Immobilization and Interaction Strategies in DFS of Biomolecular Partners - Dynamic Force Spectroscopy and Biomolecular Recognition - page 140

Maleimide compounds might couple to amines, but coupling to thiols is strongly
favored at pH below 7.5 (Hermanson, 1996; Wong et al. 2009). Also, maleimide
compounds hydrolyze in water, though hydrolysis of maleimides is slower than for
NHS esters (Hermanson, 1996). Another method to immobilize molecules by their
thiol group is coupling using iodoacetyl reactive groups. This coupling method is
less efficient than maleimide coupling (Mallik et al. 2007).
Molecules naturally displaying thiols at their surface or containing thiols that are
purposely incorporated into their structure can be coupled directly to gold substrates
or probes using high affinity of gold toward organic sulfur (Ulman, 1991; Ros et al.
1998; Sekiguchi et al. 2003; Bonanni et al. 2005; Love et al. 2005; Gilbert et al.
2007; Jiang et al. 2010; Nguyen et al. 2011). This method of immobilization involves
adsorption of molecules from solution onto gold surface; this deposition is straight-
forward to perform as it requires no prior modification of gold surface except for
initial cleaning. Possible drawbacks of this approach are that interaction with gold
might affect affinity of recognition and proximity of the surface might restrict motion
of molecules (Bizzarri & Cannistraro, 2009). Also, high density of attachment at
surfaces might promote “contamination” of single-molecule recognition events with
multiple interactions.
Carboxylic groups are often coupled by using carbodiimide coupling agent (Fig-
ure 4.2c shows coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide also
known as EDCI, EDC, or EDAC) and NHS to form an amino-reactive NHS ester
(Hermanson, 1996). This ester then can be coupled to amine groups of other
molecules (Ratto et al. 2004; Friedsam et al. 2004; Neuert et al. 2006; Odorico et al.
2007; Averett et al. 2008; Mayyas et al. 2010; Jiang et al. 2010). Hydrolysis of NHS
can be avoided by performing the first two steps at pH 5-6 and then rising pH to
7.2-7.5 to perform NHS ester coupling to amines. Another popular carbodiimide
that activates carboxyl groups for the formation of amide bonds with primary amines
and can be used for esterification of alcohols is dicyclohexylcarbodiimide (DCC)
(Hermanson, 1996; Jung et al. 2007). DCC is a known allergen and a sensitizer that
causes skin rashes; therefore, precautions should be taken when working with this
chemical to avoid skin contacts.
4.4.2 C HEMICAL M ODIFICATION OF S UBSTRATES
In DFS experiments, the recognition partners are attached to surfaces directly or via
polymeric linkers. Gold surfaces and surfaces that exhibit silanol groups are most
widely used in DFS. Gold strongly binds to organic sulfur, thus eliminating the step
of activating silanols. On the other hand, gold coating on the AFM probes makes
probes duller, thus increasing probability of multiple interactions. Silanol chemistry
is attractive because silanol groups are present on surfaces of silicon, silicon nitride,
and glass after cleaning in acids (Hinterdorfer et al. 2002). Below, we first describe
functionalization of gold surfaces and then functionalization of surfaces using silanol
groups.