Sample Handling Techniques in Biological and Environmental Applications Emphasizing Biomolecular Recognition and MALDI-TOF MS

Abstract: The use of restricted access media and other stationary phases for clean-up and trace enrichment of environmental samples coupled on-line to liquid chromatography using diode array detection as well as thermospray MS is described. This on-line configuration was compared with commercially available ELISAs for the determination of triazines in complex matrices, showing superior precision and accuracy with the chromatographic method. However, less matrix dependence and higher sample throughputs were obtained with the immunoassays. Immunoreagents were characterized for the development of competitive triazine immunoassays using fluorescence polarization. The obtained information was used for the development of a high sample throughput flow immunoassay. This assay is based on the separation of bound and free label using a restricted access column. A sample throughput of 80 samples/h was achieved with detection limits in the low ppb range. Environmental as well as biological samples such as blood and urine were successfully applied. The second part of this thesis deals with the sample handling and sample preparation of protein and peptide samples for subsequent MALDI-TOF MS analysis. Silicon micromachining has been used for the construction of a flow-through piezo-electric microdispenser, capable of handling picoliter volumes. The microdispenser is used to deposit samples onto a MALDI target plate and can be used either as a sample injector or on-line coupled to a chromatographic separation. Multiple depositions of sample was reported to increase the sensitivity. The generation of homogenous sample spots is indispensable in automated MS-analysis. Therefore, a simple method generating homogenous surfaces have been developed. These surfaces can also be obtained using the micro-dispenser, allowing high density MALDI target plates to be used. The development of a silicon micro-reactor for the enzymatic digestion of proteins coupled on-line to the microdispenser is also described. The enzymatic digestion is based on immobilized endoproteases such as trypsin and chymotrypsin. The generation of peptide maps are used for the identification of proteins through database searching. The enhanced surface area, the decreased digestion time, as well as the small dimensions are some of the advantages to the system.