Application of high-affinity recombinant binding proteins in biosensors and bioanalytical assays

Mgr. Alena SEMERÁDTOVÁ

Doctoral thesis

Abstract:

Immobilization of recognition biomolecules to the surface of the sensor is a crucial aspect of biosensors and bioanalytical assays development. The amount, the way of immobilization, the orientation of the molecules are important aspects for biosensor parameters. Substitution of commonly used antibodies by recombinant binding proteins is a promising tool for improvement of biosensors because of their higher stability and the construction that enable their repetitive use. The aim is to study the possibility of high-affinity recombinant binding proteins application in optical sensor development and in bioanalytical assays with respect to ways of immobilization to different substrates suitable for biosensor and bioassay development (glass, polymer foil, nanoparticles). Moreover, this Thesis provides a comparative study of three types of high-affinity recombinant binding proteins recognizing human serum albumin (HSA), with a potential to substitute the antibodies in diagnostic and therapeutic applications. Open glass microchip was fabricated for spatially oriented immobilization of proteins and a direct specific detection of fluorescently labelled HSA was performed. Analytical performance of optical microchip - sensitivity, LOD, and LOQ, were established. The most effective binder was selected and tested on different substrates - polyethylene naphtalate foil (PEN) and PEG/ PEG-biotin modified PAMAM dendrimer. Analytical performance of the PEN foil optical microchip was established using the direct specific detection of a fluorescently labelled analyte. The interconnection of PEG/ PEG-biotin modified PAMAM dendrimer with recombinant high-affinity binding proteins was examined by several analytical methods to inspect its potential as a dendrimer-based nanoparticle biosensing platform. The main findings of this Thesis could be employed in recombinant high-affinity binders design processes as well as in biosensor construction.

Abstract:

Immobilization of recognition biomolecules to the surface of the sensor is a crucial aspect of biosensors and bioanalytical assays development. The amount, the way of immobilization, the orientation of the molecules are important aspects for biosensor parameters. Substitution of commonly used antibodies by recombinant binding proteins is a promising tool for improvement of biosensors because of their higher stability and the construction that enable their repetitive use. The aim is to study the possibility of high-affinity recombinant binding proteins application in optical sensor development and in bioanalytical assays with respect to ways of immobilization to different substrates suitable for biosensor and bioassay development (glass, polymer foil, nanoparticles). Moreover, this Thesis provides a comparative study of three types of high-affinity recombinant binding proteins recognizing human serum albumin (HSA), with a potential to substitute the antibodies in diagnostic and therapeutic applications. Open glass microchip was fabricated for spatially oriented immobilization of proteins and a direct specific detection of fluorescently labelled HSA was performed. Analytical performance of optical microchip - sensitivity, LOD, and LOQ, were established. The most effective binder was selected and tested on different substrates - polyethylene naphtalate foil (PEN) and PEG/ PEG-biotin modified PAMAM dendrimer. Analytical performance of the PEN foil optical microchip was established using the direct specific detection of a fluorescently labelled analyte. The interconnection of PEG/ PEG-biotin modified PAMAM dendrimer with recombinant high-affinity binding proteins was examined by several analytical methods to inspect its potential as a dendrimer-based nanoparticle biosensing platform. The main findings of this Thesis could be employed in recombinant high-affinity binders design processes as well as in biosensor construction.

Keywords

Biosensor, high-affinity recombinant proteins, HSA, ABD, dendrimer, glass, PEN

Language used: English

Date on which the thesis was submitted / produced: 4. 12. 2018

Thesis defence

• Date of defence: 28. 5. 2019
• Supervisor: Mgr. Jan Malý, Ph.D., Mgr. Jan Malý, Ph.D.

JAN EVANGELISTA PURKYNĚ UNIVERSITY IN ÚSTÍ NAD LABEM

Faculty of Science

Doctoral programme / field:
Nanotechnology / Applied Nanotechnology