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"Warn, inform, and prevent" are three essential elements to remember when designing sensors for real-time and in situ monitoring of organic, inorganic, and macromolecular compounds as well as micro-nanoparticles and microorganisms [...].
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Disciplinas das Ciências Biológicas , NanopartículasRESUMO
Several studies were devoted to the design of molecularly imprinted polymer (MIP)-based sensors for the detection of a given protein. Here, we bring elements that could contribute to the understanding of the interaction mechanism involved in the recognition of a protein by an imprint. For this purpose, a polydopamine (PDA)-MIP was designed for bovine serum albumin (BSA) recognition. Prior to BSA grafting, the gold surfaces were functionalized with mixed self-assembled monolayers of (MUDA)/(MHOH) (1/9, v/v). The MIP was then elaborated by dopamine electropolymerization and further extraction of BSA templates by incubating the electrode in proteinase K solution. Three complementary techniques, electrochemistry, zetametry, and Fourier-transform infrared spectrometry, were used to investigate pH and ionic strength effects on a MIP's design and the further recognition process of the analytes by the imprints. Several MIPs were thus designed in acidic, neutral, and basic media and at various ionic strength values. Results indicate that the most appropriate conditions, to achieve a successful MIPs, were an ionic strength of 167 mM and a pH of 7.4. Sensitivity and dissociation constant of the designed sensor were of order of (3.36 ± 0.13) µA·cm-2·mg-1·mL and (8.56 ± 6.09) × 10-11 mg/mL, respectively.
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Concentração Osmolar , Concentração de Íons de Hidrogênio , Indóis , Impressão Molecular , PolímerosRESUMO
This review critically summarizes the knowledge of imprinted polymer-based electrochemical sensors for the detection of pesticides, metal ions and waterborne pathogenic bacteria, focusing on the last five years. MIP-based electrochemical sensors exhibit low limits of detection (LOD), high selectivity, high sensitivity and low cost. We put the emphasis on the design of imprinted polymers and their composites and coatings by radical polymerization, oxidative polymerization of conjugated monomers or sol-gel chemistry. Whilst most imprinted polymers are used in conjunction with differential pulse or square wave voltammetry for sensing organics and metal ions, electrochemical impedance spectroscopy (EIS) appears as the chief technique for detecting bacteria or their corresponding proteins. Interestingly, bacteria could also be probed via their quorum sensing signaling molecules or flagella proteins. If much has been developed in the past decade with glassy carbon or gold electrodes, it is clear that carbon paste electrodes of imprinted polymers are more and more investigated due to their versatility. Shortlisted case studies were critically reviewed and discussed; clearly, a plethora of tricky strategies of designing selective electrochemical sensors are offered to "Imprinters". We anticipate that this review will be of interest to experts and newcomers in the field who are paying time and effort combining electrochemical sensors with MIP technology.
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Técnicas Biossensoriais , Impressão Molecular , Técnicas Eletroquímicas , Eletrodos , Limite de Detecção , Polímeros , ÁguaRESUMO
Sensitivity is one of the most important parameters to put in the foreground in all sensing applications. Its increase is therefore an ongoing challenge, particularly for surface acoustic wave (SAW) sensors. Herein, finite element method (FEM) simulation using COMSOL Multiphysics software is first used to simulate the physical and electrical properties of SAW delay line. Results indicate that 2D configuration permits to accurately obtain all pertinent parameters, as in 3D simulation, with very substantial time saving. A good agreement between calculation and experiment, in terms of transfer functions (S21 spectra), was also shown to evaluate the dependence of the SAW sensors sensitivity on the operating frequency; 2D simulations have been conducted on 104 MHz and 208 MHz delay lines, coated with a polyisobutylene (PIB) as sensitive layer to dichloromethane (DCM). A fourfold increase in sensitivity was obtained by doubling frequency. Both sensors were then realized and tested as chem-sensors to detect zinc ions in liquid media. 9-{[4-({[4-(9anthrylmethoxy)phenyl]sulfanyl} methyl)]methyl] anthracene (TDP-AN) was selected as the sensing layer. Results show a comparable response curves for both designed sensors, in terms of limit of detection and dissociation constants Kd values. On the other hand, experimental sensitivity values were of the order of [7.0 ± 2.8] × 108 [°/M] and [16.0 ± 7.6] × 108 [°/M] for 104 MHz and 208 MHz sensors, respectively, confirming that the sensitivity increases with frequency.
