Ακινητοποίηση καρκινικών πεπτιδικών επίτοπων σε επιφάνεια βιοαισθητήρα: μελέτη της αλληλεπίδρασης των HLA-A2 μορίων με το ακινητοποιημένο στην επιφάνεια πεπτίδιο χρησιμοποιώντας ολόκληρα κύτταρα

The focus of this work was the development of a peptide-specific biorecognition surface based on self-assemble monolayers (SAMs) on gold containing mixtures of hydroxyl and carboxyl terminated alkanethiols. This surface was used for the detection of whole cell bound receptor/peptide interaction. The receptor/peptide pair was the membrane-bound HLA-A2 molecule and the antigenic nine amino-acid peptide (RLLCETELV), respectively. The detection of the binding was measured with different types of biosensors based on optical and acoustic waves. Results demonstrated specific interaction between HLA-A2 molecules/surface-immobilized peptide, and some degree of no specific binding of the cells to the surface. Biosensors are devices that combine a biochemical recognition element (ligand) with a signal conversion unit (transducer). Biosensors are mostly designed for routine analysis, such as clinical diagnosis, quality control of food, in-process control of fermentations, in environmental analysis and pharmaceuticals industry. Biosensors are used for the investigation of the interaction between proteins and biological elements in a routine basis. However, there are only few references about biosensors that can detect whole cells/ligand interactions. In this study, we tried to develop a biorecognition surface for the investigation of the interaction between HLA-A2 molecules attached on whole cells membrane, and the nine amino-acid peptide RLLCETELV, a tumor peptide epitope with an amino acid substitution (Q to C) to allow immobilization via the cysteine residue. HLA-A2 molecules belong to Class I major histocompatibility Complex (MHC) proteins which play a major role in antigen presentation and regulation of immune responses. Their function is to present short antigenic peptides (8-11 residues), derived from viral or altered proteins, to T lymphocytes. Two types of biosensors were used for detecting cells binding; one optical system based on surface plasmon resonance (SPR) and two acoustic biosensors (SAW, QCM). These techniques can measure biomolecular interactions in real-time in a label free environment. SPR direct detection is based on the difference in refractive index between water and biological molecules. The detection of acoustic biosensors is related to the velocity and energy of the wave in the sensing medium. In the case of Surface Acoustic Device (SAW), velocity and energy changes are measured as phase and amplitude, respectively4. In the case of Quartz Crystal Microbalance (QCM) the above are recorded as frequency and dissipation. Self-assemble monolayers (SAMs) containing mixtures of hydroxyl and carboxyl terminated alkanethiols were deposited on the gold-coated sensors surface and subsequently were used to produce an active surface for the covalent immobilization of the peptides6. Cells were added under flow above the surface and the interaction between HLA-A2 molecules and peptide was measured in real time through monitoring of the biosensor signal. SPR experiments demonstrated that the cells caused large changes in the refractive index in positive as well as in negative experiments. This did not allow the estimation of the interaction of HLA-A2 molecules with the peptide because of the bulk effect of the cell mass. QCM positives and negatives experiments, displayed similar kinetic pattern. However the negatives caused lower signal change (50%). This difference could be correlated with the HLA-A2 molecules/peptide interaction. In addition, at negative control experiments, there is no further change in frequency and in dissipation energy after the injection of the loading buffer, indicating that the interaction of HLA-A2 molecules with the peptides could lead to cell adhesion. The SAW device, could be appropriate for the detection due to the fact that it could sense changes occurring in a distance 50nm from the device surface. Thus the mass of the cells doesn’t affect the signal. Unfortunately, the potential of this biosensor, did not give the desire results. The composition of the surface and wettability were an important aspect in this study. In every case and in every biosensor system the cells were found to interact non specifically with the surface. This fact represented a major problem at the data analysis. So, it is important to investigate the affinity of the cells for different surfaces in order to optimize the biosensor device. Changing of composition of the alkanethiols of SAMs may be the solution