Tutorials - Surface chemistry in BioAFM

Binding of biological molecules to a solid substrate

Karrasch et al. [10] published a method to covalently bind biomolecules and supramolecular assemblies such as

• bacteriophage T4 polyheads
• eucariotic intermediate filaments
• HPI layer of deinococcus radiodurans

Their method is described as follows:
"For the covalent binding of samples to the modified glass surface, the [APTES-ANBNOS-covered] coverslips were squeezed between two glass disks (borosilicate safety sight glass; diameter = 12 cm, thickness = 2 cm) at a pressure of 100 to 5000 N/cm² to bring the hydrophilic biological structures into close contact with the hydrophobic cross-linker. Covalent coupling of the samples was induced by activating the azide with ultraviolet (UV) irradiation at 366 nm (Sylvania F8T5) at a distance of 10 cm for 3 min. The extent of the reaction was determined from the change in the absorption band of ANB-NOS at 312 nm. Coverslips were rinsed thoroughly with water to remove excess protein and stored in water or buffer. " [10]

Liu et al. bound a plant lectin (E-PHA) to ANBNOS-coated glass coverslips [24] :
"Ten microliters of E-PHA lectin (2 mg/mL in phosphate-buffered saline [PBS] buffer: 145 mM NaCl and 5 mM NaH₂PO₄/Na₂HPO₄, pH at 7.4) were compressed between tow ANBNOS-coated glass coverslips under irradiation at 302 nm (8 Watt) at 10 cm from the light source for 3 min to bring the hydrophilic lectin in close contact with the hydrophobic ANBNOS. Completion of light-activated crosslinking was confirmed spectrophotometrically. The coverslips were rinsed with PBS five times and stored in PBS [...]." [24]

The Kleinschmidt method

For sample preparation in electron microscopy Kleinschmidt in 1968 [ ] published a monolayer transfer method:
"The DNA solution is spread on the surface of a water subphase. The protein film, along with the adsorbed DNA molecules, is picked up directly by the substrate." [16]

DNA adsorption to APTES mica

The method to adsorb double stranded DNA to an APTES modified mica sheet is described in [19]:
"Modified mica strips were immersed into DNA in Tris/HCl buffer (pH 7) (10 mM Tris/HCl, 10-20 mM NaCl, 5 mM EDTA) and incubated at room temperature for between 1 and 2 h. Concentration of DNA was varied between 0.01 and 0.1 µg/mL. λ-DNA and HindIII fragments of λ-DNA were purchased from New England BioLabs and used without additional purification. After the adsorption stage had been completed, the samples were rinsed with deionized water, blotted at the edge and vacuum-dried.

Protein adsorption

AFM has the potential to make unique contributions to the study of (membrane) proteins, not only in generating nanometre resolution structures, but also in studying structural changes under various conditions.
For AFM, supported membranes have been shown to be most appropriate for achieving high resolution. Unsupported membranes, such as the plasma membrane in an intact cell, are too soft and easily deformed under the AFM tip, preventing any high-resolution imaging. So far, the best resolution achieved in solution on cell surfaces, either fixed or native, is only in the range of several 10 nm, which is insufficient to resolve membrane proteins and other membrane structures. With supported membranes on mica or glass, the best resolution is an order of magnitude higher, approaching subnanometre in some cases. [17]

Protein adsorption is a net result of various complex interactions between and within all components, including the solid surface, the protein, the solvent and any other solutes present. These interaction forces include dipole and induced dipole moments, hydrogen bond forces and electrostatic potentials. All these inter- and intramolecular forces will contribute to a decrease of the Gibbs energy during absorption. [20]

Resolution by AFM on proteins is highest when the proteins are packed into 2-dimensional arrays. The beautiful and extensive analyses of such protein arrays are included in two recent reviews [Czajkowsky, Shao, Febs. Lett. 430 (1998) 51. Müller, Fotiadis, Engel, Febs. Lett. 430 (1998) 105.], which describe the detailed substructure of protein subunits in these 2-dimensional arrays and the changes in substructure that occur when the subunits are compressed by increasing the imaging force. [21]

Substrate supported lipid membranes

For decades, the use of the Langmuir trough to transfer mono- and bilayers of phospholipids, fatty acids or other amphiphilic compounds onto glass, mica or silicon is well established. It depends on the properties of the substrate surface, if it is hydrophilic or hydrophobic, to influence the orientation of the molecular layer(s). By this method, a surface coverage of nearly 100 % can be achieved. [16]