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There is a global debate and concern about the use of glyphosate (Gly) as an herbicide. New toxicological studies will determine its use in the future under new strict conditions or its replacement by alternative synthetic or natural herbicides. In this context, we designed biomimetic polymer sensing layers for the selective molecular recognition of Gly. Towards this end, complementary surface acoustic wave (SAW) and electrochemical sensors were functionalized with polypyrrole (PPy)-imprinted polymer for the selective detection of Gly. Their corresponding limits of detection were on the order of 1 pM, which are among the lowest values ever reported in literature. The relevant dissociation constants between PPy and Gly were estimated at [Kd1 = (0.7 ± 0.3) pM and Kd2 = (1.6 ± 1.4) µM] and [Kd1 = (2.4 ± 0.9) pM and Kd2 = (0.3 ± 0.1) µM] for electrochemical and gravimetric measurements, respectively. Quantum chemical calculations permitted to estimate the interaction energy between Gly and PPy film: ΔE = -145 kJ/mol. Selectivity and competitivity tests were investigated with the most common pesticides. This work conclusively shows that gravimetric and electrochemical results indicate that both MIP-based sensors are perfectly able to detect and distinguish glyphosate without any ambiguity.
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A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈ 550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4-hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co-exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection.
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Dopamina/química , Polímeros/química , Pirróis/química , Limite de Detecção , Impressão Molecular/métodos , PolimerizaçãoRESUMO
Nanometer-scale multilayered coatings were prepared by sequential surface reactions on gold plates. First 4-ethynylphenyl organic layer was electrografted from the parent diazonium tetrafluoroborate salt providing reactive alkynylated gold plate (Au-Y). The latter served for clicking mercaptosilane via a thiol-yne photo-triggered reaction to obtain alkoxysilane-functionalized surface. The trialkoxysilane top groups in turn served as anchor sites for the final sol-gel coating resulting from the surface reaction between aminopropylsilane and tetraethoxysilane (TEOS). It is demonstrated that two coupling agents, namely, aryl diazonium salt and silane, can be coupled using photo-triggered thiol-yne click reaction, resulting in robust multilayered coatings. In addition, the process is versatile in that it offers the possibility to design patterned surfaces. The top sol-gel layer can in turn be reacted with aminosilane, therefore providing a reactive and functional surface that can be used for different applications given the reactivity of amine groups. This approach opens new avenues for photo-triggered click reactions of aryl layers from diazonium salts. It shows that the new class of surface modifiers and coupling agents has much to offer and continues to be renewed for achieving tightly bound, reactive top coatings.
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Covalent Organic Frameworks (COF) having conjugated backbone are an interesting class of metal-free, visible light active, heterogeneous photocatalysts. Interestingly, synthesis of COF using continuous flow process has emerged as an efficient, alternative method when compared to the traditional batch process. Here, we demonstrate the possibility to engineer the physical properties and hence the adsorption and catalytic activities of a ß-ketoenamine COF by varying monomer flow rate and microreactor design during the continuous flow synthesis. Crystallinity of the COF increases on varying the monomer flow rate from 100 (S-100) to 500 (S-500) and up to 1000 µLmin-1 (S-1000), in an S-shaped microreactor, resulting in an enhanced surface area: 525, 722 and 1119 m2g-1 respectively. The photophysical properties of the COF are also found to vary significantly with the change in flow synthesis conditions. S-1000 is characterized by the highest adsorption of MB, due to its high surface area and accessible pores. On the other hand, S-500 shows the highest photocurrent, a low recombination of photogenerated charges and the lowest charge transfer resistance. Thus, S-500 is found to be the best photocatalyst for the removal of a model pollutant (methylene blue, MB). Further, enhanced photocatalytic removal of MB using S-500 could be achieved by performing the photocatalysis in continuous flow.
