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1.
Anal Chim Acta ; 1147: 30-37, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33485583

RESUMO

Simple, low-cost, and sensitive new platforms for electrochemical immunosensors for virus detection have been attracted attention due to the recent pandemic caused by a new type of coronavirus (SARS-CoV-2). In the present work, we report for the first time the construction of an immunosensor using a commercial 3D conductive filament of carbon black and polylactic acid (PLA) to detect Hantavirus Araucaria nucleoprotein (Np) as a proof-of-concept. The recognition biomolecule was anchored directly at the filament surface by using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-Hydroxysuccinimide (EDC/NHS). Conductive and non-conductive composites of PLA were characterized using thermal gravimetric analysis (TGA), revealing around 30% w/w of carbon in the filament. Morphological features of composites were obtained from SEM and TEM measurements. FTIR measurement revealed that crosslinking agents were covalently bonded at the filament surface. Electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for the evaluation of each step involved in the construction of the proposed immunosensor. The results showed the potentiality of the device for the quantitative detection of Hantavirus Araucaria nucleoprotein (Np) from 30 µg mL-1 to 240 µg mL-1 with a limit of detection of 22 µg mL-1. Also, the proposed immunosensor was applied with success for virus detection in 100x diluted human serum samples. Therefore, the PLA conductive filament with carbon black is a simple and excellent platform for immunosensing, which offers naturally carboxylic groups able to anchor covalently biomolecules.


Assuntos
Anticorpos Antivirais/imunologia , Imunoensaio/métodos , Proteínas do Nucleocapsídeo/imunologia , Impressão Tridimensional , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , /virologia , Espectroscopia Dielétrica , Eletrodos , Hantavirus/isolamento & purificação , Hantavirus/metabolismo , Infecções por Hantavirus/diagnóstico , Infecções por Hantavirus/virologia , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Proteínas do Nucleocapsídeo/sangue , Fuligem/química
2.
Anal Chem ; 93(5): 3010-3017, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33499597

RESUMO

Lateral flow assay (LFA) has played pivotal roles in many emergency public safety incidents, such as coronavirus disease diagnostics; however, the present double-line (test and control line) design strategy for LFA strips greatly restricts their applications in high-throughput quantitative analysis because the limited sample diffusion distance on the strips constrains the number of test/control lines. Herein, a novel single-line-based LFA (sLFA) strip, which combines test and control line, is developed by exploiting an orthogonal emissive upconversion nanoparticle (UCNP) as a signal reporter on the test line, where one emission can be used as a reporting signal and the other as a calibrating signal. This UCNP-based test line with an interior reference also can play a validating role as a control line, and hence capturing antibodies are not needed for control lines, greatly saving fabrication costs. As a proof-of-concept, this novel sLFA strip is successfully explored to accurately and rapidly detect aflatoxin B1. Moreover, due to the removal of control lines, such a novel strategy greatly reduces the strip size, facilitating the design of a testing array for multiple detections of different samples. The test line herein is designed in a ring shape, and several test rings are assembled to be a chip for the testing of multiple samples. To our knowledge, this is the first demonstration of single-line-based LFA strips, which will significantly improve the detection capacities and accuracies and reduce the testing costs of LFA strips in real sample applications ranging from food analysis to in vitro diagnostics.


Assuntos
Aflatoxina B1/análise , Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Aflatoxina B1/imunologia , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Técnicas Biossensoriais/instrumentação , /virologia , Análise de Alimentos/métodos , Ouro/química , Humanos , Medições Luminescentes , /isolamento & purificação
3.
Viruses ; 12(11)2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33114742

RESUMO

Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals, as well as S-specific monoclonal antibodies, were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to the binding of S glycoprotein in the context of viral particles remains to be established. Here, we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA, ELISA, and neutralization assays, we observed a strong correlation between these parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of pseudoviral particles is required but not sufficient to mediate neutralization. Altogether, our results highlight the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Convalescença , Células HEK293 , Humanos , Testes de Neutralização , Pandemias , Fatores de Tempo
4.
J Chromatogr A ; 1629: 461513, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32889296

