MESA+ Institute for Nanotechnology

New detection strategies for bioassays based on liquid chromatography, fluorescence spectroscopy and mass spectrometry were developed and are presented within this thesis.

An introductory overview on different labeling strategies in enzymatic and immunoassays is presented. Direct-labeling assays as radioimmunoassays, enzyme-labeled measurement techniques as enzyme-linked immunosorbent assays, nanoparticle-labeled assays as well as methods for coupling bioassays with chromatographic methods are introduced.

Fluorescence detection schemes for the determination of two enzymes in parallel were developed for assays that yield fluorescent reaction products with non‑overlapping emission bands. The obtained results

were compared with those from the individual determination of the respective enzymes. The method was applied to the simultaneous determination of acid phosphatase and glucose oxidase in honey.

An at-line flow-injection system with fluorescence and subsequent MS detection for the simultaneous enzyme assay system was developed. The alkaline phosphatase‑catalyzed reaction of 5-FSAP to 5-FSA and the microperoxidase 11-catalyzed reaction of MNBDH to MNBDA were performed, individually and simultaneously, and the reaction progress was monitored.

The reaction product of the POD-catalyzed reaction of o-phenylenediamine with hydrogen peroxide was identified by LC/APCI-MS investigations and exact mass measurements (ESI-TOF-MS). The reaction was carried under ELISA conditions, and it was found out that 2,3-diaminophenazine is the main reaction product.

A new HPLC/post-column reaction/fluorescence detection system for the activity determination of microperoxidases was developed and applied to proteolytic digests of cytochrome c from bovine heart. Microperoxidase 9 and microperoxidase 6 were identified as products in digests using trypsin and an unspecific protease, respectively. The fluorescence-based results were confirmed by ESI-MS measurements.

Furthermore, two independent methods for the determination of the hypertension drug telmisartan were developed. The GOD-based ELISA and the TFC/LC/APCI-MS/MS set-up were applied to human plasma samples and the results were compared. Both methods showed good correlation.