Comprehensive characterization of molecular interactions based on nanomechanics

Abstract

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

Figure 1. Schematic of the homemade measurement set-up for combined mode measurements.

For more information see also Braun et al. 2007 . An array of silicon cantilevers was mounted onto a piezo element. A sinusoidal excitation signal generated from a network analyzer swept the requested frequency range vibrating the cantilevers (1). The laser beam deflection detection technique was used to monitor the response of individual cantilevers (2). A frequency analyzer (3) compared input- and output signals and continuously recorded amplitude and phase spectra (4) as well as static deflection (bending) of the cantilevers (5). NOSETools software was used to analyze the spectra and extract the mass adsorption on the cantilever (postprocessing, 6). , , , A scanning electron microscopy image of a cantilever array is shown in the supplemental Figure S1.

Figure 2. Combined mode measurements of vesicle and melittin adsorption on the cantilever sensor.

The positive controls were pre-functionalized in such a way that vesicles only bind to the upper cantilever surface and melittin does not bind at all. The lower graph displays the mass adsorption and the upper graph reveals the surface stress development measured simultaneously. Note that the surface stress represents the differential signal between the positively and negatively functionalized cantilevers (two cantilevers each). The experiment was performed in 11 sections: (I) Baseline recording in buffer. (II, VI, VIII) Injections of 500 ng/ml DOPC vesicles. (III, V, VII, IX, XI) Buffer injections. (IV, X) Melittin injections (1 µM). Note that during the injection of melittin, the adsorbed mass is initially underestimated due to the high friction of the protein-solution, see also Braun et al., 2005 for a discussion. During the subsequent buffer injection the correct mass is measured.

Figure 3. Molecular model of the nano-mechanical changes on the sensor interface explaining the data.

Without vesicles the pre-functionalized cantilever (AUT SAM on gold) is straight (a). Adsorption of vesicles on the cantilever surface bends the cantilever upwards driven by the interaction forces between the cantilever and the vesicles, which are flattened by this interaction (b). During the peptide injection, the melittin molecules first bind to the vesicle surface (c), and later insert into the membrane and form channels by oligomerization (d).