Phage display as a tool for rapid in vitro cell characterization by fluorescence imaging and Raman spectroscopy

The discovery of new markers for the identification and discrimination of cell types such as neoplastic cells is one of the principal objectives in diagnostics. Using random M13 phage display libraries on the whole U937 cells, we selected a clone, named EIII6, displaying a peptide RKIVHAQTP that preferentially recognizes these cells. The human promonocytic cell line U937 is a model for leukemia, cancer therapeutics and in vitro hematopoietic cell differentiation. Therefore, we directly labeled the phage clone EIII6 with fluo rescein isothiocyanate (FITC) for microscopy imaging application. This method allowed to identify fixed U937 cells, while preserving binding affinity of labeled phage clones. Microscopic observations showed that U937 cells were fluorescently labeled. In order to further investigate the interaction between U937 cells and EIII6, we utilized Raman Spectroscopy. Spectral peaks observed at about 980, 1008, 1185, 1215, 1320,1340,1455, 1589 and 1660 cm- 1 , are commonly assignable to proteins, saccharides and lipids components of U937. These Raman peaks in U937-EIII6 complex are shifted in position or reduced in intensity when compared to U937 alone, suggesting that phage-probe were selectively localized at cell membrane. Thereafter, we realised networks consisting of EIII6 phage clone and Ag-Nanoparticles (AgNPs) as signal reporters in Surface Enhanced Raman Spectroscopy (SERS) to better discriminate U937 cells. With this approach, U937-EIII6 Raman spectral features become sharper and more intense, confirming the selective phage-cell interaction. Both methodological approaches, proposed in this work, allows to quickly and selectively identify U937 and could be extended to the identification of other types of neoplastic cells.