Tutorials - Surface chemistry in BioAFM

Functionalized surfaces

To strengthen further the adsorption, functionalized surfaces can be introduced. The simplest method is to treat the surface with certain molecules, such as poly-L-lysine or poly-L-arginine (van Holde: Chromatin, 1989), in order to change the charge characteristics of the surface. Therefore the adsorption can be enhanced or modified. One step further is to introduce cross-linking groups to the surface. So far, several schemes have been used for AFM imaging in solution. One method is based on silanizing a solid surface with 3-aminopropyltriethoxysilane (APTES) (Lyubchenko, 1992, 1993), which protonates at neutral pH. The silane group in APTES is highly reactive and silanizes the surface by forming covalent bonds with surface atoms. Karrasch and co-workers introduced another cross-linking group at the amino end of APTES on a glass surface, N-5-azido2-nitrobenzoyloxysuccinimide (ANB-NOS) [10]. The azide group, upon ultraviolet irradiation, can make non-specific covalent bonds to proteins on contact. Since the functionalized surface becomes hydrophobic, and soluble proteins do not come close enough to be cross-linked, a squeezing pressure of 10-500 atm must be used to force macromolecules to come within reach of the azide group. In another method, an ultraflat Au(111) surface is used as a substrate for N-hydroxysuccinimide terminated self-assembled monolayers of dithio-bis(succinidylundecanoate). This monolayer readily reacts with amino groups, covalently linking the protein to the substrate." [17]

Silanized surfaces - the APTES method

AP-Mica has amino groups exposed to the surface. Aliphatic amino groups have a pK of approximately 10.6. Although the close packing of the aliphatic amino groups on the AP-mica surface decreases the pK, the surface will still be positively charged in solution at neutral pH. [8]

AP glass

The method for APTES coated glass is described in [10]:

"Silanization and derivatization of coverslips were carried out in Petri dishes that had been washed in "piranha bath" (3.5 % H₂O₂ in 18 M H₂SO₄), followed by rinsing with water and acetone. The following steps were carried out at room temperature unless stated otherwise. Coverslips were washed once with concentrated HCl/HNO₃ (3:1) and five times for 1 min with destilled water in an ultrasonic bath (50 kHz). They were etched with trifluoroacetic acid for 90 min and stored in vacuum over solid KOH for at least 10 h. Coverslips were then silanized with APTES (2 % in 95 % aqueous acetone) for 3 min followed by washing with acetone (12 times, 5 min each) [...]. Curing of the silane linkages was carried out in an oven at 110 °C for 1 h. " [10]

AP-silicon

Möller et al. [6] described a modified protocol for the APTES method: "The [ silicon] chips were immersed for 15 min in 1 % APTES solution in 95 % acetone/water. Afterwards, the chips were washed five times (5 min each) with acetone and dried for 45 min at 110 °C. They were then incubated for 2 h with a solution 0.2 % 1,4-phenylenediisothiocyanate in 10 % pyridine/dimethyl formamide and washed with methanol and acetone. The activated chips may be stored in a vacuum desiccator containing anhydrous calcium chloride for a longer time without discernible loss of activity." Möller's et al. modified procedure for the GOPS method is: "For substrate modification with 3'-glycidoxypropyl-trimethoxysilane (GOPS) the slides were suspended in dry toluene containing 1% silane at 80° C for 4-6 h, using a modified procedure from the literature [7]."

AP-mica

Method 1
"AP-mica was prepared by placing freshly cleaved mica in a 2 L glass desiccator which contained 30-100 µL of 3-aminopropyl-triethoxysilane (APTES), 98 %, for 2 h. The AP-mica was then removed and stored under argon prior to making the samples. The best AP-mica surfaces were prepared with 30 µL of APTES that had been redistilled in vacuum and stored under argon; the silylation was also done under argon. [...] AP-mica has a shelf life of approximately 1 month and has a more hydrophobic surface than bare mica, presumably due to the propyl chains that are attached to the surface amino groups." [8]

Method 2
" A desiccator was purged with argon for 2 min and 30 µL of APTES (99%, Sigma-Aldrich) placed into a small container at the bottom of the desiccator. Ten microliters of N,N-diisopropylamine (99%, destilled, Sigma-Aldrich) was placed into another small container, and the desiccator purged with argon for a further 2 min. Mica sheets were stripped on one side until smooth and immediately placed into the desiccator. The desiccator was purged for another 3 min and then sealed off, leaving the mica exposed to APTES vapor for times that were varied between 30 min and 2 h (there appeared to be no consistent effect of exposure time within this range). After this exposure, the APTES was removed, the desiccator purged, and the treated mica (AP-mica) stored in the sealed desiccator until needed. APTES was used both as received and as redistilled. Distillation was found to have no effect unless the as-received material was older than ~ 2 months or had been exposed to ambient air for some hours." [22]

ANB-NOS modified surfaces

"All subsequent steps were performed in the darkroom using red safety light. The reaction of the NH₂ groups with the succinimide ester group of ANB-NOS was carried out in 0.1 M Na₂CO₃, pH 9.0. The reaction mixture was prepared by adding 10 nmol ANB-NOS/cm² glass surface dissolved in 1 ml dioxane to 20 ml Na₂CO₃ solution. This corresponds to a 10-fold molar excess of the photocross-linker with respect to the amino groups. The coverslips were incubated for 4 h. Excess reagent was then removed by washing the coverslips three times with distilled water, and two times with acetone. Coverslips were stored under vacuum and handled in the dark." [10]