RESUMO

Extracellular vesicles (EVs) exist in biological fluids such as blood, urine, and cerebrospinal fluid, and these have shown promise for use as biomarkers of cancers. Conventional methods for determination of EVs include direct detection via enzyme-linked immunosorbent assay and detection of their membrane proteins via western blotting. These techniques, however, have individual shortcomings in terms of the need for large sample consumption, processes that are time-consuming, and a lack of the capacity for quantification. In this study, we developed a method to determine the EV membrane protein, CD63, by coupling capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF). In this process, the EVs were isolated from a culture medium and were subsequently reacted with a fluorescently labeled anti-CD63 antibody to form a CD63 complex localized on the surface of EVs. After removing the EVs containing the CD63 immune complex by centrifugation, the supernatant containing the free fluorescent antibody was injected into a capillary to serve as a sample. A decrease in the peak area of the free fluorescent antibody became apparent when the amount of EVs was increased while that of the fluorescent antibody remained constant. The peak areas were decreased proportionally against the increased amounts of EVs. The concentration of the CD63 could then be estimated based on the slope of the linear relationship. This study is the first to quantify CD63 immobilized on EVs via CEIA-LIF, which is a novel method with the potential to determine membrane proteins localized on the surface of EVs.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/análise , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Eletroforese Capilar , Corantes Fluorescentes/química , Células HeLa , Humanos , Proteínas de Membrana/imunologia , Tetraspanina 30/análise , Tetraspanina 30/imunologia
5.
Sci Adv ; 6(42)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32948512

RESUMO

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.


Assuntos
Betacoronavirus/imunologia , Peptidil Dipeptidase A/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Visualização da Superfície Celular/métodos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Dimerização , Humanos , Cinética , Pandemias , Peptídeos/química , Peptídeos/imunologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Domínios Proteicos/imunologia , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Subunidades Proteicas/metabolismo , RNA Viral/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química
6.
ACS Sens ; 5(9): 2747-2752, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32820626

RESUMO

With the current intense need for rapid and accurate detection of viruses due to COVID-19, we report on a platform technology that is well suited for this purpose, using intact measles virus for a demonstration. Cases of infection due to the measles virus are rapidly increasing, yet current diagnostic tools used to monitor for the virus rely on slow (>1 h) technologies. Here, we demonstrate the first biosensor capable of detecting the measles virus in minutes with no preprocessing steps. The key sensing element is an electrode coated with a self-assembled monolayer containing the measles antibody, immobilized through an N-heterocyclic carbene (NHC). The intact virus is detected by changes in resistance, giving a linear response to 10-100 µg/mL of the intact measles virus without the need to label or process the sample. The limit of detection is 6 µg/mL, which is at the lower limit of concentrations that can cause infections in primates. The NHC-based biosensors are shown to be superior to thiol-based systems, producing an approximately 10× larger response and significantly greater stability toward repeated measurements and long-term storage. This NHC-based biosensor thus represents an important development for both the rapid detection of the measles virus and as a platform technology for the detection of other biological targets of interest.


Assuntos
Anticorpos Imobilizados/imunologia , Benzimidazóis/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Vírus do Sarampo/isolamento & purificação , Anticorpos Imobilizados/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Limite de Detecção , Vírus do Sarampo/imunologia
7.
J Chromatogr A ; 1624: 461227, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32540069

