RESUMEN
Cytochrome c (cyt c) has been found to play a function in apoptosis in cell-free models. This work presents the creation of molecularly imprinted conducting poly(3, 4-ethylenedioxythiopene) (MIPEDOT) on the surface of a screen printed carbon electrode (SPCE) for cyt c. Cyt c was imprinted by electropolymerization due to the presence of an EDOT monomer hydrophobic functional group on SPCE, using CV to obtain highly selective materials with excellent molecular recognition ability. MIPEDOT was characterized by CV, EIS, and DPV using ferricyanide/ferrocyanide as a redox probe. Further, the characterization of the sensor was accomplished using SEM for surface morphological confirmation. Using CV, the peak current measured at the potential of +1 to -1 V (vs. Ag/AgCl) is linear in the cyt c concentration range from 1 to 1200 pM, showing a remarkably low detection limit of 0.5 pM (sensitivity:0.080 µA pM). Moreover, the applicability of the approach was successfully confirmed with the detection of cyt c in biological samples (human plasma). Similarly, our research has proven a low-cost, simple, and efficient sensing platform for cyt c detection, rendering it a viable tool for the future improvement of reliable and exact non-encroaching cell death detection.
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Compuestos Bicíclicos Heterocíclicos con Puentes , Carbono , Citocromos c , Técnicas Electroquímicas , Electrodos , Polímeros , Citocromos c/análisis , Citocromos c/química , Polímeros/química , Carbono/química , Técnicas Electroquímicas/métodos , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Polímeros Impresos Molecularmente/química , Humanos , Límite de Detección , Impresión Molecular , Técnicas Biosensibles/métodosRESUMEN
Parkinson's disease (PD) is the most prevalent type of incurable movement disorder. Recent research findings propose that the familial PD-associated molecule DJ-1 exists in cerebrospinal fluid (CSF) and that its levels may be altered as Parkinson's disease advances. By using a molecularly imprinted polymer (MIP) as an artificial receptor, it becomes possible to create a functional MIP with predetermined selectivity for various templates, particularly for the DJ-1 biomarker associated with Parkinson's disease. It mostly depends on molecular recognition via interactions between functional monomers and template molecules. So, the computational methods for the appropriate choice of functional monomers for creating molecular imprinting electropolymers (MIEPs) with particular recognition for the detection of DJ-1, a pivotal biomarker involved in PD, are undertaken in this study. Here, molecular docking, molecular dynamics simulations (MD), molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods, and quantum mechanical calculation have been applied to investigate the intermolecular interaction between DJ-1 and several functional electropentamers, viz., polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT), poly(o-aminophenol) (POAP), and polythiophene (PTS). In this context, the electropentamers were selected to mimic the imprinted electropolymer system. We analyzed the most stable configurations of the formed complexes involving DJ-1 and electropentamers as a model system for MIEPs. Among these, PEDOT exhibited a more uniform arrangement around DJ-1, engaging in numerous van der Waals, H-bond, electrostatic, and hydrophobic interactions. Hence, it can be regarded as a preferable choice for synthesizing a MIP for DJ-1 recognition. Thus, it will aid in selecting a suitable functional monomer, which is of greater significance in the design and development of selective DJ-1/MIP sensors.
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Impresión Molecular , Enfermedad de Parkinson , Humanos , Polímeros/química , Simulación del Acoplamiento Molecular , Impresión Molecular/métodos , Pirroles , Simulación de Dinámica Molecular , BiomarcadoresRESUMEN
Noradrenaline (NA), one of the important excitatory catecholamine neurotransmitters, is used as a medication for Parkinson's Disease (PD). The ß-cyclodextrin (ß-CD) is one of the most effective drug carrier & also used in chiral separation. So, in this theoretical investigation, the R/S-Noradrenaline (R/S-NA) forms binding & chiral recognition mechanisms and energies with ß-CD were explored. Using the AutoDock, R/S forms were first docked into the cavity of ß-CD giving host-guest complexes with the free energy of binding for S-NA (-4.81 kcal/mol) larger than R-NA (-4.53 kcal/mol). The host-guest inclusion 1:1 complexes between R/S-NA and ß-CD have been also modeled and optimized with ONIOM2 (B3LYP/6-31g++DP: PM6) method by using the Gaussian software. Further, frequency calculations were carried out to obtain the free energies. In comparison to the R-NA (-54.59 kcal/mol), it was observed that the S-NA (-56.48 kcal/mol) with ß-CD is more stable. Furthermore, the H-bond results from molecular dynamics simulation revealed that S-NA/ß-CD was more stable than R-NA/ß-CD. In addition, the thermodynamic properties, vibrational analysis (IR), HOMO-LUMO band gap energy, inter molecular hydrogen bond interactions, and conformational analysis were investigated for both the R/S forms to support & compare the stability of the inclusion complex. These inclusion & high stability of S-NA/ß-CD and in turn its theoretical chiral recognition behavior observed agreeing well with the reported NMR experimental data have implications in drug delivery and chiral separation research.
