Soft-probe-scanning electrochemical microscopy reveals electrochemical surface reactivity of E. coli biofilms

Sorour Darvishi, Hubert Girault, Andreas Stephan Lesch, Emadeddin Oveisi, Horst Pick
2021
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Résumé

Investigating and understanding dynamic biofilm growth mechanisms is challenging, often because state-of-theart optical characterization tools provide limited information. Micrometric electrochemical imaging of Escherichia coli biofilms using Soft-Probe-Scanning Electrochemical Microscopy (Soft-Probe-SECM) is herein presented as a complementary technique. A soft microelectrode is scanned over biofilms in a gentle contact mode, which is essential to provide a constant working distance. The on-film reduction of an electro-active compound, here the oxidized form of ferrocene methanol, is used to create in situ biofilm metabolic activity maps by applying the feedback mode of SECM. SECM approach curves of identically grown biofilms suggest that the SECM-based detection of metabolic activity is surface-confined. The analysis could therefore be carried out on entire biofilms as well as on tape-stripped biofilm surface layers. The method is further capable of distinguishing between biofilms containing E. coli cells either with or without ampicillin-resistance. Finally, the SECM detection of the degradation of an E. coli biofilm in the presence of different gentamicin concentrations is presented.

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https://infoscience.epfl.ch/record/285194?ln=fr

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Soft-probe-scanning electrochemical microscopy reveals electrochemical surface reactivity of E. coli biofilms

Sorour Darvishi, Hubert Girault, Andreas Stephan Lesch, Emadeddin Oveisi, Horst Pick

Investigating and understanding dynamic biofilm growth mechanisms is challenging, often because state-of-the-art optical characterization tools provide limited information. Micrometric electrochemical imaging of Escherichia coli biofilms using Soft-Probe-Scanning Electrochemical Microscopy (Soft-Probe-SECM) is herein presented as a complementary technique. A soft microelectrode is scanned over biofilms in a gentle contact mode, which is essential to provide a constant working distance. The on-film reduction of an electro-active compound, here the oxidized form of ferrocene methanol, is used to create in situ biofilm metabolic activity maps by applying the feedback mode of SECM. SECM approach curves of identically grown biofilms suggest that the SECM-based detection of metabolic activity is surface-confined. The analysis could therefore be carried out on entire biofilms as well as on tape-stripped biofilm surface layers. The method is further capable of distinguishing between biofilms containing E. coli cells either with or without ampicillin-resistance. Finally, the SECM detection of the degradation of an E. coli biofilm in the presence of different gentamicin concentrations is presented.

2021

Chargement

Non-invasive and semi-invasive bioimaging is of great interest for the rapid diagnosis and efficient as well as specific treatments of biological systems affecting human health, such as cancer tissue and biofilms. Electrochemical bioimaging is one of the diverse methods that can reach high sensitivities in small liquid volumes. When using (sub)micrometric electrodes, it can reach a high spatial-temporal resolution. The electrochemical processes can be based but are not limited to electron transfer reactions and the detection of metabolic activity indicators. An electrochemical scanning probe platform can be used, such as Scanning Electrochemical Microscopy (SECM), in which a micrometric electrochemical probe is translated in close proximity to a sample immersed in an electrolyte solution. In most SECM modes, the SECM tip detects locally electro-active species that are generated or consumed by active surface sites allowing to create surface reactivity maps of biological entities. It has in particular found applications for studying normal and cancer cells, yeast, and bacteria. In this thesis it was looked at utilizing Soft SECM probes as an analytical tool for sensing and analyzing complex biological samples, such as skin and three-dimensional biofilms in an as less as possible invasive manner, potentially forming the basis for the development of point of care devices for early detection and treatment. Electrochemical analysis was completed by fluorescence imaging as a state-of-the-art methodology. The study of melanoma and bacterial biofilms as the two major targets in this thesis was divided into five main chapters. The electrochemical detection of melanoma was based on the specific detection of tyrosinase, a melanoma protein marker. In Chapter 3, a non-invasive electrochemical melanoma detection approach is based on using adhesive tapes to collect and fix cells from a suspicious skin area and transfer the cells into an SECM for their analysis with soft probes. The enzyme was tagged with antibodies that contained a peroxidase label for the production of an electrochemical probe. In Chapter 4, tyrosinase was addressed with a microneedle type electrochemical biosensor containing a TYR-sensitive redox molecule for TYR sensing. Thereafter, Soft-Probe-SECM was used to investigate the electrochemical surface reactivity of Escherichia coli (E. coli) biofilms (Chapter 5) and to follow the effects of various antibiofilm treatments, such as gentamicin, sodium azide, silver nanoparticles, and flashlight (Chapter 6). Finally, in Chapter 7, an antibiofilm nanocarrier based on a DNA origami nanostructure for E. Coli biofilm treatment was studied. The DNA origami was loaded with doxorubicin (DOX) to enable the transport of DOX into the three-dimensional structure of the biofilm. In this way, the biofilm protection barrier against DOX, as generated by the extracellular polymeric matrix, was overcome. The biofilms were studied by confocal laser scanning microscopy and microelectrode arrays.

EPFL2022

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EPFL2022