. The analytically useful range of the standard curve was 0.50 ng/mL-15.00 ng/mL. The detection and quantification limit of method was 1.67 pg/mL and 5.07 pg/mL, respectively. The usefulness of the developed method was confirmed by the determination of the cathepsin L concentration in the blood plasma of some healthy persons and in the blood plasma of patients. The obtained results were compared with the results obtained by the ELISA. It was found that the correlation between these two methods was very strong, what suggest that the developed method can be used as the competitive method to the ELISA.


Assuntos
Técnicas Biossensoriais/métodos , Catepsina L/sangue , Técnicas Biossensoriais/instrumentação , Humanos , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície
16.
Appl Opt ; 58(11): 2839-2844, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-31044886

RESUMO

In this study we report the development of a novel viral pathogen immunosensor technology based on the electrochemical modulation of the optical signal from a surface plasmon wave interacting with a redox dye reporter. The device is formed by incorporating a sandwich immunoassay onto the surface of a plasmonic device mounted in a micro-electrochemical flow cell, where it is functionalized with a monoclonal antibody aimed to a specific target pathogen antigen. Once the target antigen is bound to the surface, it promotes the capturing of a secondary polyclonal antibody that has been conjugated with a redox-active methylene blue dye. The methylene blue displays a reversible change in the complex refractive index throughout a reduction-oxidation transition, which generates an optical signal that can be electrochemically modulated and detected at high sensitivity. For proof-of-principle measurements, we have targeted the hemagglutinin protein from the H5N1 avian influenza A virus to demonstrate the capabilities of our device for detection and quantification of a critical influenza antigen. Our experimental results of the EC-SPR-based immunosensor under potential modulation showed a 300 pM limit of detection for the H5N1 antigen.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/análise , Imunoensaio/instrumentação , Virus da Influenza A Subtipo H5N1/imunologia , Azul de Metileno/química , Ressonância de Plasmônio de Superfície/instrumentação , Técnicas Biossensoriais/instrumentação , Limite de Detecção
17.
Sensors (Basel) ; 19(9)2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31058824

RESUMO

Label-free evaluation and monitoring of living cell conditions or functions by means of chemical and/or physical sensors in a real-time manner are increasingly desired in the field of basic research of cells and clinical diagnosis. In order to perform multi-parametric analysis of living cells on a chip, we here developed a surface plasmon resonance (SPR) imaging (SPRI)-impedance sensor that can detect both refractive index (RI) and impedance changes on a sensor chip with comb-shaped electrodes. We then investigated the potential of the sensor for label-free and real-time analysis of living cell reactions in response to stimuli. We cultured rat basophilic leukemia (RBL)-2H3 cells on the sensor chip, which was a glass slide coated with comb-shaped electrodes, and detected activation of RBL-2H3 cells, such as degranulation and morphological changes, in response to a dinitro-phenol-conjugated human serum albumin (DNP-HSA) antigen. Moreover, impedance analysis revealed that the changes of impedance derived from RBL-2H3 cell activation appeared in the range of 1 kHz-1 MHz. Furthermore, we monitored living cell-derived RI and impedance changes simultaneously on a sensor chip using the SPRI-impedance sensor. Thus, we developed a new technique to monitor both impedance and RI derived from living cells by using a comb-shaped electrode sensor chip. This technique may enable us to clarify complex living cell functions which affect the RI and impedance and apply this to medical applications, such as accurate clinical diagnosis of type I allergy.


