The application of nanotechnology in life sciences, nanobiotechnology, is the convergence of engineering and molecular biology, leading to a new approach for biological and chemical analysis with high sensitivity, specificity and accuracy. In this trend, atomic force microscope (AFM) has been broadly employed in biological study because it has no requirement of electric conductivity or labelling process of the sample. Moreover, it can be relatively easily operated in fluids under nearly physiological conditions. Today, as there are few alternative experimental methods for protein imaging or molecular manipulation under physiological condition, AFM becomes an indispensable tool for nanobiotechnology. AFM is a versatile instrument that can image surfaces with nanoscale resolution, probe local mechanical properties, and measure a variety of interaction forces from nN to pN. For example, the observation of a single membrane protein, protein–protein interaction and the mechanical unfolding of a single protein molecule have already been reported. In addition, it has been used for bio-patterning at the submicrometer scale as well as visualization and quantitative measurement of materials, such as tip-induced local oxidation, dip-pen lithography. In-situ biological detection on the patterned local area was achieved in our previous study, which was shown to be almost impossible in other lithographic methods at this point.

Recently, the development of experimental tools allowing to measure of minute forces has opened new perspectives in material- and life sciences, and the mechanical properties of biological molecules, such as actin filaments and DNA, attracted the interest of researchers. Especially, the capability of mechanical manipulation on a limited area provides a new opportunity in the field of molecular device and biophysical phenomenon. Mechanical manipulation of selected biomolecules is a powerful approach, which has given information on the molecular behaviours. Force spectroscopy in AFM is a relatively new technique that analyzes the forces necessary for separating individually bound molecules and quantifies kinetic and thermodynamic parameters of intermolecular interactions that are unavailable by other techniques.

Here, we employed the AFM-based molecular pulling technique to demonstrate mechanical unfolding of Cu/Zn superoxide dismutase (SOD1) aggregates. SOD1 is a dimeric protein with two identical subunits arranged with eight b-stands connected by seven loops. Each subunit contains a zinc atom (Zn) whose main role is to stabilize the protein and a copper (Cu) atom responsible for its dismutase activity. Over 100 mutants of SOD1 have been implicated in the neurodegenerative disease, familial amyotrophic lateral sclerosis (FALS), such as Alzheimer's disease. Aggregation of SOD may play a causative role on FALS and it has been reported by a few researcher that subunits of SOD aggregation were cross-linked via disulfide bridges, that is, tandem repeats of a single domain. Thus characterizing mechanical properties of SOD aggregates is regarded as a meaningful approach to comprehend structural and conformational properties of SOD aggregates.

In this study, purified wild type apo SOD, that is, demetallized SOD,  was diluted with a 50 mM phosphate buffered saline (PBS) solution (pH=7.0) at a concentration of 0.1 mM. For the purpose of oxidative stress, 10 mM H₂O₂ and 1 mM CuSO₄ were added to apo SOD, dissolved solution. It has been extensively reported that aggregation of apo-type protein was readily occurred in this condition. A formation of aggregates was investigated by UV-vis spectroscopy and these were covalently coupled onto an amine activated Si surface. Topographic and lateral images of immobilized aggregates were simultaneously obtained in a single scan via an AFM, and then we obtained force distance curve by approach and retreat of AFM tip on the aggregates. Using an Au-coated AFM tip, when tip is brought into contact with the aggregate layer, several aggregates bind to the AFM tip via an affinity between Au of tip and SH-residue of free cysteine in SOD aggregates. When the tip is raised, the attached aggregates are pulled up and away from the aggregates immobilized on the substrate. The force curve was determined by measuring the deflection of the cantilever as it approached and retracted from the sample.

Therefore, we observed the morphology of immobilized aggregates and repetitive rupture peaks of a multi-domain protein aggregates bound in parallel and series via an AFM apparatus. This technique allows one to completely investigate the mechanical properties of SOD aggregates through a simple mathematical calculation. Results of this study provide the mechanical property data (base) for biomaterials and the feasibility of biomolecular-device system via a mechanical manipulation of biological macromolecules with cells.