Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 20
Filter
Add more filters











Publication year range
1.
J Neurochem ; 168(9): 3268-3283, 2024 Sep.
Article in English | MEDLINE | ID: mdl-39126680

ABSTRACT

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.


Subject(s)
Chromaffin Cells , Dynamin II , Endocytosis , Exocytosis , Mutation , Myopathies, Structural, Congenital , Chromaffin Cells/metabolism , Endocytosis/physiology , Endocytosis/genetics , Dynamin II/genetics , Dynamin II/metabolism , Animals , Exocytosis/physiology , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/pathology , Myopathies, Structural, Congenital/metabolism , Mutation/genetics , Cattle , Humans , Actins/metabolism , Actins/genetics , Cells, Cultured , Patch-Clamp Techniques , Adrenal Glands/metabolism , Adrenal Glands/pathology
2.
Mikrochim Acta ; 191(4): 197, 2024 03 14.
Article in English | MEDLINE | ID: mdl-38483622

ABSTRACT

A fully reusable electrochemical device is proposed for the first time made from laser cutting and a homemade conductive ink composed of carbon and nail polish. As a sensor substrate, we applied polymethyl methacrylate, which allows the surface to be renewed by simply removing and reapplying a new layer of ink. In addition to the ease of renewing the sensor's conductive surface, the design of the device has allowed for the integration of different forms of analysis. The determination of L-Dopa was performed using DPV, which presented a linear response range between 5.0 and 1000.0 µmol L-1, and a LOD of 0.11 µmol L-1. For dopamine, a flow injection analysis system was employed, and using the amperometric technique measurements were performed with a linear ranging from 2.0 to 100.0 µmol L-1 and a LOD of 0.26 µmol L-1. To demonstrate its applicability, the device was used in the quantification of analytes in pharmaceutical drug and synthetic urine samples.


Subject(s)
Graphite , Levodopa , Levodopa/analysis , Dopamine/analysis , Electrochemical Techniques/methods , Electrodes , Reproducibility of Results
3.
Mikrochim Acta ; 190(12): 461, 2023 Nov 06.
Article in English | MEDLINE | ID: mdl-37926729

ABSTRACT

Microfluidic cotton thread-based electroanalytical devices (µTEDs) are analytical systems with attractive features such as spontaneous passive flow, low cost, minimal waste production, and good sensitivity. Currently, sample injection in µTEDs is performed by hand using manual micropipettes, which have drawbacks such as inconstant speed and position, dependence of skilled analysts, and need of physical effort of operator during prolonged times, leading to poor reproducibility and risk of strain injury. As an alternative to these inconveniences, we propose, for the first time, the use of electronic micropipettes to carry out automated injections in µTEDs. This new approach avoids all disadvantages of manual injections, while also improving the performance, experience, and versatility of µTEDs. The platform developed here is composed by three 3D-printed electrodes (detector) attached to a 3D-printed platform containing an adjustable holder that keeps the electronic pipette in the same x/y/z position. As a proof-of-concept, both injection modes (manual and electronic) were compared using three model analytes (nitrite, paracetamol, and 5-hydroxytryptophan) on µTED with amperometric detection. As result, improved analytical performance (limits of detection between 2.5- and 5-fold lower) was obtained when using electronic injections, as well as better repeatability/reproducibility and higher analytical frequencies. In addition, the determination of paracetamol in urine samples suggested better precision and accuracy for automated injection. Thus, electronic injection is a great advance and changes the state-of-art of µTEDs, mainly considering the use of more modern and versatile electronic pipettes (wider range of pre-programmed modes), which can lead to the development of even more automated systems.

