Development of Novel ssDNA-based and PEG-based Gold-Coated Liposome (LipoAu) Nanoconjugates to Improve Sensitivity and Specificity of Vertical Flow Assays (VFAs)

Abstract

Point of Care (POC) diagnostic tests, also known as at-home tests have proven exceptional tools for both professionals and non-professionals alike to rapidly detect target analytes (i.e. disease markers) without the need for laboratory equipment. Common immunodiagnostic POC tests, such as the lateral flow assay (LFA) have seen extensive usage across a variety of fields and played a critical role in controlling the spread of SARS-CoV-2 virus in recent years. Despite clear benefits LFA technology provides, however, these types of assays suffer from limitations in terms of sample volume capacity, response time and false negatives via the hook effect, which impacts sensitivity, specificity, and user friendliness. Detection agents used to emit colorimetric qualitative signals in these assays, such as colloidal gold nanoparticles (AuNPs) also impact performance, and continuing to improve such nanomaterials are critical to improving such technologies. More recent developments in immunodiagnostics, such as the rapid vertical flow assay (VFA), coupled with improved nanoconjugates, have great potential to negate shortcomings seen in currently available LFAs. For this thesis, two experiments were performed. In the first, novel Protein A-based nanoconjugates utilizing gold-coated liposomes (LipoAu) were developed: the first incorporating biotinylated ssDNA (SH-15A-Biotin) as a spacer between avidin and biotinylated analyte (LipoAu-DNA-Avidin-Biotinylated Protein A) and the second containing no ssDNA (LipoAu-Avidin-Biotinylated Protein A). These nanoconjugates were then applied to MedMira rapid VFAs to test if incorporating ssDNA as a spacer would provide better sensitivity and specificity to VFAs. In a separate second experiment, LipoAu nanoconjugates utilizing carboxy-PEG-thiol (SH-PEG-COOH) to conjugate Protein A (LipoAu-PEG-Protein A) to the nanoparticle surface were developed to test their performance in VFAs. This study will provide insight into how to improve qualitative results of VFAs, which could lead to improved POC testing across at-home and industry settings.