Nanoscale AFM Calibration Standard Using DNA Origami Nanotechnology

Atomic Force Microscopy (AFM) is one of the important tools in the field of nanotechnology, facilitating both the fabrication and analysis of nanostructures. Precise calibration of AFM instruments is imperative to guarantee measurement accuracy. Currently, lateral calibration standards for a range of several tens of nanometers are hardly commercially available, an essential range for many nanotechnological applications. Furthermore, there is a lack of reference standards for validating accurate measurements in the liquid state, crucial for biological and chemical samples in AFM. This study addresses these challenges by proposing a novel approach to develop a calibration standard for AFMs at the nanoscale, compatible with both air and liquid environments. The method is based on DNA origami nanotechnology. DNA origami nanostructures are recognized as ideal candidates for engineering precise nanostructures because of their unique properties. These features include the precise addressability of DNA origami structures and the potential for atomic-scale labeling. In this study, the rectangular DNA origami was chosen for its simplicity and ease of modification. To minimize AFM tip-sample convolution effects, well-defined protrusions were placed onto the origami, providing clearer reference points for the AFM tip. These precisely positioned protrusions, define the final standard measurand. To ensure that the designed structure aligns with metrological requirements, a series of crucial assessments were performed on it in both air and liquid environments. These assessments focused on stability, reproducibility, and durability, which are significant factors for the nanostructures if they are used as dimensional calibration standards. The outcomes confirm the robustness of DNA origami structures, establishing them as a viable option for reliable calibration standards. After successful testing of the designed structure, an innovative AI-based measurement tool was developed to automate data analysis efficiently, ensuring the reliability of measurement results across a large number of nanostructures. This nanoscale reference standard, created by leveraging simple equipment and materials, opens doors to affordable and precise calibration, transforming the landscape for research and industry.