Screen-printed Working Electrode Research Articles

An Immunosensor for Microcystins Based on Fe 3 O 4 @Au Magnetic Nanoparticle Modified Screen-Printed Electrode

The coreshell Fe3O4@Au magnetic nanoparticles were modified onto the screen-printed working electrode surface,and the antibody of microcystin-(leucine-arginine)(anti-MCLR) was immobilized on the modified electrode surface by the adsorption between Au nanoparticles and the antibody.Subsequently,bovine serum albumin(BSA) was used to block the non-specific adsorption sites of the immunosensor to obtain immunosensor for the detection of MCLR.The immunosensor was based on the direct competitive immunoassay format between the labeled agent of horseradish peroxidase-conjugated MCLR(MCLR-HRP) and the analyte.MCLR was determined by differential pulse voltammetry(DPV).Under the optimal experimental conditions,the peak current response decreased proportionally with the concentration of MCLR in the range of 0.79-12.90 μg/L with a detection limit of 0.38 μg/L.The developed biosensor was used to determine MCLR in real water samples,and the recoveries of standard additions were in the range of 95% to 107%.The proposed immunosensor possesses the properties of fast,sensitive and portable,etc.

Novel Electrochemical Biosensor for Simultaneous Detection of Adenine and Guanine Based on Cu2O Nanoparticles

In this paper Cu2O nanoparticles were prepared and used for the construction of novel electrochemical voltammetric biosensor for the simultaneous detection of adenine and guanine. Cu2O nanoparticles were synthesized via simple wet chemical route, where glucose was used as a reductant. The nanoparticles were characterized by SEM and XRD analysis, which showed the presence of spherical aggregations with diameter of 1000 nm. These nanoparticles were successfully used for fabrication of spray-coated and screen-printed working electrodes on alumina substrate for the electrochemical detection of purine bases. We observed that Cu(I) reacts with adenine to form insoluble complex that accumulates on the electrode surface and causes the decrease of current response. In the case of guanine, we did not observed any significant decrease of current response which is probably caused by adsorption of guanine on the electrode surface.

Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.

A Novel Amperometric Immunosensor for Detection of Alpha-Fetoprotein Based on Magnetic and Electroactive Nanoprobes

A novel three-dimensional, magnetic and electroactive nanoprobes were constructed for the first time. Using hemin (TPP) as electron mediator, mutli-walled carbon nanotubes with carboxyl groups (MCNTs) as supporter, a novel (MCNTs-TPP-Fe3O4)magnetic nanocomposites were first prepared. Then using alpha-fetoprotein (AFP) as model system, gold nanoparticles (Au NPs) as immobilizing matrix, the AFP/anti-AFP/Au NPs/ MCNTs-TPP-Fe3O4nanoprobes were prepared and then dropped on the surface of screen-printed working electrode (SPCE) to construct a new amperometric immunosensors for detecting biomakers. The microstructure of different nanoparticles were observed by transmission electron microscopy (TEM) andX-ray fluorescence spectrometery (XRFS). Under optimal experimental conditions, the logarithm of response signal was proportional to the logarithm of AFP concentration from 0.1 to 200 ng/mL, with a correlation coefficient of 0.998. The detection limit was 0.04 ng/mL at a signal-to-noise ratio of 3. The proposed method offered a platform for fast, sensitive and simultaneous determination for serum samples.

A Three-Dimensional, Magnetic and Electroactive Nanoprobe for Amperometric Determination of Tumor Biomarkers