4.
Biosensors (Basel) ; 13(7)2023 Jun 29.
Article in English | MEDLINE | ID: mdl-37504089

ABSTRACT

The present study reports the development and application of a flow injection analysis (FIA) system for the simultaneous determination of uric acid (UA) and caffeine (CAF) using cathodically pretreated boron-doped diamond electrode (CPT-BDD) and multiple-pulse amperometry (MPA). The electrochemical profiles of UA and CAF were analyzed via cyclic voltammetry in the potential range of 0.20-1.7 V using 0.10 mol L-1 H2SO4 solution as supporting electrolyte. Under optimized conditions, two oxidation peaks at potentials of 0.80 V (UA) and 1.4 V (CAF) were observed; the application of these potentials using multiple-pulse amperometry yielded concentration linear ranges of 5.0 × 10-8-2.2 × 10-5 mol L-1 (UA) and 5.0 × 10-8-1.9 × 10-5 mol L-1 (CAF) and limits of detection of 1.1 × 10-8 and 1.3 × 10-8 mol L-1 for UA and CAF, respectively. The proposed method exhibited good repeatability and stability, and no interference was detected in the electrochemical signals of UA and CAF in the presence of glucose, NaCl, KH2PO4, CaCl2, urea, Pb, Ni, and Cd. The application of the FIA-MPA method for the analysis of environmental samples resulted in recovery rates ranging between 98 and 104%. The results obtained showed that the BDD sensor exhibited a good analytical performance when applied for CAF and UA determination, especially when compared to other sensors reported in the literature.


Subject(s)
Caffeine , Uric Acid , Caffeine/analysis , Oxidation-Reduction , Electrodes , Electrochemical Techniques/methods
5.
Talanta ; 259: 124536, 2023 Jul 01.
Article in English | MEDLINE | ID: mdl-37062090

ABSTRACT

Antibiotics such as tetracycline (TC) are widely prescribed to treat humans or dairy animals. Therefore, it is important to establish affordable devices in laboratories with minimal infrastructure. 3D printing has proven to be a powerful and cost-effective tool that revolutionizes many applications in electrochemical sensing. In this work, we employ a conductive filament based on graphite (Gr) and polylactic acid (PLA) (40:60; w/w; synthesized in our lab) to manufacture 3D-printed electrodes. This electrode was used "as printed" and coupled to batch injection analysis with amperometric detection (BIA-AD) for TC sensing. Preliminary studies by cyclic voltammetry and differential pulse voltammetry revealed a mass transport governed by adsorption of the species and consequent fouling of the redox products on the 3D printed surface. Thus, a simple strategy (solution stirring and application of successive potentials, +0.95 V followed by +1.2 V) was associated with the BIA-AD system to solve this effect. The proposed electrode showed analytical performance comparable to costly conventional electrodes with linear response ranging from 0.5 to 50 µmol L-1 and a detection limit of 0.19 µmol L-1. Additionally, the developed method was applied to pharmaceutical, tap water, and milk samples, which required minimal sample preparation (simple dilution). Recovery values of 92-117% were obtained for tap water and milk samples, while the content found of TC in the capsule was close to the value reported by the manufacturer. These results indicate the feasibility of the method for routine analysis involving environmental, pharmaceutical, and food samples.


Subject(s)
Laboratories , Tetracycline , Animals , Humans , Anti-Bacterial Agents , Printing, Three-Dimensional , Electrodes , Water , Pharmaceutical Preparations , Electrochemical Techniques
6.
Molecules ; 28(1)2023 Jan 02.
Article in English | MEDLINE | ID: mdl-36615580

ABSTRACT

Nitrite is a ubiquitous pollutant in modern society. Developing new strategies for its determination is very important, and electroanalytical methods present outstanding performance on this task. However, the use of bare electrodes is not recommended because of their predisposition to poisoning and passivation. We herein report a procedure to overcome these limitations on carbon fiber microelectrodes through pulsed amperometry. A three-pulse amperometry approach was used to reduce the current decay from 47% (after 20 min under constant potential) to virtually 0%. Repeatability and reproducibility were found to have an RSD lower than 0.5% and 7%, respectively. Tap water and synthetic inorganic saliva samples were fortified with nitrite, and the results obtained with the proposed sensor were in good agreement with the amount added.