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Estruturas Metalorgânicas , Catálise , Estruturas Metalorgânicas/química , Adsorção , Luz , Processos Fotoquímicos , Poluentes Químicos da Água/química , Aminas/química , CetonasRESUMO
This study investigates the feasibility of a simple electrochemical detection of Prostate Cancer Antigen 3 (PCA3) fragments extracted from patients' urine, using a thiolated single-strand DNA probe immobilized on a gold surface without using a redox probe. To enhance the PCA3 recognition process, we conducted a comparative analysis of the hybridization location using two thiolated DNA probes: Probe 1 targets the first 40 bases, while Probe 2 targets the fragment from bases 47 to 86. Hybridization with PCA3 followed, using square wave voltammetry. The limit of detection of the designed genosenors were of the order of (2.2 ng/mL), and (1.6 ng/mL) for Probes 1 and 2, respectively, and the subsequent sensitivities were of the order of (0.09 ± 0.01) µA-1 · µg-1 · mL and (0.10 ± 0.01) µA-1 · µg-1 · mL. Specificity tests were then conducted with the sensor functionalized with Probe 2, as it presents better analytical performances. The electrochemical results indicate that the designed sensor can clearly discriminate a complementary target from a non-complementary one. A further modeling of the calibration curves with the Power Law/Hill model indicates that the dissociation constant increases by one order of magnitude, confirming the ability of the designed sensor to perfectly discriminate complementary targets from non-complementary ones.
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Helicobacter pylori (H. pylori) is a highly contagious pathogenic bacterium that can cause gastrointestinal ulcers and may gradually lead to gastric cancer. H. pylori expresses the outer membrane HopQ protein at the earliest stages of infection. Therefore, HopQ is a highly reliable candidate as a biomarker for H. pylori detection in saliva samples. In this work, an H. pylori immunosensor is based on detecting HopQ as an H. pylori biomarker in saliva. The immunosensor was developed by surface modification of screen-printed carbon electrodes (SPCE) with MWCNT-COOH decorated with gold nanoparticles (AuNP) followed by HopQ capture antibody grafting on SPCE/MWCNT/AuNP surface using EDC/S-NHS chemistry. The sensor performance was investigated utilizing various methods, such as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscope (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX). H. pylori detection performance in spiked saliva samples was evaluated by square wave voltammetry (SWV). The sensor is suitable for HopQ detection with excellent sensitivity and linearity in the 10 pg/mL-100 ng/mL range, with a 2.0 pg/mL limit of detection (LOD) and an 8.6 pg/mL limit of quantification (LOQ). The sensor was tested in saliva at 10 ng/mL, and recovery of 107.6% was obtained by SWV. From Hill's model, the dissociation constant Kd for HopQ/HopQ antibody interaction is estimated to be 4.60 × 10-10 mg/mL. The fabricated platform shows high selectivity, good stability, reproducibility, and cost-effectiveness for H. pylori early detection due to the proper choice of biomarker, the nanocomposite material utilization to boost the SPCE electrical performance, and the intrinsic selectivity of the antibody-antigen approach. Additionally, we provide insight into possible future aspects that researchers are recommended to focus on.
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Técnicas Biossensoriais , Helicobacter pylori , Nanopartículas Metálicas , Ouro , Técnicas Biossensoriais/métodos , Reprodutibilidade dos Testes , Imunoensaio , Biomarcadores , Limite de Detecção , Técnicas Eletroquímicas/métodos , EletrodosRESUMO
Engrailed 2 (EN2) is a homeodomain-containing transcription factor expressed in prostate cancer (PCa) cell lines and is secreted into the urines. It is nowadays considered as a promising non-invasive biomarker for PCa early diagnosis. Herein, we report the design of an electrochemical immunosensor for EN2 detection. The biosensor fabrication involved a covalent immobilization of anti-EN2 antibodies onto a poly para amino benzoic acid (PABA) film electropolymerized on a gold electrode. Square wave voltammetry was investigated for EN2 detection in a phosphate buffer solution in a concentration range of 10-5 ng/mL to 1 µg/mL. The limit of detection of the designed sensor was equal to 10-5 ng/mL and the sensitivity was of order of (29 ± 2) µL/ng. The dissociation constant Kd of the "complex" EN2/anti-EN2, estimated from a Hill model, was of order of (0.9 ± 0.2) fM. Experimental results revealed that the immunosensor enabled selective detection of EN2 in a mixture of three proteins which can be found in men' urine: human serum albumin (HSA), prostate-specific antigen (PSA) and immunoglobulin G (IgG). Tests in artificial urine, with an ionic strength of 0.18 M, have been done and results were found comparable to those obtained in PBS (0.16 M). These encouraging results show a potentially promising future for the development of an electrochemical biosensor for robust and accurate urinary biomarkers detection.
