Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.

AbstractThe cover image is based on the Advanced Review From stars to stripes: RNA‐directed shaping of plant viral protein templates ‐ structural synthetic virology for smart biohybrid nanostructures, by Christina Wege and Claudia Koch. https://doi.org/10.1002/wnan.1591. image

Nanomaterials composed of plant viral components are finding their way into medical technology and health care, as they offer singular properties. Precisely shaped, tailored virus nanoparticles (VNPs) with multivalent protein surfaces are efficiently loaded with functional compounds such as contrast agents and drugs, and serve as carrier templates and targeting vehicles displaying e.g. peptides and synthetic molecules. Multiple modifications enable uses including vaccination, biosensing, tissue engineering, intravital delivery and theranostics. Novel concepts exploit self-organization capacities of viral building blocks into hierarchical 2D and 3D structures, and their conversion into biocompatible, biodegradable units. High yields of VNPs and proteins can be harvested from plants after a few days so that various products have reached or are close to commercialization. The article delineates potentials and limitations of biomedical plant VNP uses, integrating perspectives of chemistry, biomaterials sciences, molecular plant virology and process engineering.

Nanomaterials composed of plant viral components are finding their way into medical technology and health care, as they offer singular properties. Precisely shaped, tailored virus nanoparticles (VNPs) with multivalent protein surfaces are efficiently loaded with functional compounds such as contrast agents and drugs, and serve as carrier templates and targeting vehicles displaying e.g. peptides and synthetic molecules. Multiple modifications enable uses including vaccination, biosensing, tissue engineering, intravital delivery and theranostics. Novel concepts exploit self-organization capacities of viral building blocks into hierarchical 2D and 3D structures, and their conversion into biocompatible, biodegradable units. High yields of VNPs and proteins can be harvested from plants after a few days so that various products have reached or are close to commercialization. The article delineates potentials and limitations of biomedical plant VNP uses, integrating perspectives of chemistry, biomaterials sciences, molecular plant virology and process engineering. (C) 2018 Elsevier B.V. All rights reserved.

The self-assembly of viral building blocks bears exciting prospects for fabricating new types of bionanoparticles with multivalent protein shells. These enable a spatially controlled immobilization of functionalities at highest surface densities-an increasing demand worldwide for applications from vaccination to tissue engineering, biocatalysis, and sensing. Certain plant viruses hold particular promise because they are sustainably available, biodegradable, nonpathogenic for mammals, and amenable to in vitro self-organization of virus-like particles. This offers great opportunities for their redesign into novel "green" carrier systems by spatial and structural synthetic biology approaches, as worked out here for the robust nanotubular tobacco mosaic virus (TMV) as prime example. Natural TMV of 300 x 18 nm is built from more than 2,100 identical coat proteins (CPs) helically arranged around a 6,395 nucleotides ssRNA. In vitro, TMV-like particles (TLPs) may self-assemble also from modified CPs and RNAs if the latter contain an Origin of Assembly structure, which initiates a bidirectional encapsidation. By way of tailored RNA, the process can be reprogrammed to yield uncommon shapes such as branched nanoobjects. The nonsymmetric mechanism also proceeds on 3'-terminally immobilized RNA and can integrate distinct CP types in blends or serially. Other emerging plant virus-deduced systems include the usually isometric cowpea chlorotic mottle virus (CCMV) with further strikingly altered structures up to "cherrybombs" with protruding nucleic acids. Cartoon strips and pictorial descriptions of major RNA-based strategies induct the reader into a rare field of nanoconstruction that can give rise to utile soft-matter architectures for complex tasks. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures

Nanotubular tobacco mosaic virus (TMV) particles and RNA-free lower-order coat protein (CP) aggregates have been employed as enzyme carriers in different diagnostic layouts and compared for their influence on biosensor performance. In the following, we describe a label-free electrochemical biosensor for improved glucose detection by use of TMV adapters and the enzyme glucose oxidase (GOD). A specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CP aggregates was achieved via bioaffinity binding. Glucose sensors with adsorptively immobilized [SA]-GOD, and with [SA]-GOD cross-linked with glutardialdehyde, respectively, were tested in parallel on the same sensor chip. Comparison of these sensors revealed that TMV adapters enhanced the amperometric glucose detection remarkably, conveying highest sensitivity, an extended linear detection range and fastest response times. These results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for applications in biosensorics and biochips. Here, we describe the fabrication and use of amperometric sensor chips combining an array of circular Pt electrodes, their loading with GOD-modified TMV nanotubes (and other GOD immobilization methods), and the subsequent investigations of the sensor performance.

The presentation of enzymes on viral scaffolds has beneficial effects such as an increased enzyme loading and a prolonged reusability in comparison to conventional immobilization platforms.Here, we used modified tobacco mosaic virus (TMV) nanorods as enzyme carriers in penicillin G detection for the first time.Penicillinase enzymes were conjugated with streptavidin and coupled to TMV rods by use of a bifunctional biotin-linker.Penicillinase-decorated TMV particles were characterized extensively in halochromic dye-based biosensing.Acidometric analyte detection was performed with bromcresol purple as pH indicator and spectrophotometry.The TMV-assisted sensors exhibited increased enzyme loading and strongly improved reusability, and higher analysis rates compared to layouts without viral adapters.They extended the half-life of the sensors from 4 -6 days to 5 weeks and thus allowed an at least 8-fold longer use of the sensors.Using a commercial budget-priced penicillinase preparation, a detection limit of 100 µM penicillin was obtained.Initial experiments also indicate that the system may be transferred to label-free detection layouts.

