Impedimetric immunosensor for electronegative low density lipoprotein (LDL(-)) based on monoclonal antibody adsorbed on (polyvinyl formal)-gold nanoparticles matrix

Monoclonal antibodies (MAb) have been commonly applied to measure LDL in vivo and to characterize modifications of the lipids and apoprotein of the LDL particles. The electronegative low density lipoprotein (LDL(-)) has an apolipoprotein B-100 modified at oxidized events in vivo. In this work, a novel LDL-electrochemical biosensor was developed by adsorption of anti-LDL(-) MAb on an (polyvinyl formal)-gold nanoparticles (PVF-AuNPs)-modified gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the recognition of LDL-. The interaction between MAb-LDL(-) leads to a blockage in the electron transfer of the [Fe(CN)(6)](4-)/K(4)[Fe(CN)(6)](3-) redox couple, which may could result in high change in the electron transfer resistance (R(CT)) and decrease in the amperometric responses in CV analysis. The compact antibody-antigen complex introduces the insulating layer on the assembled surface, which increases the diameter of the semicircle, resulting in a high R(CT), and the charge transferring rate constant k(0) decreases from 18.2 x 10(-6) m/s to 4.6 x 10(-6) m/s. Our results suggest that the interaction between MAb and lipoprotein can be quantitatively assessed by the modified electrode. The PVF-AuNPs-MAb system exhibited a sensitive response to LDL(-), which could be used as a biosensor to quantify plasmatic levels of LDL(-). (C) 2011 Elsevier B.V. All rights reserved.

Development of instrumentation for amperometric and coulometric detection using ultramicroelectrodes

In this work it is presented the development of a simple, portable and inexpensive instrumentation for amperometric and coulometric detection in different analytical instrumentation systems utilizing ultramicroelectrodes. The software, developed in LabVIEW 7.1TM, is capable to carry out three main detection techniques (amperometric, pulsed amperometric and coulometric detection) and a voltammetric technique (cyclic voltammetry). The instrumentation was successfully evaluated using the following systems: cyclic voltammograms of metallic electrodes in alkaline solutions, flow electrochemical detection of glucose and glycine and direct determination of herbicide glyphosate (electrochemical detection coupled to HPLC).

Determination of paracetamol at a graphite-polyurethane composite electrode as an amperometric flow detector

A bare graphite-polyurethane composite was evaluated as an amperometric flow injection detector in the determination of paracetamol (APAP) in pharmaceutical formulations. A linear analytical curve was observed in the 5.00 x 10-5 to 5.00 x 10-3 mol L-1 range with a minimum detectable net concentration of 18.9 µmol L-1 and 180 determinations h-1, after optimization of parameters such as the detection potential, sample loop volume, and carrier solution flow rate. Interference of ascorbic acid was observed, however, it was possible overcome the interference, reaching results that agreed with HPLC within 95% confidence level. These results showed that the graphite-polyurethane composite can be used as an amperometric detector for flow analysis in the determination of APAP.

Flow injection analysis of paracetamol using a biomimetic sensor as a sensitive and selective amperometric detector

This work describes the coupling of a biomimetic sensor to a flow injection system for the sensitive determination of paracetamol. The sensor was prepared as previously described in the literature (M. D. P. T. Sotomayor, A. Sigoli, M. R. V. Lanza, A. A. Tanaka and L. T. Kubota, J. Braz. Chem. Soc., 2008, 19, 734) by modifying a glassy carbon electrode surface with a Nafion (R) membrane doped with iron tetrapyridinoporphyrazine (FeTPyPz), a biomimetic catalyst of the P450 enzyme. The performance of the sensor for paracetamol detection was investigated and optimized in a flow injection system (FIA) using a wall jet electrochemical cell. Under optimized conditions a wide linear response range (1.0 x 10(-5) to 5.0 x 10(-2) mol L(-1)) was obtained, with a sensitivity of 2579 (+/- 129) mu A L mu mol(-1). The detection and quantification limits of the sensor for paracetamol in the FIA system were 1.0 and 3.5 mu mol L(-1), respectively. The analytical frequency was 51 samples h(-1), and over a period of five days (320 determinations) the biosensor maintained practically the same response. The system was successfully applied to paracetamol quantification in seven pharmaceutical formulations and in water samples from six rivers in Sao Paulo State, Brazil.

