Biomedical Engineering Reference
In-Depth Information
The most common physical method of attaching biomolecules utilizes strong spe-
cific bonds formed between biotin and avidin (other proteins that can be used in this
approach are streptavidin and neutravidin) (Chilkoti et al. 1995). Because avidin is
tetrameric, upto four biotin molecules can be bound to one protein molecule simul-
taneously. Therefore, avidin strongly binds to surfaces that display biotin groups
and is yet capable of binding to additional biotin molecules. Consequently, this sys-
tem can be used as a universal platform to attach molecules to surfaces because
biotin that attaches to the surface-immobilized avidin can be chemically conjugated
to other molecules of interest. Typical sample preparation consists of three steps: (1)
preparing surface with attached biotin molecules; (2) coating surface with avidin;
and (3) coupling biotin-conjugated molecule of interest to the avidinated substrate.
The initial biotin-coated surface can be prepared by adsorption of biotinylated bovine
serum albumin (BSA) (Lee et al. 1994; Moy et al. 1994; Florin et al. 1994; Chilkoti
et al. 1995) or by chemical methods (Evans et al. 2001; Evans et al. 2004; Pincet &
Husson, 2005).
Universality and relative simplicity of sample preparation using biotin-avidin
platform are the main merits of this approach. However, there are significant
drawbacks that preclude the widespread use of this approach. The most significant
drawbacks are (1) the relative weakness of the biotin-avidin bond in comparison to
covalent bonds and (2) difficulty in preparing low-attachment density of molecules
on surfaces. As a result of the first aspect, the rate of dissociation under applied
force is the sum of dissociation rates of molecules under study and the dissocia-
tion rate of biotin-avidin bond (Patel et al. 2004). Therefore, if the force-dependent
dissociation rate of molecular bond under study is comparable to the dissociation
rate of biotin-avidin bond, at some fraction of experiments the latter might be bro-
ken, thus distorting the distribution of rupture forces. Such distortion might result
in significant errors in extracted kinetic parameters of bond dissociation. However,
this aspect might be of minor importance when studying bonds that are consider-
ably weaker than biotin-avidin bonds. The second aspect mentioned above stems
from the tetrameric nature of avidin. Consequently, it is likely that more than one
biotin molecule will attach to one avidin molecule and thus probability to form two
molecular bonds instead of one becomes significant (Guo et al. 2010a). Moreover,
if the surface density of avidin molecules is not low, even larger number of molecu-
lar bonds might be formed. This effects result in large number of rupture transitions
detected in a single force curve and in wide distribution of rupture forces (Lee et al.
1994; Moy et al. 1994; Zhang & Moy, 2003). Consequently, distribution of rupture
forces coming from a single molecular bond cannot be extracted reliably from the
data.
Physisorption of molecules directly to the substrate is an alternative to using
biotin-avidin linking described above. Typically, molecules are adsorbed on flat mica
or other substrates without making a covalent bond (Radmacher et al. 1994; Fritz
et al. 1998; Willemsen et al. 1998; Baumgartner et al. 2000; Averett et al. 2008). If
physisorbed molecules strongly adhere to the substrate and are significantly diluted
on the surface, two drawbacks that are mentioned above can be avoided. For example,
it has been shown that specific knob-hole interaction between different parts of fibrin
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