84 An Application of Atomic Force Microscopy to Measure the Spring Constant of Single Antigen-antibody Complex L. A. Chtcheglova*, G. T. Shubeita, S. K. Sekatskii and G. Dietler *Institut de Physique de la Matière Condensée, University of Lausanne, CH-1015 Lausanne, Switzerland e-mail address corresponding author: Lilia.Chtcheglova@ipmc.unil.ch fax: +41 (0)21 692 3635 Aim of our study is to use Atomic Force Microscopy (AFM) to measure the spring constant for single bovine serum albumin (BSA) – polyclonal antibody to BSA (Ab-BSA) complex (k complex ) as a function of pulling off force and complex extension. By the combination of AFM operated in the force-spectroscopy mode with the small dithering of the AFM tip we are able to detect the change of the dithering amplitude upon the pulling off process and to derive the value of k complex . At the moment of single specific bond rupture the spring constant value of 0.017 ± 0.003 N/m is determined. 1. Introduction Over the last decade, the AFM has been widely extended to measure interaction forces at the molecular level (Force Spectroscopy) [1]. By biological functionalization of tip and sample it becomes possible to investigate molecular forces, like forces between a receptor and a ligand including antigen-antibody (Ag-Ab) interactions [2]. Recently, Schindler et al. [3] proposed to use in addition a small modulation of the tip- sample distance at a selected optimal driving frequency ω. Recording the amplitude of the tip vibrations at this driving frequency leaded to an improved molecular recognition fidelity. This method opens also the possibility to measure the dynamical properties of single molecules. Here we developed this approach for the measurement of the spring constant of a stretched BSA – Ab- BSA complex. A complete study of specific unbinding forces between BSA and poly- and two monoclonal antibodies to BSA were earlier accomplished in our laboratory. 2. Materials and Methods Standard Si 3 N 4 cantilevers with a nominal spring constant of 0.06 N/m (Digital Instruments (DI), Santa Barbara, CA) and with range of resonant frequency in liquid from 8.5 to 10 kHz were functionalized with polyclonal rabbit anti-bovine serum albumin antibody (Ab- BSA). Before biological functionalizion of substrates (mica) with BSA molecules chemical coating of mica was done by silanization using 3-aminopropyltriethoxysilane (APTES). BSA and antibodies were purched from Sigma and used without further purification. All experiments were performed in a phosphate buffer solution (PBS) (50 mM, 150 mM NaCl, pH 7.4). Experimental setup is presented in self-exploratory Fig. 1. A commercial scanning probe microscope (Nanoscope IIIa; DI) operated in “force-volume” mode was used. A small sine wave modulation voltage U mod with a peak-to-peak amplitude of 20-80 mV at frequency of ω=2π*3.2 kHz was additionally applied on the piezocrystal (normally used in tapping mode option). This modulation was equivalent to application of a driving force F ω onto the cantilever, and caused a tip dithering with the amplitude of 0.5-2 nm. This resulted in the modulation of the photodiode current at the same frequency ω. The signal from the photodiode was measured by a SR750 digital lock in amplifier. Therefore, force-distance cycles and amplitude-distance cycles were recorded simultaneously. We have not detected a dependence of the results on the dithering amplitude. The smallest reasonable loading rate value of 0.7 nN/s was used. 3. Results and Discussion Fig. 2A shows our typical experimental data. The recording of the data starts when the tip and sample are in the non-contact region (interval A-B). When the tip is in contact with the surface (B-C approaching, C-D retracting), the cantilever deflection is a linear function of the displacement of the sample.