Dual contactless conductivity and amperometric detection on hybrid PDMS/glass electrophoresis microchips

A new approach for the integration of dual contactless conductivity and amperometric detection with an electrophoresis microchip system is presented. The PDMS layer with the embedded channels was reversibly sealed to a thin glass substrate (400 mu m), on top of which a palladium electrode had been previously fabricated enabling end-channel amperometric detection. The thin glass substrate served also as a physical wall between the separation channel and the sensing copper electrodes for contactless conductivity detection. The latter were not integrated in the microfluidic device, but fabricated on an independent plastic substrate allowing a simpler and more cost-effective fabrication of the chip. PDMS/glass chips with merely contactless conductivity detection were first characterized in terms of sensitivity, efficiency and reproducibility. The separation efficiency of this system was found to be similar or slightly superior to other systems reported in the literature. The simultaneous determination of ionic and electroactive species was illustrated by the separation of peroxynitrite degradation products, i.e. NO(3)(-) (non-electroactive) and NO(2)(-) (electroactive), using hybrid PDMS/glass chips with dual contactless conductivity and amperometric detection. While both ions were detected by contactless conductivity detection with good efficiency, NO(2)(-) was also simultaneously detected amperometrically with a significant enhancement in sensitivity compared to contactless conductivity detection.

Carbon nanotubes in nanostructured films: Potential application as amperometric and potentiometric field-effect (bio-)chemical sensors

The assembly of carbon nanotubes (CNTs) into nanostructured films is attractive for producing functionalized hybrid materials and (bio-)chemical sensors, but this requires experimental methods that allow for control of molecular architecturcs. In this study, we exploit the layer-by-layer (LbL) technique to obtain two types of sensors incorporating CNTs. In the first, LbL films of alternating layers of multi-walled carbon nanotubes (MWNTs) dispersed in polyarninoamide (PAMAM) dendrimers and nickel phthalocyanine (NiTsPc) were used in amperometric detection of the neurotransmitter dopamine (DA). The electrochemical properties evaluated with cyclic voltammetry indicated that the incorporation of MWNTs in the PAMAM-NT/NiTsPc LbL films led to a 3-fold increase in the peak current, in addition to a decrease of 50 mV in the oxidation potential of DA. The latter allowed detection of DA even in the presence of ascorbic acid (AA), a typical interferent for DA. Another LbL film was obtained with layers of PAMAM and single-walled carbon nanotubes (SWNTs) employed in field-effect-devices using a capacitive electrolyte-insulator-semiconductor structure (EIS). The adsorption of the film components was monitored by measuring the flat-band voltage shift in capacitance-voltage (C-P) curves, caused by the charges from the components. Constant capacitance (ConCap) measurements showed that the EISPAMAM/SWNT film displayed a high pH sensitivity (ca. 54.5 mV/pH), being capable of detecting penicillin G between 10(-4) mol L(-1) and 10(-2) mol L-1, when a layer of penicillinase was adsorbed atop the PAMAM/SWNT film. (C) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phytase immobilization on modified electrodes for amperometric biosensing

Phytase (myo-inositol hexaphosphate phosphohydrolase) and phytic acid (myo-inositol hexaphosphate) play an important environmental role, in addition to being a health issue in food industry. Phytic acid is antinutritional due to its ability to chelate metal ions and may also react with proteins decreasing their bioavailability. In this work, we produced biosensors with phytase immobilized in Layer-by-Layer (LbL) films, which could detect phytic acid with a detection limit of 0.19 mmol L-1, which is sufficient to detect phytic acid in seeds of grains and vegetables. The biosensosrs consisted of LbL films containing up to eight bilayers of phytase alternated with poly(allylamine) hydrochloride (PAH) deposited onto an indium-tin oxide (ITO) substrate modified with Prussian Blue. Amperometric detection was conducted in an acetate buffer solution (at pH 5.5) at room temperature, with the biosensor response attributed to the formation of phosphate ions. In subsidiary experiments with the currents measured at 0.0 V (vs. SCE), we demonstrated the absence of effects from some interferents, pointing to a good selectivity of the biosensor. (c) 2007 Elsevier B.V. All rights reserved.

