15 min to get an antibody layer. At this point the gold surfaces are ready to detect the
antigens [41]. In Fig. 11, a schematic representation of the functionalization of gold
surfaces with the method above descripted is shown.
In addition, the crystallizable fragment (Fc)-binding proteins such as protein A,
protein G, and protein A/G can be employed for an oriented immobilization of
antibodies on the gold-coated QCM surface such that their antigen-binding sites (Fab
region) are completely free for binding antigens. Protein A and Protein G are small
proteins, derived from bacteria, which can specifically bind the Fc portion of
antibodies allowing oriented systems to be obtained [42]. The IgG binding domain
of Protein A, known as the Z-domain or ZZ-domain, is also used as a smaller
synthetic option for Fc binding. This technique offers a method for truly obtaining
oriented antibodies as binding can only occur via the Fc portion. Due to its
effectiveness, protein A, or its derivatives, has been exploited with many surface
immobilization strategies including biotin–streptavidin [43], SAMs [25, 26],
EDC/NHS chemistry [44–46], glutaraldehyde [47], tyrosinase chemistry [47],
non-natural amino acid insertion, gold-binding peptide or polystyrene affinity ligand
fusion, and additional protein linkers [48]. It is worth to note that several issues may
arise:
• the Protein A capture of the Fc is reversible, protein A has been reported to bind
Fab regions and albumin (although to a much lesser extent);
• it is required that the Fc binding site of the Protein A is correctly oriented at the
substrate to permit antibody binding [49].
Another method to immobilize the IgG antibodies onto gold surface is by using
the interactions between thiols and gold, which are very strong and have been
exploited for antibody binding by employing self-assembled layers of thiols and
sulfides. One approach for site-oriented immobilization of immunoglobulins onto
the gold supports consists in using the native immunoglobulin thiol groups, which
are free after the splitting of the intact antibody into two half-IgG fragments without
the destruction of the binding site of the antibody (Fig. 12). The half-IgG fragments
can be immobilized onto gold supports by simple adsorption. The proposed
approach is advantageous over existing methods because the immobilized antibodies
maintain both high antigen-binding constants and high stability.
Another very elegant technique called photochemical immobilization technique
(PIT) consists in a light-assisted approach for Ab immobilization and was adopted
by Della Ventura et al. [51, 52]. Disulfide bonds are broken upon absorption of UV
light by nearby aromatic amino acids, yielding reactive thiol groups that are effective
for oriented binding onto gold electrodes. A working scheme of this technique is
shown in Fig. 13.
The authors affirm that PIT preserves the native structure and the functional
properties of the immobilized proteins while favoring the proper orientation of the
biomolecule on the support. This is achieved by avoiding any chemical and thermal
treatment. One of the advantages of PIT relies in the wide field of application since
the closely spaced triad of residues Trp/Cys-Cys is present in all members of the
immunoglobulin superfamily. Every IgG has 12 intradomain disulfide bridges near a
Quartz Crystal Microbalance Sensors: New Tools for the Assessment of. . .
333
antigens [41]. In Fig. 11, a schematic representation of the functionalization of gold
surfaces with the method above descripted is shown.
In addition, the crystallizable fragment (Fc)-binding proteins such as protein A,
protein G, and protein A/G can be employed for an oriented immobilization of
antibodies on the gold-coated QCM surface such that their antigen-binding sites (Fab
region) are completely free for binding antigens. Protein A and Protein G are small
proteins, derived from bacteria, which can specifically bind the Fc portion of
antibodies allowing oriented systems to be obtained [42]. The IgG binding domain
of Protein A, known as the Z-domain or ZZ-domain, is also used as a smaller
synthetic option for Fc binding. This technique offers a method for truly obtaining
oriented antibodies as binding can only occur via the Fc portion. Due to its
effectiveness, protein A, or its derivatives, has been exploited with many surface
immobilization strategies including biotin–streptavidin [43], SAMs [25, 26],
EDC/NHS chemistry [44–46], glutaraldehyde [47], tyrosinase chemistry [47],
non-natural amino acid insertion, gold-binding peptide or polystyrene affinity ligand
fusion, and additional protein linkers [48]. It is worth to note that several issues may
arise:
• the Protein A capture of the Fc is reversible, protein A has been reported to bind
Fab regions and albumin (although to a much lesser extent);
• it is required that the Fc binding site of the Protein A is correctly oriented at the
substrate to permit antibody binding [49].
Another method to immobilize the IgG antibodies onto gold surface is by using
the interactions between thiols and gold, which are very strong and have been
exploited for antibody binding by employing self-assembled layers of thiols and
sulfides. One approach for site-oriented immobilization of immunoglobulins onto
the gold supports consists in using the native immunoglobulin thiol groups, which
are free after the splitting of the intact antibody into two half-IgG fragments without
the destruction of the binding site of the antibody (Fig. 12). The half-IgG fragments
can be immobilized onto gold supports by simple adsorption. The proposed
approach is advantageous over existing methods because the immobilized antibodies
maintain both high antigen-binding constants and high stability.
Another very elegant technique called photochemical immobilization technique
(PIT) consists in a light-assisted approach for Ab immobilization and was adopted
by Della Ventura et al. [51, 52]. Disulfide bonds are broken upon absorption of UV
light by nearby aromatic amino acids, yielding reactive thiol groups that are effective
for oriented binding onto gold electrodes. A working scheme of this technique is
shown in Fig. 13.
The authors affirm that PIT preserves the native structure and the functional
properties of the immobilized proteins while favoring the proper orientation of the
biomolecule on the support. This is achieved by avoiding any chemical and thermal
treatment. One of the advantages of PIT relies in the wide field of application since
the closely spaced triad of residues Trp/Cys-Cys is present in all members of the
immunoglobulin superfamily. Every IgG has 12 intradomain disulfide bridges near a
Quartz Crystal Microbalance Sensors: New Tools for the Assessment of. . .
333