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Técnicas Biossensoriais , Neoplasias da Próstata , Ácido 4-Aminobenzoico , Biomarcadores Tumorais , Detecção Precoce de Câncer , Técnicas Eletroquímicas , Ouro , Proteínas de Homeodomínio , Humanos , Imunoensaio , Imunoglobulina G , Limite de Detecção , Masculino , Proteínas do Tecido Nervoso , Fosfatos , Próstata , Antígeno Prostático Específico , Neoplasias da Próstata/diagnóstico , Albumina Sérica Humana , Fatores de TranscriçãoRESUMO
Plants are an important source of pharmacologically active compounds. In the present work, we characterize the impact of black cumin (Nigella sativa L.) aqueous extracts on a yeast model of p53-dependent apoptosis. To this end, the Saccharomyces cerevisiae recombinant strain over-expressing p53 was used. The over-expression of p53 triggers the expression of apoptotic markers: the externalization of phosphatidylserine, mitochondrial defect associated with cytochrome-c release and the induction of DNA strand breaks. These different effects were attenuated by Nigella sativa L. aqueous extracts, whereas these extracts have no effect on the level of p53 expression. Thus, we focus on the anti-apoptotic molecules present in the aqueous extract of Nigella sativa L. These extracts were purified and characterized by complementary chromatographic methods. Specific fluorescent probes were used to determine the effect of the extracts on yeast apoptosis. Yeast cells over-expressing p53 decrease in relative size and have lower mitochondrial content. The decrease in cell size was proportional to the decrease in mitochondrial content and of mitochondrial membrane potential (ΔΨm). These effects were prevented by the purified aqueous fraction obtained by fractionation with different columns, named C4 fraction. Yeast cell death was also characterized by reactive oxygen species (ROS) overproduction. In the presence of the C4 fraction, ROS overproduction was strongly reduced. We also noted that the C4 fraction promotes the cell growth of control yeast cells, which do not express p53, supporting the fact that this purified extract acts on cellular mediators activating cell proliferation independently of p53. Altogether, our data obtained on yeast cells over-expressing p53 demonstrate that anti-apoptotic molecules targeting p53-induced apoptosis associated with mitochondrial dysfunction and ROS overproduction are present in the aqueous extracts of Nigella seeds and in the purified aqueous C4 fraction.
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Nigella sativa , Apoptose , Nigella sativa/química , Nigella sativa/metabolismo , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Sementes/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The data presented here are related to the article entitled "New contributions to the drug profile of TNFα inhibitor SPD304: affinity, selectivity ADMET considerations" published in the European Journal of pharmacology. As DMSO is usually used as a co-solvent to dissolve low aqueous soluble small molecules, such as SPD304, we have investigated the influence of its concentration on the kinetic of interaction between tumor necrosis factor α (TNF-α) and its inhibitor, SPD304. The presented data, acquired using a surface acoustic wave sensor, can be used in further biological studies to compare the kinetic of interaction between proteins/small molecules in general and TNFα/inhibitors in particular. The estimated dissociation constant can be compared to other ones to statute on the degree of affinity between a protein and a given molecule.