Subject(s)
Biofouling , Nitrites , Microelectrodes , Carbon Fiber , Nitrites/analysis , Reproducibility of Results , Biofouling/prevention & control , Electrodes , Carbon , Electrochemical Techniques/methods
7.
Mikrochim Acta ; 189(5): 185, 2022 04 09.
Article in English | MEDLINE | ID: mdl-35396635

ABSTRACT

Graphene-based materials present unique properties for electrochemical applications, and laser-induced conversion of polyimide to graphene is an emerging route to obtain a high-quality material for sensing. Herein we present compact and low-cost equipment constructed from an open-source 3D printer at which a 3.5-W visible (449 nm) laser was adapted to fabricate laser-induced graphene (LIG) electrodes from commercial polyimide, which resulted in electron transfer kinetic (k0) of 5.6 × 10-3 cm s-1 and reproducibility calculated by relative standard deviation (RSD < 5%) from cyclic voltammograms of [Fe(CN)6]3-/4- using 5 different electrodes. LIG electrodes enabled the simultaneous voltammetric determination of uric acid (+ 0.1 V vs. pseudo-reference) and nitrite (+ 0.4 V vs pseudo-reference), with limit of detection (LOD) values of 0.07 and 0.27 µmol L-1, respectively. Amperometric measurements for the detection of H2O2 (applying + 0.0 V vs. Ag|AgCl|KCl(sat.)) after Prussian blue (PB) modification and ciprofloxacin (applying + 1.2 V vs. Ag|AgCl|KCl(sat.)) were performed under flow conditions, which confirmed the high stability of LIG and LIG-PB surfaces. The LOD values were 1.0 and 0.2 µmol L-1 for H2O2 and ciprofloxacin, respectively. The RSD values (< 12%) obtained for the analysis using three different electrodes attested the precision of LIG electrodes manufactured in two designs. No sample matrix effects on the determination of ciprofloxacin in milk samples were observed  (recoveries between 84 and 96%). The equipment can be built with less than $300 and each LIG electrode costs less than $0.01.


Subject(s)
Graphite , Ciprofloxacin , Electrodes , Graphite/chemistry , Hydrogen Peroxide , Lasers , Reproducibility of Results
8.
Talanta ; 246: 123477, 2022 Aug 15.
Article in English | MEDLINE | ID: mdl-35462247

ABSTRACT

This work presents, for the first time, a fast and highly sensitive electrochemical method for determination of three organophosphorus compounds (OPs), diazinon (DZN), malathion (MLT), and chlorpyrifos (CLPF), using a modified pyrolytic graphite electrode (PGE) coupled to batch injection analysis system with multiple pulse amperometric detection (BIA-MPA). The PGE was modified by a nanocomposite based on functionalized carbon nanotubes (CNTf) and silver nanoparticles (AgNPs). The OPs samples were directly analyzed on the modified working electrode surface by BIA-MPA system in Britton-Robinson (BR) buffer 0.15 mol L-1 at pH 6.0. The MPA detection of DZN, MLT and CLPF was performed using two potential pulses, which were sequentially applied on modified PGE at -1.3 V (100 ms) and +0.8 V (100 ms) for selective determination of these three OPs and working electrode cleaning, respectively. Under optimized conditions, the sensor presented a linear range of 0.1-20 µmol L-1 for DZN, 1.0-30 µmol L-1 for MLT and from 0.25 to 50 µmol L-1 for CLPF. The limits of detection (LOD) and quantification (LOQ) of 0.35 and 1.18 µmol L-1 for DZN, 0.89 and 2.98 µmol L-1 for MLT, and 0.53 and 1.78 µmol L-1 for CLPF were obtained. The proposed method exhibited high sensitivity of 0.068, 0.030 and 0.043 mA L µmol-1 for DZN, MLT and CLPF detection, respectively. Furthermore, the BIA-MPA system provided an analytical frequency of 71 determinations per hour for direct determination of these OPs in water and food samples. The modified PGE coupled to BIA-MPA system showed a high stability of electrochemical response for OPs detection with relative standard deviation (RSD) of 1.60% (n = 20). The addition-recovery studies of the proposed method were carried out in tap water, orange juice, and apple fruit real samples, which showed suitable recovery values between 77 and 124%. The analytical performance of the developed sensor provides an attractive alternative method for OPs determination with great potential for a fast and sensitive application in contaminated samples with these pesticides.


