Final Report Summary - VIROMA (Design of a versatile and fast colloidal sensor based on virus modified particles)
The VIROMA project aims to fabricate multiplexed bead-based arrays as sensing devices on the basis of virus capsids and virosomes Layer by Layer-assisted assembly on colloidal carriers. The virosome based platform technology will be suitable for the detection of several analytes ranging from comparatively small molecules, of the size of dioxins to larger biomolecules such as small proteins.
Such systems are fast and sensitive, and require, due to multiplexing and single particle based fluorometric read-outs only a very small amount of sample for analysis and is, suitable for high throughput analysis. The uniqueness of the approach developed in VIROMA is that the virus particles will retain their specific recognition properties provided by the virus capsides and transfer this specificity to the colloidal carrier. Moreover, the possibility of assembling different virus nanoparticles and virosomes on top of the colloids will result in particles with multiple recognition capabilities, going a step ahead nature.
Colloidal particles have been engineered with polyelectrolytes to produce the multiplex systems with multiple fluorescence labels and coated with lipid layers to which immunostimulating reconstituted influenza virosomes (IRIVs) have been fused. The mechanism of IRIVs fusio to artificial supported lipid membranes assembled on polyelectrolyte multilayer on both colloidal particles and planar substrates has been studied in detail. The R18 assay has been used to prove the IRIVs fusion in dependence of pH, temperature and HA concentration. IRIVs display a pH-dependent fusion mechanism, fusing at low pH in analogy to the influenza virus. The pH dependence has been confirmed by the Quartz Crystal Microbalance technique. Atomic Force Microscopy imaging shows that at low pH virosomes are integrated in the supported membrane displaying flattered features and a reduced vertical thickness.
IRIVs entailing anti troponin were fabricated and assembled on supported lipid membranes following the protocols previously developed. The specific recognition properties for cardic tropoinin of the antitroponin carried in the IRIVs fused on supported liped bilayers were tested with the Quartz Crystal Microbalance and flowcytometry.
The knowledge generated throughout the Project has been finallyintegrated in the fabrication of a coloidal sensor for cardiac troponin.
VIROMA achieved for the first time the fabrication of a colloidal device based on LbL engineered beads with supported lipid membranes and fused IRIVs carrying anti troponin for troponin detection. After the binding of the protein to the antitroponin on the beads a detecting fluorescent antibody was added to form a sandwich structure. Finally, the red-out was performed by flow cytometer using a colloidal dispersion of the beads.
The results of VIROMA offer a novel approach for the biofunctionalization of colloidal beads and engineering of interfaces for sensing applications.
The project has involved a synergic collaboration between two academic institutions: CIC biomaGUNE and the University of Leipzig with the company SURFLAY. The project has achieved a close working interaction between the academic and non-academic partners on the basis of the secondments of researchers between both sectors. The intersectoral secondments have contributed to a mutual exchange of ideas and knowledge for the common benefit. The secondment of Early Stage Researchers is a particularly important goal for the consortium in order to strengthen their career development with an industrial experience.
Project website: http://personal.cicbiomagune.es/smoya/viroma/index.html
Project facebook page: www.facebook.com/Viroma-1619629061588317/?ref=hl
Such systems are fast and sensitive, and require, due to multiplexing and single particle based fluorometric read-outs only a very small amount of sample for analysis and is, suitable for high throughput analysis. The uniqueness of the approach developed in VIROMA is that the virus particles will retain their specific recognition properties provided by the virus capsides and transfer this specificity to the colloidal carrier. Moreover, the possibility of assembling different virus nanoparticles and virosomes on top of the colloids will result in particles with multiple recognition capabilities, going a step ahead nature.
Colloidal particles have been engineered with polyelectrolytes to produce the multiplex systems with multiple fluorescence labels and coated with lipid layers to which immunostimulating reconstituted influenza virosomes (IRIVs) have been fused. The mechanism of IRIVs fusio to artificial supported lipid membranes assembled on polyelectrolyte multilayer on both colloidal particles and planar substrates has been studied in detail. The R18 assay has been used to prove the IRIVs fusion in dependence of pH, temperature and HA concentration. IRIVs display a pH-dependent fusion mechanism, fusing at low pH in analogy to the influenza virus. The pH dependence has been confirmed by the Quartz Crystal Microbalance technique. Atomic Force Microscopy imaging shows that at low pH virosomes are integrated in the supported membrane displaying flattered features and a reduced vertical thickness.
IRIVs entailing anti troponin were fabricated and assembled on supported lipid membranes following the protocols previously developed. The specific recognition properties for cardic tropoinin of the antitroponin carried in the IRIVs fused on supported liped bilayers were tested with the Quartz Crystal Microbalance and flowcytometry.
The knowledge generated throughout the Project has been finallyintegrated in the fabrication of a coloidal sensor for cardiac troponin.
VIROMA achieved for the first time the fabrication of a colloidal device based on LbL engineered beads with supported lipid membranes and fused IRIVs carrying anti troponin for troponin detection. After the binding of the protein to the antitroponin on the beads a detecting fluorescent antibody was added to form a sandwich structure. Finally, the red-out was performed by flow cytometer using a colloidal dispersion of the beads.
The results of VIROMA offer a novel approach for the biofunctionalization of colloidal beads and engineering of interfaces for sensing applications.
The project has involved a synergic collaboration between two academic institutions: CIC biomaGUNE and the University of Leipzig with the company SURFLAY. The project has achieved a close working interaction between the academic and non-academic partners on the basis of the secondments of researchers between both sectors. The intersectoral secondments have contributed to a mutual exchange of ideas and knowledge for the common benefit. The secondment of Early Stage Researchers is a particularly important goal for the consortium in order to strengthen their career development with an industrial experience.
Project website: http://personal.cicbiomagune.es/smoya/viroma/index.html
Project facebook page: www.facebook.com/Viroma-1619629061588317/?ref=hl