Tutorials - Surface chemistry in BioAFM

Silanized surfaces - the APS method

APS-mica

"Unlike the (3-aminopropyl)triethoxysilane (APTES) commonly used for silica surface modifications, 1-(3-aminopropyl)silatrane (APS) is not very reactive and is extremely resistant to hydrolysis and polymerisation at neutral pH. " [9]

APS synthesis:
1-(3-Aminopropyl)silatrane was prepared by vacuum evaporation of a mixture of 4.13 g of triethanolamine containing 1 mg of sodium as a catalyst and 6.12 g of (3-aminopropyl)triethoxysilane at 60 °C to constant weight (6.4 g). The compound obtained by this procedure can be used directly without purification for surface modification. A 50 mM solution of APS in deionised water was used as a stock solution. The stock was stored in refrigerator at 4 °C with no special precautions." [9]

"Preparation of APS mica for AFM: The APS stock solution diluted 1: 300 in deionised water was used for APS-mica preparation. Freshly cleaved mica strips of appropriate sizes were immersed into APS working solution for 30 min, rinsed thoroughly with deionised water, Ar dried and stored in Ar filled tubes." [9]

Immobilization on poly-L-lysine-coated surface

Poly-L-lysine is a positively charged polymer with adsorbs very well to negatively charged glass or silicon dioxide leading to positively charged surfaces.

"Acid-washed coverslips were coated with a poly-L-lysine solution (10 mg/mL ; Mr 1000-4000), washed with water after 1 min and dried in air. The protein solution (3 mg/mL) was deposited on the dry surface and washed off after 15 min." [10]

Hydrophobic /hydrophilic surfaces

"For AFM in solution, hydrophobic surfaces do not appear to be a very useful substrate, not only because many proteins could denature upon contact with a hydrophobic surface, but also because the Si₃N₄ tip interacts strongly with a hydrophobic surface, resulting in a noticeable adhesion force in solution. The exact nature of these interactions is not fully understood, and reliable results cannot be obtained. However, for AFM in air, hydrophobic surfaces can be adequate, and sometimes superior. For example, carbon-coated mica was shown to be a good substrate for imaging DNA with a very good lateral resolution and specimen stability. One problem with a hydrophilic surface in air is that a thin layer of water will be condensed onto the surface for medium to high humidity. This condensed water can interact with the tip giving rise to a quite large adhesion force (capillary force) and can reduce the stability of adsorbed macromolecules. " [17]

Glass Hydrophobization

Method 1 - Silanization with dichlorodimethylsilane
The method for silanization of glass is described in [11]:
"A freshly cleaned cover slip is incubated in dichlorodimethylsilane vapor (CH₃)₂SiCl₂ for 3 seconds up to 5 minutes. It is sufficient to place the surface over the mouth of a bottle with the silane. Afterwards the surface is washed with ethanol. The silanized surface has a higher roughness than the pure glass." [11]

Method 2 - Silanization with n-octadecyltrimethoxysilane
"n-octadecyltrimethoxysilane (Gelest or United Chemical), methanol (Optima Grade, Fisher), and heptane (Optima Grade, Fisher) were used as received. Water was deionized, distilled over quartz, and filtered by a Milli-Q reagent water system (Millipore Co.), which resulted in a resistivity of 18 MΩ*cm.
Glass microscope slides (Fisher Scientific; rms surface roughness measured by AFM was 1.5 (0.2 Å) were soaked in concentrated NH₄OH for 1 h and washed 5 times by ultrasonication in 18 MΩ*cm water. The surfaces were then dried for 1 h at 140 °C and placed directly in methanol to keep the surface free from water vapor and other unwanted adsorbates. Glass surfaces and SiO₂-coated cantilevers were silanized by immersion in a 5 mM silane solution in heptane for 24 h. The silanized surfaces were washed 3 times with heptane to remove unreacted silane materials. The treated surfaces were then heated for 1 h at 70 °C. After being coated, they were rinsed with acetone to remove any unreacted silane and then stored in methanol until used." [23]

N-Hydroxysuccinimide ester functionalization

"The approach is based on the ex-situ synthesis of dithiobis(succinimidyl-undecanoate) DSU providing a long-chain N-hydroxysuccinimide-ester functionalized dialkyldisulfide, which is accessible for nucleophilic attack (e.g. amide bond formation with amino-group- containing molecules)." [12]

Synthesis of 3
"Sodium thiosulfate (55.3 g, 350 mmol) was added to a suspension of 11-bromo-undecanoic acid 1 (92.8 g, 350 mmol) in 50 % aqueous 1,4-dioxane (1000 ml). The mixture was heated at reflux (90 °C) for 2 h until the reaction to the intermediate Bunte salt 2 was complete (clear solution). The oxidation to the corresponding disulfide was carried out in situ by addin iodine in portions until the reaction retitrated with 15 % sodium pyrosulfite in water. After removal of 1,4 dioxane by rotary evaporation the creamy acetate/tetrahydrofuran (THF) provided 3 as a white solid (73.4 g, 96.5 %): mp 94 °C." [5]

Synthesis of 4
"To a solution of 3 (1.0 g, 2.3 mmol) in THF (50 ml) was added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by dicyclohexylcarbodiimide (DCC, 1.03 g, 5 mmol) at 0 °C. After the reaction mixture was allowed to warm to 23 °C and was stirred for 36 h at room temperature, the dicyclohexylurea (DCU) was filtered. Removal of the solvent under reduced pressure and recrystallization from acetone/hexane provided 4 as a white solid. Final purification was achieved by medium-pressure liquid chromatography (9 bar) using silica gel and a 2:1 mixture of ethyl acetate and hexane. The organic phase was concentrated and dried in vacuum to yield 4 (1.12 g, 78 %): mp 95 °C." [5]

Preparation of ultraflat gold

The preparation of ultraflat gold is described in [5]:
"[...] gold was evaporated onto mica and glued upside down to a silicon wafer or glass coverslip; the mica was then removed by soaking in tetrahydrofuran. These "template-stripped" gold surfaces (TSG) consisted, therefore, of the gold atom layer that had been deposited on the mica template first."

More detailed instructions for the preparation of ultraflat gold are described in [13], [14], [15] .

Adhesive tape

Commercially available adhesive tape can be used for immobilization of powders or even biological samples. First a strip of tape is superglued with its uncovered side to a microscope glass slide.
When an inorganic powder has to be imaged it is trickled onto the glue side of the tape and the supernatant is removed with a stream of pure nitrogen.
Also red blood cells have been successfully immobilized from suspensions. In this case the suspension has been poured onto the tape. After a short incubation time of some minutes the cells adhered. In our lab, red blood cells and polyelectrolyte shells could be successfully adsorbed onto adhesive tape, even in aqueous solution.

Superglue

A drop of superglue is dabbed onto a glass slide and smeared like a blood smear. When an inorganic powder has to be imaged it is trickled onto the glue and the supernatant is removed with a stream of pure nitrogen after drying.
Note: Superglue cannot be used when AFM experiment is performed in aqueous environment.