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Tumor necrosis factor alpha (TNFα) is a relevant clinical target for the treatment of chronic inflammatory diseases. Currently, only few small molecules are known as direct inhibitors of TNFα. To date, none of these molecules has shown both an efficient activity and a low toxicity to be considered for clinical trials. The SPD304 is considered as a reference of direct inhibitors of TNFα because of its well demonstrated mechanism (He et al., 2005). Herein, we provide new insights regarding the drug profile, selectivity and absorption, distribution, metabolism, excretion and toxicity (ADMET) considerations of SPD304 to evaluate its potential as a hit for the structure-based design of novel TNFα inhibitors. ELISA experiments confirmed the inhibition of TNFα/TNF receptor 1 binding (IC50 = 12 µM). Cellular-based assays highlighted the cytotoxicity of SPD304, as well as its ability to inhibit TNFα signaling pathways at non-cytotoxic concentrations. A surface acoustic wave (SAW) experiment highlighted only one binding site with a dissociation constant of 6.1 ± 4.7 nM. SPD304 inhibited the binding of the cytokines like interleukins (IL)-4 and IL-13 to their receptors and showed no direct inhibition on proteins involved in the TNFα pathway. Finally, the thermodynamic solubility and Caco-2 cells permeability of SPD304 were experimentally evaluated and ADMET in silico predictions are also discussed. The physicochemical, pharmacological and ADMET studies of SPD304 have shown that is not an ideal hit for a drug optimization program based on its chemical structure.
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Fator de Necrose Tumoral alfa , Células CACO-2 , Humanos , Transdução de SinaisRESUMO
Rapid and accurate detection of proteins in biological fluids is increasingly required in the biomedical environment. Actually, it is performed with conventional techniques, which are generally run by robotized platforms at centralized laboratories. In this work, molecular dynamics calculations and an experimental procedure were conducted to set up electrochemical sensors based on polypyrrol (PPy) molecular imprinted polymers (MIP) for proteins detection. Here, prostate-specific antigen (PSA) was selected as a template model. Computational calculations indicate that for any PPy conformation and any amino-acid location in the protein, PSA molecules remain strongly inserted in the PPy polymer without biological alterations. One from possible orientations, appeared to be most probable as it presents the lowest absorption energy (-363â¯kcalâ¯mol-1) and largest contact area (4034.1â¯Å2). The device was then elaborated by in situ electropolymerization of PPy films. MIP's thickness and extraction duration were optimized by chronoamperometry. Square wave voltammetry technique was investigated for PSA detection in standard solution in the concentration range of 3x10 -8 ng.ml-1- 300â¯ngâ¯ml-1. According to the Hill equation, the equilibrium dissociation constant Kdbetween PSA and its imprint was estimated at Kd = (1.02⯱â¯0.54)â¯×â¯10-14â¯M, confirming the strong binding between the designed MIP and the protein as predicted by the computational study. PSA concentration values directly measured in 35 human serum samples were found closely correlated to those measured by the ELISA technique. The promising fast and low-cost sensor might be used successfully for proteins detection at low concentrations with high selectivity and reproducibility.
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Técnicas Biossensoriais , Impressão Molecular , Antígeno Prostático Específico/isolamento & purificação , Proteínas/isolamento & purificação , Humanos , Limite de Detecção , Conformação Molecular , Polímeros/química , Antígeno Prostático Específico/genética , Proteínas/genéticaRESUMO
Prostate cancer is the most common male cancer in the world. The diagnosis, staging, prognosis and monitoring are usually done with Prostate Specific Antigen (PSA). Biosensors are emerging as a novel analytical technology for PSA detection. They provide several advantages for clinical applications and will benefit clinicians, patients and forensic workers in the future. Among them, electrochemical immunosensors have gained growing interests. Hence, their sensitivity is often improved by modifying them with nanoparticles especially iron oxide (IONP). Functionalized IONP attracted much attention in the fabrication of biosensing systems, due to their multiple properties, such as biocompatibility and signal amplification, and their ability to bind covalently to antibodies via their functional groups. In the present study, two electrochemical immunosensors were investigated for PSA detection. The first one was functionalized with 3- glycidoxypropyltrimethoxysilane self-assembled monolayer, while the second one was based on iron oxide nanoparticles functionalized with 3-aminopropyltriethoxysilane. Square wave voltammetry (SWV) has been investigated to follow-up the PSA detection in a phosphate buffer solution, in an artificial serum and in a human serum. The limit of detection (LOD) of both immunosensors was found of order of 10 fg/ml. When estimated in human serum this value increases up to 50 pg/ml.
