Biology Reference
In-Depth Information
3. Methods
3.1. Crosslinking
1.
Attach cantilevers and freshly cleaved mica 1.5
1.5 cm with double-stick Scotch
tape into a holder of a turbo vacuum evaporator equipped with a quartz crystal
for monitoring thickness of gold (
see
Note 1
). Place gold wire in an evaporation
slot, turn vacuum on and adjust current of turbo vacuum evaporator. For a
designated time, coat cantilevers and freshly cleaved mica to give thickness of
10-20 nm gold.
×
2.
Immediately after gold coating, mount cantilevers in the teflon holders and place
mica in 1-5 mL glass beaker with a shift glass cap. (
See
Note 5
.)
3.
Add 1-2 mL of freshly prepared 1 m
M
solution of 11-thio-undodecanol in ana-
lytical-grade methanol and incubate for 12 h at room temperature to form
selfassembled monolayer.
4.
Wash cantilevers and mica five times with analytical grade methanol.
5.
Add 1-2 mL of 50 mg/mL of freshly prepared carbonyldiimidazole in analytical
grade methanol and incubate for 20 min at room temperature.
6.
Wash four times with 1 mL of methanol.
7.
Dismount cantilever from teflon holder and place them on stretched parafilm in
plastic dish humid chamber with tip facing upwards. Immediately add 10-20
µ
L
of 1-20
g of molecules to be crosslinked in 0.1-0.5
M
NaCl 10 m
M
HEPES
buffer pH 7.4. With forceps, take mica and do same as with cantilevers. Incubate
30 min to 1 h at room temperature in humid chamber.
µ
8.
Wash five times with buffer to be used for measurements.
3.2. AFM Measurements
1.
Mount by double Scotch tape the back side of mica crosslinked with adhesion
molecule to the AFM base. The upper part of mica with crosslinked molecules
should always be covered wit 10-20
µ
L buffer.
2.
Rapidly mount cantilevers with crosslinked molecules on a AFM equipped with
a liquid cell filled with buffer to prevent drying.
3.
Assure that no bubbles were in the liquid cell between cantilever tip and mica.
4.
Use contact mode soft approach.
5.
Perform about 50 force distance measurements of intermolecular binding strength
at 0.01-1 Hz on 5-10 different locations (molecules on mica). Change solution to
use blockers and/or activators of binding and repeat the same number of mea-
surements. Return to original solution and repeat to test reversibility of blocking
or activation.
6.
At the end of the experiment measure the spring constant of the cantilever.
4. Notes
1.
The author has used three following types of commercial AFMs equipped with a
liquid cell: Digital Instruments Nanoscope III, TopoMetrix, and Park Instrument
AutoProbe CP.
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