A field-effect biosensor employing tobacco mosaic virus (TMV) particles as scaffolds for enzyme immobilization is presented. Nanotubular TMV scaffolds allow a dense immobilization of precisely positioned enzymes with retained activity. To demonstrate feasibility of this new strategy, a penicillin sensor has been developed by coupling a penicillinase with virus particles as a model system. The developed field-effect penicillin biosensor consists of an Al-p-Si-SiO2-Ta2O5-TMV structure and has been electrochemically characterized in buffer solutions containing different concentrations of penicillin G. In addition, the morphology of the biosensor surface with virus particles was characterized by scanning electron microscopy and atomic force microscopy methods. The sensors possessed a high penicillin sensitivity of ~ 92 mV/dec in a nearly-linear range from 0.1 mM to 10 mM, and a low detection limit of about 50 µM. The long-term stability of the penicillin biosensor was periodically tested over a time period of about one year without any significant loss of sensitivity. The biosensor has also been successfully applied for penicillin detection in bovine milk samples.

A novel approach for the development of enzyme biosensors based on field-effect structures modified with tobacco mosaic virus (TMV) as enzyme nanocarrier is presented.TMV nanoparticles serve as scaffolds for precisely positioned high-density enzyme immobilization with retained enzyme activity.To prove this new concept, a capacitive penicillin biosensor based on an electrolyte-insulatorsemiconductor structure modified with penicillinase-functionalized TMV nanotubes was realized as model system.The fabricated sensor has been electrochemically characterized by capacitancevoltage and constant-capacitance measurements.The sensor surface morphology with TMV nanotubes was physically investigated by scanning electron microscopy and atomic force microscopy.The results of this study demonstrate the promising potential of virus particles as universal scaffolds for the immobilization of biomolecules on sensor surfaces and for the development of highly sensitive enzyme biosensors.

A new concept for the development of semiconductor field-effect biosensors by modification of a gate surface with tobacco mosaic virus (TMV) nanotubes, serving as enzyme nanocarrier, is presented. TMV nanotubes enable an immobilization of a high amount of enzymes without substantial loss of their activity, resulting in an enhanced biosensor performance. This approach has been experimentally demonstrated by realizing a capacitive field-effect penicillin biosensor using TMV nanotubes functionalized with the enzyme penicillinase as model system.

A new approach for the development of electrochemical biosensors using tobacco mosaic virus (TMV) nanotubes and coat protein (CP) aggregates as enzyme nanocarriers is presented. The generic approach has been demonstrated by realizing amperometric glucose sensors combining an array of Pt electrodes loaded with glucose oxidase (GOD)-modified TMV nanotubes and CPs as a model system. The presence of TMV nanotubes or CPs on the sensor surface allows binding of a high amount of enzymes without substantial loss of their activity. The obtained results underline the potential of integrating biomolecule/virus hybrids with electronic transducers for applications in biosensorics.

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

We introduce an extremely simple and highly stable system for stimulated Raman scattering (SRS) microscopy. An 8-W, 450-fs Yb:KGW bulk oscillator with 41 MHz repetition rate pumps an optical parametric amplifier, which is seeded by a cw tunable external cavity diode laser. The output radiation is frequency doubled in a long PPLN crystal and generates 1.5-ps long narrowband pump pulses that are tunable between 760 and 820 nm with >50 mW average power. Part of the oscillator output is sent through an etalon and creates Stokes pulses with 100 mW average power and 1.7 ps duration. We demonstrate SRS microscopy at a 30-μs pixel dwell time with high chemical contrast, signal-to-noise ratio in excess of 45 and no need for balanced detection, thanks to the favorable noise properties of the bulk solid-state system. Cw seeding intrinsically ensures low spectral drift. We discuss its application to chemical contrast microscopy of freshly prepared plant tissue sections at different vibrational bands.

The conjunction of (bio-)chemical recognition elements with nanoscale biological building blocks such as virus particles is considered as a very promising strategy for the creation of biohybrids opening novel opportunities for label-free biosensing. This work presents a new approach for the development of biosensors using tobacco mosaic virus (TMV) nanotubes or coat proteins (CPs) as enzyme nanocarriers. Sensor chips combining an array of Pt electrodes loaded with glucose oxidase (GOD)-modified TMV nanotubes or CP aggregates were used for amperometric detection of glucose as a model system for the first time. The presence of TMV nanotubes or CPs on the sensor surface allows binding of a high amount of precisely positioned enzymes without substantial loss of their activity, and may also ensure accessibility of their active centers for analyte molecules. Specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CPs was achieved via bioaffinity binding. These layouts were tested in parallel with glucose sensors with adsorptively immobilized [SA]-GOD, as well as [SA]-GOD crosslinked with glutardialdehyde, and came out to exhibit superior sensor performance. The achieved results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for future applications in biosensorics and biochips. (C) 2016 Elsevier B.V. All rights reserved.

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.