Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes

Abstract

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.

Tobacco Mosaic Virus as a Versatile Platform for Molecular Assembly and Device Fabrication

“…To expand TMV's functional capability, several modifications have introduced new functional groups to the TMV CP. The insertion of cysteine residues near the N‐terminus of the CP enables well‐established thiol‐to‐maleimide bioconjugations including fluorescent dyes (Texas Red and Atto 488) and biotinylated PEG, which allowed the subsequent attachment of streptavidin‐labeled enzymes including horseradish peroxidase and glucose oxidase with a 50% conjugation efficiency . A lysine inserted at the N‐terminus or C‐terminus similarly allows for bioconjugation to the exterior of TMV through a reaction with an N‐hydroxysuccinimide (NHS)‐ester‐labeled target.…”

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…HRP is a popular enzyme for ELISAs due to its ability to convert chromogenic substrates into colored products and chemiluminescent substrates into fluorescent products. 382 Immobilization of HRP, as well as glucose oxidase (GOX), has been explored using CPMV 386 and TMV 387 platforms. While the addition of sensing molecules to the virions will be required for their use in ELISAs, the display of the enzymes themselves on the particles can be directly used for sensing.…”

Micro‐ and Nanoscopic Imaging of Enzymatic Electrodes: A Review

“…In the same article, the use of transmission electron microscopy is worth mentioning since patterns corresponding to HRP could clearly be distinguished on the nanotubes (Figure ). The presence of individual enzymes on nanotubular nucleoproteins could also be visualized by SEM …”