Biosensor based on DNA directed immobilization of enzymes onto optically sensitive porous Si
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Biosensor based on DNA directed immobilization of enzymes onto optically sensitive porous Si Giorgi Shtenberg1, Naama Massad-Ivanir2, Oren Moscovitz3, Sinem Engin4, Michal Sharon3, Ljiljana Fruk4 and Ester Segal2,5 1 The Inter-Departmental Program of Biotechnology, 2Department of Biotechnology and Food Engineering, 5The Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israel, 3Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, 4Karlsruhe Institute of Technology, DFG – Center for Functional Nanostructures, Karlsruhe 76131, Germany ABSTRACT Optical biosensor for monitoring proteolytic activity is constructed by DNA-directed immobilization of enzymes onto porous Silicon nanostructures. This sensor configuration allows both protease recycling and easy surface regeneration for subsequent biosensing analysis by means of mild dehybridization conditions. We demonstrate real-time analysis of minute quantities of proteases paving the way for substrate profiling and the identification of cleavage sites. The biosensor is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products. INTRODUCTION The catalytic activity of proteases is crucial for life. These enzymes regulate a multitude of biological processes through the modification of protein activity or by controlling protein turnover [1]. They have the unique ability to irreversibly hydrolyze peptide bonds, which results not only in protein degradation, but also in the introduction of new levels of information content into the signaling pathways [2]. Identifying protease substrate repertoire is important for understanding the functions of these enzymes, revealing their biological roles, and generating lead compounds for new therapeutic strategies [3]. Most common methods to assess protease activity suffer from low efficiencies and extended pretreatments that can easily affect the native activity of the desired enzyme. Even the rapid technologies e.g., chromatography and massspectrometry, that possess high selectivity, reproducibility and sensitivity, have drawbacks associated with the meticulous sample preparation prior to analysis [4]. Thus, the need for rapid information screening requires the development of new and improved bioanalytical tools, allowing for fast and high-throughput analysis of minute samples with minimal losses. In recent years, porous Si (PSi) has emerged as a promising nanomaterial for optical biosensing applications due to its large internal surface area and tunable optical properties [5-9]. It has been demonstrated that PSi-based interferometers, which operate by measuring a change in refractive index in a volume of solution contained within the porous nanostructure, allows for label-free detection of a variety of biomolecular interactions, including enzymes, DNA fragments, proteins, and antibodies [5]. In this work, we report on a generic approach for reversible enzyme conjugation to porous SiO2
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