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Simulación de Dinámica Molecular , Norepinefrina/química , beta-Ciclodextrinas/química , Conformación Molecular , Modelos Moleculares , Enlace de Hidrógeno , Termodinámica , VibraciónRESUMEN
Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.
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Técnicas Biosensibles/tendencias , Técnicas Electroquímicas/tendencias , Inmunoensayo/tendencias , Superóxido Dismutasa/análisis , Animales , Humanos , Ratones , Óxido Nítrico , Estrés Oxidativo , Sistemas de Atención de Punto , Especies Reactivas de Oxígeno , Compuestos de SulfhidriloRESUMEN
Heat, electric shock, and burn injuries induce apoptosis by releasing cytochrome c (cyt-c) from mitochondria and by subsequently activating the death protease, caspases-3. During apoptosis, cyt-c undergoes changes in the secondary structure that have been suggested to increase its peroxidase activity. Information about these structural changes will provide better understanding of the apoptotic mechanism. Hence, temperature-dependent conformational dynamics of cyt-c has been investigated through molecular dynamics (MD) simulations to explain the structural changes and to correlate them with its apoptotic behavior. We observe that, at lower temperatures (223, 248, and 300K), the secondary structure of cyt-c, remains stable, while at higher temperatures (323, 373, 423, and 473K), the secondary structural regions change significantly. Further, our MD results indicate that these structural changes are mainly localized on α-helices, turns, ß-sheets, and important loops that were involved in the stabilization of the heme conformation. This conformational transition between specific regions of secondary structure of cyt-c directly affects the electron tunneling properties of the proteins as observed experimentally. We quantify and compare these changes and explain that the temperature plays a vital role in assuring the structural stability of cyt-c and thus its functions. Our findings from this MD study reproduce experimental results at high temperatures and provide evidence for the alteration of the heme through the disruption of the H-bonding interactions between specific regions of cyt-c, thereby enhancing its peroxidase activity which plays a crucial role in the apoptotic process.
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Citocromos c/química , Simulación de Dinámica Molecular , Conformación Proteica , Termodinámica , Animales , Apoptosis , Caballos , Enlace de Hidrógeno , Oxidación-Reducción , Pliegue de Proteína , Estructura Secundaria de Proteína , Desplegamiento Proteico , TemperaturaRESUMEN
Cytochrome c (cyt-c) upon binding with cardiolipin acquires peroxidase activity and is strictly connected to the pathogenesis of many human diseases including neurodegenerative and cardiovascular diseases. Interaction of cyt-c with cardiolipin mimics partial unfolding/conformational changes of cyt-c in different solvent environments. Dynamic pictures of these conformational changes of cyt-c are crucial in understanding their physiological roles in mitochondrial functions. Therefore, atomistic molecular dynamics (MD) simulations have been carried out to investigate the effect of different solvents (water, urea/water, MeOH and DMSO) on the structure and conformations of apoptotic cyt-c (Fe3+). Our study demonstrates that the structural changes in the protein are solvent dependent. The structural differences are observed majorly on the ß-sheets and α-helical conformations and the degree of their perturbation are specific to the solvent. Although a complete loss of ß-sheets (0%) is observed in MeOH and DMSO, by contrast, well preserved ß-sheets (3.84%) are observed in water and urea/water. A significant decrease in the α-helical contents is observed in MeOH (41.34%) and water (42.46%), a negligible alteration in DMSO (44.25%) and well preserved α-helical (45.19%) contents in urea/water. The distances between the residues critical for electron transfer are decreased considerably for DMSO. Further, the reduction in residue flexibility and the conformational space indicate that the collective motions of cyt-c are reduced when compared to other cosolvents. Essential dynamics analysis implies that the overall motions of cyt-c in water, MeOH and urea/water are involved in three to four eigenvectors and in first eigenvector in DMSO. Overall, we believe that MD simulations of cyt-c in different solvents can provide a detailed microscopic understanding of the physiological roles, electron transport and peroxidase function in the early events of apoptosis which are hard to probe experiments.
