Functionalizing silica sol-gel with entrapped plant virus-based immunosorbent nanoparticles

Abstract

Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol-gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol-gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol-gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications.

Keywords: Antibody purification; Biosensing; Bioseparations; Molecular pharming; Monoclonal antibody; Nanobiotechnology; Plant-made pharmaceuticals; Silica sol–gel; Tobamovirus; Virus-based nanomaterial.

Fig. 1

Illustrated schematics of a synthesis of sol–gel functionalized with entrapped virus-based nanomaterials (including example photographs of gels synthesized in this study), and b the example use case presented in this work of gel-entrapped plant virus-based immunosorbent nanoparticles for affinity purification of plant-made antibodies

Fig. 2

Purification and immobilization of fluorescently labelled plant virions a SDS-PAGE results of an ultracentrifuge-based purification of wt-TMV from Nicotiana benthamiana leaf tissue, which is then chemically conjugated with Cy5 dye in a two-step reaction to generate Cy5-TMV. Lane definitions: I—filtered plant extract, II—PEG-precipitated pellet resuspension, III—microfuge pellet, IV—ultracentrifuge pellet resuspension, V—ultracentrifuge wash, VI—initial wt-TMV pre-conjugation, VII—Cy5-TMV post-conjugation. b UV–Vis absorbance spectra of wt-TMV and Cy5-TMV. c Negative stain transmission electron microscope image of the Cy5-TMV. d Normalized areal fluorescence per 2 µL volume silica bead containing Cy5-TMV over 48 h. Beads were exchanged into fresh PBS buffer after each measurement. Error bars represent one standard deviation with biological triplicate. e Fluorescent microscope images of the silica sol–gel beads containing Cy5-TMV over 48 h of submerged gel wash. Scale bars represent 500 µm

Fig. 3

Use of silica sol–gel functionalized with entrapped plant virus-based immunosorbent nanoparticles (VIN) to purify monoclonal antibodies. Reducing condition SDS-PAGE (upper) and western blot (lower) results of the non-bound liquid sample after loading and low pH elution of human immunoglobulin G for a first and second use cycle shown for a sample loading consisting of human immunoglobulin G spiked into a solution of a clean PBS and b sterile-filtered Nicotiana benthamiana extract. Lanes are loaded with fixed volume. HC, heavy chain of the human immunoglobulin G