Subject(s)
Chlorpyrifos , Graphite , Metal Nanoparticles , Nanocomposites , Nanotubes, Carbon , Chlorpyrifos/analysis , Diazinon/analysis , Electrochemical Techniques/methods , Electrodes , Graphite/chemistry , Malathion , Metal Nanoparticles/chemistry , Nanotubes, Carbon/chemistry , Organophosphorus Compounds , Silver/chemistry , Water/analysis
9.
Diagnosis (Berl) ; 8(4): 510-514, 2021 11 25.
Article in English | MEDLINE | ID: mdl-32857713

ABSTRACT

OBJECTIVES: The differential diagnosis between acute bacterial meningitis (BM) and viral meningitis (VM) is crucial for treatment and prognosis. Cerebrospinal fluid (CSF) lactate (LA) is considered a good biomarker for differentiating BM from VM. The objective of this study was to compare the clinical performance of amperometry, which is not validated for measurement of LA in CSF samples, with a validated method (enzymatic ultra violet), for their ability to discriminate between acute BM and VM. METHODS: It was a retrospective, descriptive comparative study, 320 CSF reports were included; LA was quantified in CSF using either Dimension AR machine (Dade Behring) or amperometry (RAPID Point 500, Siemens). All samples with bacteria (n=54) or virus (n=139) identified, compared with a control with normal CSF (n=127). RESULTS: CSF LA levels were comparable for amperometry or enzymatic methods on each group studied, in a wide range of LA levels; it was capable to distinguish BM from VM independent of the method used to quantify. CONCLUSIONS: The findings support the use of the amperometric method in measuring LA concentrations in CSF in a wide range of values. Amperometry is a less expensive method, validated for blood, easily available in small laboratories including in limited resources countries.


Subject(s)
Meningitis, Bacterial , Meningitis, Viral , Biomarkers , Humans , Lactic Acid , Meningitis, Bacterial/diagnosis , Retrospective Studies
10.
Anal Bioanal Chem ; 412(26): 7123-7130, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32737552

ABSTRACT

This work exploits the applicability of a chemically reduced graphene oxide (CRGO) modification on the electrochemical response of a glassy carbon electrode (GCE) for the first-time sensitive determination of furosemide in natural waters. The batch injection analysis (BIA) is proposed as an analytical method, where CRGO-GCE is coupled to a BIA cell for amperometric measurements. Acetate buffer (0.1 µmol L-1, pH 5.2) was used as the background electrolyte. The modification provided an increase in sensitivity (0.024 µA/µmol L-1), low limit of detection (0.7 µmol L-1), RSD (< 4%), and broad linear range (1-600 µmol L-1). Recovery tests performed in two different concentration ranges resulted in values between 89 and 99%. Recovery tests were performed and compared with high-performance liquid chromatography (HPLC) with UV-Vis detection using Student's t test at a 95% significance level, and no significant differences were found, confirming the accuracy of the method. The developed method is proven faster (169 h-1) compared with the HPLC analysis (5 h-1), also comparable with other flow procedures hereby described, offering a low-cost strategy suitable to quantify an emerging pharmaceutical pollutant. Graphical abstract.


