Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core-Shell Matrixes.

Polymer particles with antibody-like affinity, i.e., molecularly imprinted polymers, offer an ideal platform for biopharmaceutical virus purification. In recent years, attempts combining molecular imprinting technology with a variety of visualization and detection techniques have been reported for directly confirming the localized presence of the template. Direct target visualization is crucial for the characterization of molecularly imprinted polymers, especially if biological templates such as viruses are used. In the present study, for the first time the viral binding behavior at virus-imprinted polymers (VIPs) via stimulated emission depletion (STED) microscopy is shown by imaging individual, fluorescently labeled virus particles. STED microscopy achieves among various other super-resolution techniques the best temporal resolution at high spatial resolution. An innovative virus purification material selective for human adenovirus type 5 (AdV5) offered highly purified virus for the subsequent fluorescent labeling procedure, thus enabling STED imaging. Excellent binding affinities (150-fold higher versus control particles) and high selectivity toward the target virus (AdV5) were observed at those VIPs, even in competitive binding experiments with minute virus of mice using dual-label STED microscopy.

Nanoparticle Tracking of Adenovirus by Light Scattering and Fluorescence Detection.

The detailed characterization of biological nanoparticles is of paramount importance for various industrial sectors, as for production of viral therapeutics. More recently, technologies that allow real-time quantification with simultaneous sizing and determination of surface potentials of virus particles in solution have been developed. In this study, nanoparticle tracking analysis (NTA) was applied to determine the size and the zeta potential of human adenovirus type 5 (AdV5), one the most frequently used therapeutic/oncolytic agents and viral vectors. Virus aggregation was detected, and the kinetics of the dissolution of virus aggregates were studied in real time. In addition, advanced fluorescence detection of AdV5 was performed enabling the measurements in matrices and discrimination of viral subpopulations. It was shown that NTA is an efficient approach for investigating infectious viruses in a live viewing mode. Consequently, NTA provides a promising methodology for virus particle detection and analysis in real time beyond assays requiring nucleic acids or infectivity.

Selective virus capture via hexon imprinting.

An imprinting technique has been developed to generate synthetic polymer beads suitable for selectively binding a supramolecular target. The viral hexon protein, which is the most abundant and accessible surface protein component of the human Adenovirus type 5 (hAdV5) icosahedral capsid, was applied as the template molecule to generate functional polymer beads entailing selectivity for the entire virus. Individual and competitive rebinding studies using two different viruses (i.e. hAdV5 and Minute Virus of Mice - MVM) revealed exquisite selectivity of the imprinted beads for the target hAdV5. Additionally, the morphology of thus imprinted beads was checked via scanning electron microscopy (SEM).

Enhanced Selectivity by Passivation: Molecular Imprints for Viruses with Exceptional Binding Properties.

Inspired by the recognition processes found in biology such as enzyme-substrate and antibody-antigen interactions, synthetic systems with comparable molecular recognition properties have been investigated during recent years based on molecular imprinting strategies. While materials with recognition capabilities for small molecules (i.e., with low molecular weight) have achieved substantial advancements, the synthesis of molecularly imprinted materials with virus recognition properties remains challenging to date. Likewise, protein-surface and protein-protein interactions are essential for a wide variety of biological applications in biotechnology. In biological sensor technology the coating of surfaces to prevent nonspecific adsorption interactions plays an important role. Particularly, polyethylene glycol (PEG) stands out for its high performance in preventing proteins from nonspecifically interactions. However, blocking agents such as the protein bovine serum albumin (BSA) can also be useful as unspecific binding prevention agents for passivation, without modification of the surface. Herein the influence of blocking agents as unspecific reaction components is investigated on the enhancements of selectivity from adenovirus-imprinted particles, whereas adenovirus was used as target species in molecular imprinting. Furthermore, quantitative polymerase chain reaction (qPCR) was used for the first time as virus quantification approach in this context.