RESUMO

Affinity chromatography is generally regarded as a powerful tool allowing the single step purification of recombinant proteins with high purity and yields. However, for most protein products, affinity purification methods for industrial applications are not readily available, mainly due to the lack of specific and robust natural counterparts that could function as affinity ligands. In this study, we explored the applicability of nanobody-based peptide-tag immunorecognition systems as a platform for affinity chromatography. Two typical nanobodies (BC2-nb and Syn2-nb) that are capable of recognizing specifically a particular peptide-tag, were prepared through prokaryotic expression and proved to be able to bind with nanomolar affinity to their cognate tag fused to enhanced green fluorescent protein (eGFP). Through an epoxy-based immobilization reaction, the two nanobodies were coupled on a Sepharose CL-6B matrix under the same conditions. The remaining antigen binding activity of the immobilized BC2-nb and Syn2-nb was determined to be 83.1% and 42.9%, yielding the resins with the dynamic binding capacity (DBC) of 21.4 mg/mL and 5.9 mg/mL, respectively. The immobilized affinity ligands exhibited high binding specificity towards their respective target peptides, yielding a product purity above 90% directly from crude bacterial lysates in one single chromatographic step. However, for the both affinity complexes, desorption has been found difficult, and effective recovery of the bound products could be only achieved with competitive elution or after employing harsh conditions such as 10 mM NaOH solution, which will compromise the reuse cycles of the affinity resins. This study shows the potential of nanobody-based affinity chromatography for efficient purification of recombinant proteins especially from complex feedstocks and reveals the primary issues to be addressed to develop a successful application.


Assuntos
Cromatografia de Afinidade/métodos , Peptídeos/imunologia , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Cromatografia Líquida de Alta Pressão , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Peptídeos/química , Peptídeos/isolamento & purificação , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo
8.
Mikrochim Acta ; 187(5): 268, 2020 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-32285207

RESUMO

The influence of Au@Pt nanoparticles' composition, morphology, and peroxidase-mimicking activity on the limit of detection (LOD) of lateral flow immunoassay (LFIA) has been investigated. Fourteen types of nanoparticles were synthesized by varying the concentration of Pt4+ (20-2000 µM), using gold nanoparticles (GNP, diameter 20.0 ± 2.6 nm) as the seeds and ascorbic acid as a reducing agent. Au@Pt nanoparticles and GNPs were conjugated with antibodies specific to the target analyte, a widespread and dangerous phytopathogenic bacteria species (Clavibacter michiganensis). We found that the 100-fold growth of the Pt4+ concentration was accompanied by an increase of the Au@Pt nanoparticle diameter (24-55 nm) and surface area with the formation of urchin-shaped morphology. These changes led to a 70-fold increase in peroxidase-mimicking activity in the solution (specific activity 0.06-4.4 U mg-1) and a 30-fold decrease in LOD using the catalytic activity of Au@Pt. The Au@Pt nanoparticles synthesized at 1000-2000 µM of Pt4+ demonstrated statistically indistinguishable catalytic activity. The highest sensitivity of LFIA was reached for Au@Pt nanoparticles synthesized at Pt4+ concentration equal to 1000 µM. Au@Pt nanoparticles saved most of their peroxidase-mimicking activity, whereas endogenous plant peroxidases were completely inhibited by sodium azide. The LOD of LFIA with Au@Pt nanoparticles synthesized at 1200 µM of Pt4+ was 300 colony-forming units (CFU) per mL of buffer and 500 CFU per mL of potato tuber extract, which provides 330- and 200-fold improvement compared to the conventional LFIA with GNPs. The assay consists of three rapid 5-min stages, namely, extraction, lateral flow, and color enhancement (oxidation of diaminobenzidine by Au@Pt nanoparticles). LFIA with the urchin Au@Pt nanoparticles allows the detection of latent bacterial infections rapidly without equipment or special skills. Graphical abstract.


Assuntos
Actinobacteria/isolamento & purificação , Imunoensaio/métodos , Nanopartículas Metálicas/química , Actinobacteria/imunologia , Anticorpos Imobilizados/imunologia , Benzidinas/química , Catálise , Corantes/química , Ouro/química , Limite de Detecção , Oxirredução , Platina/química
9.
Mikrochim Acta ; 187(5): 264, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32270338