Subject(s)
Carbon/chemistry , Diuretics/analysis , Electrochemical Techniques/methods , Electrodes , Furosemide/analysis , Graphite/chemistry , Water Pollutants, Chemical/analysis , Chromatography, High Pressure Liquid/methods , Limit of Detection , Oxidation-Reduction , Reproducibility of Results , Spectrophotometry, Ultraviolet/methods
11.
Talanta ; 216: 120957, 2020 Aug 15.
Article in English | MEDLINE | ID: mdl-32456891

ABSTRACT

The aim of this paper was the development of a method for the determination of six strobilurins (fungicides) using boron-doped diamond (BDD) electrode with amperometric detection (AD) homemade coupled to high performance liquid chromatography (HPLC/UV-Vis). HPLC separation of fungicides was performed in a C18 reverse phase column using both UV and AD detectors at 200 mn and 1.9 V, respectively. The linear range for each strobilurin was from 5 to 15 mg L-1 and the correlation coefficients for all the compounds were above 0.997. Both detectors presented adequate detectability (LOD ranging from 1.33 to 1.57 µg kg-1) respecting the limits pre-established by regulatory agencies. The method was validated presenting good values of recovery and accuracy. In the spiked samples the recoveries ranged from 61.6% (trifloxystrobin) to 98.8% (azoxystrobin) for UV and 62.3% (trifloxystrobin) to 95.2% (azoxystrobin) for AD. In blanks spikes the recovery varied from 77.8% (picoxystrobin) to 88.4% (kresoxim-methyl) for UV and 76.7% (picoxystrobin) to 87.1% (dimoxystrobin) for AD. The method showed good precision (RSD < 10%). The results obtained by amperometric and UV detections were statistically comparable. Seven bean samples were analyzed to detect fungicide residues.


Subject(s)
Boron/chemistry , Fungicides, Industrial/analysis , Strobilurins/analysis , Vigna/chemistry , Chromatography, High Pressure Liquid , Electrodes , Spectrophotometry, Ultraviolet
12.
Acta Physiol (Oxf) ; 228(4): e13417, 2020 04.
Article in English | MEDLINE | ID: mdl-31769918

ABSTRACT

AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.


Subject(s)
Calcium Signaling/physiology , Chromaffin Cells/physiology , Exocytosis/physiology , Animals , Calcium/metabolism , Calcium Channels, P-Type/metabolism , Calcium Channels, Q-Type/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/metabolism , Female , Male , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Patch-Clamp Techniques/methods
13.
Clin Chim Acta ; 486: 59-65, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30006287

ABSTRACT

INTRODUCTION: The PKU is an inborn error of amino acid metabolism, in which phenylalanine (Phe) accumulated in the blood causing alterations at the central nervous system. We report a novel paper-based enzymatic platform coupled to screen printed graphene-modified electrode for the neonatal screening of phenylketonuria (PKU0. METHODS: The paper-based analytical device coupled to electrochemical detection (EPAD) is based on the use of paper microzones modified with phenylalanine dehydrogenase enzyme (PheDH). The modified PADs were placed on the surface of an electrode modified with electrochemically reduced graphene (ERGO). PheDH in the presence of NAD+ catalyzes the reversible deamination of Phe to form phenylpyruvate, ammonia, and NADH. The electrochemical oxidation of NADH was monitored by differential pulse amperometry (DPA) at 0.6 V. The method was linear in the concentration range from 1 to 600 µmol/L of Phe with a LOQ of 1 µmol/L and LOD of 0.2 µmol/L. Within day precision was 5.7% across 3 levels of control samples. Between-day precision was 8.3%. The comparison with the standard Phe enzyme assay kit showed good agreement. The time required for the overall assay was <5 min. The non-sophisticated equipment required, the short assay time and the appropriate LOQ and LOD achieved by our EPAD make it an attractive and easy to use alternative compared to existing methods applied to the screening of PKU in neonatal samples.


