Bacterial Adhesion and Interaction with Biomaterial Surfaces - Biointerfaces: Where Material Meets Biology - page 389

A.2 Immobilization of Bacterial Cells on an AFM Probe
Probe: commercially available AFM tips can be utilized, including sil-
icon, silicon nitride and gold-coated tips. Curvature of the AFM tips is
preferred to be around 50 nm or tipless probe may be used. Before use,
AFM probes are cleaned in acetone for 15 min and then treated in glow
discharge air plasma at 100 W power for 30 min to remove any organic
contaminants.
Harvest of bacterial cells: bacteria are cultured in media at 37 1C with
shaking, and harvested in mid-exponential phase by centrifuging at
1360 g for 10 min. Wash the cell pellet with buffer for three times and
transfer the pellet onto a clean glass slide, forming a thin film of
bacteria.
Modification of an AFM probe: place one drop of 1% polyethyleneimine
(PEI, MW ¼ 1200) onto the AFM probe and allow to adsorb for 2-3 h.
The probe can also be treated with 0.1% (w/v) poly- L -lysine for 5 min.
Remove the excess solution, and rinse the probe with water.
Immobilization of cells on the probe: place the probe in the tip
holder; position the probe over the bacterial film using an optical
microscope. Set the scan size to 0 nm and scan rate to 0.1 Hz. Engage to
allow the tip to approach the bacterial layer. Allow the tip to come into
contact with the bacteria for 1-3 min. Withdraw the probe and rinse in
buffer.
Check the probe: the presence of bacteria on the AFM tip is checked by
scanning electron microscopy or inspection of characteristic force
curves for coated and uncoated tips.
Note: bacterial cells may be treated in 2.5% glutaraldehyde for fixation
before and after immobilization on the probe, but lose the activity of
live cells.

d n 3 r 4 n g | 3

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A.3 Functionalization of an AFM Probe with Antibodies
AFM probe: silicon nitride probes with nominal spring constant of
0.06 N m 1 .
Probes are treated by glow discharge plasma at 100 W power for
30 min.
Incubate the probe in a 1% (v/v) solution of aminopropyltriethoxysilane
in ethanol for 1 h to provide reactive amine groups on the tip.
Thoroughly rinse with Millipore water three times. Then the probes are
soaked in 10% glutaraldehyde in aqueous solution for 1 h. Rinse the
probes with Millipore water three times.
Incubate the probes in antibodies solution ( 25 mgmL 1 ) for 1 h.

The probes are rinsed with buffer after removal from protein solution
and are stored in buffer at 4 1C until use within 2 days.
Multiple probes should be prepared together to improve consistency
between experiments.

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