Covalent bonding, nonspecific methods

To demonstrate the application of these butterfly wing SERS substrates to the problem of protein-binding detection, Garrett et al. devised a protein-binding assay. There are a variety of methods for binding proteins to a metal surface, some of which depend on the amino acid composition of the protein, others of which do not. Direct adsorption via covalent bonds between sulfur groups in proteins and metal surfaces has been used with some success [43] however, this method is unsuitable for use in Raman spectroscopy-based assays, as nonspecific binding may result in a wide variety of conformational orientations of the molecules with respect to the metal s surface. This can lead to Raman spectra that are difficult to reproduce. Not only that, but the analyte may bind to the metal as well as the antibody, which can lead to noisy spectra. 

For immobilization studies to date, two distinct modes of immobilization have been used. The first utilizes nonspecific covalent bonding to CNBr-activated Sepharose via primary amino groups on the antibody molecule. Since there are many of these available on the antibody, this is expected to result in random orientation of antibody molecules on the support. The other method involves linkage through immobilized protein A, a protein which binds immunoglobulins in the structural F portion of the molecule. 

Pinto et al. reported the PDMS surface functionalization for the development of an immune-sensor for salivary cortisol analysis, using three different antibodies immobilization methods immobilization by passive adsorption on pristine PDMS silaniza-tion of PDMS surface with (3-aminopropyl)-triethoxysilane (APTES) to generate amino groups and posterior covalent immobilization of antibodies on APTES-PDMS using crosslinker glutaraldehyde (GA) coating the PDMS surface with BSA to block nonspecific protein adsorption, and then covalent bond of the protein A via GA (Pinto etal.,2015). 

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