Subject(s)
Amino Acid Oxidoreductases/metabolism , Graphite/chemistry , Neonatal Screening , Paper , Phenylketonurias/diagnosis , Electrodes , Humans , Infant, Newborn , Phenylalanine/blood , Phenylalanine/metabolism , Phenylketonurias/blood , Phenylketonurias/metabolism
14.
Talanta ; 174: 420-427, 2017 Nov 01.
Article in English | MEDLINE | ID: mdl-28738602

ABSTRACT

This work presents a portable electrochemical system for the continuous monitoring of corrosion inhibitors in a wide range of matrices including ethanol, seawater and mineral oil following simple dilution of the samples. Proof-of-concept is demonstrated for the sensing of 2,5-dimercapto-1,3,5-thiadiazole (DMCT), an important corrosion inhibitor. Disposable screen-printed graphitic electrodes (SPGEs) associated with a portable batch-injection cell are proposed for the amperometric determination of DMCT following sample dilution with electrolyte (95% v/v ethanol + 5% v/v 0.1molL-1 H2SO4 solution). This electrolyte was compatible with all samples and the organic-resistant SPGE could be used continuously for more than 200 injections (100µL injected at 193µLs-1) free from effects of adsorption of DMCT, which have a great affinity for metallic surfaces, and dissolution of the other reported SPGE inks which has hampered prior research efforts. Fast (180h-1) and precise responses (RSD < 3% n = 10) with a detection limit of 0.3µmolL-1 was obtained. The accuracy of the proposed method was attested through recovery tests (93-106%) and the reasonable agreement of results of DMCT concentrations in samples analyzed by both proposed and spectrophotometric (comparative) methods.

15.
Anal Chim Acta ; 928: 32-38, 2016 Jul 20.
Article in English | MEDLINE | ID: mdl-27251854

ABSTRACT

In this study, homemade disposable gold electrodes made from recordable compact disks were modified with carbon nanotubes for amperometric quantification of terbutaline sulfate in pharmaceutical products. A flow cell using an impingent jet of solution on the electrode surface was build and used for amperometric detection, and a series of experiments were carried out to find the best experimental conditions for the new electrode in a specially designed cell. A linear response for terbutaline was obtained in the range from 3.0 × 10(-6) to 5.0 × 10(-4) mol L(-1) (at 0.63 V vs. Ag/AgCl). The limits of detection and quantification were calculated as 5.8 × 10(-7) mol L(-1) (S/N = 3) and 1.9 × 10(-6) mol L(-1) (S/N = 10), respectively. A frequency of 30 injections h(-1) was attained. The proposed method was successfully applied to the analyses of commercial syrup samples, and all results were in good agreement with those obtained by using high performance liquid chromatography and capillary electrophoresis-tandem mass spectrometry.


Subject(s)
Compact Disks , Electrochemistry/instrumentation , Limit of Detection , Nanotubes, Carbon/chemistry , Pharmaceutical Preparations/chemistry , Terbutaline/analysis , Electrodes , Gold/chemistry , Terbutaline/chemistry , Time Factors
16.
Food Chem ; 192: 691-7, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26304399

ABSTRACT

This work proposes the application of batch-injection analysis with amperometric detection to determine the antioxidant capacity of real samples based on the measurement of DPPH radical consumption. The efficient concentration or EC50 value corresponds to the concentration of sample or standard required to scavenge 50% DPPH radicals. For the accurate determination of EC50, samples were incubated with DPPH radical for 1h because many polyphenolic compounds typically found in plants and responsible for the antioxidant activity exhibit slow kinetics. The BIA system with amperometric detection using a glassy-carbon electrode presented high precision (RSD = 0.7%, n = 12), low detection limit (1 µmol L(-1)) and selective detection of DPPH (free of interferences from antioxidants). These contributed to low detection limits for the antioxidant (0.015 and 0.19 µmol L(-1) for gallic acid and butylated hydroxytoluene, respectively). Moreover, BIA methods show great promise for portable analysis because battery-powered instrumentation (electronic micropipette and potentiostats) is commercially available.


