טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShtenberg Giorgi
SubjectDevelopment of Optical Biosensors for Monitoring Proteins
Activity
DepartmentDepartment of Biotechnology
Supervisor Professor Ester H. Segal
Full Thesis textFull thesis text - English Version


Abstract

Proteases clearly do a lot more than digest your food; they act on substrates that are fundamental to a raft of physiological processes, from generalized protein digestion to more specific regulated processes. However, despite major advances in our understanding of proteases, their substrates and biological functions are not fully understood. The objective of this research is to develop a generic biosensor system for real-time monitoring of protease activity and defining these enzymes substrate specificity. Our biosensor system is based on a multifunctional porous Si (PSi) nanostructure, which is designed to optically monitor the proteolytic activity and also function as microaffinity purification method to recover peptide fragments for downstream proteomics analysis. First, an oxidized PSi (PSiO2) optical nanostructure, a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element. Immobilization of the protease onto the nanostructure is performed through DNA-Directed Immobilization. We demonstrate high enzymatic activity of the immobilized proteases (more than 80%), while maintaining their specificity. The catalytic activity of the proteases immobilized within the porous nanostructure is monitored in real-time by reflective interferometric Fourier transform spectroscopy (RIFTS), allowing us to both concentrate and quantify the reaction products. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The biosensor configuration is compatible with common proteomic methods and allows for downstream mass spectrometry analysis of the reaction products for substrate profiling and cleavage sites identification. The generic design of this biosensor allows tailoring unlimited experimental setups by varying the model enzymes. For example: nanostructured PSiO2 is utilized for systematic analysis of heavy metal ions in real surface water samples by enzymatic activity inhibition. We exploit the specificity of enzymes to heavy metal ions and hence monitor the catalytic activity in real-time by RIFTS technique. First, we show a general detection assay by immobilizing horseradish peroxidase (HRP) within the PSiO2 thin-film, revealing high sensitivity towards three metal ions (Ag>Pb2>Cu2). Next, we demonstrate the concept of specific detection for Cu2 (as a model heavy metal), by immobilizing Laccase within the PSiO2 scaffold. The biosensor detects the presence of trace levels of Cu2 in real water samples, with values identical to those obtained by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), a gold standard analytical technique. Another example is the influence of thermal oxidation conditions on the performance of PSi based biosensors for label-free monitoring of enzymatic activity. We compare three oxidation temperatures and their effect on the enzyme immobilization efficiency and the PSiO2 intrinsic stability. Despite the significant decrease in porous volume and specific surface area with elevating the oxidation temperature, higher content and surface coverage of the immobilized enzymes is attained. Thus, by proper control of the oxide layer formation, we can eliminate the aging effect; thus achieving efficient immobilization of different biomolecules, optical signal stability and sensitivity.