Assuntos
Técnicas Biossensoriais , Fenômenos Fisiológicos Celulares , Rastreamento de Células/métodos , Diagnóstico por Imagem/métodos , Animais , Humanos , Leucemia/diagnóstico , Leucemia/patologia , Ratos , Ressonância de Plasmônio de Superfície
18.
Anal Chim Acta ; 1065: 12-20, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31005144

RESUMO

We are reporting an innovative building-block for the development of biosensors based on the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with avidin (MWCNTs-avidin). In this work, at variance with previous reports, avidin has the double role of simultaneously being the exfoliating agent of MWCNTs and the platform for anchoring different biotinylated biomolecules. The optimum dispersion was obtained by sonicating for 5.0 min 0.50 mgmL-1 MWCNTs with 1.00 mgmL-1 avidin solution prepared in 50:50 v/v ethanol/water. As proof-of-concept, we immobilized biotinylated horseradish peroxidase (b-HRP) at glassy carbon electrodes (GCE) modified with MWCNTs-avidin to develop a hydrogen peroxide biosensor using hydroquinone as redox mediator. Surface plasmon resonance, electrochemical impedance spectroscopy, cyclic voltammetry and amperometry demonstrated that, even after the partial denaturation of avidin due to the drastic conditions used to functionalize the MWCNTs, it preserves the biorecognition properties and efficiently interacts with biotinylated horseradish peroxidase (b-HRP). The analytical characteristics of the resulting hydrogen peroxide biosensor are the following: linear range between 1.0 × 10-6 M and 1.4 × 10-5 M, sensitivity of (1.37 ±â€¯0.04) x 105 µAM-1, detection limit of 24 nM and reproducibility of 2.9%. The sensor was challenged with different samples, a mouthwash solution, human blood serum and milk, with very good performance.


Assuntos
Avidina/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/análise , Nanotubos de Carbono/química , Animais , Espectroscopia Dielétrica , Eletrodos , Peroxidase do Rábano Silvestre/química , Humanos , Leite/química , Ressonância de Plasmônio de Superfície
19.
Anal Bioanal Chem ; 411(16): 3543-3552, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025179

RESUMO

α-Cyclopiazonic acid (CPA) is a tremorgenic mycotoxin produced by Aspergillus and Penicillium fungal species, commonly found on agricultural commodities or fermented food products. A sensitive and rapid imaging surface plasmon resonance (iSPR) assay was developed to detect CPA in maize and cheese by combining an indirect competitive immunoassay and signal amplification based upon a secondary antibody (Ab2) conjugated with gold nanoparticles. Matrix-matched calibration curves were used to determine CPA content in maize and cheese samples. Recoveries, at two spiking levels in maize and cheese, were 89 to 126%, with standard deviations of repeatability (RSDr) of less than 16%. The limits of detection were 17 and 6 µg/kg in maize and cheese, respectively. To separate the CPA-contaminated samples from uncontaminated samples, a cutoff validation level of 40 µg/kg was introduced. The assay was applied to samples of naturally contaminated maize and was compared with competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA). This is the first report to detect CPA using an immuno-biosensor iSPR format.


Assuntos
Queijo/análise , Imunoensaio/métodos , Indóis/análise , Ressonância de Plasmônio de Superfície/métodos , Zea mays/química , Técnicas Biossensoriais , Calibragem , Limite de Detecção , Reprodutibilidade dos Testes
20.
Talanta ; 199: 556-566, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952298

RESUMO

Aptamers are nucleic acid-based molecular recognition elements that are specific and have high binding affinity against their respective targets. On account of their target recognition capacity, aptamers are widely utilized in a number of applications including diagnostics. This review aims to highlight the recent developments of aptasensors expedient for point-of-care (POC) diagnostics. Significant focus is given on the primary assay formats of aptamers such as fluorescence, electrochemical, surface plasmon resonance (SPR) and colorimetric assays. A potpourri of platforms such as paper-based device, lateral flow assay, portable electrodes, portable SPR and smart phones expedient for point-of-care (POC) diagnostics are discussed. Emphasis is also given on the technicalities and assay configurations associated with the sensors.


Assuntos
Aptâmeros de Nucleotídeos/química , Sistemas Automatizados de Assistência Junto ao Leito , Aptâmeros de Nucleotídeos/síntese química , Técnicas Biossensoriais , Colorimetria , Técnicas Eletroquímicas , Fluorescência , Humanos , Ressonância de Plasmônio de Superfície
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