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Citocromos c/química , Simulación de Dinámica Molecular , Conformación Proteica , Solventes/química , Apoptosis , Peroxidasa/química , Peroxidasa/metabolismo , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Over the past several decades, nanotechnology has contributed to the progress of biomedicine, biomarker discovery, and the development of highly sensitive electroanalytical / electrochemical biosensors for inâ vitro and inâ vivo monitoring, and quantification of oxidative and nitrosative stress markers like reactive oxygen species (ROS) and reactive nitrogen species (RNS). A major source of ROS and RNS is oxidative stress in cells, which can cause many human diseases, including cancer. Therefore, the detection of local concentrations of ROS (e. g. superoxide anion radical; O2â¢- ) and RNS (e. g. nitric oxide radical; NO⢠and its metabolites) released from biological systems is increasingly important and needs a sophisticated detection strategy to monitor ROS and RNS inâ vitro and inâ vivo. In this review, we discuss the nanomaterials-based ROS and RNS biosensors utilizing electrochemical techniques with emphasis on their biomedical applications.
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Técnicas Biosensibles , Nanoestructuras/química , Especies de Nitrógeno Reactivo/análisis , Especies Reactivas de Oxígeno/análisis , Técnicas Electroquímicas , Humanos , Estrés Oxidativo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa-1/metabolismoRESUMEN
Inkjet printed graphene (IPG) has recently shown tremendous promise in reducing the cost and complexity of graphene circuit fabrication. Herein we demonstrate, for the first time, the fabrication of an ion selective electrode (ISE) with IPG. A thermal annealing process in a nitrogen ambient environment converts the IPG into a highly conductive electrode (sheet resistance changes from 52.8 ± 7.4 MΩ/â¡ for unannealed graphene to 172.7 ± 33.3 Ω/â¡ for graphene annealed at 950 °C). Raman spectroscopy and field emission scanning electron microscopy (FESEM) analysis reveals that the printed graphene flakes begin to smooth at an annealing temperature of 500 °C and then become more porous and more electrically conductive when annealed at temperatures of 650 °C and above. The resultant thermally annealed, IPG electrodes are converted into potassium ISEs via functionalization with a poly(vinyl chloride) (PVC) membrane and valinomycin ionophore. The developed potassium ISE displays a wide linear sensing range (0.01-100 mM), a low detection limit (7 µM), minimal drift (8.6 × 10-6 V/s), and a negligible interference during electrochemical potassium sensing against the backdrop of interfering ions [i.e., sodium (Na), magnesium (Mg), and calcium (Ca)] and artificial eccrine perspiration. Thus, the IPG ISE shows potential for potassium detection in a wide variety of human fluids including plasma, serum, and sweat.
RESUMEN
This review is an attempt, for the first time, to describe advancements in sensing technology for cytochrome c (cyt c) detection, at point-of-care (POC) application. Cyt c, a heme containing metalloprotein is located in the intermembrane space of mitochondria and released into bloodstream during pathological conditions. The release of cyt c from mitochondria is a key initiative step in the activation of cell death pathways. Circulating cyt c levels represents a novel in-vivo marker of mitochondrial injury after resuscitation from heart failure and chemotherapy. Thus, cyt c detection is not only serving as an apoptosis biomarker, but also is of great importance to understand certain diseases at cellular level. Various existing techniques such as enzyme-linked immunosorbent assays (ELISA), Western blot, high performance liquid chromatography (HPLC), spectrophotometry and flow cytometry have been used to estimate cyt c. However, the implementation of these techniques at POC application is limited due to longer analysis time, expensive instruments and expertise needed for operation. To overcome these challenges, significant efforts are being made to develop electrochemical biosensing technologies for fast, accurate, selective, and sensitive detection of cyt c. Presented review describes the cutting edge technologies available in the laboratories to detect cyt c. The recent advancements in designing and development of electrochemical cyt c biosensors for the quantification of cyt c are also discussed. This review also highlights the POC cyt c biosensors developed recently, that would prove of interest to biologist and therapist to get real time informatics needed to evaluate death process, diseases progression, therapeutics and processes related with mitochondrial injury.
