Measuring the force of single protein molecule detachment from surfaces with AFM.

Atomic force microscopy (AFM) was used to measure the non-specific detachment force of single fibrinogen molecules from glass surfaces. The identification of single unbinding events was based on the characteristics of the parabolic curves, recorded during the stretching of protein molecules. Fibrinogen molecules were covalently bound to Si(3)N(4) AFM tips, previously modified with 3-aminopropyl-dimethyl-ethoxysilane, through a homobifunctional poly(ethylene glycol) linker bearing two hydroxysulfosuccinimide esters. The most probable detachment force was found to be 210 pN, when the tip was retracting with a velocity of 1400 nm/s, while the distribution of the detachment distances indicated that the fibrinogen chain can be elongated beyond the length of the physical conformation before detachment. The dependence of the most probable detachment force on the loading rate was examined and the dynamics of fibrinogen binding to the surface were found amenable to the simple expression of the Bell-Evans theory. The theory's expansion, however, by incorporating the concept of the rupture of parallel residue-surface bonds could only describe the detachment of fibrinogen for a small number of such bonds. Finally, the mathematical expression of the Worm-Like Chain model was used to fit the stretching curves before rupture and two interpretations are suggested for the description of the AFM curves with multiple detachment events.

Measurement of interaction forces between fibrinogen coated probes and mica surface with the atomic force microscope: The pH and ionic strength effect.

The study of protein-surface interactions is of great significance in the design of biomaterials and the evaluation of molecular processes in tissue engineering. The authors have used atomic force microscopy (AFM) to directly measure the force of attraction/adhesion of fibrinogen coated tips to mica surfaces and reveal the effect of the surrounding solution pH and ionic strength on this interaction. Silica colloid spheres were attached to the AFM cantilevers and, after plasma deposition of poly(acrylic acid), fibrinogen molecules were covalently bound on them with the help of the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of N-hydroxysulfosuccinimide (sulfo-NHS). The measurements suggest that fibrinogen adsorption is controlled by the screening of electrostatic repulsion as the salt concentration increases from 15 to 150 mM, whereas at higher ionic strength (500 mM) the hydration forces and the compact molecular conformation become crucial, restricting adsorption. The protein attraction to the surface increases at the isoelectric point of fibrinogen (pH 5.8), compared with the physiological pH. At pH 3.5, apart from fibrinogen attraction to the surface, evidence of fibrinogen conformational changes is observed, as the pH and the ionic strength are set back and forth, and these changes may account for fibrinogen aggregation in the protein solution at this pH.

pH and ionic strength effect on single fibrinogen molecule adsorption on mica studied with AFM.

Although several investigations have been reported on the effect of pH or ionic strength on protein adsorption, most of them have been carried out with protein monolayers and not with single molecules. We have used atomic force microscopy to image, in phosphate buffer, single fibrinogen molecules adsorbed on mica and compare the surface coverage at variable pH (7.4, 5.8, 3.5) or ionic strength (15, 150, 500 mM) conditions. The images obtained and the statistical analysis of the surface coverage indicate adsorption enhancement at the IEP of fibrinogen (pH 5.8) and minimum adsorption at pH 3.5. On the other hand, more protein was adsorbed when the salt concentration of the buffer at pH 7.4 was increased from 15 to 150 mM. However, further increase of salt concentration up to 500 mM resulted in decreased adsorption. To confirm the aforementioned results an approaching bare Si(3)N(4) tip was used as an electrostatic analogue to a protein molecule and interaction force curves between it and the substrate were recorded. The results were in consistence with the double layer theory which justifies the screening of electrostatic repulsion as the salt concentration increases.