Tutorials - Surface chemistry in BioAFM

Substrates

Since AFM does not require the substrate to be conductive, the choice of substrates is almost unlimited. Both glass cover slips and cleaved mica sheets have been used to adsorb various macromolecules and organelles with varying degrees of success. However, the functionality of the adsorbed macromolecules on these surfaces has not been characterized by biochemical experiments. Within the resolution achieved by AFM so far, structural changes have not been observed when compared with that obtained by electron microscopy. Imaging of living cells in buffer solutions did not appear to affect the integrity of the cells either. This, of course, does not imply that structural changes at higher resolutions can be entirely ruled out, nor that the function of theses molecules is not in some way affected. Detailed comparison with X-ray structures or cryo-electron microscopy should be invaluable in addressing this question. [17]

Today, a number of specimen supports are used, among which the most important are epitaxially grown gold films, layered transition metal dichalcogenides, amorphous thin films, glass, silicon and mica. The support used depends on the specimens and on how they are to be immobilized: by physisorption, covalent bonding, self-assembly on van der Waals surfaces or the Langmuir-Blodgett technique. In the following, frequently used specimen supports and the most important immobilization techniques are described.[4]

Many sample preparation techniques used for AFM are derived from sample preparation in electron and light microscopy. For example samples are prepared on glass slides for light microscopy or on iron stubs used for electron microscopy. In 1968 Kleinschmidt [18] published a method for DNA preparation, long time before the AFM was invented and which today is still in use. The basics about substrates used for AFM sample preparation are described in [4]:

"All surfaces become immediately covered with hydrocarbons when exposed to ambient air. Even bidistilled water can be a source of organic contaminants. A layer of these hydrocarbons on the sample or the probe can be most disturbing for the AFM. As a result, the sample supports should be prepared or activated immediately before use. Ultrapure water should be used to prepare all buffer and rinsing solutions. Since it contains fewer hydrocarbons and macroscopic contaminants than conventional bidis-tilled water, it is less likely to influence the imaging procedure by either damaging the probe or contaminating the specimen being scanned. Organic contamination layers can be removed from the probes and the specimen supports by exposure to plasma or to UV light."

Substrates: preferences

The biopolymers to be imaged in the AFM are normally dissolved in aqueous solution and then deposited onto the substrate. Compared with the size of the sample molecule the substrate has to be flat and easy to prepare.

Mica

da Silva and Kindt described the properties of mica in [3, 20]:
The most commonly used substrate is mica. Mica is a non-conducting layered material. It is cheap and can easily be cleaved, usually with a pin or sometimes cellotape, to produce clean, atomically flat surfaces up to even millimetres in size. The commonest form of mica is Muscovite KAl₂(OH)₂AlSi₃O₁₀. The minimum step size which can be observed on the surface is the thickness of an individual layer (1 nm) and the hexagonal lattice constant within the layers, which can be used for calibration, is 0.52 nm." "The root-mean-square roughness is 0.06 ± 0.01 nm." [3] "Mica has been successfully used in numberless studies especially for AFM imaging of double stranded DNA and DNA-protein complexes, protein arrays, and densely packed proteins. Although the mechanism by which macromolecules absorb to this substrate still remains poorly understood, a large number of protein samples adhere tightly to this surface." [20]

Mica's property to be highly charged at the surface leads to the fact that it is always covered with a thin (0.5 nm) layer of water when exposed to ambient air. This water layer leads to a continuous adhesion between AFM tip and sample.

Despite the widespread use of these substrates, the mechanism of adsorption is not well understood. For both glass and mica in aqueous media, it is known that positive ions tend to dissociate from the surface to make them negatively charged. It is also known that most protein surfaces contain both positively and negatively charged residues at neutral pH and that the neutrality can be altered by changing the pH of the buffer solution. For example, ferritin has an isoelectric point of about 5 and is positively charged at pH < 5 and negatively charged at pH > 5. Therefore it seems plausible to assume that electrostatic interaction is primarily responsible for adsorption. However, we must also realize that most of these charged groups are shielded by counter-ions in solution. It is not clear whether it is the direct interaction between the oppositely charged groups o the salt bridges between like charged groups that is responsible for surface adsorption in each case. [17]