Biomedical Engineering Reference
In-Depth Information
Biomolecules
-
DNA and proteins
Oligonucleotides, especially DNA are very popular samples for biological AFM studies.
DNA is usually deposited on mica [294], although HOPG has also been used [295, 296].
The negative charge of as-cleaved mica is a disadvantage for this application, as DNA Is
also usually negatively charged. Typically this is overcome by treatment of the mica with a
solution containing divalent cations, or deposition or imaging of the DNA in a solution
containing such cations (typically MgCl
2
or NiCl
2
containing buffers) [297-299]. Alter-
natively, a procedure to bind oligonucleotides to mica with the aminosilane APTES has
been thoroughly described [300]. The procedure should be followed carefully so that this
does not increase the roughness of the surface. As with oligonucleotides, mica is the most
commonly used substrate for protein absorption [294, 301, 302], but HOPG can also be
used [303]. Again, divalent cations are commonly used to encourage protein binding to
mica, if the proteins are negatively charged [301, 304]. The presence of monovalent cations
in the buffer solution can compete with the divalent cations, and prevent the adhesion of a
number of proteins to mica [305]. Other methods to fix proteins onto mica include covalent
binding, although this may change the protein structure somewhat [306], and for membrane
proteins, insertion into a lipid layer is a very suitable strategy [307, 308].
Cell cultures
Cultured cells are typically grown on some sort of support such as a Petri dish or glass
microscope slide [309], to be directly mounted into the AFM. Where the instrument does
not support such large substrates, microscope slides may be simply cut to size, or small
cover slips used [310]. Cells may be fixed and dried before analysis or imaged
in situ
either
in cell culture medium, or in a filtered buffer solution. In combination with temperature
control, such a preparation can lead to the ability to image live cells [309-311].
Bacteria
Bacterial cells are common samples for AFM, see Section 7.3.2. Typically for imaging in
air, bacteria are transferred to a clean buffer, dried onto a surface and extensively washed
[169, 312]. For imaging in liquid, several procedures to adhere the cells to the substrate
have been described [302, 313]. Without these treatments, the cells will normally be
removed by the probe while scanning in liquid. Immobilization strategies include the use
of gelatin coated mica to mechanically trap bacteria on the surface [6]. This has
the advantage of not inducing chemical changes in the cells, as could be the case for
binding the cells to the substrate with poly-l-lysine or other chemical treatments [184, 314,
315]. Another technique that might reduce the changes caused to the bacteria is allowing
the formation of a biofilm on the substrate surface [184, 316]. For those bacteria which do
form biofilms, this is the best way to adhere them to a surface for imaging in liquid. For
spherical cells, physical immobilization in a solid substrate with holes (such as a mem-
brane or lithographically patterned surface) has been reported to be very successful,
although this is not appropriate for rod-shaped bacteria [317].
Nanotubes
Carbon nanotubes, nanowires and whiskers are a subset of nanoparticles. These particles
are normally produced in large quantities as powders or are grown directly on a substrate.
