Amyloid fibrils are insoluble protein aggregates that have been associated with a range of neurodegenerative diseases, including Huntington, Alzheimer's, Parkinson's, and Creutzfeldt-Jakob disease . The fibrils typically have a diameter ranging from 4 to 12 nm, and lengths from 100 nm up to several microns [2–4]. In this study, we investigated the nanomechanical properties of amyloid fibrils formed from the human α-synuclein protein, which is associated with Parkinson's disease. α-Synuclein amyloid fibrils are found in the brains of Parkinson's disease patients as components of larger plaques called Lewy bodies [5, 6].
Atomic force microscopy (AFM) has been primarily used as an imaging tool to determine morphological parameters such as height and length of amyloid fibrils, such as those formed from α-synuclein [2–4], insulin , and β-lactoglobulin . AFM is also a powerful technique for characterizing mechanical properties. With the ability to exert and measure forces up to the piconewton range, AFM is a particularly suitable tool to determine the nanomechanical properties of nanometer-sized biological structures, such as amyloid fibrils. Mechanical properties such as stiffness, rigidity, resistance to breakage or adhesive properties of these fibrils or individual monomers are interesting for the use of these fibrils as nanomaterials, for getting a better understanding of the physico-chemical properties of these fibrils, and to get more insight into their structure and growth [9–14].
Indentation-type AFM or single-point nanoindentation (SPI), for example, implemented as 'Point-and-Shoot' in the Veeco operating software, is the most widely used method to measure nanomechanical properties of a sample. In this mode, the tip approaches and indents the sample until a certain predefined force is reached. At this point the tip is retracted again. During this approach and retract cycle the force is continuously measured, resulting in a force versus distance graph. AFM nanoindentation has been performed on different biological substrates such as collagen , insulin fibrils, and crystals , but also on different polymeric materials, such as fibrils used for biodegradable scaffolds . The approach-retract cycle is typically performed at a rate of 0.5 to 10 Hz, which makes this method inherently slow. To get an overview of the mechanical properties of a sample, nanoindentation can be used in a force-volume mode. Here, for every pixel in an image a complete force curve is recorded, which results in data acquisition times of up to hours for a single image.
Recently, several different surface property mapping techniques have become available that work at much higher speeds, leading to significantly increased data throughput [18–20]. Two commercially available approaches are PeakForce QNM and Harmonic force microscopy or HarmoniX (Veeco, Santa Barbara, CA, USA). PeakForce QNM is based on the force-volume approach; however, the speed of taking the force curves is significantly increased (either at 1 or 2 kHz). In this mode the maximum force exerted on the sample is maintained constant, which is beneficial for soft delicate biological samples. Because of the recent introduction of the Peakforce QNM method, only a few studies have been reported, such as the stiffness mapping of polymer blends .
HarmoniX is another surface property mapping technique based on the nonlinear dynamic behavior of a cantilever in tapping mode due to repulsive and attractive forces caused by the specific material characteristics of the sample acting on the tip [21, 22]. Because of the low bandwidth of the cantilever response, this information ends up in the phase image as obtained during tapping mode imaging. This phase signal is related to energy dissipation, which is determined by the viscoelastic and adhesive properties of the sample [21, 23]. However, because of the convolution of multiple physical properties into one signal, interpretation of these images is not straightforward. The higher harmonic vibrations of the cantilever excited by these material properties can provide more information, but they are heavily suppressed and are difficult to measure [21, 24]. In HarmoniX, a torsional cantilever with the tip positioned off-axis solves this problem and acts as a high bandwidth force sensor . HarmoniX has been applied to both polymers and biological features, for example, DNA [25, 26].
We used these three different methods, SPI, PeakForce QNM, and HarmoniX, to determine the modulus of elasticity of protein nanofibrils, generated from the E46K mutant of the human α-synuclein protein. The resulting values for the elastic modulus are in the range between 1.3 and 2.1 GPa. We discuss the relative merits of the application of these three methods specifically for the determination of the elastic properties of protein fibrils in more detail, with particular emphasis on the differences and difficulties of each method.