RESUMO

An electrochemical immunoassay for the carcinoembryonic antigen is described. It is based on the use of Au NPs modified zeolitic imidazolate framework (ZIF-8) and ordered mesoporous carbon (OMC). Au NPs@ZIF-8 was synthesized by reduction of chloroauric acid. It serves as immobilization support nanocarrier to increase antibody loading due to its large surface area. OMC was dropped on a glassy carbon electrode to improve electrochemical signals due to enhanced electrical conductivity. Differential pulse voltammetry was carried out to record electrochemical responses (best measured at 0.26 V vs. Ag/AgCl). The immunosensor demonstrated excellent electrochemical performance with a linear determination range of 5 pg mL-1 to 400 ng mL-1 and a determination limit of 1.3 pg mL-1 (S/N = 3). The sensor also exhibited high selectivity, good stability, and acceptable reproducibility. Graphical abstract Scheme 1 Schematic representation of fabrication of the immunosensor for CEA determination.


Assuntos
Carbono/química , Antígeno Carcinoembrionário/sangue , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Nanopartículas Metálicas/química , Estruturas Metalorgânicas/química , Anticorpos Imobilizados/imunologia , Antígeno Carcinoembrionário/imunologia , Ouro/química , Humanos , Limite de Detecção , Porosidade , Reprodutibilidade dos Testes
10.
Talanta ; 212: 120781, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113544

RESUMO

Existing techniques for the detection of Group A Streptococcus pyogenes (GAS) have drawbacks in rapidness, accuracy or in high-cost. Considering the clinical importance of GAS, we have developed a culture-free detection method based on pyrrolidonyl arylamidase (PYR) activity with the aid of magnetic gold nanoparticles (AuNPs). GAS is the reason for pharyngitis and sampling starts from the throat with cotton swabs. After swab sampling, the target was collected with antibody modified magnetic AuNPs and transferred into 500 µL of PYR-broth without any antigen extraction or pure colony isolation. Then, the assay was finished by adding 25 µL of 4-(dimethylamino)-cinnamaldehyde (DMACA) reagent after 4-h incubation. A red color formation was evaluated as the presence of GAS comparing to blank, however, image analysis was employed for the interpretation of color changes clearly. For this purpose, a formula related to image data was proposed and analytical validation parameters were defined. Thus, the correlation was found to be linear with the R2 of 0.9685 between the log of bacteria concentration and the image data with the limit of detection of 3.3 × 102 CFU/mL of GAS. In addition, the assay worked efficiently in the abundance interference of Enterococcus faecalis. The results represent a new feature to nanoparticles eliminating the selective growth media for a bacteria and this study provided a detection with intact cells of bacteria without any antigen or DNA/RNA extraction. The proposed work has been the most similar to the gold standard but a faster method in this field.


Assuntos
Aminopeptidases/análise , Proteínas de Bactérias/análise , Ensaios Enzimáticos/métodos , Nanopartículas de Magnetita/química , Streptococcus pyogenes/isolamento & purificação , Anticorpos Imobilizados/imunologia , Técnicas de Tipagem Bacteriana/métodos , Ouro/química , Imunoensaio/métodos , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/imunologia
11.
Talanta ; 212: 120792, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113554

RESUMO

Surface plasmon resonance (SPR) biosensors are often used in the detection of solid, liquid or gaseous samples in diagnostics, pharmaceutics and military defense. Plasmon waveguide resonance (PWR) mode is obtained when a dielectric waveguide layer is added to the metal film. In this study, a self-referenced PWR (SRPWR) silicon dioxide (SiO2) chip was examined. The self-referenced measurement is important to compensate for temperature fluctuations, other instabilities and allows RI signal measurement without an additional reference sample, thus minimising the sample volume needed. The chip was fabricated with a multi-layer of metals and dielectrics, consisting of a 420 nm SiO2 layer, a 40 nm Ag layer and another 480 nm SiO2 layer. This chip was shown to give one internal plasmon excited on the bottom interface SiO2/Ag, which is used as self-reference in the detection. The top layer acts as a waveguide layer and can be designed to give modes with ultrahigh penetration depth. A direct assay was developed, where the recognition molecule (specific antibody) was immobilized onto the SiO2 plasmonic chip surface, via a covalent coupling protocol based on 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde. The SRPWR biosensor was developed for the sensing of two chosen stroke biomarkers: NT-proBNP and S100ß, which are sensitive and specific for stroke diagnostics. For both biomarkers, a linear decreasing pattern in the RI signal was recognized with the increasing biomarkers concentrations. Biomarkers detection was conducted in deionized water and validation was done in spiked porcine plasma. The SiO2 based plasmonic chip demonstrates a limit-of-detection of less than 1 ng/mL that is clinically relevant for both stroke biomarkers.