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Técnicas Biosensibles/métodos , Citocromos c/análisis , Animales , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Humanos , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Mitocondrias/química , Modelos Moleculares , Sistemas de Atención de PuntoRESUMEN
Nitrite (NO2-) supplementation limits hypoxia-induced oxidative stress and activates the alternate NO pathway which may partially account for the nitrite-mediated cardioprotection. So, sensitive and selective biosensors with point-of-care devices need to be explored to detect the physiological nitrite level due to its important role in human pathophysiology. In this work, cytochrome c reductase (CcR) biofunctionalized self assembled monolayer (SAM) functionalized on gold nanoparticles (GNPs) in polypyrrole (PPy) nanocomposite onto the screen printed carbon electrode (SPCE) was investigated as a biosensor for the detection of nitrite based on its electrochemical and catalytic properties. CcR was covalently coupled with SAM layers on GNPs by using EDC and NHS. Direct electrochemical response of CcR biofunctionalized electrodes showed a couple of well-defined and nearly reversible cyclic voltammetric peaks at -0.34 and -0.45 vs. Ag/AgCl. Under optimal conditions, the biosensor could be used for the determination of NO2- with a linear range from 0.1-1600µm and a detection limit of 60nM with a sensitivity of 0.172µAµM-1cm-2. Further, we have designed and developed a novel and cost effective portable electrochemical analyzer for the measurement of NO2- in hypoxia induced H9c2 cardiac cells using ARM microcontroller. The results obtained here using the developed portable electrochemical nitrite analyzer were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.
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Técnicas Biosensibles , Técnicas Electroquímicas , Nitritos/aislamiento & purificación , Citocromos c/química , Electrodos , Enzimas Inmovilizadas/química , Oro/química , Humanos , Límite de Detección , Nanotubos de Carbono/química , Nitritos/química , Oxidorreductasas/química , Polímeros/química , Pirroles/químicaRESUMEN
This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO2(-)) and nitrate (NO3(-)) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP-ZnO-SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 µM with a detection limit of 100 nM and sensitivity of 85.4 nA µM(-1). Furthermore, NO2(-) measurement showed linearity of 100 nM to 1mM with a detection limit of 100 nM for NO2(-) and sensitivity of 96.4 nA µM(-1). Then, the concentration of NO3(-) was measured after its enzymatic conversion into NO2(-). Using this assay, the concentrations of NO, NO2(-), and NO3(-) present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP-ZnO-SPCE and validated with the standard Griess method.
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Clorofilidas/química , Cobre/química , Técnicas Electroquímicas/instrumentación , Nitratos/sangre , Óxido Nítrico/sangre , Nitritos/sangre , Técnicas Biosensibles/instrumentación , Electrodos , Diseño de Equipo , Humanos , Límite de DetecciónRESUMEN
In the present study, the fruiting body extracts of Xylaria sp. strain R006 were obtained from hexane, ethyl acetate and methanol. Among them, the ethyl acetate extract exhibited significant antimicrobial activities against bacterial and fungal pathogens. Based on the effective antimicrobial activity, the crude ethyl acetate extract was fractionized by two-step siliga gel column chromatography. All the fractions were tested for antibacterial activity against drug resistant Staphylococcus aureus strains (1-10) and Pseudomonas aeruginosa strains (1-8). The fraction E showed a maximum inhibition zone of 27.9 mm against drug resistant S. aureus strain 3 and 29.4 mm against drug resistant P. aeruginosa strain 4. Minimal inhibitory concentration of fraction E showed potential result against all the drug resistant strains however, the lowest concentration of 75 µg/mL-1 was observed against S. aureus strains 1 and 6 and P. aeruginosa strain 3. Further, 60 µg/mL of fraction E had significant cytotoxic activity of 54.9, 55.1 and 54.9% against MDA-MB-231 (breast carcinoma cells), A-549 (lung carcinoma cells) and MCF-7 (breast carcinoma cells) human cancer cell lines, respectively. The spectral data revealed that the fraction E has chromophoric groups in it and had the C = O stretching, C-C = C asymmetric stretch, N-H stretch and C-O stretch as functional groups. The results indicate that the metabolites of fruiting bodies of Xylaria sp. R006 are the potential natural source for the development of new anticancer agents.
