Biomedical Engineering Reference
In-Depth Information
0.5 M
OH
Protein
Protein
O
NalO
4
NaCNBH
3
H
2
SO
4
NH
2
-protein
OH
O
N
NH
Epoxy
Diol
Aldehyde
Schiff base
Secondary amine
FIGURE 4.5
Immobilization of molecules with amine functional group onto surface with
epoxy or aldehyde functional group. Reaction proceeds through formation of Schiff base that
is subsequently reduced into a stable secondary amine bond.
Schiff base is susceptible to hydrolysis and thus it is reduced into a stable bond using
sodium cyanoborohydride (Larsson, 1984; Hermanson, 1996; Gong et al. 2000).
Many different cross linkers with different functional groups and with differ-
ent spacer lengths (including polymeric cross linkers) are commercially available.
Table 4.3 lists several typical reactive cross-linkers and reactive polymeric linkers
that can be used to couple to amines and thiols. Coupling reactions are shown in
Figures 4.2 and 4.5. In addition to bifunctional tethers listed in Table 4.3, mono-
functional tethers can be mixed with bifunctional tethers to block reactive groups
on the surface. Therefore, surface density of attached molecules decreases facili-
tating single-molecule detection. Some molecules listed in Table 4.3 are homo-bi-
functional. Therefore, bridging between functional groups on surfaces is possible.
However, this is not detrimental to use of these tethers because this effect helps
decreasing nonspecific adhesion to surfaces (Gong et al. 2000).
Functionalized polymeric tethers that can be used with activated substrates and
biomolecules are listed in Table 4.4. These tethers are also commercially available
in a wide range of molecular weights. In this table, possible uses of such tethers
are listed instead of providing chemical formula that is evident from the name.
Aforementioned comments about mono-functional tethers and about bridging of
homo-bi-functional tethers between surface groups also apply here.
Although there are many more commercially available chemical coupling
reagents and cross-linking tethers than are mentioned in this section, those indicated
here are likely to be sufficient to immobilize biorecognition partners in DFS exper-
iments. In the following sections, two sample preparations for DFS measurements
are described, one method uses gold-thiol chemistry and another method uses silanol
chemistry.
4.4.2.4 Example of Chemical Immobilization on Gold Surfaces
Here we describe sample preparation for investigation of “A-a” knob-hole interac-
tion of fibrinogen. This interaction is involved in the early stages of blood clotting
and provides mechanical stability of fibrinogen network before chemical cross link-
ing of fibrin occurs (Averett et al. 2008; Averett et al. 2009). Fibrinogen consists
of linear array of three globular-like units held together by thin connecting threads
(Hall & Slayter, 1959). The “a” holes are located at both terminal units that are
spaced
∼
50 nm apart. The “A” knobs are located in the central unit of fibrinogen
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