Immobilization of Bioengineered Portal Protein Within a Solid-State Nanopore for Molecular Sensing

Abstract

Site-specific, reproducible, and uniform-orientation protein immobilization on an inorganic surface while preserving protein conformation and activity is of wide interest for applications in biosensing, protein microarrays, and enzymology, among others. Successful immobilization requires understanding of material's surface chemistry, protein properties, and nature of their interaction to eliminate non-specific binding. Portal protein is a naturally occurring pore (biological nanopore) and part of the bacteriophage packaging machine that pumps the viral genome inside its capsid. In this work, we have explored different approaches to immobilize G20c portal protein from double-stranded bacteriophage G20c into a thin (∼30 nm) silicon nitride (SiNx) membrane embedded in a silicon chip. Desired orientation of the immobilized protein is achieved by maintaining a voltage bias across the membrane to electrokinetically drive a single protein into the synthetic nanopore. The nanopore geometry dictates a predominant favorable protein orientation while the portal preserves its conformation, as indicated by ion current measurements through the “hybrid nanopore”. Application of nanopores (both synthetic and biological) as label-free, single-molecule biosensors for electrical and/or optical probing of structural features in biomolecules have been widely explored. Confirmed by our single-molecule electrical sensing results, our hybrid nanopore system provides mechanically robust and chemically compatible synthetic protein framework, superior to its natural counterparts such as organic membranes (lipid bilayer, for example), and exploits tunable and engineerable characteristics of thermostable G20c portal protein rendering an active, high-resolution biomolecule sensing platform. We demonstrate here through chemical functionalization of synthetic nanopores and/or engineering G20c portal protein assemblies, a protein nanopore chemically linked to a synthetic nanopore, rendering considerably improved protein stability, sensing lifetime, and signal-to-noise ratio compared to our previous hybrid system. This development will be widely applicable to coupled nanopore sensor arrangements such as electro-optical and electro-pressure systems.