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Hetzelt, Helen Anna (2005): Entwicklung eines Biosensors zum simultanen Nachweis von Mikroorganismen und Toxinen. Dissertation, LMU München: Tierärztliche Fakultät



Development of a biosensor for the simultaneous detection of microorganisms and toxins This project aimed at the development of a novel test system (parallel affinity sensor array, PASA) enabling the rapid, parallel and automated detection of microorganisms and toxins (Y. pestis, F. tularensis, C. burnetii, shigatoxins 1 und 2, Staphylococcal enterotoxin B und orthopoxvirus). For this purpose the classical sandwich-EIA principle was combined with the innovative microarray technology. The core of the biosensor is a planar biochip represented by a modified microscope slide on which the different tests run in parallel. The detection is enabled by specific antibodies attached to the slides surface by covalent or adsorptive binding. All further incubation steps are performed in a flow-through cell, reagents are automatically supplied by computer controlled pumps. With respect to the implementation of the detection methods in the PASA system each single-analyte assay was optimized by using classical EIA techniques and then adapted to a uniform test procedure. To enhance assay sensitivity the Biotin/ExtrAvidin, the Digoxigenin/anti-digoxigenin and partially fluorescence detection methods were checked. The most sensitive assays could be established by using detection antibodies labelled with digoxigenin. The detection limits of the optimized assays were in the range of 103 - 104 cfu/ml, microbial toxins could be detected at the pg-level. For transferring these optimized assays from microtiter plates to the biosensor platform a broad range of different biochip surfaces were tested. With some of the tested materials major problems encountered either due to inactivation of the antibodies during the immobilization on covalently binding solid phases or due to bleaching-out of antibody-coated adsorptive surfaces. The use of antibodies labelled with fluorochromes proved to be a failure. Two chemiluminescent microarray tests which complied with the basic requirements adherent to the establishment of an automated rapid detection method were further optimized and evaluated for robustness. Both assays enabled the reliable and reproducible detection of bacteria (105 – 107 cfu/ml) and purified toxins (lower ng-range) within 25 min. Thus, these microarrays rank among the most sensitive rapid tests described so far.