Assuntos
Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Dióxido de Silício/química , Acidente Vascular Cerebral/sangue , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Anticorpos Imobilizados/imunologia , Biomarcadores/sangue , Humanos , Peptídeo Natriurético Encefálico/imunologia , Fragmentos de Peptídeos/imunologia , Testes Imediatos , Propilaminas/química , Subunidade beta da Proteína Ligante de Cálcio S100/imunologia , Silanos/química , Ressonância de Plasmônio de Superfície/métodos , Suínos
12.
Talanta ; 212: 120794, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113556

RESUMO

A new type of nanocomposite composed of carboxylated single-walled carbon nanotubes (CNTs-COOH), reduced graphene oxide (rGO), bovine serum albumin-Ag hybride (Ag@BSA), and poly(3,4-ethylenedioxythiophene) (PEDOT) was fabricated to develop an ultrasensitive electrochemical platform for the detection of carcinoembryonic antigen (CEA) as a model of biomarkers. Two steps are involved for the fabrication of the organic/inorganic nanocomposites. The Ag@BSA nanoflowers were first synthesized to be doped with CNTs-COOH and rGO followed by the adsorption of PEDOT resulting in CNTs-COOH/rGO/Ag@BSA/PEDOT. The as-prepared nanocomposites were then deposited onto an Au electrode together with subsequent immobilization of CEA antibody (anti-CEA) to construct the electrochemical immunosensor. This unique structure and composition of the developed immunosensor can expect an excellent electrochemical response. The immunosensor offers a linear relationship between the electrochemical responses and the CEA concentrations from 0.002 to 50 ng∙mL-1 with a detection limit of 1 × 10-4 ng∙mL-1. Moreover, the ultrasensitive immunoassay can detect CEA in real human serum samples, and the results are comparable to those obtained from the commercial ELISA. Therefore, this strategy can monitor diseases, offer clinical diagnosis, and may be valuable for the development of new biomedical devices.


Assuntos
Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/sangue , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Nanocompostos/química , Animais , Anticorpos Imobilizados/imunologia , Biomarcadores Tumorais/imunologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Antígeno Carcinoembrionário/imunologia , Bovinos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Grafite/química , Humanos , Limite de Detecção , Nanotubos de Carbono/química , Polímeros/química , Soroalbumina Bovina/química , Prata/química
13.
Talanta ; 212: 120797, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113559

RESUMO

We report here sensitive photoelectrochemical immunosensing of Staphylococcus aureus (S. aureus) using ZnS-Ag2S/polydopamine (PDA) as a novel photoelectric material and Cu2O as the peroxidase mimic tag. ZnS-Ag2S heterojunctions were prepared on indium tin oxide (ITO) via electrodeposition of ZnS nanoparticles, followed by silver ion exchange. To prepare a PDA/ZnS-Ag2S/ITO, the ZnS-Ag2S/ITO electrode was coated with PDA by self-polymerization of dopamine. The photocurrent of the PDA/ZnS-Ag2S/ITO is 1.55 times that of the ZnS-Ag2S/ITO and 7.87 times that of the ZnS/ITO, indicating a high-performance photoelectric material. A sandwiched-type photoelectrochemical immunosensor was constructed by using PDA/ZnS-Ag2S/ITO as the photoelectrode and Cu2O nanocubes as the labels. Cu2O nanocubes can serve as peroxidase mimic to generate catalytic precipitates on the immunoelectrodes, and both the Cu2O nanocubes and the generated precipitates can decrease the photocurrents of the immunoelectrodes, so a photoelectrochemical immunosensor for detecting S. aureus was constructed, showing a linear range between 10 and 107 CFU mL-1 and a low detection limit of 2 CFU mL-1. Owing to the signal amplification of Cu2O labeling, the sensitivity of the Cu2O-labeled immunosensor is 4 times that of a label-free immunosensor for detecting S. aureus, and the detection limit (2 CFU mL-1) is lower than that of a label-free immunosensor (10 CFU mL-1). This work not only provides a new and efficient photoelectric material but also demonstrated an efficient signal-amplification strategy for photoelectrochemical biosensing.


