‌‌‌‌‌‌‌‌Scanning probe microscopy (SPM) can be successfully used in biological investigations. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) are two main SPM techniques and they are capable of imaging biological objects at single 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 tunneling 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 tunneling 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 (Figure 1). 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 potential.


Figure 1. EC-STM and SECPM setup scheme.


In our research, SPM techniques are used to study various biological objects, from nanometer-sized DNA and redox proteins to micrometer-sized bacteria spores.

Horseradish peroxidase (HRP), a redox enzyme, was studied using both, EC-STM and SECPM under in situ conditions. 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 (Figure 2 and 3) [1]. Additionally, SECPM can map the potential profile of the electrochemical double layers (EDLs) at the electrode/electrolyte interfaces. Since EDLs are easily influenced by any electrochemical reactions on the interfaces, SECPM can be a promising tool to offer a brand-new perspective on the ET reactions in redox enzymes.


Figure 2. A) SECPM image and B) EC-STM image of horseradish peroxidase on a HOPG electrode



Figure 3. A) 3D SECPM image of HRP on HOPG. B) Contour plot of (A). C) and D) xare PyMOL simulations of HRP.



SPM is also a powerful tool for the visualization of intercalation/de-intercalation process in Li-ion batteries (LIBs) and Na-ion batteries (NIB or SIB, S for sodium).


Structural changes of a battery electrode active material during charging/discharging are visualized using EC-STM on atomic scale in the previous works. By employing the tunneling current that is highly sensitive to tip-sample distance, the EC-STM was able to capture the reversible and irreversible profile changes upon ion intercalation/de-intercalation.


Currently, we are focused in understanding the behavior of the battery cathode material during cycling using in-situ EC-STM. Our initial experiments on NMC 811 cathode active material using STM inside the nitrogen filled glove box shows atomic resolution see the Figure 4


Figure 4. Atomically resolved STM image of NMC 811 and the profile of a single atom which has a size of about 2.6Å                      


High-resolution imaging of DNA origami can be achieved by AFM. We characterized the morphologies of different scaffolded DNA origami nanostructures (Figure 5 to 7) and the results helped to confirm the synthetic scaffold design with DNA origamis[2].


Figure 5. AFM imaging of the square DNA origami based on a DBS scaffold.


Figure 6. AFM imaging of the square DNA origami based on the pUC19 scaffold.



Figure 7. AFM imaging of triangle RNA-DNA hybrid origami based on a DBS scaffold.



AFM in liquid was also used to investigate the morphology and cellular process of Streptomycetes venezuelae both the wild type and the mutant 6. 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 (Figure 8). 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 potentials, and we found the morphology of the spore didn’t change in the wide potential window.



Figure 9. EC-AFM imaging bacterial spores trapped in a patterned hole on SiC surface.


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

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