Tutorials - Surface chemistry in BioAFM

Glass

Glass is flat enough for imaging cells or other large and relatively high samples. Apart from cells, also large molecules like tubulin molecules, chromosomes or cell organels. On the other hand glass is generally too rough for reliable visualization of DNA, especially under fluid.[3]

For preparation of AFM samples on glass coverslips, the round ones with a diameter of 25 mm and a thickness of 0.17 mm are widely used. Also the square cover slides are easily obtainable. Amrein describes a method for cleaning glass in [4]: "The slides can be used either unmodified or altered to change their physisorption or chemical properties. The surface can be almost featureless on the scale of macromolecular specimens. They are best suited for all experiments in which visible light is transmitted across the sample, as in scanning near field optical microscopy (SNOM) or in the combined light- microscopy and SPM. [...] Before use, organic contaminants, dust or other particles are removed by washing on time with concentrated HCl/HNO₃ (3:1 v/v) and 5 times for 1 min with Millipore water in an ultrasonic bath (50 kHz). This process makes the coverslips clean and smooth (rms-roughness ~ 0.5 nm). They show a mottled background with less than one particle/µm² (particle size ~ 0.3 µm²)."

Glass cleaning

Using glass as a solid support in AFM experiments it requires cleaning. In [11] two glass cleaning methods are recommended:

• rinse with ultrapure (Milli-Q) water several times
• rinse with ethanol
• rinse with Milli-Q- water
• air plasma treatment for 20 min
• dry in a nitrogen flow
• additional treatment in the plasma cleaner increases the charge density on the surface

Precleaning with detergent

• glass is pre-cleaned with commercial detergent
• rinse with Milli-Q water

Removal of organic contamination

• incubation for 5-10 min. in RCA1 solution at 70 deg C. RCA1 solution is a mixture of Milli-Q, ammonia (25%), hydrogenperoxide (30%) in 5:1:1 ratio.
• thoroughly rinse with Milli-Q water

Removal of inorganic contamination

• incubation for 5-10 min in RCA2 solution at 70 deg C. RCA2 solution is a mixture of Milli-Q, hydrochloric acid (37%), hydrogenperoxide (30%) in 5:1:1 ratio.
• thoroughly rinse with Milli-Q-water
• dry in nitrogen flow

Gold

The properties of gold surfaces used for AFM sample preparation are described in [4]:
Gold surfaces can be easily prepared by vapor deposition onto glass and mica. Gold is chemically inert against oxygen and stable against radicals. It binds organic thiols or bifunctional disulfides with high affinity, which can be used to covalently attach biological macromolecules.

Epitaxially grown gold surface Au(111)

Special preparation techniques deliver ultraflat gold surfaces as described in [17] and [5]: "For epitaxially grown gold surfaces, atomically flat areas of up to several micrometers can be found and the surface is hydrophilic if clean." "Ultraflat Au(111) surfaces have a mean roughness of 0.2-0.5 nm over areas larger than 25 µm². "

Silicon

Also parts of silicon wafers used in semiconductor industry can be used as substrate. They have a thin oxide layer on their surface which makes them hydrophilic because of the OH groups on the surface.

Graphite - HOPG

HOPG is not widely spread among AFM sample techniques. Its chemical properties and its disadvantages are described in [4] and [5]:
The sheets were of HOPG were flat on an atomic scale over areas of microns. They were also well conductive, which was a prerequisite for STM. However, the adsorption of most biological specimens was very poor. Moreover, defects and the fine structure of step edges of pure graphite were very misleading, since they often resembled the expected structure of the specimen. Graphite is now used exclusively as a support for the investigation of self-assembly monolayers of organic molecules. " [4] "Furthermore, HOPG bind macromolecules by way of weak electrostatic or "adsorption" forces, and, moreover, it has fallen into, perhaps excessive disrepute when it was shown to yield artifactual images mimicking DNA [in STM investigations]." [5]

Thermanox

A rough polymer like Thermanox is only suitable for AFM imaging of large structures like cells as described in [11]. Thermanox is hydrophobic and available from different distributors [11].