A novel electrochemical immunosensor for tumor biomarker detection based on three-dimensional, magnetic and electroactive nanoprobes was developed in this study. To fabricate the nanoprobes, negatively charged Fe3O4 nanoparticles (Fe3O4 NPs) and gold nanoparticles (Au NPs) were first loaded on the surface of multiple wall carbon nanotubes (MCNTs) which were functioned with redox-active hemin and cationic polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA). Using alpha fetoprotein (AFP) as a model analyte, AFP antibody (anti-AFP) was absorbed on the surface of Au NPs, bovine serum albumin (BSA) was then used to block sites against non-specific binding, and finally formed anti-AFP/Au NPs/Fe3O4/hemin/MCNTs named anti-AFP nanoprobes. When the target antigen AFP was present, it interacted with anti-AFP and formed an antigen-antibody complex on the nanoprobe interface. This resulted in a decreased electrochemical signal of hemin for quantitative determination of AFP when immobilized onto the screen-printed working electrode (SPCE). The results showed that the nanoprobe-based electrochemical immunosensor was sensitive to AFP detection at a concentration of 0.1 to 200 ng·mL−1 with a detection limit of 0.04 ng·mL−1, it also demonstrated good selectivity against other interferential substances. The electroactive nanoprobes can be massively prepared, easily immobilized on the SPCE for target detection and rapidly renewed with a magnet. The proposed immunosensor is fast, simple, sensitive, stable, magnet-controlled, nontoxic, label-free and reproducible.

Laccase-Nafion Based Biosensor for the Determination of Polyphenolic Secondary Metabolites

A laccase-based biosensor was developed by specific enzyme adsorption on screen-printed working electrodes of DROPSENS cells, and stabilized with Nafion 0.1% membrane. The electrode was characterized with respect to response time, sensitivity, linear range, detection limit, pH dependence, interferences, and long-term stability. The tested substrates were catechol, rosmarinic acid, caffeic acid, chlorogenic acid, and gallic acid. The optimized biosensor proved the following characteristic performances: the apparent Michaelis Menten calculated considering rosmarinic acid substrate 8.3 × 10−6 mol L−1 (r = 0.995, n = 6); the dynamic range of biosensor response for rosmarinic acid 7 × 10−7 − 1.5 × 10−6 mol L−1; the detection limit for rosmarinic acid 1.19 × 10−7 mol L−1 (RSD = 1.08%, n = 3). It was noticed that the biosensor reaches systematically 90% to 94.3% from the response obtained by LC-DAD-ESI-MS for real samples.

Detection of Adenosine Deaminase Activity with a Thick-Film Screen-Printed Ir/C Sensor to Detect Liver Disease

An electrochemical biosensor, comprising a thick-film screen-printed Ir/C working and counter electrodes, was developed for the detection of the enzymatic activity of adenosine deaminase for potential patient management with liver disease. Present methods of detecting adenosine deaminase involve spectrometric methods that require costly, nonportable equipment that is ill-suited for a point-of-care testing. Studies dealing with the electrochemical detection of adenosine deaminase intermediates such as xanthine and hypoxanthine required the use of , vs a Ag/AgCl reference electrode, and a large sample volume, 1 mL or more. The proposed biosensor used a sample volume of and a potential of vs Ag/AgCl. This biosensor used the enzymes xanthine oxidase (EC 1.1.3.22) and purine nucleoside phosphorylase (EC 2.4.2.1) coimmobilized on the thick-film screen-printed working electrode to detect enzymatically generated . Constant potential measurements showed that this biosensor had good linear performance over a 0–36 units/L of adenosine deaminase concentration range in phosphate buffer, mouse plasma, and mouse whole blood. The effect of the variation in the quantity of enzyme on the current output and the interference from intermediate-formed compounds were also explored.

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC(1, 2) methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain(3) and screen printed electrodes as sensing platform. Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health(4). The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity(5). The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution. At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution. A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform. Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5. The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution. At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution. A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

Easy to use and rapid isolation and detection of a viral nucleic acid by using paramagnetic microparticles and carbon nanotubes-based screen-printed electrodes

A method that is easy to use, rapid, with a low cost of detecting viral nucleic acid in a biological sample represents the essential tool in targeted therapy. In this study, we report the use of paramagnetic microparticles covered by streptavidin and modified by an oligonucleotide probe with a specific viral sequence labeled by biotin to detect human immunodeficiency virus (HIV) and influenza virus subtype H5N1. The viral nucleic acids were primarily detected by adsorptive transfer stripping technique coupled with square wave voltammetry using carbon paste, hanging mercury drop or carbon nanotubes-based screen-printed working electrodes. Detection limits were estimated for both sequences down to picograms per 3 μl. To isolate the viral sequences, paramagnetic microparticles covered with biotin-labeled oligonucleotides were used. We calculated the yield of isolation for H5N1 and/or HIV sequences, which was defined as “isolated concentration of viral nucleic acid sequence”/“given viral nucleic acid sequence” × 100. We estimated the yield for both sequences as 59%. Moreover, we studied the influence of human serum, dsDNA and non-complementary sequence of nucleic acids on isolation of viral nucleic acids. We also used carbon nanotubes-based screen-printed electrodes coupled with micro-flow instrument to detect viral nucleic acids. We were able to isolate and detect nanogram amounts of nucleic acids.

