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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - VIRUSCAN (Optomechanics for Virology)

Teaser

Summary

The scientific communities gathered around the VIRUSCAN project target the goal of first time development and validation of optonanomechanical systems for spectrometry of viruses, bringing optomechanical devices to a clinical application for the first time. The proposed technology aims to the identification and detection of viruses on clinical samples based on their intrinsic physical properties of viruses, without any dependence on prior amplification by Polymerase Chain Reaction (PCR), nor demand for the development of antibodies for biochemical assays. In particular, the technology shifts the classical paradigm in spectrometry of identification of biological constituents by the mass-to-charge signature towards the identification by two orthogonal physical coordinates: the mass and the stiffness. This concept brings more selectivity and it may provide relevant information on the physical properties of viruses such as the infectivity potential and maturation state.

Current molecular biology techniques for virus detection are continuously challenged by the capability of the RNA viruses to mutate. Therefore, diagnostic tools based on recognition of viral nucleic acid sequences or antibody-based detection of viral antigens may encounter problems due to the virus evolution during epidemics. VIRUSCAN presents an integrated approach for direct biophysical detection of viruses from human samples through the measurement of their physical properties: mass and stiffness. These physical properties, contrary to nucleic acid content, are highly conserved during the virus evolution because they are required for viral fitness. In addition, addressing the stiffness of viruses also opens a new route to assess the infectiveness of viral traces in human samples, which is a difficult objective to tackle with present technologies.

Obj 1. Design and development of optomechanical devices to measure mass and stiffness of single viral particles at 100-1000 Da and 1-100 ppm resolution, respectively, and with 40 dB dynamic range.
Obj 2. Build the most complete to date database of biophysical properties of viruses and understand the relation among virion stiffness and its infective potential.
Obj 3. Isolation and concentration of virions from samples at early stages of infection, which implies low viral loads, in the range of 10 virions/mL.
Obj 4. Combine ion and hydrodynamic guiding for soft-landing of viral particles to a small surface detection area of 100-500 micrometer diameter.
Obj 5. Advanced engineering and integration of the novel technologies into an instrument complying with BSL-II safety standards and benchmarking to actual technologies.

Work performed

Work performed during the second period is summarised as follows:

WP1 has achieved successful cleanroom fabrication of the first DLD modules for virus isolation and concentration. The DLD module has been tested with submicrometric polystyrene beads and has demonstrated the isolation of bacteria from blood samples.

WP2 devoted to particle delivery and soft landing has advanced considerably. A beta prototype has been developed and delivered to partner CSIC, after successful demonstration of superior levels of sensitivity and highly efficient particle focusing of the new design. The RF interface for the mass spectrometer has been constructed and installed in Hamburg (HPI) and a first set of successful experiments has been completed.

WP3 has advanced the design and fabrication of the optomechanical devices. The design of the resonator devices has followed an evolution that now guarantees optimized opto-mechanical performances for sensing of virus-like particles. The design has a sensitivity that can go down to 10-6 of its own mass, allowing for the detection of large viruses, while the collection area is compatible with electro-spraying with the current figures of merit of FASMATECH developments. Optomechanical chips fabricated by UPD were provided to TNO partner for the integration in the prototype.

WP4 has focused in the systems engineering and risk management within this period and the most important functional system requirements have been identified. Also, a short list of particles and viruses has been selected for the proof of concept and a preliminary error budget has been depicted and major risks identified as part of the Viruscan project risk matrix.

In WP5 the method to disentangle the mass, the stiffness, the position and the angle of orientation of a non-symmetric virus from the relative frequency shifts of different vibration modes has been optimized. Also, a software based on the cited models to automatically extract the mass and stiffness of the landing particles from the measured relative frequency shifts has been developed and experiments with nanoparticles and bacteria performed.

In WP6 the work to create a virus library collecting virus mass and stiffness and understand the relation among virion stiffness and its infective potential has advanced considerably. In this period a shortlist of initially chosen viruses as well as a growing set of samples available to the consortium resulting from a close collaboration of SERMAS, RUG and HPI has been studied including on virus-like particles (VLPs) of noroviruses (NoV), human papilloma virus (HPV) and VLPs of ebola virus (EboV).

In WP7 the virus purification and transfer of samples to WP1-6 for validation and proof of principle experiments has been done. The tasks to find a correlation of mass/stiffness parameters with infectivity of viral particles has started with discussions with partner HPI-RUG about the samples to be used to stablish the link between infectivity and stiffness. Selection of a panel of viruses and virus-like VLPs that can be worked in standard BSL1 level laboratories, production of those virus and VLPs and transfer to the other groups to use them as test samples has been done.

In WP8 has continued the dissemination of the project and project results. The project dissemination has already had an impact in the general public with dissemination in mass media. The VIRUSCAN concept of biophysically characterizing viral particles obtained broad approval during dissemination activities of partner HPI on virology conferences.

Final results

VIRUSCAN will allow developing all the needed technological advancements to effectively take current knowledge in optomechanics, NEMs, native mass spectrometry and biophysics to clinical settings. VIRUSCAN will provide an extensive database of biophysical properties of viruses allowing clinicians a rapid diagnosis of a large number of viruses. The five years goal of VIRUSCAN is to detect and identify virus like particles in complex media and virions in human samples from their mass and stiffness and asses its infectiveness.
VIRUSCAN will initiate a radically new line of research and technology development whose transformational impact will not be limited to viral infections but it could also be extended later to other areas such as: nanoparticle/container based drug delivery; study of physical properties of a broad range of naturally occurring and synthetic nanoparticles relevant for materials science, energy and environment research; for food, pharma and agro applications (monitoring medium-large molecules, e.g. enzymes, proteins, etc). Advancements in the mass and stiffness spectrometry approach will nurture those areas in the future, thanks to the breakthrough and pioneering advancements made in VIRUSCAN