Scanning probe microscopy (SPM) can be successfully used in biological investigations¹. Scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) are two main SPM techniques and they are capable of imaging biological objects at molecular level under in situ conditions. With this advantage, STM and AFM are applied to study not only the topographical structures of bio-samples in their natural environment, but also their electrochemical properties at liquid-solid interfaces.

The electrochemical properties of biomolecules are usually studied by integral electrochemical methods, like cyclic voltammetry. The degree of surface coverage, the surface orientation of enzymes and the activity of a single enzyme remain a question of dispute, however, electrochemical SPM techniques can bring clarification. Electrochemical scanning tunnelling microscopy (EC-STM) and electrochemical atomic force microscopy (EC-AFM) provide visual topological information of biomolecules adsorbed on the electrode surface at single-molecular level. Some experimental issues, such as the requirement of the sample being conductive, potential damage to samples caused by the tunnelling current or tip-sample force, may limit their application. Scanning electrochemical potential microscopy (SECPM), a technique that measures the surface potential distribution at zero current, can be a promising alternative. SECPM shares most of the hardware setup with EC-STM except for the current amplifier being substituted by a potentiometer. It is easy to switch between SECPM and EC-STM to compare the topographical profile of the same area by mapping electron density and surface potential².

In our study, SPM techniques are used to study various biological objects, from nanometer-sized DNA and redox proteins to micrometer-sized bacteria spores. Since high-resolution images of DNA origami nanostructures can be achieved by using AFM³, we use both EC-AFM and SECPM techniques to investigate the morphology and potential-dependent adsorption behaviour of DNA scaffold and origamis⁴ on the modified HOPG surface. We found DNA scaffold can adsorb on HOPG in a certain potential range, and the adsorption structure changes with the applied electrochemical potential.

Horseradish peroxidase (HRP), a redox enzyme, was studied using both, EC-STM and SECPM under in situ conditions⁵. HRP molecules were adsorbed on highly oriented pyrolytic graphite (HOPG) surfaces and imaged in phosphate buffered saline. We found that SECPM shows a better resolution than EC-STM in imaging this biomolecule. SECPM is also capable of measuring the surface charge distribution of the adsorbed biomolecules at the electrochemical interfaces.

AFM in liquid was used to investigate the morphology and cellular process of Streptomycetes venezuelae both the wild type and the mutant⁶. To immobilize the bacteria on the surface for imaging, we use SiC patterned by photolithography with hole arrays (1.5×1.5×0.6 µm) to trap the bacteria and then measure under the physiological condition. The designed hole arrays trap the bacteria without causing any denaturation and due to the high electronic conductivity, they can serve as the working electrode under electrochemical conditions for EC-AFM and SECPM studies. EC-AFM was applied to image the morphology of the bacterial spore under different potential, and we found the morphology of the spore didn’t change in the wide potential window.

(1) Choi, E.; Kim, A.; Son, H.; Pyo, S. G. J. Nanosci. Nanotechnol. 2014, 14 (1), 924.

(2) Herpich, M.; Friedl, J.; Stimming, U. In Surface Science Tools for Nanomaterials Characterization; Springer Berlin Heidelberg: Berlin, Heidelberg, 2015; pp 1–67.

(3) Bald, I.; Keller, A. Molecules 2014, 19 (9), 13803.

(4) Kozyra, J.; Ceccarelli, A.; Torelli, E.; Lopiccolo, A.; Gu, J.-Y.; Fellermann, H.; Stimming, U.; Krasnogor, N. ACS Synth. Biol. 2017, 6 (7), 1140.

(5) Baier, C.; Stimming, U. Angew. Chem. Int. Ed. Engl. 2009, 48 (30), 5542.

(6) Santos-Beneit, F.; Gu, J.-Y.; Stimming, U.; Errington, J. Heliyon. Accepted