Soft Sub-Structured Multi-Material Biosensor Hydrogels with Enzymes Retained by Plant Viral Scaffolds.

An all-soft multi-material combination consisting of a hydrogel based on poly(ethylene glycol) (PEG) coated with spatially defined spots of gelatin methacryloyl (GM) containing selectively addressable viral nanorods is presented, and its basic application as a qualitative biosensor with reporter enzymes displayed on the tobacco mosaic virus (TMV) bioscaffolds within the GM is demonstrated. Biologically inert PEG supports are equipped with GM spots serving as biological matrix for enzymes clustered on TMV particles preventing diffusion out of the gel. For this multi-material combination, i) the PEG-based hydrogel surface is modified to achieve a clear boundary between coated and non-coated regions by introducing either isothiouronium or thiol groups. ii) Cross-linking of the GM spots is studied to achieve anchoring to the hydrogel surface. iii) The enzymes horseradish peroxidase or penicillinase (Pen) are conjugated to TMV and integrated into the GM matrix. In contrast to free enzymes, enzyme-decorated TMVs persist in GM spots and show sustained enzyme activity as evidenced by specific color reaction after 7 days of washing, and for Pen after 22 months after dry storage. Therefore, the integration of enzyme-coupled TMV into hydrogel matrices is a promising and versatile approach to obtaining reusable and analyte-specific sensor components.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System.

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.

Differential Effects of RNA-Dependent RNA Polymerase 6 (RDR6) Silencing on New and Old World Begomoviruses in Nicotiana benthamiana.

RNA-dependent RNA polymerases (RDRs) are key players in the antiviral defence mediated by RNA silencing in plants. RDR6 is one of the major components of the process, regulating the infection of certain RNA viruses. To better clarify its function against DNA viruses, we analyzed the effect of RDR6 inactivation (RDR6i) in N. benthamiana plants on two phloem-limited begomoviruses, the bipartite Abutilon mosaic virus (AbMV) and the monopartite tomato yellow leaf curl Sardinia virus (TYLCSV). We observed exacerbated symptoms and DNA accumulation for the New World virus AbMV in RDR6i plants, varying with the plant growth temperature (ranging from 16 °C to 33 °C). However, for the TYLCSV of Old World origin, RDR6 depletion only affected symptom expression at elevated temperatures and to a minor extent; it did not affect the viral titre. The accumulation of viral siRNA differed between the two begomoviruses, being increased in RDR6i plants infected by AbMV but decreased in those infected by TYLCSV compared to wild-type plants. In situ hybridization revealed a 6.5-fold increase in the number of AbMV-infected nuclei in RDR6i plants but without egress from the phloem tissues. These results support the concept that begomoviruses adopt different strategies to counteract plant defences and that TYLCSV evades the functions exerted by RDR6 in this host.

Convenient site-selective protein coupling from bacterial raw lysates to coenzyme A-modified tobacco mosaic virus (TMV) by Bacillus subtilis Sfp phosphopantetheinyl transferase.

A facile enzyme-mediated strategy enables site-specific covalent one-step coupling of genetically tagged luciferase molecules to coenzyme A-modified tobacco mosaic virus (TMV-CoA) both in solution and on solid supports. Bacillus subtilis surfactin phosphopantetheinyl transferase Sfp produced in E. coli mediated the conjugation of firefly luciferase N-terminally extended by eleven amino acids forming a 'ybbR tag' as Sfp-selective substrate, which even worked in bacterial raw lysates. The enzymes displayed on the protein coat of the TMV nanocarriers exhibited high activity. As TMV has proven a beneficial high surface-area adapter template stabilizing enzymes in different biosensing layouts in recent years, the use of TMV-CoA for fishing ybbR-tagged proteins from complex mixtures might become an advantageous concept for the versatile equipment of miniaturized devices with biologically active proteins. It comes along with new opportunities for immobilizing multiple functionalities on TMV adapter coatings, as desired, e.g., in handheld systems for point-of-care detection.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1-3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Enzymatic activity of individual bioelectrocatalytic viral nanoparticles: dependence of catalysis on the viral scaffold and its length.

