Genetically engineered bio-nanoparticles with co-expressed enzyme reporter and recognition element for IgG immunoassay

Abstract

A bioluminescence-based immunoassay, relying on genetically engineered multifunctional outer membrane vesicles (OMVs, ∼50 nm) with co-expressed enzyme reporter and IgG-binding domain, has been developed for detection of immunoglobulin G (IgG), an important biomacromolecule in humoral immunity. OMVs with co-expressed IgG-binding domain (Z domain) and ATP-independent NanoLuc luciferase (∼150-fold more active than either the firefly (Photinus pyralis) or Renilla reniformis luciferase) were produced through simple fermentation of genetically engineered nano-vesicle-forming E. coli. The size and concentration of the as-produced multifunctional OMVs were determined by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis, respectively. To reduce the non-specific binding of OMVs during immunoassay, blocking agent test was systematically carried out before IgG detection. Sensitive detection of IgG was further demonstrated with the limit of detection of 5 ng/mL IgG, comparable to that of commercial IgG ELISA kit. The advantages of genetically engineered bio-nanoparticles (bio-NPs) containing both reporter and recognition element are cost-effective and scalable through simple cell cultures with high reproducibility and pre-determined biological functions, in comparison with the conventional costly enzyme-antibody conjugation used in immunoassay. This study opens a new pathway on the low-cost immunoassay by rational design of genetically engineered bio-NPs with integrated reporter/recognition function.