Assuntos
Cobre/química , Imunoensaio/métodos , Indóis/química , Nanopartículas Metálicas/química , Polímeros/química , Staphylococcus aureus/isolamento & purificação , Anticorpos Imobilizados/imunologia , Técnicas Biossensoriais/métodos , Catálise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Indóis/efeitos da radiação , Luz , Limite de Detecção , Nanopartículas Metálicas/efeitos da radiação , Polímeros/efeitos da radiação , Compostos de Prata/química , Compostos de Prata/efeitos da radiação , Staphylococcus aureus/imunologia , Sulfetos/química , Sulfetos/efeitos da radiação , Compostos de Estanho/química , Compostos de Zinco/química , Compostos de Zinco/efeitos da radiação
14.
Anal Chim Acta ; 1101: 58-64, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32029119

RESUMO

An original dual signal amplification immunoassay for N-Terminal pro-brain natriuretic peptide (NT-proBNP) detection is developed based on Au NPs and Zn doped CdS nanoparticles (Zn0.02Cd0.98S) co-sensitized Bi2MoO6 nanosheet photoelectrochemical (PEC) platform, the target is a good myocardial marker for diagnosing heart failure (HF). Bi2MoO6 as an outstanding photocatalysis material is successfully used in PEC analysis in this work, and the nanosheet structure provide a preponderance to capture superior Zn0.02Cd0.98S in order to achieve anticipant PEC response. The doping of Zn makes the energy band of CdS matched more compatible with Bi2MoO6, and improves the narrow band gap of CdS, making the surface plasma resonance (SPR) effect of Au NPs more significant, thus further improving the PEC response, as well as elevated the detection sensitivity of biological targets. The constructed PEC platform for NT-proBNP provides a wide detection range of 0.0001-1000 ng mL-1 and gives the minimum detection value of 0.037 pg mL-1. Great stability, high selectivity, and good reproducibility are also achieved. The proposed PEC immunoassay provides more possibilities for other protein ultra-sensitivity detection.


Assuntos
Bismuto/química , Imunoensaio/métodos , Molibdênio/química , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Imobilizados/imunologia , Cádmio/química , Técnicas Eletroquímicas , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Peptídeo Natriurético Encefálico/imunologia , Fragmentos de Peptídeos/imunologia , Reprodutibilidade dos Testes , Zinco/química
15.
Anal Chim Acta ; 1102: 109-118, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32043989

RESUMO

The combination of isothermal nucleic acid amplification and lateral flow assay (LFA) provides highly sensitive non-laboratory ("point-of-care") detection. The aim of this study is to investigate the recognition on lateral flow membranes of DNA targets with different lengths as products of recombinase polymerase amplification (RPA). We produced double-stranded DNA with lengths of 50, 100, 150, 200, and 300 bp. Each DNA target was functionalized with biotin and fluorescein (FAM). Kinetic and equilibrium constants of the interaction of FAM at the 5'-end of DNA with anti-FAM antibodies did not depend on DNA length. Gold nanoparticles (GNPs) with diameters of 17.4 ± 1.0 nm were conjugated with anti-FAM antibodies and streptavidin. LFA was performed in two schemes: 1) anti-FAM antibodies immobilized in the test zone, GNP-streptavidin conjugates recognized as DNA; 2) streptavidin immobilized in the test zone, GNP‒anti-FAM antibodies conjugates recognized as DNA. Considering that the components of the RPA mixture caused the aggregation of the GNP-streptavidin conjugate in contradistinction to conjugate with anti-FAM antibodies, we found that 150 bp was the most promising length for the DNA target. For this length, a detection limit was achieved up to 70 pM that was approximately 10 times lower than for 50-bp DNA in the same scheme. Moreover, we showed that high concentrations of primers containing FAM or biotin competed with the DNA target on lateral flow membranes. These results demonstrated that a DNA length should be considered when designing RPA-LFA systems to detect DNA targets with high sensitivity.


