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High throughput mass spectrometry of single proteins in liquid environment

Periodic Reporting for period 4 - LIQUIDMASS (High throughput mass spectrometry of single proteins in liquid environment)

Reporting period: 2021-05-01 to 2022-10-31

Although mass spectrometry has brought about major advancements in proteomics in the last decade, protein mass spectrometers still have important limitations. One fundamental limitation is that they require sample ionization, desorption into the gas phase and fragmentation, clearly leading to protein denaturation. Since relevant protein complexes are unstable or transient, their characterization in its native state and physiological environment remains an unexplored route towards the full understanding of protein function and protein interactions. In Liquidmass, we envisioned that a high throughput characterization technology capable of addressing single proteins in its native state would have a large impact in proteomics. We have successfully developed in LIQUIDMASS a high throughput spectrometric technique able to address single proteins in liquids. We have successfully developed two technologies that are complementary: Technology 1) Hollow transparent resonator sensors and Technology 2) Open nanofluidic resonator sensors based on nanowires and ionic liquids. We have been able to demonstrate the usefulness of the technologies for biomedicine, not only in the initially intended field of proteomics, but also in other fields not initially envisioned, as the classification of healthy and cancerous cells, detection of bacteria and nanoparticle characterization. We expect LIQUIDMASS will have a positive impact in society through the advancement of precision medicine that demands such technologies.
LIQUIDMASS has allowed the team to advance in two fronts towards protein characterization in liquids. Activities have been devoted to the development of two complementary technologies, hollow resonators and silicon nanowire resonators. We have successfully developed both technologies: Technology 1) Hollow transparent resonator sensors and Technology 2) Open nanofluidic resonator sensors based on nanowires and ionic liquids. We have patented both technologies and the results have afterwards been disseminated to scientists and the general public. We have demonstrated applicability in biomedicine of the first technology in fields not envisioned in the DoA, in the classification of cancerous cells (ACS Sens. 12, 3325, 2019), detection of bacteria from liquid samples (licensing agreement with Company Nanological, Nat. Nanotechnol. 15, 469, 2020) and nanoparticle characterization (Sci Rep 11, 3535, 2021). The second technology has provided the first mass flow controller and sensor for open nanofluidic channels (registered pending patent1). We have also developed novel optomechanical transduction methods for these devices. Optomechanical transduction of vertical nanowires has been attained for the first time in Liquidmass (Nano Lett. 20, 4, 2359, 2020) and large dynamic range nanowires designed, fabricated and characterized (Nano Lett. 21, 15, 6617, 2021). These advancements have been very relevant for the application of the technology in biomedicine, as we can address now large arrays of vertical nanowires having a large dynamic range for mass detection of a wide range of nanoparticles, as viruses or biomolecules. We have simulated the transport of proteins along the open nanofluidic devices based on nanowires and ionic liquids and separation of proteins has been demonstrated in these simulations. As an alternative to the initially envisioned nanowires, we have also developed membrane resonator devices for the analysis of nanoparticles and protected the technology and methods through registered pending patent2.
LIQUIDMASS was proposing the development of technologies based in innovative concepts merging highly sensitive nano mechanical sensors and liquid transport. The LIQUIDMASS team has worked intensively in the integration of nanomechanical resonators, nano-optics and nanofluidics. The disruptive approach proposed has brought about new knowledge and new technologies that will impact diverse fields, from nano mechanics to proteomics, oncology and microbiology fields. We have developed novel methodologies for the fabrication of long nanowires with a high dynamic range, optimized for the biomedical applications envisioned. This methodology has been protected through a pending divisional patent. We have also developed a novel methodology for the classification of cancerous and healthy cells from the same tissue type and label-free with sensitivity of 80% and precision of 76%. This had never been achieved before by nanomechanical sensors. This technology and the associated methodology has been protected through PCT/ES2021/070765. This patent has been licensed to the spin-off Nanological and this Company will use the devices for an unforeseen application in the Project proposal: detection of bacteria from patient samples for sepsis diagnosis.
We have developed a novel methodology for the transport of liquids along nanowires that has been protected by a pending patent.
Nanomechanical sensors based on silicon nanowires for proteomics