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Antibacterianos/farmacología , Antineoplásicos/farmacología , Xylariales , Línea Celular Tumoral , Farmacorresistencia Bacteriana Múltiple , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
Recently, mutations in copper-zinc superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (fALS), a progressive neurodegenerative disease involving motor neuron loss, paralysis and death. It is mainly due to protein misfolding and aggregation resulting from the enhanced peroxidase activity of SOD1 mutants. In this study, we have carried out a 20 ns molecular dynamics simulation for wild type (WT), H43R and W32F mutated SOD1's dimer and compared their structure and conformational properties by extracting several quantitative properties from the trajectory to understand the pathology of fALS disease. Our results show considerable differences in H43R compared to WT and W32F mutated SOD1, such as increasing distances between the critical residues results in open conformation at the active site, strong fluctuations in the important loops (Zinc and electrostatic loops) and weakening of important hydrogen bonds especially between N (His 43/Arg 43) and carbonyl oxygen (His 120) in agreement with the experimental report. The calculated buried surface area of dimer interface for WT, H43R and W32F are 682, 726 and 657 Å(2) respectively, representing the loss of dimerization in H43R. Essential dynamics reveal that overall motions of WT and W32F are mainly involved in three to four eigenvectors, but in H43R the overall motions are mainly in the first eigenvector. These data thus provide a unifying description for the structural destabilization, enhanced peroxidase activity, loss of dismutation activity and increase in aggregation propensity in the pathology of fALS diseases.
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Esclerosis Amiotrófica Lateral/enzimología , Cobre/química , Simulación de Dinámica Molecular , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Zinc/química , Esclerosis Amiotrófica Lateral/genética , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Multimerización de Proteína , Electricidad Estática , Superóxido Dismutasa-1RESUMEN
This work presents a novel bienzymatic biosensor for the simultaneous determination of nitrite (NO2(-)) and nitrate (NO3(-)) ions using copper, zinc superoxide dismutase (SOD1) and nitrate reductase (NaR) coimmobilized on carbon nanotubes (CNT)-polypyrrole (PPy) nanocomposite modified platinum electrode. Morphological changes of the PPy and CNT modified electrodes were investigated using scanning electron microscopy. The electrochemical behavior of the bienzymatic electrode (NaR-SOD1-CNT-PPy-Pt) was characterized by cyclic voltammetry exhibiting quasi-reversible redox peak at +0.06 V and reversible redox peaks at -0.76 and -0.62V vs. Ag/AgCl, for the immobilized SOD1 and NaR respectively. The electrocatalytic activity of SOD1 towards NO2(-) oxidation observed at +0.8 V was linear from 100 nM to 1mM with a detection limit of 50 nM and sensitivity of 98.5 ± 1.7 nA µM(-1)cm(-2). Similarly, the coimmobilized NaR showed its electrocatalytic activity towards NO3(-) reduction at -0.76 V exhibiting linear response from 500 nM to 10mM NO3(-) with a detection limit of 200 nM and sensitivity of 84.5 ± 1.56 nA µM(-1)cm(-2). Further, the present bienzymatic biosensor coated with cellulose acetate membrane for the removal of non-specific proteins was used for the sensitive and selective determinations of NO2(-) and NO3(-) present in human plasma, whole blood and saliva samples.
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Técnicas Biosensibles/métodos , Nitratos/aislamiento & purificación , Nitritos/aislamiento & purificación , Superóxido Dismutasa/química , Enzimas Inmovilizadas/química , Humanos , Nitrato-Reductasa/química , Nitratos/sangre , Nitritos/sangreRESUMEN
We have designed here a label-free direct electrochemical immunosensor for the detection of cytochrome c (cyt c), a heme containing metalloprotein using its specific monoclonal antibody. Two nanocomposite-based electrochemical immunosensor platforms were evaluated for the detection of cyt c; (i) self-assembled monolayer (SAM) on gold nanoparticles (GNP) in polypyrrole (PPy) grafted screen printed electrodes (SPE) and (ii) carbon nanotubes (CNT) integrated PPy/SPE. The nanotopologies of the modified electrodes were confirmed by scanning electron microscopy. Electrochemical impedance spectroscopy and cyclic voltammetry were employed to monitor the stepwise fabrication of the nanocomposite immunosensor platforms. In the present method, the label-free quantification of cyt c is based on the direct electron transfer between Fe (III)/Fe (II)-heme redox active site of cyt c selectively bound to anti-cyt c nanocomposite modified SPE. GNP/PPy and CNT/PPy nanocomposites promoted the electron transportation through the conductive pore channels. The overall analytical performance of GNP/PPy based immunosensor (detection limit 2 nM; linear range: 2 nM to 150 µM) was better than the anti-cyt c/CNT/PPy (detection limit 10 nM; linear range: 10 nM to 50 µM). Further, the measurement of cyt c release in cell lysates of cardiomyocytes using the GNP/PPy based immunosensor gave an excellent correlation with standard ELISA.