Assuntos
DNA/análise , Anticorpos Imobilizados/imunologia , Biotina/química , DNA/química , Fluoresceínas/química , Corantes Fluorescentes/química , Ouro/química , Imunoensaio/métodos , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Estreptavidina/química
16.
Sensors (Basel) ; 20(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054015

RESUMO

Exosomes are cell-derived nanovesicles released into biological fluids, which are involved in cell-to-cell communication. The analysis of the content and the surface of the exosomes allow conclusions about the cells they are originating from and the underlying condition, pathology or disease. Therefore, the exosomes are currently considered good candidates as biomarkers to improve the current methods for clinical diagnosis, including cancer. However, due to their low concentration, conventional procedures for exosome detection including biosensing usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. In this paper, the immunomagnetic separation is presented as an alternative method for the specific isolation of exosomes in serum. To achieve that, a rational study of the surface proteins in exosomes, which can be recognized by magnetic particles, is presented. The characterization was performed in exosomes obtained from cell culture supernatants of MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines, including TEM and nanoparticle tracking analysis (NTA). For the specific characterization by flow cytometry and confocal microscopy, different commercial antibodies against selected receptors were used, including the general tetraspanins CD9, CD63 and CD81, and cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). The effect of the serum matrix on the immunomagnetic separation was then carefully evaluated by spiking the exosomes in depleted human serum. Based on this study, the exosomes were preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further magnetic actuation on the surface of the electrode for the electrochemical readout. The performance of this approach is discussed and compared with classical characterization methods.


Assuntos
Exossomos/metabolismo , Separação Imunomagnética/métodos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Exossomos/química , Feminino , Humanos , Biópsia Líquida , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Tetraspanina 28/imunologia
17.
Mikrochim Acta ; 187(1): 95, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31903507

RESUMO

To increase the sensitivity of electrochemical sensor, Fe-MIL-88B-NH2 (Fe-MOF) with peroxidase-like activity is designed for the construction of immunoprobe. The Fe-MOF was prepared by one-step hydrothermalf method using 2-aminoterephthalic acid and iron(III) chloride. For the immunoprobe, it was fabricated by gold nanocomposite/Fe-MOF (Au/Fe-MOF) for the immobilization of labeling antibody (the antibody was used to conjuncting with label materials). The thin layer of Methylene Blue (MB) covered by reduced graphene oxide-gold nanocomposites (Au-rGO) serves as a substrate to covalently fix coating antibodies. The MB as a redox-active species was modified on the glass carbon electrode that can give a strong amperometric signal at 0.18 V (vs. Ag/AgCl). With the participation of H2O2, Fe-MOF can induce the Fenton reaction which degrades MB covered by Au-rGO on the substrate. The rest of MB on the surface of electrode becomes oxidized thereby generating a current signal. Square wave voltammetry (SWV) was used to quantify PSA. Under optimal conditions, the immunoassay is stable, specific and reproducible. It has a lower detection limit of 0.13 pg mL-1 (S/N = 3) and a wide analytical range that extends from 0.001 to 100 ng mL-1. Graphical abstractA sandwich-type amperometric immunoassay based on Fe-MOF-induced Fenton reaction was designed for sensitive determination of prostate specific antigen.


