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Biosensors are nowadays ubiquitous in medical research as well as in environmental monitoring, food control, drug discovery and military applications. Protein-based electrochemical biosensors are based on the detection of an electrical signal produced after a biochemical reaction between the target analyte and the adsorbed proteins on the biosensor. The coupling of biosensors to heme proteins allows a highly sensitive and selective detection of a range of small molecules such as H2O2, H2S, NO and phenolic compounds. These small molecules are typical byproducts of many industrial processes and are extremely toxic at high concentration. The heme iron of the heme protein changes its oxidation state upon binding with the target molecule which can be detected by the electrode of the biosensor. However, proteins are not stable outside their physiological environment, and therefore immobilization of the heme proteins on the electrode surface is necessary. Ordered mesoporous materials, such as mesoporous titania (TiO2) and silica (SiO2), have been widely considered for the immobilization of proteins because of their advantageous properties that can be fine-tuned for interaction with specific proteins to enhance protein activity. The characterization of new heme proteins is essential and is therefore the focus of the first part of the thesis. The 34 heme proteins of the nematode Caenorhabditis elegans are found to possess widely divergent functions, several participating in redox and signaling reactions which are interesting for future biosensing applications. Since knockout of glb-3 resulted in a severe reduction in fertility and motility of the nematode, a biophysical characterization of GLB-3 is performed for the first time in this work. In the second part, the correlation between the structural changes and the activity of the heme proteins upon adsorption is unraveled by means of several techniques. Horse heart myoglobin, human neuroglobin and horseradish peroxidase are well-characterized heme proteins and were immobilized in different mesoporous materials. Electron paramagnetic resonance spectroscopy (EPR) was the main technique used in this part to study the active site, i.e. the heme group, of the heme proteins before and after immobilization. Moreover, a first immobilization test was performed on GLB-3 in TiO2 to illustrate the importance of optimizing each adsorption experiment when working with a different protein. Additionally, to obtain more insight in the structural changes of neuroglobin after immobilization, site-directed spin labelling in combination with EPR was performed. Finally, the generation of reactive oxygen species on the titania surface was analyzed via EPR. |