Amperometric detection of benzoyl peroxide in pharmaceutical preparations using carbon paste electrodes with peroxidases naturally immobilized on coconut fibers

This paper describes the applications of anew carbon paste electrode containing fibers of coconut (Cocus nucifera L) fruit, which are very rich in peroxidase enzymes naturally immobilized on its structure. The new sensor was applied for the amperometric quantification of benzoyl peroxide in facial creams and dermatological shampoos. The amperometric measurements were performed in 0.1 mol L(-1) phosphate buffer (pH 5.2), at 0.0 V (versus Ag/AgCl). On these conditions, benzoyl peroxide was rapidly determined in the 5.0-55 mu mol L(-1), with a detection limit of 2.5 mu mol L(-1) (s/n = 3), response time of 4.1 s (90% of the steady state) and sensitivity limit of 0.33 A mol L(-1) cm(-2). The amperometric results are in good agreement with those obtained by spectrophotometric technique, used as a standard method. (C) 2009 Elsevier B.V. All rights reserved.

A renewable copper electrode as an amperometric flow detector for nitrate determination in mineral water and soft drink samples

A novel approach was developed for nitrate analysis in a FIA configuration with amperometric detection (E=-0.48 V). Sensitive and reproducible current measurements were achieved by using a copper electrode activated with a controlled potential protocol. The response of the FIA amperometric method was linear over the range from 0.1 to 2.5 mmol L(-1) nitrate with a detection limit of 4.2 mu mol L(-1) (S/N = 3). The repeatability of measurements was determined as 4.7% (n=9) at the best conditions (flow rate: 3.0 mL min(-1), sample volume: 150 mu L and nitrate concentration: 0.5 mmol L(-1)) with a sampling rate of 60 samples h(-1). The method was employed for the determination of nitrate in mineral water and soft drink samples and the results were in agreement with those obtained by using a recommended procedure. Studies towards a selective monitoring of nitrite were also performed in samples containing nitrate by carrying out measurements at a less negative potential (-0.20 V). (C) 2009 Elsevier B.V. All rights reserved.

Nickel hydroxide electrodes as amperometric detectors for carbohydrates in flow injection analysis and liquid chromatography

The present paper describes the utilization of nickel hydroxide modified electrodes toward the catalytic oxidation of carbohydrates (glucose, fructose, lactose and sucrose) and their utilization as electrochemical sensor. The modified electrodes were employed as a detector in flow injection analysis for individual carbohydrate detection, and to an ionic column chromatography system for multi-analyte samples aiming a prior separation step. Kinetic studies were performed on a rotating disk electrode (RDE) in order to determine both the heterogeneous rate constant and number of electrons transferred for each carbohydrate. Many advantages were found for the proposed system including fast and easy handling of the electrode modification, low cost procedure, a wide range of linearity (0.5-50 ppm), low detection limits (ppb level) and high sensitivities. (C) 2009 Elsevier B.V. All rights reserved.

Flow injection amperometric determination of hydrogen peroxide in household commercial products with a ruthenium oxide hexacyanoferrate modified electrode

Hydrogen peroxide was determined in oral antiseptic and bleach samples using a flow-injection system with amperometric detection. A glassy carbon electrode modified by electrochemical deposition of ruthenium oxide hexacyanoferrate was used as working electrode and a homemade Ag/AgCl (saturated KCl) electrode and a platinum wire were used as reference and counter electrodes, respectively. The electrocatalytic reduction process allowed the determination of hydrogen peroxide at 0.0 V. A linear relationship between the cathodic peak current and concentration of hydrogen peroxide was obtained in the range 10-5000 mu mol L(-1) with detection and quantification limits of 1.7 (S/N = 3) and 5.9 (S/N = 10) mu mol L(-1), respectively. The repeatability of the method was evaluated using a 500 mu mol L(-1) hydrogen peroxide solution, the value obtained being 1.6% (n = 14). A sampling rate of 112 samples h(-1) was achieved at optimised conditions. The method was employed for the quantification of hydrogen peroxide in two commercial samples and the results were in agreement with those obtained by using a recommended procedure.

Urea amperometric biosensors based on a multifunctional bipolymeric layer: Comparing enzyme immobilization methods

This paper outlines the results obtained with biosensors designed for urea amperometric detection. The incorporation of urease into a bipolymeric substrate consisting of poly(pyrrole) and poly(5-amino-1-naphthol) was performed through four different approaches: direct adsorption, entrapment in cellulose acetate layer. cross-linking with glutaraldehyde, and also covalent attachment to the polymeric matrix. Poly(pyrrole) acts as amperometric transducer in these biosensors, while poly(5-amino-1-naphthol) drastically reduces the interference signal of agents such as ascorbic and uric acids. The biosensors containing urease covalently attached to the substrate provided interesting results in terms of sensitivity towards urea (0.50 mu A cm(-2) mmol(-1) L), lifetime (20 days) and short response times, due to the enzyme immobilization method used. All biosensors analyzed showed also a wide linear concentration range (up to 100 mmol L(-1)) and low detection limits (0.22-0.58 mmol L(-1)). (C) 2009 Elsevier B.V. All rights reserved.

