Virus‐Tethered Magnetic Gold Microspheres with Biomimetic Architectures for Enhanced Immunoassays

Abstract

In immunoassays, non‐specific bindings to biosensing surfaces can be effectively prevented by formation of biocompatible and hydrophilic self‐assembled monolayer (SAM) on the surfaces. A thin gold (Au) layer on magnetic microspheres, 15 μm in diameter, enables facile SAM formation and thereby accepts second layer of filamentous virus scaffolds for the immobilization of functional proteins. The merger of the virus and SAM‐Au protected microspheres not only provides exceptionlly dense antibody loading, but also resembles biological cellular structures that enhance ligand‐receptor interactions. Site‐specific biotinylation of filamenous viruses allows formation of free‐standing virus threads (>1.0 × 1010) on streptavidin‐modified SAM‐Au microspheres. The augmented yield of antibody loading, due to the increased surface to volume ratio, on virus‐modified Au microspheres is confirmed by measuring fluorescence intensities. The bead‐based immunoassays for the detection of cardiac marker proteins exhibit increased sensitivity of virus‐Au microspheres, as low as 20 pg mL−1 of cardiac troponin I in serum, and extremely low non‐specific adsorption when compared with bare polymer beads. This increased sensitivity due to filamentous morphology and SAM‐Au layer demonstrates the feasibility of merging viruses with non‐biological materials to yield biomimetic tools for the enhanced bead‐based immunoassays.

Translocation Pathway‐Dependent Assembly of Streptavidin‐ and Antibody‐Binding Filamentous Virus‐Like Particles

“…Production of BirA : His‐tagged BirA was prepared as previously described . Briefly, the pET21a‐BirA plasmid was transformed into E. coli BL21(DE3)pLysS, plated on LB‐agar with carbenicillin (100 µg mL −1 ).…”

“…Previously, we showed that AP (AviTag, GLNDIFEAQK-IEWHE) fused to the p3 of fd-tet can be in vitro biotinylated by bacterial biotin ligase BirA, rendering site-specific and vertical immobilization of the virions on SAV microbeads. [4] Although high-density probe antibodies can be chemically conjugated on the virion surface using bioorthogonal Staudinger ligation, chemical modification of antibodies and virions inevitably decreased antigen binding capacity and increased hydrophobicity of the virion surface, necessitating extensive blocking steps to prevent nonspecific bindings. We thus seek to display genetically encoded IgGbinding domains while keeping the biotinylation motif at one of the virion tips for vertical immobilization.…”

“…To construct bioinspired cellular microvilli-like architecture, we immobilized the p3-biotinylated f88 virions on SAV-coated microbeads, such as commercially available Dynabeads (1.0 and 2.8 µm in diameter) and custom-made magnetic gold microshells (MGM, 15 µm in diameter). [4,60] We then stained the phage particles with Alexa488 and obtained optical images using the 3D Nikon super-resolution structured illumination microscope (N-SIM) to verify the end-on immobilization of the virions (Figure 5 and Movies S1 and S2 (Supporting Information)). The microvilli-like structure made of filamentous phage particles was embossed as the size of the microbeads decreased.…”

“…First, if immobilized vertically, the filamentous virions can offer unique opportunities in constructing bioinspired structures owing to their similar morphological dimensions to many cellular threadlike structures including, but not limited to, microvilli and neurites of differentiated mammalian cells, as well as bacterial pili and fimbriae. [4] These long flexible tethers of cells dramatically increase surface-to-volume ratio and serve as multivalent tentacles that enhance the selectivity in ligand-receptor interactions related to cellular movement, adhesion, endocytosis, and mating. [73][74][75] Second, if the surface of the virion nanowires is functionalized with target-binding molecules or antibodies with controlled orientation, the target-binding sites or antigen binding pocket, a complementarity-determining region, can remain exposed, which dramatically improves biosensor performance.…”

“…Phage display provides one of the most remarkable biomedical tools in human medicine, dairy and food industry, and plant science, as well as noncanonical opportunities in the field of materials science, biotechnology, and nanotechnology . Among others, Ff class of filamentous bacteriophages (f1, fd, and M13) garner much attention in immunoassays owing to their cellular microvilli‐like structures with extreme length–width ratio (≈1 µm long, ≈5 nm wide) that can significantly increase the surface area for antibody immobilization . In contrast to minor capsid proteins (p3 and p6 are on one end, p7 and p9 are on the other) with low copy numbers ranging 3–5, the major capsid protein p8 (50 amino acid‐long) counts up to ≈4000 copies.…”