Development and application of a poly(2,2′-dithiodianiline) (PDTDA)-coated screen-printed carbon electrode in inorganic mercury determination

In this work, monomer solutions of aniline (ANI) and 2,2′-dithiodianiline (DTDA), an aniline derivative containing –S–S– links, were prepared and used in the electrochemical copolymerisation of ANI and DTDA by cyclic voltammetry on a screen-printed electrode (SPE) in 1 M HCl. Electropolymerisation of aniline on the surface of the screen-printed working electrode was performed by sweeping the potential between −500 and + 1100 mV (vs. Ag/AgCl) at a sweep rate of 100 mV/s. Electrocopolymerisation was performed with a mixture of ANI and DTDA by sweeping the potential between −200 and + 1100 mV (vs. Ag/AgCl) at a sweep rate of 100 mV/s [J.L. Hobman, J.R. Wilson, N.L. Brown, in: D.R. Lovley (Ed.), Environmental Microbe Metal Interactions, ASM Press, Herndon, Va, 2000, p. 177]. The cyclic voltammogram (CV) for each of the electrochemically deposited polyaniline (PANI) and the mixture of ANI and DTDA for the co-polymer polymerisation on SPCE were recorded for electrochemical analysis of the peak potential data for the mono and copolymer. Anodic stripping voltammetry (ASV) was used to evaluate a solution composed of (1 × 10 −6 M HgCl 2, 0.1 M H 2SO 4, 0.5 M HCl), in the presence of the co-polymer sensor electrode. The Hg 2+ ions were determined as follows: (i) pre-concentration and reduction on the modified electrode surface and (ii) subsequent stripping from the electrode surface during the positive potential sweep. The experimental conditions optimised for Hg 2+ determination included the supporting electrolyte concentration and the accumulation time. The results of the study have shown the use of a conducting polymer modified SPCE as an alternative transducer for the voltammetric stripping and analysis of inorganic Hg 2+ ions.

Determination of total bile acid levels using a thick-film screen-printed Ir/C sensor for the detection of liver disease

An electrochemical sensor, based on thick-film screen-printed Ir/C working and counter electrodes, was developed for the detection of total bile acid concentration in a physiological fluid for potential patient management in patients with liver disease. Current electrochemical methods of detecting total bile acid levels involve the use of potentials greater than +0.45 V, versus an Ag/AgCl reference electrode, and require a selectively permeable membrane. The proposed detection method did not require any membrane and used a potential of +0.27 V versus Ag/AgCl. This biosensor used 3-alpha-hydroxysteroid dehydrogenase (3alpha-HSD) (EC 1.1.1.50) immobilized on the thick-film screen-printed working electrode to detect the enzymatically generated NADH. The production of the NADH resulted from the reaction of the enzyme with bile acids such as sodium cholate, taurocholic and taurochenodeoxycholic acid, which could then be used to quantify the total bile acid. Constant potential measurements showed that this biosensor had good linear performance over a 0-200 microM concentration range in the phosphate buffer and the bovine serum. The sensor performance was also examined at different temperatures and pH conditions. This sensor prototype could be used for single use, disposable detection of total bile acids, extending its applicability for simple and early detection of liver disease.

Quantitation of bacteria through adsorption of intracellular biomolecules on carbon paste and screen-printed carbon electrodes and voltammetry of redox-active probes

A new electrochemical method for the quantitation of bacteria that is rapid, inexpensive, and amenable to miniaturization is reported. Cyclic voltammetry was used to quantitate M. luteus, C. sporogenes, and E. coli JM105 in exponential and stationary phases, following exposure of screen-printed carbon working electrodes (SPCEs) to lysed culture samples. Ferricyanide was used as a probe. The detection limits (3s) were calculated and the dynamic ranges for E. coli (exponential and stationary phases), M. luteus (exponential and stationary phases), and C. sporogenes (exponential phase) lysed by lysozyme were 3 x 10(4) to 5 x 10(6) colony-forming units (CFU) mL(-1), 5 x 10(6) to 2 x 10(8) CFU mL(-1) and 3 x 10(3) to 3 x 10(5) CFU mL(-1), respectively. Good overlap was obtained between the calibration curves when the electrochemical signal was plotted against the dry bacterial weight, or between the protein concentration in the bacterial lysate. In contrast, unlysed bacteria did not change the electrochemical signal of ferricyanide. The results indicate that the reduction of the electrochemical signal in the presence of the lysate is mainly due to the fouling of the electrode by proteins. Similar results were obtained with carbon-paste electrodes although detection limits were better with SPCEs. The method described herein was applied to quantitation of bacteria in a cooling tower water sample.

Stripping chronopotentiometric measurements of lead(II) and cadmium(II) in soils extracts and wastewaters using a bismuth film screen-printed electrode assembly.

The key to remediative processes is the ability to measure toxic contaminants on-site using simple and cheap sensing devices, which are field-portable and can facilitate more rapid decision-making. A three-electrode configuration system has been fabricated using low-cost screen-printing (thick-film) technology and this coupled with a portable electrochemical instrument has provided a a relatively inexpensive on-site detector for trace levels of toxic metals. The carbon surface of the screen-printed working electrode is used as a substrate for in situ deposition of a metallic film of bismuth, which allows the electrochemical preconcentration of metal ions. Lead and cadmium were simultaneously detected using stripping chronopotentiometry at the bismuth film electrode. Detection limits of 8 and 10 ppb were obtained for cadmium(II) and lead(II), respectively, for a deposition time of 120 s. The developed method was applied to the determination of lead and cadmium in soils extracts and wastewaters obtained from polluted sites. For comparison purposes, a mercury film electrode and ICP-MS were also used for validation.

Analytical performance characteristics of thin and thick film amperometric microcells.

The analytical performance of amperometric microcells with different electrode geometries is compared for enzyme activity measurements. The microcells were fabricated with thin film photolithography or thick film screen-printing in four different designs. The cells made with the thin film process used flexible substrate with microelectrode array or a circular, disk-shaped working electrode. The screen-printed working electrodes had semicircle or disk shape on ceramic chips. Putrescine oxidase (PUO) activity measurement was used as a model. The determination of PUO activity is important in the clinical diagnosis of premature rupture of the amniotic membrane. An electropolymerized m-phenylenediamine size-exclusion layer was used to eliminate common interferences. The size exclusion layer revealed also to be advantageous in protecting the electrodes from fouling by putrescine (enzyme substrate). The electrode fouling of bare electrodes was insignificant for screen-printed electrodes, but very severe for electroplated platinum working electrodes. The microelectrode array electrodes demonstrated smaller RSD and higher normalized sensitivities for hydrogen peroxide and PUO activity. All the other electrodes were demonstrating comparable analytical performances.

Immunosensor for 2,4-dichlorophenoxyacetic acid in aqueous/organic solvent soil extracts.

The development of a simple electrochemical immunoassay procedure for the field-based quantification of the herbicide 2,4-D in methanolic soil extracts is presented. The sensor utilizes a competitive immunoassay format incorporating an immobilized antigen complex at the surface of a disposable screen-printed working electrode element. The extent of glucose oxidase-labeled antibody binding to the antigen--electrode is determined amperometrically and is related to sample analyte concentration. The performance of the sensor is assessed in buffer, 30% methanol, and methanolic soil extracts. The device is capable of quantifying 2,4-D in all three matrixes at the low ppm level with coefficient of variation values of 6.2-33.6%. The causes of the variation observed in the sensor response in different soil matrixes are examined and improvements proposed. The sensor, tested in parallel with a commercial 2,4-D immunoassay test kit, yields comparable quantitative data and detection limits while exhibiting greater assay simplicity.