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Citocromos c/análisis , Técnicas Electroquímicas/instrumentación , Oro/química , Nanocompuestos/química , Nanotubos de Carbono/química , Polímeros/química , Pirroles/química , Animales , Técnicas Biosensibles/instrumentación , Bovinos , Electrodos , Diseño de Equipo , Límite de Detección , Modelos MolecularesRESUMEN
Emerging evidences have pointed out that the release of cytochrome c (cyt c) from mitochondria into cytosol is a critical step in the activation of apoptosis. This article presents a novel approach for the detection of mitochondrial cyt c release for the first time using cytochrome c reductase (CcR) immobilized on nanoparticles decorated electrodes. Two kinds of nanomaterial-based biosensor platforms were used: (a) carbon nanotubes (CNT) incorporated polypyrrole (PPy) matrix on Pt electrode and (b) self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in PPy-Pt. Scanning electron microscope was used to characterize the surface morphologies of the nanomaterial modified electrodes. Cyclic voltammograms of both the biosensors showed reversible redox peaks at -0.45 and -0.34 V vs Ag/AgCl, characteristic of CcR. In comparison, the CcR-CNT biosensor gave a detection limit of 0.5±0.03 µM cyt c, which was 4-fold better than the CcR-GNP biosensor (2±0.03 µM). Moreover, the CcR-CNT biosensor achieved a much larger linear range (1-1000 µM) over the CcR-GNP biosensor (5-600 µM) with 2-fold better sensitivity. The CcR-CNT-PPy-Pt biosensor was further applied to quantify the mitochondrial cyt c released in cytosol of A549 cells upon induction of apoptosis with doxorubicin, the results agreed well with standard western blot analysis.
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Técnicas Biosensibles/métodos , Citocromos c/análisis , Enzimas Inmovilizadas/química , NADPH-Ferrihemoproteína Reductasa/química , Animales , Bovinos , Línea Celular Tumoral , Citocromos c/metabolismo , Técnicas Electroquímicas , Electrodos , Enzimas Inmovilizadas/metabolismo , Oro , Humanos , Límite de Detección , NADPH-Ferrihemoproteína Reductasa/metabolismo , Nanopartículas/química , Nanotubos de Carbono/química , Polímeros/química , Pirroles/química , PorcinosRESUMEN
In this work, we have designed and developed a novel and cost effective virtual electrochemical analyzer for the measurement of NO in exhaled breath and from hydrogen peroxide stimulated endothelial cells using home-made potentiostat. Here, data acquisition system (NI MyDAQ) was used to acquire the data from the electrochemical oxidation of NO mediated by copper, zinc superoxide dismutase (Cu,ZnSOD). The electrochemical control programs (graphical user-interface software) were developed using LabVIEW 10.0 to sweep the potential, acquire the current response and process the acquired current signal. The Cu,ZnSOD (SOD1) immobilized on the carbon nanotubes in polypyrrole modified platinum electrode was used as the NO biosensor. The electrochemical behavior of the SOD1 modified electrode exhibited the characteristic quasi-reversible redox peak at the potential, +0.06 V vs. Ag/AgCl. The biological interferences were eliminated by nafion coated SOD1 electrode and then NO was measured selectively. Further, this biosensor showed a wide linear range of response over the concentration of NO from 0.1 µM to 1 mM with a detection limit of 0.1 µM and high sensitivity of 1.1 µA µM(-1). The electroanalytical results obtained here using the developed virtual electrochemical instrument were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.