Subject(s)
Antioxidants/chemistry , Biphenyl Compounds/analysis , Free Radicals/analysis , Picrates/analysis , Antioxidants/analysis , Butylated Hydroxytoluene/analysis , Electrodes , Gallic Acid/analysis , Oxidation-Reduction
17.
Mater Sci Eng C Mater Biol Appl ; 58: 310-5, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26478315

ABSTRACT

In this work nanostructured film composites of the monoamine oxidase B (MAO-B) enzyme, free or encapsulated in liposomes, were fabricated by the layer-by-layer (LbL) self-assembly technique, employing polyethylene imine (PEI) as polycation. Initially, the MAO-B enzyme was incorporated into liposomes in order to preserve its enzymatic structure ensuring their activity and catalytic stability. The LbL film growth was monitored by surface plasmon resonance (SPR) by gold resonance angle shift analysis after each bilayer deposition. Subsequently, the films were applied as amperometric biosensors for dopamine detection using Prussian Blue (PB) as the electron mediator. The biosensor fabricated by MAO-B incorporated into liposomes composed of DPPG:POPG in the ratio (1:4) (w/w) showed the best performance with a sensitivity of 0.86 (µA cm(-2))/(mmol L(-1)) and a detection limit of 0.33 mmol L(-1).


Subject(s)
Biosensing Techniques , Dopamine/analysis , Monoamine Oxidase/metabolism , Nanostructures/chemistry , Circular Dichroism , Electrochemical Techniques , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Ferrocyanides/chemistry , Humans , Limit of Detection , Liposomes/chemistry , Monoamine Oxidase/chemistry , Polyethyleneimine/chemistry , Surface Plasmon Resonance
18.
Pflugers Arch ; 467(11): 2307-23, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25791627

ABSTRACT

Adrenal chromaffin cells (CCs) from spontaneously hypertensive rats (SHRs) secrete more catecholamine (CA) upon stimulation than CCs from normotensive Wistar Kyoto rats (WKY). Unitary CA exocytosis events, both spontaneous and stimulated, were amperometrically recorded from cultured WKY and SHR CCs. Both strains display spontaneous amperometric spikes but SHR CCs produce more spikes and of higher mean amplitude. After a brief stimulation with high K(+) or caffeine which produces voltage-gated Ca(2+) influx or intracellular Ca(2+) release, respectively, more spikes and of greater mean amplitude and unitary charge were recorded in SHR CCs. Consequently, peak cumulative charge was ~2-fold higher in SHR CCs. Ryanodine (10 µM), a specific blocker of the ryanodine receptors reduced depolarization-induced peak cumulative charge by ~10 % in WKY and ~77 % in SHR CCs, suggesting, a larger contribution of Ca(2+)-induced Ca(2+) release to CA exocytosis in SHR CCs. Accordingly, Ca(2+) imaging showed larger [Ca(2+)]i signals induced both by depolarization and caffeine in SHR CCs. Distribution amplitude histograms showed that small amperometric spikes (0-50 pA) are more frequent in WKY than in SHR CCs. Conversely, medium (50-190 pA) and large (190-290 pA) spikes are more numerous in SHR than in WKY CCs. This study reveals that the enhanced CA secretion in SHR CCs results from a combination of (1) larger depolarization-induced Ca(2+) transients, due to a greater Ca(2+)-induced intracellular Ca(2+) release, (2) more exocytosis events per time unit, and (3) a greater proportion of medium and large amperometric spikes probably due to a higher mean CA content per granule. Enhanced CA release by excessive amplification by Ca(2+) induced Ca(2+) release and larger granule catecholamine content contributes to the increased CA plasma levels and vasomotor tone in SHRs.


