Handheld Device for Rapid In-Field Assay of Listeria Monocytogenes Using Designed Supercharged Binding Proteins for Enhanced Surface Plasmon Resonance (SPR)

Project Abstract The proposed project aims to create a new weapon in the arsenal of the CDC for use in the `winnable battleandapos for improved food safety a low cost hand held high sensitivity rapid sensing platform to detect pathogens and biological and chemical toxins in the field such as on farms at food processing plants and in medical facilities This sensor device incorporates a new class of supercharged binding proteins created through newly developed biodesign technologies onto a sensor chip with a metastructured gold film to produce a one thousandfold enhancement in the surface plasmon resonance SPR shift upon binding of the target pathogen Other than the engineered binding protein the overall device uses only low cost disposable detector chips fabricated via standard semiconductor fabrication processes and off the shelf optical components arranged in a more compact and robust geometry than any other known commercially available SPR detector This research will initially target the detection of Listeria monocytogenes serotype b which is responsible for an estimated deaths and $ billion in outbreak containment costs each year The broader underlying principles can be applied to tap into the rapidly growing In Vitro Diagnostics IVD market valued globally at $ billion in to achieve the long term goal of the rapid sensitive highly specific detection of most viral and gram negative bacterial pathogens which are relevant to public health both domestically and abroad The proposed research will proceed with specific aims Design and optimize short chain variable fragment ScFv proteins based upon wild type monoclonal antibodies to L monocytogenes serotype b the target pathogen Supercharge the designed ScFv protein by mutating antibody residues not essential for target binding Use standard semiconductor fabrication techniques to fabricate a gold plasmonic metasurface on a transparent fused silica substrate and attach the designed ScFv protein to it using standard gold thiol and biotin streptavidin chemistries and Measure the SPR resonance shift as ScFv binds to its target ie Listeria As a proof of concept of the highest risk highest reward component of the device i e development of the supercharged binding protein a model heme binding protein named Mega was designed synthesized and characterized Mega demonstrated a large binding induced conformational change which caused a refractive index change which is calculated to correspond to a fold enhancement in SPR signal relative to commercially available ligand antibody detection schemes Structural elements of Mega were shown to undergo a phase transition from an ensemble of random coil states into a more restricted ensemble of structured states by tuning solution properties and thus the range of electrostatic effects in the structure with or without the presence of ligand Thus the designed binding interaction was shown to be largely insensitive to folding unfolding phase equilibria two features essential to SPR signal enhancement Project Narrative Our research aims to develop a new weapon in the winnable battle for improved food safety an inexpensive handheld device to optically detect foodborne pathogens within a matter of minutes We combine recent developments in the disciplines of protein engineering and surface plasmon resonance SPR with off the shelf electronic components to create a device ideal for proactive monitoring on site eg at a farm food processing facility or restaurant Although our initial efforts will focus on the detection of Listeria Monocytogenes a difficult to detect pathogen which is responsible for an estimated deaths annually in the US the principles underlying our technology can be applied more broadly to the quantitative assay of a broad range of small molecules proteins viruses and bacteria relevant to public health and food safety