Seminarium Oddziału Spektroskopii Optycznej
sala 6 (bud. VIII)
Electrophoretic purification and characterization of single antibody modified photon-upconverting nanoparticles
dr Antonin Hlaváček
Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Single molecule methods (SMMs) have unraveled details on the dynamics of biomolecular interactions, conformational states and subpopulations of proteins and other biomolecules that are hidden in classical bulk experiments. [1,2] Furthermore, SMMs allowed for reaching the ultimate limit of detection of a single molecule. Fluorescence microscopy of luminescence-labeled biomolecules is one of the most sensitive SMM. The limited availability of bright and photostable labels, however, are great challenges for SMMs. Photon-upconverting nanoparticles (UCNPs) with their capability to be excited by near-infrared light and emit light of shorter wavelengths (anti-Stokes emission) belong to new generation of luminescence label for SMMs. [3] While the physicochemical properties of nanoparticle surfaces have been studied in detail, the properties of more complex bioconjugates, which are needed for SMMs, are less understood. For instance, the control of aggregation and the number of attached biomolecules is needed for better understanding of their properties.
For the first time, we have used gel electrophoresis for the characterization of silica-coated UCNPs and their bioconjugates. [4] The inevitable presence of monomers, dimers, trimers and higher oligomers in the samples of as prepared silica coated UCNPs was reported. Preparative gel electrophoresis with continuous elution and upconversion luminescence flow detector were used to produce truly monodisperse (monomeric) silica coated UCNPs with a diameter of 10 nm. Those purified particles were used for bioconjugation of discrete antibody molecules onto the surface of UCNPs. Monoclonal mouse antibody with specificity towards human serum albumin was used as a model system in this study. Fractions of UCNPs substituted by one, two and three antibody molecules were separated. The prepared well defined UCNP-antibody conjugates will allow for studies of specific interactions (antibody-antigen) and non-specific interactions (protein “corona” formation). The understanding of these properties is crucial for single biomolecule tracking experiments, single molecule visualization and single biomolecule interaction studies.
Literature:
[1] Tracking single molecules at work in living cells. Kusumi, A., Tsunoyama, T.A., Hirosawa, K.M., Kasai, R.S., Fujiwara, T.K. Nat. Chem. Biol. 2014, 10, 524.
[2] Analytical chemistry on the femtoliter scale. Gorris, H.H., Walt, D.R. Angew. Chem. Int. Ed. 2010, 49, 3880.
[3] Photon-upconverting nanoparticles for optical encoding and multiplexing of cells, biomolecules, and microspheres. Gorris, H.H., Wolfbeis, O.S. Angew. Chem. Int. Ed. 2013, 52, 3584.
[4] Electrophoretic characterization and purification of silica-coated photon-upconverting nanoparticles and their bioconjugates. Hlaváček, A., Sedlmeier, A., Skládal, P., Gorris, H.H. ACS Appl. Mater. Interfaces 2014, 6, 6930.
