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- Davidsson, Richard
(författare)
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Microfluidic biosensing systems based on enzymes, antibodies and cells
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rapid developments within the life science and biotechnology areas put up ever-new demands on more sophisticated techniques and methods for chemical and biological analysis to reach deeper insight into the events at molecular level. Miniaturisation of assays and systems on microchips is one way to increase throughput and advance through parallel analysis lines, multiplexing and integration. This thesis shows some aspects on the use of silicon microchips as a platform for immobilisation of enzymes, antibodies and cells for chemiluminometric biosensing assays. For antibody- and enzyme-based systems, special attention was paid to the immobilisation chemistry due to its influence on stability and activity of the attached biomolecules. In general it was found that surface coatings with a layer of polymer, e.g., polyethylenimine or dextran, increased both stability and signal intensity of the systems, compared to ordinary silanisation-based attachment chemistry. Furthermore, two cell-based monitoring systems, using either yeast cells or a reporter gene modified human (HeLa) cell line, were developed. These systems demonstrated the usefulness and advantages of microfluidics, e.g. in continuous monitoring of cellular events in real time, which stands in great contrast to the common end-point microtiter plate assays. The demands on yeast compared to human cells are however quite different, where the latter has highly specialised requirements on the physical and chemical environment. Special attention was thus paid on investigating different factors that can lead to unspecific and stress-related expression of the reporter gene, hence affecting the quality of the data and overall performance of the system. In conclusion, the main focus of this thesis has been to develop and apply analytical techniques and methods on microchips on flow-format. During the period the which this research was conducted, many interesting and elegant microfluidic analytical systems have been reported in the literature, however, the attention is often drawn mainly to the hardware set-up and construction. The systems developed in this thesis (paper I to V) are technically simple but show real applications in which biological elements, such as living cells, enzymes and antibodies, are handled on silicon microchips.
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- 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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- 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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- 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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- 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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- Sparr Eskilsson, Cecilia, et al.
(författare)
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Harmful azo colorants in leather. Determination based on their cleavage and extraction of corresponding carcinogenic aromatic amines using modern extraction techniques.
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 955:2, s. 215-227
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Tidskriftsartikel (refereegranskat)abstract
- This study concerns the possibilities of using microwave-assisted extraction (MAE) or supercritical fluid extraction (SFE) for detection of harmful azo colorants in leather. After degreasing of the leather sample with SFE there follows a reductive cleavage of the azo colorants to their corresponding aromatic amines in the MAE or SFE equipment. The aromatic amines are subsequently extracted using either MAE or SFE and then finally determined by liquid chromatography with diode-array detection. The results have been compared with recoveries obtained using the German DIN method 53316. This standard method, based on conventional solvent extraction, is used in several European countries. Overall much better recoveries were obtained using MAE or SFE. With both MAE and SFE the amine recoveries of spiked leather samples were generally above 50%. The average recoveries were 62% for MAE and 60% for SFE (solvent collection) compared to 24% with the DIN method. For genuine leather samples the recoveries decreased, especially for benzidine. In this case the average values for MAE, SFE and DIN were 54, 38 and 19%, respectively. The quantification limits in leather samples using MAE or SFE were below 1 mg/kg for all amines investigated. The within-laboratory precision was generally better than 10%, varying somewhat with the analyte considered. With the proposed methodology, the amount of hazardous organic solvents used could be decreased and the sample throughput increased with at least a factor of two with less manual handling compared to the DIN method.
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