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Técnicas Biosensibles/métodos , Enzimas Inmovilizadas/química , Nanotubos de Carbono/química , Óxido Nítrico/metabolismo , Polímeros/química , Lenguajes de Programación , Pirroles/química , Superóxido Dismutasa/química , Adulto , Pruebas Respiratorias , Electroquímica , Electrodos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Enzimas Inmovilizadas/metabolismo , Espiración , Humanos , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Platino (Metal)/química , Superóxido Dismutasa/metabolismoRESUMEN
An electrochemical NO(x) sensor was fabricated based on the incorporation of hemin on a ZnO-PPy nanocomposite modified Pt electrode. Scanning electron microscopy, energy dispersive X-ray analysis and cyclic voltammetry were used to confirm the successful stepwise assembly procedure for the sensor. The electrocatalytical behavior of the sensor was investigated by cyclic voltammetry. The hemin-ZnO-PPy-Pt electrode exhibited characteristic hemin reversible redox peaks at 0.035 V and -0.11 V vs. Ag/AgCl respectively. The hemin-ZnO-PPy-Pt electrode exhibited 3-fold enhanced electrocatalytic activity towards NO(x) compared to the hemin-PPy-Pt electrode. The electrocatalytic response of the sensor was proportional to the NO(x) concentration in the range of 0.8 to 2000 µM (r(2) = 0.9974) with a sensitivity of 0.04 µA µM(-1) cm(-2) and detection limit of 0.8 µM for the hemin-ZnO-PPy-Pt electrode. The low detection limit, wide linear range and enhanced sensitivity of the present sensor make it valuable for potential applications. In addition, this sensor exhibited good reproducibility and stability.
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Técnicas de Química Analítica/instrumentación , Hemina/química , Nanocompuestos/química , Óxidos de Nitrógeno/análisis , Platino (Metal)/química , Polímeros/química , Pirroles/química , Óxido de Zinc/química , Electroquímica , Electrodos , Óxidos de Nitrógeno/química , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Dopamine (3,4-dihydroxyphenylethylamine, DA), an important neurotransmitter, exists in the cell bodies of the dopaminergic neurons of the substantia nigra. Oxidation of DA to its quinone and subsequent reaction with Adenine and Guanine in DNA result in the formation of depurinating adducts, thus causing DNA damage. In this article, we investigate the interaction of quinone metabolites of dopamine (DMQ) with models representing the structure of DNA using dispersion corrected density functional theory with an aim to evaluate the associated structural changes in DNA upon their interaction. Various binding sites for the DA metabolite on these DNA models have been considered and our computations on the activation barriers allowed us to identify preferential bonding sites for these metabolites analogous to experiments. Analysis of the geometry of these adducts in comparison to free base pairs reveals that the attack of DMQ causes remarkable changes in the structural properties. With our calculations, we propose that these structural alterations induce mutations by favoring the formation of depurinating adducts leading to mutagenic effects such as base mispairing, explaining the toxicological (carcinogenic and neurotoxic) behavior of DMQ.
Asunto(s)
Dopamina/análogos & derivados , Dopamina/química , Modelos Moleculares , Mutágenos/química , Quinonas/química , Sitios de Unión , Simulación por Computador , ADN/química , Aductos de ADN/química , Daño del ADN , Enlace de Hidrógeno , TermodinámicaRESUMEN
There is a growing need for new and effective antibiotic agents due to the recent emergence of life-threatening, multidrug-resistant bacterial infections such as methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. In the present study, the antimicrobial potential of mushroom was investigated against multidrug-resistant bacterial strains. The mushroom was identified as Xylaria sp. strain R005 based on the morphological characteristics and confirmed by 18S ribosomal RNA sequence comparisons. The crude ethyl acetate extracts of culture filtrate and fruiting bodies of Xylaria sp. showed significant antibacterial activity against multidrug-resistant S. aureus strains (1-10) and P. aeruginosa strains (1-8). The minimum inhibitory concentration of the ethyl acetate extracts of culture filtrate and fruiting bodies ranged from 225 µg/mL to 625 µg/mL, and 120 µg/mL to 625 µg/mL, respectively, against clinical strains of S. aurues and P. aeruginosa. The synergistic action of extracts of Xylaria sp. with vancomycin and ciprofloxacin was observed against S. aureus strain 6 and P. aeruginosa strain 3, respectively. The fractional inhibitory concentration indices (FICIs) of culture filtrate extract with vancomycin and ciprofloxacin were 0.5 and 0.18, respectively. The FICI of fruiting body extract with vancomycin and ciprofloxacin were 0.5 and 0.375, respectively. These results clearly indicate that the metabolites of culture filtrate and fruiting bodies of Xylaria sp. are the potential source for production of new antimicrobial compounds.