Handheld Device For Rapid In-field Assay of Listeria Monocytogenes Using Designed Supercharged Binding Proteins For Enhanced Surface Plasmon Resonance (spr)

Project AbstractThe proposed project aims to create a new weapon in the arsenal of the CDC, for use in the `winnable battle'for improved food safety: a low-cost, hand-held, high-sensitivity, rapid-sensing platform to detect pathogensand biological and chemical toxins in the field, such as on farms, at food processing plants, and in medicalfacilities. This sensor device incorporates a new class of supercharged binding proteins, created throughnewly 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 chipsfabricated via standard semiconductor fabrication processes, and off-the-shelf optical components, arranged ina more compact and robust geometry than any other known commercially available SPR detector.This research will initially target the detection of Listeria monocytogenes serotype 4b, which is responsible foran estimated 260 deaths and $2.8 billion in outbreak containment costs each year. The broader underlyingprinciples can be applied to tap into the rapidly growing In Vitro Diagnostics (IVD) market (valued globally at$50 billion in 2014), to achieve the long-term goal of the rapid, sensitive, highly specific detection of most viraland gram-negative bacterial pathogens which are relevant to public health, both domestically and abroad.The proposed research will proceed with 4 specific aims:1) Design and optimize short-chain variable fragment(ScFv) proteins, based upon wild-type monoclonal antibodies to L. monocytogenes serotype 4b (the targetpathogen) 2) Supercharge the designed ScFv protein by mutating antibody residues not essential for targetbinding, 3) Use standard semiconductor fabrication techniques to fabricate a gold plasmonic metasurface on atransparent fused silica substrate, and attach the designed ScFv protein to it using standard gold-thiol andbiotin-streptavidin chemistries, and 4) 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 thesupercharged binding protein), a model heme-binding protein named Mega(-) was designed, synthesized, andcharacterized. Mega(-) demonstrated a large, binding-induced conformational change, which caused arefractive index change, which is calculated to correspond to a 100-1000fold enhancement in SPR signal,relative to commercially available ligand-antibody detection schemes. Structural elements of Mega(-) wereshown to undergo a phase transition from an ensemble of random-coil states into a more restricted ensembleof 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 largelyinsensitive to folding/unfolding phase equilibria-two features essential to SPR signal enhancement.