The enzymatic activity of tobacco mosaic virus (TMV) nanorod particles decorated with an integrated electro-catalytic system, comprising the quinoprotein glucose-dehydrogenase (PQQ-GDH) enzyme and ferrocenylated PEG chains as redox mediators, is probed at the individual virion scale by atomic force microscopy-scanning electrochemical atomic force microscopy (AFM-SECM). A marked dependence of the catalytic activity on the particle length is observed. This finding can be explained by electron propagation along the viral backbone, resulting from electron exchange between ferrocene moieties, coupled with enzymatic catalysis. Thus, the use of a simple 1D diffusion/reaction model allows the determination of the kinetic parameters of the virus-supported enzyme. Comparative analysis of the catalytic behavior of the Fc-PEG/PQQ-GDH system assembled on two differing viral scaffolds, TMV (this work) and bacteriophage-fd (previous work), reveals two distinct kinetic effects of scaffolding: An enhancement of catalysis that does not depend on the virus type and a modulation of substrate inhibition that depends on the virus type. AFM-SECM detection of the enzymatic activity of a few tens of PQQ-GDH molecules, decorating a 40 nm-long viral domain, is also demonstrated, a record in terms of the lowest number of enzyme molecules interrogated by an electrochemical imaging technique.

The Effect of Pooling on the Detection of the Nucleocapsid Protein of SARS-CoV-2 with Rapid Antigen Tests.

The COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen. The holder of the device can be produced locally through 3D printing. The extraction and the elution can be performed with the entire set-up encapsulated in a transparent bag, minimizing the risk of infection for the operator. With 0.35 mL extraction buffer and six swabs, including a positive control swab, 43 ± 6% (n = 8) of the signal for an individual extraction of a positive control standard was obtained. Image analysis still showed a signal-to-noise ratio of approximately 2:1 at 32-fold dilution of the extract from a single positive control swab. The relative signal from the test line versus the control line was found to scale linearly upon dilution (R2 = 0.98), indicating that other pooling regimes are conceivable. A pilot project involving 14 participants and 18 pooled tests in a laboratory course at our university did not give any false positives, and an individual case study confirmed the ability to detect a SARS-CoV-2 infection with five-fold or six-fold pooling, including one swab from a PCR-confirmed COVID patient. These findings suggest that pooling can make frequent testing more affordable for schools, universities, and similar institutions, without decreasing sensitivity to an unacceptable level.

Detection of plant virus particles with a capacitive field-effect sensor.

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.

Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte's pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.

Capacitive Field-Effect Biosensor Studying Adsorption of Tobacco Mosaic Virus Particles.

Plant virus-like particles, and in particular, tobacco mosaic virus (TMV) particles, are increasingly being used in nano- and biotechnology as well as for biochemical sensing purposes as nanoscaffolds for the high-density immobilization of receptor molecules. The sensitive parameters of TMV-assisted biosensors depend, among others, on the density of adsorbed TMV particles on the sensor surface, which is affected by both the adsorption conditions and surface properties of the sensor. In this work, Ta2O5-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption. The impact of the TMV concentration on both the sensor signal and the density of TMV particles adsorbed onto the Ta2O5-gate surface has been studied systematically by means of field-effect and scanning electron microscopy methods. In addition, the surface density of TMV particles loaded under different incubation times has been investigated. Finally, the field-effect sensor also demonstrates the label-free detection of penicillinase immobilization as model bioreceptor on TMV particles.

Field-Effect Sensors for Virus Detection: From Ebola to SARS-CoV-2 and Plant Viral Enhancers.