Assuntos
Técnicas Eletroquímicas/métodos , Calicreínas/análise , Estruturas Metalorgânicas/química , Nanocompostos/química , Peroxidase/metabolismo , Antígeno Prostático Específico/análise , Anticorpos Imobilizados/imunologia , Técnicas Eletroquímicas/normas , Eletrodos , Ouro , Humanos , Peróxido de Hidrogênio/química , Ferro , Calicreínas/imunologia , Azul de Metileno/química , Mimetismo Molecular , Oxirredução , Antígeno Prostático Específico/imunologia
18.
Talanta ; 209: 120465, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892037

RESUMO

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L-1), below the maximum levels allowed by the legislation, with a wide response range (50-10000 pmol L-1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Nanopartículas Metálicas/química , Neonicotinoides/análise , Nitrocompostos/análise , Praguicidas/análise , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Armoracia/enzimologia , Benzidinas/química , Citrullus/química , Água Potável/análise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Contaminação de Alimentos/análise , Ouro/química , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Lycopersicon esculentum/química , Neonicotinoides/imunologia , Nitrocompostos/imunologia , Praguicidas/imunologia , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/imunologia
19.
Talanta ; 209: 120501, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892087

RESUMO

In the present study, we have developed a capacitance electrochemical biosensor based on silicon nitride substrate (Si3N4/SiO2/Si[P]/Al) for Tumour Necrosis Factor Alpha (TNF-α) cytokines detection. Micro-contact printing, Fluorescence microscopy characterization and contact angle measurement (CAM) were carried out during the bio-functionalization of the biosensor surface. Mott-Schottky analyses were applied for TNF-α detection within the range of 1 pg/mL to 30 pg/mL in which the immunosensor has exhibited a good linearity, a sensitivity of 4 mV.pM-1 and 4.4 mV.pM-1 in PBS and artificial saliva (AS) respectively. While the LOD was found at 0.38 pg/mL and 1 pg/mL in PBS and AS respectively. The developed immunosensor has also demonstrated a high and good selectivity for TNF-α detection in human AS when compared to other interferences like Cortisol and Interleukin-10. The performances of the developed biosensor are very promising for biomedical application to predict the first sign of inflammation.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Saliva Artificial/química , Compostos de Silício/química , Transdutores , Fator de Necrose Tumoral alfa/análise , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Capacitância Elétrica , Técnicas Eletroquímicas/instrumentação , Eletrodos , Corantes Fluorescentes/química , Humanos , Imunoensaio/métodos , Limite de Detecção , Rodaminas/química , Dióxido de Silício/química , Fator de Necrose Tumoral alfa/imunologia
20.
Talanta ; 206: 120187, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514860

RESUMO

CA125/MUC16 is an ovarian tumor cell marker widely used as a biomarker in epithelial ovarian carcinoma. CA125/MUC16 is also used for evaluation of the ROMA (Risk of Ovarian Malignancy Algorithm) value. In this work, a Surface Plasmon Resonance Imaging (SPRI) biosensor for circulating CA125/MUC16 has been developed. The anti-MUC16 antibody was attached to a gold chip via a cysteamine linker. The EDS/NHS protocol was used for the covalent attachment of the antibody. The developed biosensor is specific for CA125/MUC16, and exhibits good recovery and acceptable precision. Its linear response range (2.2-150 U/ml) is well suited to determination of the marker in the blood serum of a healthy control group and, after appropriate dilution, of patients with ovarian cancer. CA125/MUC16 was determined in two series of real samples: blood serum from patients with ovarian cancer and endometrial cysts. The method was validated by parallel determination of the samples using the chemiluminescent Architect i2000 method.


Assuntos
Biomarcadores Tumorais/sangue , Técnicas Biossensoriais/métodos , Antígeno Ca-125/sangue , Proteínas de Membrana/sangue , Animais , Anticorpos Imobilizados/imunologia , Biomarcadores Tumorais/imunologia , Antígeno Ca-125/imunologia , Cistos/sangue , Endométrio/patologia , Feminino , Humanos , Limite de Detecção , Proteínas de Membrana/imunologia , Neoplasias Ovarianas/sangue , Coelhos , Ressonância de Plasmônio de Superfície/métodos
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