Subject(s)
Adrenal Glands/metabolism , Calcium Signaling/drug effects , Calcium/metabolism , Calcium/pharmacology , Catecholamines/metabolism , Chromaffin Cells/metabolism , Adrenal Glands/drug effects , Animals , Blood Pressure/drug effects , Caffeine/pharmacology , Cells, Cultured , Chromaffin Cells/drug effects , Exocytosis , Heart Rate/drug effects , Male , Phosphodiesterase Inhibitors/pharmacology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/drug effects
19.
Talanta ; 126: 20-6, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24881530

ABSTRACT

Different materials like glass, silicon and poly(methyl methacrylate) (PMMA) are being used to immobilise enzymes in microchannels. PMMA shows advantages such as its low price, biocompatibility and attractive mechanical and chemical properties. Despite this, the introduction of reactive functional groups on PMMA is still problematic, either because of the complex chemistry or extended reaction time involved. In this paper, a new methodology was developed to immobilise glucose oxidase (GOx) in PMMA microchannels, with the benefit of a rapid immobilisation process and a very simple route. The new procedure involves only two steps, based on the reaction of 5.0% (w/w) polyethyleneimine (PEI) with PMMA in a dimethyl sulphoxide medium, followed by the immobilisation of glucose oxidase using a solution containing 100U enzymes and 1.0% (v/v) glutaraldehyde. The reactors prepared in this way were evaluated by a flowing system with amperometric detection (+0.60V) based on the oxidation of the H2O2 produced by the reactor. The microreactor proposed here was able to work with high bioconversion and a frequency of 60 samples h(-1), with detection and quantification limits of 0.50 and 1.66µmol L(-1), respectively. Michaelis-Menten parameters (Vmax and KM) were calculated as 449±47.7nmol min(-1) and 7.79±0.98mmol. Statistical evaluations were done to validate the proposed methodology. The content of glucose in natural and commercial coconut water samples was evaluated using the developed method. Comparison with spectrophotometric measurements showed that both methodologies have a very good correlation (tcalculated, 0.05, 4=1.35

Subject(s)
Electrochemical Techniques/methods , Enzymes, Immobilized/chemistry , Glucose Oxidase/chemistry , Polymethyl Methacrylate/chemistry , Dimethyl Sulfoxide/chemistry , Electrochemical Techniques/instrumentation , Enzymes, Immobilized/metabolism , Glucose/chemistry , Glucose/metabolism , Glucose Oxidase/metabolism , Glutaral/chemistry , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Kinetics , Oxidation-Reduction , Polyethyleneimine/chemistry , Reproducibility of Results , Spectrophotometry/methods
20.
Talanta ; 116: 1026-32, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24148511

ABSTRACT

In this paper, we present two new electrochemical methods for fast and simultaneous determination of codeine (CO) and diclofenac (DCF). The first one is based on batch injection analysis with amperometric detection (BIA-MPA) and the second one is based on capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4)D). The proposed BIA-MPA method is highly-precise (RSD of 1.1% and 0.9% for DCF and CO, respectively; n=10), fast (300 injections h(-1)) and has low detection limits (1.1 and 1.0 µmol L(-1) for DCF and CO, respectively). The proposed CE-C(4)D method allows the determination of CO and DFC in less than 1 min with high precision (RSD of 0.3% and 0.7% for DCF and CO, respectively; n=10) and low detection limits (11 and 21 µmol L(-1) for DCF and CO, respectively). Both proposed methods were applied to the determination of CO and DCF in pharmaceutical samples with similar results to those achieved by high-performance liquid chromatography (HPLC) at a 95% confidence level.


Subject(s)
Codeine/analysis , Diclofenac/analysis , Electrochemical Techniques/instrumentation , Electrophoresis, Capillary/instrumentation , Tablets/chemistry , Chromatography, High Pressure Liquid , Electric Conductivity , Flow Injection Analysis , Limit of Detection , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL