| 1. |
- Badea, M, et al.
(författare)
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A flow immunoassay for alkylphenol ethoxylate surfactants and their metabolites - questions associated with cross-reactivity, matrix effects, and validation by chromatographic techniques
- 2003
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Ingår i: Analyst. - : Royal Society of Chemistry (RSC). - 1364-5528. ; 128:7, s. 849-856
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes the application and evaluation of a competitive enzyme flow injection immunoassay (EFIIA) for screening of alkylphenol ethoxylate (APEO) surfactants in different water samples based on a generic immunoassay system previously developed ( see E. Burestedt, C Nistor, U. Schagerlof and J. Emneus, Anal. Chem., 2000, 72, 4171 - 4177). The detection limits for octylphenol ethoxylates (OPEOs), nonylphenol ethoxylates (NPEOs), and nonylphenol (NP) were 0.5 mug l(-1), between 2 and 3 mug l(-1), and 50 mug l(-1), respectively, with a sample throughput of 6 h(-1) (i.e., for triplicate analysis of each sample). Different OPEOs and NPEOs were highly cross-reactive within the assay, with sensitivities in the same order of magnitude for all the ethoxylates tested, thus the result obtained by the EFIIA method could be used as an "alkylphenol ethoxylate index". No or minor matrix effects with recoveries between 70 - 120% for the reference analyte NPEO10 in tap, and surface water, and acceptable for rainwater, were observed. Influent and effluent surfactant containing wastewater samples were analysed by EFIIA, LC-MS, LC-Fluoresence (LC-FL), and a commercial microplate ELISA. High recoveries for different concentrations of APEO(10) spiked into a 200 times diluted raw influent and effluent wastewater were achieved with the EFIIA method, however, the found APEO content of the same diluted wastewater samples, before spiking, could not be correlated directly to the chromatographic result by any of the immunoassays, and the possible reasons for this are discussed. The same trend of decreasing APEO content from influent to effluent wastewater could, however, be followed for all methods employed.
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| 2. |
- Bjarnason, Bjarni, et al.
(författare)
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Enzyme flow immunoassay using a Protein G column for the screening of triazine herbicides in surface and waste water
- 2001
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Ingår i: Analytica Chimica Acta. - 0003-2670. ; 426:2, s. 197-207
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Tidskriftsartikel (refereegranskat)abstract
- A method for screening of triazine herbicides in surface and waste water is presented. The method is based on an enzyme flow immunoassay (EFIA) for the detection of the free fraction of a horse radish peroxidase (HRP)-labelled antigen (tracer). This was accomplished by trapping the bound tracer fraction in a Protein G column, allowing the residual free tracer fraction to pass and be detected spectrophotometrically after incubation with an enzyme substrate. As compared with detecting the bound tracer fraction this reduces the regeneration requirements of the Protein G column used for capturing the bound fraction and, therefore, reduces assay time. A polyclonal antibody directed against simazine showed no reactivity towards tracers that were thiopropionic acid derivatives of atrazine, simazine and terbutylazine. It had good sensitivity towards tracers using derivatives of 2-chloro-4,6-(alkylamino)-s-triazines such as atrazine and simazine. The highest sensitivity was obtained with an Et/Cl/N-C5-HRP tracer because this tracer could be used in combination with the lowest concentration of antibody. The detection limit was 0.1 μgl-1 with a linear range between 0.1 and 10 μgl-1 and an assay throughput of 12 h-1. Natural water samples from various locations in Russia were analysed for triazines and the results were compared with a previously developed fluorescein flow immunoassay for triazines. The results were further verified by supported liquid membrane (SLM) extraction combined with HPLC. The results show that the two immunoassays behave differently and that the sample matrix influences their performance, however, no false negative results were obtained. The possible reasons for the different results between the two immunoassays are discussed. (C) 2001 Elsevier Science B.V.
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| 3. |
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| 4. |
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| 5. |
- Davidsson, Richard, et al.
(författare)
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Microfluidic biosensing systems - Part I. Development and optimisation of enzymatic chemiluminescent mu-biosensors based on silicon microchips
- 2004
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189. ; 4:5, s. 481-487
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Tidskriftsartikel (refereegranskat)abstract
- Chemiluminescent (CL) enzyme-based flow-through microchip biosensors (mu-biosensors) for detection of glucose and ethanol were developed for the purpose of monitoring real-time production and release of glucose and ethanol from microchip immobilised yeast cells. Part I of this study focuses on the development and optimisation of the mu-biosensors in a microfluidic sequential injection analysis (muSIA) system. Glucose oxidase (GOX) or alcohol oxidase (AOX) was co-immobilised with horseradish peroxidase (HRP) on porous silicon flow through microchips. The hydrogen peroxide ;produced from oxidation of the corresponding analyte (glucose or ethanol) took part in the chemiluminescent (CL) oxidation of luminol catalysed by HRP enhanced by addition of p-iodophenol ( PIP). All steps in the mSIA system, including control of syringe pump, multiposition valve (MPV) and data readout, were computer controlled. The influence of flow rate and luminol- and PIP concentration were investigated using a 2(3)-factor experiment using the GOX-HRP sensor. It was found that all estimated single factors and the highest order of interaction were significant. The optimum was found at 250 muM luminol and 150 muM PIP at a flow rate of 18 mul min(-1), the latter as a compromise between signal intensity and analysis time. Using the optimised system settings one sample was processed within 5 min. Two different immobilisation chemistries were investigated for both m-biosensors based on 3-aminopropyltriethoxsilane (APTS)- or polyethylenimine (PEI) functionalisation followed by glutaraldehyde (GA) activation. GOX-HRP mu-biosensors responded linear in a log-log format within the range 10-1000 mM glucose. Both had an operational stability of at least 8 days, but the PEI-GOX-HRP sensor was more sensitive. The AOX-HRP mu-biosensors responded linear (log-log) in the range between 1 and 10 mM ethanol, but the PEI-AOX-HRP sensor was in general more sensitive. Both sensors had an operational stability of at least 8 h, but with a half-life of 2-3 days.
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| 6. |
- Davidsson, Richard, et al.
(författare)
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Microfluidic biosensing systems - Part II. Monitoring the dynamic production of glucose and ethanol from microchip-immobilised yeast cells using enzymatic chemiluminescent mu-biosensors
- 2004
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189. ; 4:5, s. 488-494
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Tidskriftsartikel (refereegranskat)abstract
- A microfluidic flow injection (muFIA) system was employed for handling and monitoring of cell-released products from living cells immobilised on silicon microchips. The dynamic release of glucose and ethanol produced from sucrose by immobilised Saccharomyces cerevisiae cells was determined using microchip biosensors (mu-biosensors) with either co-immobilised glucose oxidase-horseradish peroxidase (GOX-HRP), or alcohol oxidase-horseradish peroxidase (AOX-HRP), catalysing a series of reactions ending up with chemiluminescence (CL) generated from HRP-catalysed oxidation of luminol in presence of p-iodophenol (PIP). The yeast cells were attached by first treating them with polyethylenimine (PEI) followed by adsorption to the microchip surface. The cell loss during assaying was evaluated qualitatively using scanning electron microscopy (SEM), showing that no cells were lost after 35 min liquid handling of the cell chip at 10 mul min(-1). The enzymes were immobilised on microchips via PEI-treatment followed by glutaraldehyde (GA) activation. The GOX-HRP mu-biosensors could be used during five days without any noticeable decrease in response, while the AOX-HRP mu-biosensors showed continuously decreasing activity, but could still be used employing calibration correction. The glucose and ethanol released from the immobilised yeast chips were quantitatively monitored, by varying the incubation time with sucrose, showing the possibilities and advantages of using a microfluidic system set-up for cell-based assays.
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| 7. |
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| 8. |
- Heiskanen, Arto, et al.
(författare)
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Amperometric monitoring of redox activity in living yeast cells: comparison of menadione and menadione sodium bisulfite as electron transfer mediators
- 2004
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Ingår i: Electrochemistry Communications. - : Elsevier BV. - 1388-2481. ; 6:2, s. 219-224
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Tidskriftsartikel (refereegranskat)abstract
- An amperometric method was applied for real-time monitoring of intracellular redox enzyme activity. Baker’s yeast (Saccharomyces cerevisiae) cells were immobilized on platinum microband electrodes and mediated anodic currents were measured. The currents were observed in the absence and in the presence of glucose as a source of reducing equivalents, NADH and NADPH. 2-Methyl-1,4-naphthoquinone (menadione, vitamin K3) and water soluble 2-methyl-1,4-naphthoquinone sodium bisulfite (menadione sodium bisulfite MSB) were compared as artificial electron acceptors for their ability to transduce internal cellular redox activity into electrode current. It was found that hydrophobic menadione was superior to its water-soluble bisulfite derivative for probing whole intact cells.
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| 9. |
- Jain, Seema Rani, et al.
(författare)
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A chemiluminescence flow immunosensor based on a porous monolithic metacrylate and polyethylene composite disc modified with Protein G
- 2004
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Ingår i: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 19:8, s. 795-803
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Tidskriftsartikel (refereegranskat)abstract
- A generic, fast, sensitive and new type of flow immunosensor has been developed. The basis is a monolithic porous poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) polymer disc modified with protein G, placed in a fountain type flow cell compartment, in close proximity to a photomultiplier tube (PMT). Analyte and HRP labelled analyte derivative (tracer) compete for anti-analyte antibody binding sites. The mixture is then injected into the flow immunosensor system where the formed analyte- and tracer-antibody complexes are trapped by the monolithic protein G disc. The amount of bound tracer, inversely related to the concentration of analyte in the sample, is determined in a second step by injection of luminol, p-iodophenol and H2O2, generating enhanced chemiluminescence (CL) with horseradish peroxidase (HRP). A third and final step is need for regeneration of the protein G disc so that a new analysis cycle can take place. The performance of the disc immunosensor system was compared with a one step continuous flow injection immunoassay (FIIA) system, using the same reagents and a protein G column, in terms of assay sensitivity and influence of matrix effects from various water samples (millipore-, tap- and surface water). The detection limit for the analyte atrazine in PBS and surface water (SW) was 0.208±0.004 g l−1 (PBS) and 0.59±0.120 g l−1 (SW) for the FIIA and 0.033±0.003 g l−1 (PBS) and 0.038±0.003 g l−1 (SW) for the disc immunosensor. Statistical comparison of the two systems shows that the disc immunosensor results were significantly less influenced by the sample matrix, which is explained by the fact that the sample in the FIIA arrives simultaneously with the matrix to the detector, whereas these are separated in time in the disc immunosensor system.
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| 10. |
- Khampha, Wanida, et al.
(författare)
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Specific detection of L-glutamate in food using flow-injection analysis and enzymatic recycling of substrate
- 2004
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Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 1873-4324 .- 0003-2670. ; 518:1-2, s. 127-135
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Tidskriftsartikel (refereegranskat)abstract
- A flow injection analysis (FIA) system for specific determination of L-glutamate in food samples based on a bi-enzymatic amplification system has been developed. The content of L-glutamate in the sample was amplified by cycling between L-glutamate dehydrogenase (GIDH) and a novel enzyme, D-phenylglycine aminotransferase (D-PhgAT). In this system, GIDH converts L-glutamate to 2-oxoglutarate with concomitant reduction of NAD(+) to NADH. D-PhgAT transfers an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate, thus recycling L-glutamate. Accumulation of NADH in the course of the enzymatic recycling was monitored both by fluorescence and UV absorbance and used for quantification of L-glutamate. The assay was characterized by high long-term stability (at least 70 days) and good reproducibility (within-day and between-day RSDs were 4.3-7.3% and 8.9%). The fluorimetric assay was slightly more sensitive with a L-glutamate detection limit of 0.4 muM and linear range of 2.5-50 muM. The assay was specific for L-glutamate, with recoveries between 95-103% in the presence of 17 different amino acids tested one by one. The method was applied to analysis of real food samples and results were correlated with a commercial Boehringer Mannheim assay kit. (C) 2004 Elsevier B.V. All rights reserved.
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| 11. |
- Nistor, Catalin, et al.
(författare)
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A capillary-based amperometric flow immunoassay for 2,4,6-trichlorophenol
- 2003
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642. ; 375:1, s. 125-132
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes the development of two different capillary-based heterogeneous competitive flow immunoassay formats (capillary flow injection immunoassay (CFIIA) and capillary sequential injection immunoassay (CSIIA)) for the determination of 2,4,6-trichlorophenol (2,4,6-TCP). The assays are based on the competition between the analyte and an analyte derivative labelled with the enzyme #-galactosidase, for an anti-TCP antibody, followed by the injection of the mixture at equilibrium into a flow stream, where separation between the fractions bound and unbound to the antibody is performed in a glass capillary containing immobilised protein A. The antibody-tracer fraction retained inside the protein A capillary was measured by injection of 4-aminophenyl-#-D-galactoside (4-APG), followed by amperometric detection of the enzymatically generated 4-aminophenol (4-AP), leading to a negative correlation between the signal and the analyte concentration.
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| 12. |
- Nistor, Catalin, et al.
(författare)
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A glucose dehydrogenase biosensor as an additional signal amplification step in an enzyme-flow immunoassay.
- 2002
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Ingår i: Analyst. - : Royal Society of Chemistry (RSC). - 1364-5528. ; 127:8, s. 1076-1081
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Tidskriftsartikel (refereegranskat)abstract
- Both the antibody affinity and the detectability of the label are essential in deciding the final characteristics of a heterogeneous immunoassay. This paper describes an approach to obtain a supplementary enhancement of the signal generated by using an enzyme label, e.g., by including the product of the enzymatic reaction in an additional amplification cycle during the detection step performed with an amperometric biosensor based on glucose dehydrogenase (GDH). An immunoassay format with a labelled analyte derivative that competes with the analyte present in the sample for a limited amount of antibody binding sites was employed. The beta-galactosidase label hydrolyses the substrate aminophenyl-beta-galactopyranoside, and the generated aminophenol enters then into a bioelectrocatalytic amplification cycle at the GDH biosensor. The principle was applied for determination of 4-nitrophenol, with the best minimal concentration of 1.5 microM and a midpoint of the calibration of 24 microM. The potentials and limitations of such a system are discussed.
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| 13. |
- Nistor, Catalin, et al.
(författare)
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Detection of Escherichia coli in water by culture-based amperometric and luminometric methods.
- 2002
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Ingår i: Water Science and Technology. - 0273-1223. ; 45:4-5, s. 191-199
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Tidskriftsartikel (refereegranskat)abstract
- The application of amperometric biosensor- and chemiluminiscence based methods for rapid detection of viable E. coli in water has been investigated. An amplification of the amperometric signal by a factor of 4 was obtained when the cellobiose dehydrogenase (CDH) biosensor was used instead of a plain graphite electrode for detection of b-galactosidase (b-GAL) activity at 22.5 degrees C. A linear correlation was demonstrated for detection time (DT) vs. initial concentrations (logarithmic units) of E. coli IT1 and E. coli in environmental samples, respectively, by use of the CDH biosensor or a chemiluminometric technique. The study has shown that an E. coli concentration > or = 10(4) cfu/100 mL in environmental samples was determined by the CDH biosensor within one working day. However, further reduction of the DT can be obtained, e.g. by increasing the signal amplification factor using other biosensors.
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| 14. |
- Nistor, Catalin, et al.
(författare)
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In-field monitoring of cleaning efficiency in waste water treatment plants using two phenol-sensitive biosensors
- 2002
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Ingår i: Analytica Chimica Acta. - 1873-4324. ; 456:1, s. 3-17
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Tidskriftsartikel (refereegranskat)abstract
- Two amperometric biosensors based on the enzymes cellobiose dehydrogenase (CDH) and quinoprotein-dependent glucose dehydrogenase (GDH), have been applied for monitoring the phenolic content in water samples, collected at different stages of a waste water treatment process, thus representing different cleaning levels of two waste water treatment plants (WWTPs). The biosensor measurements were performed in-field, compared with the results obtained by liquid chromatography-mass spectrometry and were further correlated with the cleaning efficiencies of the WWTPs. The effect of several potentially interfering compounds on the sensor response was also studied. The general purpose of the study was to evaluate the potential use of biosensors, not as quantitative tools for phenol analysis, but rather as screening tools indicating a certain trend, i.e. compounds present or not present, and potential correlation with sample toxicity. It was found that the biosensors and LC-MS results were not quantitatively comparable, however, both sensors could follow the decrease of the phenol content from the influent, primary treated and effluent waters. In addition, the correlation between biosensor inhibition and sample toxicity is discussed.
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| 15. |
- Nistor, Catalin, et al.
(författare)
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Multivariate analysis to separate the signal given by cross-reactants in immunoassay with sample matrix dilution
- 2004
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 380:7-8, s. 898-907
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes a new approach to achieve selectivity in an immunoassay by separating the signals given by two cross-reactive compounds present simultaneously in a complex sample matrix. The method is based on the sequential dilution of the sample containing a mixture of the two analytes, spiking each diluted sample with a reference compound, and the detection by enzyme-linked immunosorbent assay (ELISA). The obtained multivariate response was used for the individual calibrations of the assay for each. of the two cross-reactants simultaneously by using principal component analysis (PCA) and partial least squares regression (PLSR) data modeling. The calibration models showed. that the signal separation due the analytes 2,4-dinitro-phenol (2,4-DNP) and 4-nitrophenol (4-NP) was possible with a prediction concentration error of 1.4 muM and 72 muM, respectively.
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| 16. |
- Rose, A, et al.
(författare)
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GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates
- 2002
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Ingår i: Biosensors & Bioelectronics. - 1873-4235. ; 17:11-12, s. 1033-1043
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Tidskriftsartikel (refereegranskat)abstract
- The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the mug/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.
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| 17. |
- Sapelnikova, Svetlana, et al.
(författare)
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Amperometric sensors based on tyro sinase-modified screenprinted arrays
- 2003
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Ingår i: Talanta. - 1873-3573. ; 61:4, s. 473-483
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes the design, development and characteristics of a tyrosinase (polyphenol oxidase) modified amperometric screen-printed biosensor array, with the enzyme cross-linked in a redox-hydrogel namely the PVI13-dmeOs polymer. Two types of Au-screen-printed four-channel electrode arrays, differing in design and insulating layer, were compared and investigated. Au-, graphite-coated-Au- and Carbopack C-coated-Au-surfaces, serving as the basis for tyrosinase immobilisation, were investigated and the performances of the different arrays were evaluated and compared in terms of their electrocatalytic characteristics, as well as operational- and storage stability using catechol as model substrate. It was found that the Carbopack C-coated array was the best choice for tyrosinase immobilisation procedure mainly due to a higher mechanical stability of the deposited enzyme layer, combined with good sensitivity and stability for up to 6 months of use. In the batch mode the biosensors responded linearly to catechol up to 30 muM with limits of detection from 0.14 muM. Parameters from cyclic voltammograms indicated that the reversibility of the direct electrochemical reaction for catechol on the three types of electrode surfaces (no tyrosinase modification) was not the limiting factor for the construction and performance of tyrosinase biosensors. (C) 2003 Elsevier B.V. All rights reserved.
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| 18. |
- Sapelnikova, Svetlana, et al.
(författare)
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Screen-printed multienzyme arrays for use in amperometric batch and flow systems
- 2003
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 376:7, s. 1098-1103
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Tidskriftsartikel (refereegranskat)abstract
- Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.
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| 19. |
- Taranova, L A, et al.
(författare)
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Comamonas testosteroni strain TI as a potential base for a microbial sensor detecting surfactants
- 2004
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Ingår i: Applied Biochemistry and Microbiology. - 0003-6838. ; 40:4, s. 404-408
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Tidskriftsartikel (refereegranskat)abstract
- Strain Comamonas testosteroni TI, capable of degrading the nonionic surfactant (NIS) nonylphenolethoxylate (OP-10), was used for constructing a pilot cellular biosensor. The lower NIS detection limit for the biosensor was 0.25 mg/l. We studied the substrate specificity of the biosensor with respect to a wide range of organic compounds: surfactants, polyaromatic compounds (PAC), carbohydrates, alcohols, etc. It was shown that the biosensor based on Comamonas testosteroni TI did not respond to glucose, which was an advantage over the formerly described biosensor based on Pseudomonas rathonis T. The amplitude of the sensor response remained stable for 10 days.
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| 20. |
- Tudorache, Madalina, et al.
(författare)
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Immuno-SLM—a combined sample handling and analytical technique
- 2004
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 1872-7905 .- 0022-1759. ; 284:1-2, s. 107-118
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Tidskriftsartikel (refereegranskat)abstract
- Immuno-supported liquid membrane (immuno-SLM) extraction is a new technique that makes use of antibody (Ab)–antigen interactions as the "extraction force" to drive the mass transfer in a selective way. In immuno-SLM, anti-analyte (Ag) Abs are introduced into the acceptor phase of the SLM unit to trap the Ag that passes from the flowing donor through the SLM into the stagnant acceptor. The amount of immuno-extracted analyte (AbAg) is quantified by connecting the immuno-SLM unit on-line with a non-competitive heterogeneous fluorescence flow immunoassay (FFIA) that makes use of a fluorescein-labeled analyte tracer that titrates the residual excess of Ab present in the acceptor. A restricted access (RA) column is used for the separation of the two tracer fractions (Ag* and AbAg*) formed, and the eluted AbAg* fraction is measured downstream by a fluorescence detector. Factors influencing the optimum immuno-SLM extraction parameters, i.e., donor flow rate, extraction time and type of Ab, were investigated for immuno extraction of the model analyte atrazine. Immuno-SLM coupled to FFIA (immuno-SLM–FFIA) and FFIA alone were compared in terms of the assay sensitivities obtained and the sample matrix influence. The concentration at the mid-point of the calibration curve (IC50) was 16.0±1.4 and 36±16 g/l, the limit of detection (LOD) was 2.0±1.1 and 20±10 g/l, and the dynamic range was 2–100 and 20–500 g/l atrazine for immuno-SLM–FFIA and FFIA, respectively. The matrix influence on the FFIA was significant in orange juice and surface water, whereas the influence was minor for immuno-SLM–FFIA with recoveries between 104% and 115% for 5 g/l atrazine in tap water, orange juice and river water.
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| 21. |
- Yakovleva, J, et al.
(författare)
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Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection
- 2002
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 74:13, s. 2994-3004
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Tidskriftsartikel (refereegranskat)abstract
- Silicon microchips with immobilized antibodies were used to develop microfluidic enzyme immunoassays using chemiluminescence detection and horseradish peroxidase (HRP) as the enzyme label. Polyclonal anti-atrazine antibodies were coupled to the silicon microchip surface with an overall dimension of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235-mum depth and 25-mum width. Different immobilization protocols based on covalent or noncovalent modification of the silica surface with 3-aminopropyltriethoxysilane (APTES) or 3-glycidoxypropyltrimethoxysilane (GOPS), linear polyethylenimine (LPEI, MW 750 000), or branched polyethylenimine (BPEI, MW 25 000), followed by adsorption or covalent attachment of the antibody, were evaluated to reach the best reusability, stability, and sensitivity of the microfluidic enzyme immunoassay (muFEIA). Adsorption of antibodies on a LPEI-modified silica surface and covalent attachment to physically adsorbed BPEI lead to unstable antibody coatings. Covalent coupling of antibodies via glutaraldehyde (GA) to three different functionalized silica surfaces (APTES-GA, LPEI-GA, and GOPS-BPEI-GA) resulted in antibody coatings that could be completely regenerated using 0.4 M glycine/HCl, pH 2.2. The buffer composition was shown to have a dramatic effect on the assay stability, where the commonly used phosphate buffer saline was proved to be the least suitable choice. The best long-term stability was obtained for the LPEI-GA surface with no loss of antibody activity during one month. The detection limits in the muFEIA for the three different immuno surfaces were 45, 3.8, and 0.80 ng/L (209, 17.7, and 3.7 pM) for APTES-GA, LPEI-GA, and GOPS-BPEI-GA, respectively.
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| 22. |
- Yakovleva, J, et al.
(författare)
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Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection
- 2003
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Ingår i: Biosensors & Bioelectronics. - 1873-4235. ; 19:1, s. 21-34
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Tidskriftsartikel (refereegranskat)abstract
- Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 mum depth and 25 pm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-mug 1(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 mug 1-1, and IC50 values of 0.096 and 0.130 mug 1(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCI buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124%, at atrazine fortification levels of 0.5-3 and 80-240 mug 1(-1), respectively. (C) 2003 Elsevier Science B.V. All rights reserved.
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