Targeted Immobilization of Globins on Mesoporous Materials: A spectroscopist's View
14 mei 2018
Campus Middelheim, G0.10 - Middelheimlaan 1 - 2020 Antwerpen (route: UAntwerpen, Campus Middelheim
Organisatie / co-organisatie:
Sabine Van Doorslaer & Pegie Cool
Doctoraatsverdediging Bert Cuypers - Faculteit Wetenschappen, Departement Fysica
For many years now, there is a rising demand for the immobilization of biomolecules in and onto synthetic matrices for biosensor applications. Targeted incorporation requires characterization tools that allow identification of the biomolecule outside its natural environment. This PhD thesis describes the contributions of a spectroscopist towards the development of such a characterization methodology. Several spectroscopic techniques were used throughout this thesis, including electron paramagnetic resonance (EPR) techniques (both continuous-wave and pulsed EPR) and optical spectroscopy techniques such as optical absorption spectroscopy, resonance Raman (RR) spectroscopy and laser flash photolysis. The characterization methodology is applied to the incorporation of globin proteins in mesoporous Ti-containing materials. Globins are iron-containing proteins that are of interest for biosensor applications because of their redox properties, but they require immobilization on the electrodes of a biosensor.
The first part of this thesis consists of the spectroscopic characterization of different globins using a generic approach. The cytoglobins of two Antarctic fish, Dissostichus mawsoni and Chaenocephalus aceratus, were thoroughly characterized. Interestingly, the latter lacks hemoglobin (Hb) in the blood and as such is thus a white-blooded fish. Although both fish Cygbs have many features in common with human Cygb, there are some striking differences in CO binding and in multimerization. Furthermore, the non-symbiotic hemoglobins (nsHbs) from the model legume Lotus japonicus were characterized. Interestingly, Lotus japonicus expresses two class-1 nsHbs and only one class-2 nsHbs. Using different spectroscopic techniques, I identified the differences and similarities between the different nsHbs. The characterizations performed in the first part of this thesis are an important step towards the immobilization of globins on mesoporous materials: they provide fingerprint spectra of the globins in their natural environment which can be compared with the spectral data obtained after immobilization.
The second part of this thesis describes how EPR is a valuable tool for the characterization of Ti-containing materials. A series of ZnTi layered double hydroxides with different Zn/Ti ratios were used as catalysts for the photodegradation of salicylic acid under visible light. The mechanism of this photodegradation was studied here.
Finally, the impact of the buffer solution on the globin immobilization onto mesoporous titania was systematically studied. Commercially available materials (horse heart mygolobin and mesoporous titania) were used to test the influence of different buffers on the protein incorporation. The buffers show differing interactions with the TiO2 surface, which drastically affects the adsorption of myoglobin as well as its structure, as can be observed by EPR.