Biomedical Engineering Reference
In-Depth Information
(FMOC-PEG-SCM) with a mass-average molecular mass of 3400 Da (Laysan
Bio Inc.) for 24 h (10 mg of FMOC-PEG-SCM were dissolved in 300
lofDMF
with 10% pyridine [v/v]). Next, the substrates were treated with poly(ethylene
glycol)mono-methyl ether mono(succinimidyl succinate) ester (PEG-SS) (Poly-
sciences Inc., Warrington, PA ) with a mass-average molecular mass of 1900 Da
for 24 h (10 mg of PEG-SS were dissolved in 300
μ
l of DMF with 10% pyridine
[v/v]). This step was aimed to block unreacted amines and hinder adsorption of
streptavidin on surface of glass substrate. After that, the substrates were thor-
oughly rinsed with DMF and activated in DMF and piperidine mixture solution
(v/v, 4:1) for 30 min to remove FMOC group. After removing FMOC group,
the substrates were further reacted with solution of 10 mg of homo-bi-functional
amine-reactive cross-linker 1,4-phenylenediisothiocyanate (PDITC) in 300
μ
lof
DMF with 5% pyridine (v/v) for 2 h. Activated slides were cleaned by ultrason-
ication in DMF and ethanol twice for 10 min each. Then, 200
μ
g/ml
solution of streptavidin in phosphate buffer (PBS, VWR international, 0.05 M
pH 7 and pH 10 PBS were mixed together to reach pH 8) was deposited on the
PDITC-activated substrates. The covalent attachment of streptavidin was per-
formed in a dark environment for 2 h. Here, pH 8 PBS solution was chosen to
optimize the PDITC-streptavidin reaction but keep streptavidin stable (Weber
et al. 1989; Hermanson, 1996). Substrates with attached streptavidin were thor-
oughly rinsed with pH 7 PBS buffer solution and used immediately for data col-
lection.
μ
l of 100
μ
Figure 4.7 compares force curves with rupture events measured when streptavidin
is attached directly to the surface and when it is tethered using PEG linker. In the
double-tether experiment, ruptures occur consistently at larger tip-sample separation
when in the single-tether experiment. This is consistent with streptavidin attachment
to the end of polymeric tether.
4.5 MULTIPLE INTERACTIONS BETWEEN IMMOBILIZED
MOLECULES
Covalent immobilization of molecules with polymeric tethers removes problems
associated with potentially weak physical attachment, avoids effects of nonspecific
probe adhesion to the substrate, and decreases steric hindrance. However, using teth-
ers does not eliminate possibility of multiple interactions as illustrated in Figure 4.1.
Feasibility of multiple bonds forming during the probe-sample contact is illustrated
in Figure 4.8. Simple estimate of possible number of bonds between the probe and
the substrate can be obtained assuming high density of attachments of interacting
molecules to surfaces. If we further assume that biomolecules tethered to the probe
can bind to the partners immobilized on the substrate at separation that is similar in
magnitude to the end-to-end distance
l
rms
of a free tether in solution, then the largest
number of bonds during one tip-sample contact would be approximately equal to the
ratio of the surface area of spherical cap shown in Figure 4.8 to the area occupied by
Search WWH ::
Custom Search