Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy

• Published in: Analyst (London) 2019-11, Vol.144 (23), p.6871-688
• Main Authors: Della Ventura, Bartolomeo, Banchelli, Martina, Funari, Riccardo, Illiano, Anna, De Angelis, Marella, Taroni, Paola, Amoresano, Angela, Matteini, Paolo, Velotta, Raffaele
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies; Antibodies, Immobilized - chemistry; Antibodies, Immobilized - radiation effects; Antibodies, Monoclonal, Murine-Derived - chemistry; Antibodies, Monoclonal, Murine-Derived - radiation effects; Antigens; Binding; Biosensing Techniques - instrumentation; Biosensors; Disulfides; Disulfides - radiation effects; Immobilization; Immunoglobulin Constant Regions - chemistry; Immunoglobulin Constant Regions - radiation effects; Immunoglobulins; Mass spectrometry; Metal Nanoparticles - chemistry; Morphology; Protein Conformation; Raman spectroscopy; Recognition; Scientific imaging; Silver - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis, Raman; Surface Properties; Thiols; Tryptophan; Ultraviolet Rays
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c9an00443b

Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab ( i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications. Thirty seconds of appropriate UV irradiation brings about specific thiol formation in IgGs that allows them to bind "side on" on a thiol reactive surface, thereby making such a photochemical approach a valuable tool for surface functionalization.