Coronavirus disease 2019 (COVID-19) is a novel human infectious disease provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific vaccines or drugs against COVID-19 are available. Therefore, early diagnosis and treatment are essential in order to slow the virus spread and to contain the disease outbreak. Hence, new diagnostic tests and devices for virus detection in clinical samples that are faster, more accurate and reliable, easier and cost-efficient than existing ones are needed. Due to the small sizes, fast response time, label-free operation without the need for expensive and time-consuming labeling steps, the possibility of real-time and multiplexed measurements, robustness and portability (point-of-care and on-site testing), biosensors based on semiconductor field-effect devices (FEDs) are one of the most attractive platforms for an electrical detection of charged biomolecules and bioparticles by their intrinsic charge. In this review, recent advances and key developments in the field of label-free detection of viruses (including plant viruses) with various types of FEDs are presented. In recent years, however, certain plant viruses have also attracted additional interest for biosensor layouts: Their repetitive protein subunits arranged at nanometric spacing can be employed for coupling functional molecules. If used as adapters on sensor chip surfaces, they allow an efficient immobilization of analyte-specific recognition and detector elements such as antibodies and enzymes at highest surface densities. The display on plant viral bionanoparticles may also lead to long-time stabilization of sensor molecules upon repeated uses and has the potential to increase sensor performance substantially, compared to conventional layouts. This has been demonstrated in different proof-of-concept biosensor devices. Therefore, richly available plant viral particles, non-pathogenic for animals or humans, might gain novel importance if applied in receptor layers of FEDs. These perspectives are explained and discussed with regard to future detection strategies for COVID-19 and related viral diseases.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus Begomovirus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer. For Abutilon mosaic virus (AbMV), phosphorylation of MP expressed in bacteria, yeast, and Nicotiana benthamiana plants, respectively, has been demonstrated in previous studies. Three phosphorylation sites (T221, S223, and S250) were identified in its C-terminal oligomerization domain by mass spectrometry, suggesting a regulation of MP by posttranslational modification. To examine the influence of the three sites on the self-interaction in more detail, MP mutants were tested for their interaction in yeast by two-hybrid assays, or by Förster resonance energy transfer (FRET) techniques in planta. Expression constructs with point mutations leading to simultaneous (triple) exchange of T221, S223, and S250 to either uncharged alanine (MPAAA), or phosphorylation charge-mimicking aspartate residues (MPDDD) were compared. MPDDD interfered with MP-MP binding in contrast to MPAAA. The roles of the phosphorylation sites for the viral life cycle were studied further, using plant-infectious AbMV DNA-B variants with the same triple mutants each. When co-inoculated with wild-type DNA-A, both mutants infected N. benthamiana plants systemically, but were unable to do so for some other plant species of the families Solanaceae or Malvaceae. Systemically infected plants developed symptoms and viral DNA levels different from those of wild-type AbMV for most virus-plant combinations. The results indicate a regulation of diverse MP functions by posttranslational modifications and underscore their biological relevance for a complex host plant-geminivirus interaction.

From stars to stripes: RNA-directed shaping of plant viral protein templates-structural synthetic virology for smart biohybrid nanostructures.

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.

Hydrophobization of Tobacco Mosaic Virus to Control the Mineralization of Organic Templates.

The robust, anisotropic tobacco mosaic virus (TMV) provides a monodisperse particle size and defined surface chemistry. Owing to these properties, it became an excellent bio-template for the synthesis of diverse nanostructured organic/inorganic functional materials. For selective mineralization of the bio-template, specific functional groups were introduced by means of different genetically encoded amino acids or peptide sequences into the polar virus surface. An alternative approach for TMV surface functionalization is chemical coupling of organic molecules. To achieve mineralization control in this work, we developed a synthetic strategy to manipulate the surface hydrophilicity of the virus through covalent coupling of polymer molecules. Three different types of polymers, namely the perfluorinated (poly(pentafluorostyrene) (PFS)), the thermo-responsive poly(propylene glycol) acrylate (PPGA), and the block-copolymer polyethylene-block-poly(ethylene glycol) were examined. We have demonstrated that covalent attachment of hydrophobic polymer molecules with proper features retains the integrity of the virus structure. In addition, it was found that the degree of the virus hydrophobicity, examined via a ZnS mineralization test, could be tuned by the polymer properties.

Plant virus-based materials for biomedical applications: Trends and prospects.

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.

Precise Assembly of Genetically Functionalized Magnetosomes and Tobacco Mosaic Virus Particles Generates a Magnetic Biocomposite.

