Use of Biorecognition Elements or Their Mimics in New Flow Analysis Systems

Detta är en avhandling från Dep. of Analytical Chemisry, Box 124, Lunds University, 221 00 Lund, Sweden

Sammanfattning: We have evaluated capacitive and thermal transducers for recognition of bio- molecular interactions with different analytes. These assays were made in flow analysis systems because of the simplicity such methods provide. A capacitive transducer, based on a surface modified gold electrode, has the ability to monitor affinity based interactions directly, provided that the interactions introduce measurable changes in the dielectric layer on the surface of the electrode. The method is capable of very low detection limits for various analytes. Interleukin 6 has, for example, been detected down to femtomolar concentrations . For the determination of urea in serum we have applied a thermal transducer fitted in a flow cell. The enzyme urease was used to selectively hydrolyze urea in an injected serum sample. In the hydrolysis process, heat is produced that can be used for quantifying urea in serum. The approach applied here was heat conduction calorimetry where a reactor has been placed on the surface of a thermocouple plate (TC-plate). This approach allowed detection of urea in serum in the range of 0.5-10 mM. One of the greatest advantages of the method is the insensitivity of the sensor towards changes in the flow rate. The method does thus not require careful optimization of the flow rate. Changes in flow rate during analysis are also not expected to influence the quality of the results. This was also reflected in the low standard deviation (< 2%) of repeatedly injected samples. Immunoassays for pesticide determinations have also been evaluated in this work. The common way of conducting an immunoassay is by the use of ELISA plates. Efforts have been made, however, to simplify the assay procedure by applying flow injection techniques. This will simplify the automation protocol for such assays as well as reduce analysis time per sample. The difference between the various flow injection immuno analysis formats is often how the free un-reacted tracer is separated from the tracer-antibody complex. Results have shown that immobilization of the antibody may not always be the most suitable method of analysis because of the hostile environment the regeneration solution provides for the antibody. Therefore immobilization of antibody receptors like protein-A or G have been considered. The regeneration of the protein instead of the antibody appearers to be a suitable solution to the problem. We have furthermore applied molecular imprinted polymers for selective, on-line, extraction of triazines from contaminated water samples, which allowed simultaneous determination of low concentrations (at least 0.5 ng/ml) of several triazines in solutions containing 20 ppm humic acid.

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