Evaluation of SERS labeling of CD20 on CLL cells using optical microscopy and fluorescence flow cytometry

Immunophenotyping of lymphoproliferative disorders depends on the effective measurement of cell surface markers. The inherent light-scattering properties of plasmonic nanoparticles (NPs) combined with recent developments in NP design may confer significant advantages over traditional fluorescence probes. We report and evaluate the use of surface-enhanced Raman scattering (SERS) gold NPs (AuNPs) conjugated to therapeutic rituximab antibodies for selective targeting of CD20 molecules. SERS AuNPs were prepared by adsorbing a Raman-active dye onto the surface of 60 nm spherical AuNPs, coating the particles with 5 kDa polyethylene glycol, and conjugating rituximab to functional groups on polyethylene glycol. The effective targeting of CD20 on chronic lymphocytic leukemia cells by rituximab-conjugated SERS AuNPs was evaluated by dark-field imaging, Raman spectroscopy, and flow cytometry with both competitive binding and fluorescence detection procedures. Evidence of CD20 clustering within approximately 100 nm was observed.From the Clinical EditorThis study discusses the use of surface enhancement Raman scattering (SERS)-based plasmonic gold nanoparticles, which can be used for cell specific labeling. In this example rituximab, a commercially available CD20 humanized monoclonal antibody is used. Dark field imaging, Raman spectroscopy and flow cytometry was utilized to demonstrate the sensitive labeling capability of these gold nanoparticle based hybrid nanodevices.

Graphical AbstractPolymer-coated SERS-active gold nanoparticles bound to anti-CD20 rituximab antibodies (center) specifically targeted CD20 receptor proteins on the surface of B lymphocytes in chronic lymphocytic leukemia samples. The binding of SERS probes to CD20 was detected using Raman spectroscopy to determine the presence of the particles'SERS signature in drops of cell suspension (left). Dark field microscopy was used to image intense Rayleigh scattering of the particles. The 60 nm SERS gold particles appear as green-gold dots bound over the surface of B-cells (right). SERS gold nanoparticle labeling was also compatible with, and had its specificity evaluated by fluorescence flow cytometry.Figure optionsDownload high-quality image (93 K)Download as PowerPoint slide