Magnetosomes represent magnetic nanoparticles with unprecedented characteristics. Both their crystal morphology and the composition of the enveloping membrane can be manipulated by genetic means, allowing the display of functional moieties on the particle surface. In this study, we explore the generation of a new biomaterial assembly by coupling magnetosomes with tobacco mosaic virus (TMV) particles, both functionalized with complementary recognition sites. TMV consists of single-stranded RNA encapsidated by more than 2100 coat proteins, which enable chemical modification via functional groups. Incubation of EmGFP- or biotin-decorated TMV particles with magnetosomes genetically functionalized with GFP-binding nanobodies or streptavidin, respectively, results in the formation of magnetic, mesoscopic, strand-like biocomposites. TMV facilitates the agglomeration of magnetosomes by providing a scaffold. The size of the TMV-magnetosome mesostrands can be adjusted by varying the TMV-magnetosome particle ratios. The versatility of this novel material combination is furthermore demonstrated by coupling magnetosomes and terminal, 5'-functionalized TMV particles with high molecular precision, which results in "drumstick"-like TMV-magnetosome complexes. In summary, our approaches provide promising strategies for the generation of new biomaterial assemblies that could be used as scaffold for the introduction of further functionalities, and we foresee a broad application potential in the biomedical and biotechnological field.

TMV Particles: The Journey From Fundamental Studies to Bionanotechnology Applications.

Ever since its initial characterization in the 19th century, tobacco mosaic virus (TMV) has played a prominent role in the development of modern virology and molecular biology. In particular, research on the three-dimensional structure of the virus particles and the mechanism by which these assemble from their constituent protein and RNA components has made TMV a paradigm for our current view of the morphogenesis of self-assembling structures, including viral particles. More recently, this knowledge has been applied to the development of novel reagents and structures for applications in biomedicine and bionanotechnology. In this article, we review how fundamental science has led to TMV being at the vanguard of these new technologies.

Bottom-Up Assembly of TMV-Based Nucleoprotein Architectures on Solid Supports.

RNA-guided self-assembly of tobacco mosaic virus (TMV)-like nucleoprotein nanotubes is possible using 3'-terminally surface-linked scaffold RNAs containing the viral origin of assembly (OAS). In combination with TMV coat protein (CP) preparations, these scaffold RNAs can direct the growth of selectively addressable multivalent carrier particles directly at sites of interest on demand. Serving as adapter templates for the installation of functional molecules, they may promote an integration of active units into miniaturized technical devices, or enable their presentation on soft-matter nanotube systems at high surface densities advantageous for, for example, biodetection or purification applications. This chapter describes all procedures essential for the bottom-up fabrication of "nanostar" colloids with gold cores and multiple TMV-like arms, immobilized in a programmable manner by way of hybridization of the RNA scaffolds to oligodeoxynucleotides exposed on the gold beads.

Bioinspired Silica Mineralization on Viral Templates.

Plant virus capsids are attractive entities for nanotechnological applications because of their variation in shape and natural assembly ability. This chapter describes the production and modification of three differently shaped plant virus capsids for silica mineralization purposes. The chosen plant viruses exhibit either an icosahedral (cowpea mosaic virus, CPMV), or a flexuous rod-like structure (potato virus X, PVX), or a rigid rod-like shape (tobacco mosaic virus, TMV), and are well-known and frequently used plant viruses for biotechnological applications. We describe the production (including genetic or chemical modification) and purification of the plant viruses or of empty virus-like particles in the case of CPMV, as well as the characterization of these harvested templates. The mineralization procedures and differences in the protocols specific to the distinct viruses are described, and the analyses of the mineralization results are explained.

Dual Functionalization of Rod-Shaped Viruses on Single Coat Protein Subunits.

Plant viruses are emerging as versatile tools for nanotechnology applications since it is possible to modify their multivalent protein surfaces and thereby introduce and display new functionalities. In this chapter, we describe a tobacco mosaic virus (TMV) variant that exposes two selectively addressable amino acid moieties on each of its 2130 coat protein (CP) subunits. A lysine as well as a cysteine introduced at accessible sites of every CP can be modified with amino- and/or thiol-reactive chemistry such as N-hydroxysuccinimide esters (NHS ester) and maleimide containing reagents alone or simultaneously. This enables the pairwise immobilization of distinct molecules in close vicinity to each other on the TMV surface by simple standard conjugation protocols. We describe the generation of the mutations, the virus propagation and isolation as well as the dual functionalization of the TMV variant with two fluorescent dyes. The labeling is evaluated by SDS-PAGE and spectrophotometry and the degree of labeling (DOL) calculated.