Periodic Reporting for period 4 - VIRUSCAN (Optomechanics for Virology)
Período documentado: 2021-05-01 hasta 2022-04-30
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.
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.
As result of VIRUSCAN implementation, we have improved the architecture of the final integrated microfluidic platform based on DLD system and we have advanced in the development of an automated DLD system. The system can efficiently sort beads with of Dc~140nm and we expect to extend this study to virions and implement it on the integrated fluidic platform.
The final prototype has been installed at CSIC laboratory and it has demonstrated a high efficiency in particle transportation and a focusing behavior with gold and polystyrene nanoparticles. The prototype has also demonstrated the soft-lander behaviour by decelerating the particles velocity with an optimized aerolens system. The system was tested with ad-hoc developed optomechanical disk resonator and with microcantilever resonators, demonstrating that multimode weighing of individual nanoparticles can be achieved with these sensors. These devices have been characterized under the vacuum and temperature conditions of Soft-Lander 3 and used for the detection of G-Phage, T5-Phage and 140 nm polystyrene nanoparticle with promising results. We have developed a theoretical framework that can extract the mass, position and stiffness from the relative frequency shifts.
On the other hand, we measured the mass of 70 nm and 100 nm gold nanoparticles and the mass of G-Phage by applying the invers problem algorithm developed during the project. The viral stiffness of bacteriophages (e.g. M13, MS2) and virus-like particles (e.g. NoroVLPs, HPVLPs) have been collected using AFM. We also take viral mechanical properties calculated from AFM nanoindentation measurements as a tool to explore the hidden effects of genome-capsid interactions, non-viral molecule binding, and strain-dependent structure on viruses. We utilized several techniques, amongst others native MS, CDMS and AFM for the characterization of viruses. The results show that while either parameter is not sufficient to differentiate between viruses, differences in solution stability are well reflected in mechanical properties suggesting that distinction between viruses using multiple parameters could be feasible. We have created a virus database that will be available to the public in the project website.
During this project, we have also measured for the first time the vibration of a single bacterial cell with an optomechanical disk resonator. Finally, we have also demonstrated the high-throughput dry mass measurement of bacterial cells.
Two patents have been properly registered, protecting results achieved in the framework of VIRUSCAN.
The final prototype has been installed at CSIC laboratory and it has demonstrated a high efficiency in particle transportation and a focusing behavior with gold and polystyrene nanoparticles. The prototype has also demonstrated the soft-lander behaviour by decelerating the particles velocity with an optimized aerolens system. The system was tested with ad-hoc developed optomechanical disk resonator and with microcantilever resonators, demonstrating that multimode weighing of individual nanoparticles can be achieved with these sensors. These devices have been characterized under the vacuum and temperature conditions of Soft-Lander 3 and used for the detection of G-Phage, T5-Phage and 140 nm polystyrene nanoparticle with promising results. We have developed a theoretical framework that can extract the mass, position and stiffness from the relative frequency shifts.
On the other hand, we measured the mass of 70 nm and 100 nm gold nanoparticles and the mass of G-Phage by applying the invers problem algorithm developed during the project. The viral stiffness of bacteriophages (e.g. M13, MS2) and virus-like particles (e.g. NoroVLPs, HPVLPs) have been collected using AFM. We also take viral mechanical properties calculated from AFM nanoindentation measurements as a tool to explore the hidden effects of genome-capsid interactions, non-viral molecule binding, and strain-dependent structure on viruses. We utilized several techniques, amongst others native MS, CDMS and AFM for the characterization of viruses. The results show that while either parameter is not sufficient to differentiate between viruses, differences in solution stability are well reflected in mechanical properties suggesting that distinction between viruses using multiple parameters could be feasible. We have created a virus database that will be available to the public in the project website.
During this project, we have also measured for the first time the vibration of a single bacterial cell with an optomechanical disk resonator. Finally, we have also demonstrated the high-throughput dry mass measurement of bacterial cells.
Two patents have been properly registered, protecting results achieved in the framework of VIRUSCAN.
VIRUSCAN implementation has allowed to develop all the needed technological advancements to gather current knowledge in optomechanics, NEMs, native mass spectrometry and biophysics to current VIRUSCAN prototype, which still needs some adjustments for being installed in the clinical settings. Nevertheless, the stage of development achieved has allowed to deeply advance in the knowledge of the biophysical properties of the viruses. As result, an extensive database of biophysical properties of virus like particles that will allow clinicians a rapid diagnosis of a large number of viruses has been openly set. The goal of VIRUSCAN beyond this five years and a half it would be achieve the final integration of the whole technology, allowing to detect and identify virions in human samples from their mass and stiffness and asses its infectiveness.
Definitely, VIRUSCAN has led to 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, that could be crucial in a possible future bacterian pandemic due the current problem with the antibiotic resistance, one of the biggest threats to global health, food security, and development today.
Definitely, VIRUSCAN has led to 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, that could be crucial in a possible future bacterian pandemic due the current problem with the antibiotic resistance, one of the biggest threats to global health, food security, and development today.