Fast and reliable analyses of sulphite in fruit juices using a supramolecular amperometric detector encompassing in flow gas diffusion unit

A new compact system encompassing in flow gas diffusion unit and a wall-jet amperometric FIA detector, coated with a supramolecular porphyrin film, was specially designed as an alternative to the time-consuming Monier-Williams method, allowing fast, reproducible and accurate analyses of free sulphite species in fruit juices. In fact, a linear response between 0.64 and 6.4 ppm of sodium sulphite. LOD = 0.043 ppm, relative standard deviation of +/- 1.5% (n = 10) and analytical frequency of 85 analyses/h were obtained utilising optimised conditions. That superior analytical performance allows the precise evaluation of the amount of free sulphite present in foods, providing an important comparison between the standard addition and the standard injection methods. Although the first one is most frequently used, it was strongly influenced by matrix effects because of the unexpected reactivity of sulphite ions with the juice matrixes, leading to its partial consumption soon after addition. In contrast, the last method was not susceptible to matrix effects yielding accurate results, being more reliable for analytical purposes. (C) 2011 Elsevier Ltd. All rights reserved.

Flow injection analysis using carbon film resistor electrodes for amperometric determination of ambroxol

Flow injection analysis (FIA) using a carbon film sensor for amperometric detection was explored for ambroxol analysis in pharmaceutical formulations. The specially designed flow cell designed in the lab generated sharp and reproducible current peaks, with a wide linear dynamic range from 5 x 10(-7) to 3.5 x 10(-4) mol L-1, in 0.1 mol L-1 sulfuric acid electrolyte, as well as high sensitivity, 0.110 A mol(-1) L cm(-2) at the optimized flow rate. A detection limit of 7.6 x 10(-8) mol L-1 and a sampling frequency of 50 determinations per hour were achieved, employing injected volumes of 100 mu L and a flow rate of 2.0 mL min(-1). The repeatability, expressed as R.S.D. for successive and alternated injections of 6.0 x 10(-6) and 6.0 x 10(-5) mol L-1 ambroxol solutions, was 3.0 and 1.5%, respectively, without any noticeable memory effect between injections. The proposed method was applied to the analysis of ambroxol in pharmaceutical samples and the results obtained were compared with UV spectrophotometric and acid-base titrimetric methods. Good agreement between the results utilizing the three methods and the labeled values was achieved, corroborating the good performance of the proposed electrochemical methodology for ambroxol analysis. (C) 2008 Elsevier B.V. All rights reserved.

Determination of carbaryl in tomato ""in natura"" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity

This work reports the utilization of two methodologies for carbaryl determination in tomatoes. The measurements were carried out using an amperometric biosensor technique based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption and a HPLC procedure. The electrochemical experiments were performed in 0.1 mol L-1 phosphate buffer solutions at pH 7.4 with an incubation time of 8 min. The analytical curve obtained in pure solutions showed excellent linearity in the 5.0 x 10(-5) to 75 x 10(-5) mol L-1 range, with the limit of detection at 0.4 x 10(-3) gL(-1). The application of such a methodology in tomato samples involved solely liquidising the samples, which were spiked with 6.0 x 10(-6) and 5.0 x 10(-5) mol L-1 carbaryl. Recovery in such samples presented values of 99.0 and 92.4%, respectively. In order to obtain a comparison, HPLC experiments were also conducted under similar conditions. However, the tomato samples have to be manipulated by an extraction procedure (MSPD), which yielded much lower recovery values (78.3 and 84.8%, respectively). On the other hand, the detection limit obtained was much lower than that for the biosensor, i.e., 3.2 x 10(-6) g L-1. Finally, the biosensor methodology was employed to analyze carbaryl directly inside the tomato, without any previous manipulation. In this case, the biosensor was immersed in the tomato pulp, which had previously been spiked with the pesticide for 8 min, removed and inserted in the electrochemical cell. A recovery of 83.4% was obtained, showing very low interference of the matrix constituents. (C) 2007 Elsevier B.V. All rights reserved.