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Técnicas Eletroquímicas/instrumentação , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Humanos , Masculino , Sensibilidade e EspecificidadeRESUMO
TNFα is a homotrimeric pro-inflammatory cytokine, whose direct targeting by protein biotherapies has been an undeniable success for the treatment of chronic inflammatory diseases. Despite many efforts, no orally active drug targeting TNFα has been identified so far. In the present work, we identified through combined in silico/in vitro/in vivo approaches a TNFα direct inhibitor, compound 1, displaying nanomolar and micromolar range bindings to TNFα. Compound 1 inhibits the binding of TNFα with both its receptors TNFRI and TNFRII. Compound 1 inhibits the TNFα induced apoptosis on L929 cells and the TNFα induced NF-κB activation in HEK cells. In vivo, oral administration of compound 1 displays a significant protection in a murine TNFα-dependent hepatic shock model. This work illustrates the ability of low-cost combined in silico/in vitro/in vivo screening approaches to identify orally available small-molecules targeting challenging protein-protein interactions such as homotrimeric TNFα.
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Anti-Inflamatórios/farmacologia , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A highly selective and sensitive surface acoustic wave (SAW) sensor of dopamine (DA) was developed by depositing cobalt phthalocyanine (CoPc) nanopillars on gold-coated sensing platform of SAW sensor. The developed biosensor presents a sensitivity of 1.6°/nM, has a low limit of detection (LOD) on the order of 0.1 nM, and imparts more selectivity toward DA, since the detection limit of the interfering ascorbic acid (AA) is as high as 1 mM. To understand the selectivity mechanisms of this sensor toward DA, density functional theory-based chemical calculations were carried out. Calculations suggest two different types of interactions: dative bond with a very strong character for DA-CoPc complexes, and significant ionic character in the case of AA-CoPc ones. The interaction energies, in liquid phase, were estimated to be equal to -81 kJ mol(-1) and -38 kJ mol(-1) for DA-CoPc and AA-CoPc complexes, respectively, therefore accounting for the selective detection of DA over AA using tandem CoPc nanopillar-based SAW sensor device. This work demonstrates a simple and efficient design of SAW sensors employing thin nanostructured CoPc biomolecular recognition layers for DA detection.
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Cobalto/química , Dopamina/análise , Indóis/química , Nanopartículas Metálicas/química , Sistemas Microeletromecânicos/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Técnicas Biossensoriais/instrumentação , Simulação por Computador , Dopamina/química , Desenho de Equipamento , Análise de Falha de Equipamento , Isoindóis , Nanopartículas Metálicas/ultraestrutura , Modelos Químicos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SomRESUMO
A silicon nitride functionalized electrode and a 104 MHz lithium tantalate (LiTaO3) surface acoustic wave (SAW) sensor have been used to investigate target-probe recognition processes. Electrochemical and gravimetric measurements have been considered to monitor hybridization of single base mismatch (SBM) in synthetic oligonucleotides and single-nucleotide polymorphisms ApoE in real clinical genotypes. Obvious discrimination of SBM in nucleotides has been shown by both gravimetric and electrochemical techniques, without labeling nor amplification. Investigations on mismatches nature and position have also been considered. For guanine-adenine (GA), guanine-thymine (GT) and guanine-guanine (GG) mismatches, the sensors responses present a dependence upon positions. Considering the capacitance variations and hybridization rates, results showed that gravimetric transduction is more sensitive than electrochemical one. Moreover, the highest value of GT hybridization rate (in the middle position) was found in accordance with the nearest-neighbor model, where the considered configuration appears as the most thermodynamically stable. For the real samples, where the electrochemical transduction, by combining capacitance and flat-band potential measurements, were found more sensitive, the results show that the realized sensor permits an unambiguous discrimination of recognition between fully complementary, non-complementary and single base mismatched targets, and even between the combination of differently matched strands.