Advancing atomic force microscopy-scanning electrochemical microscopy based sensing platforms for biological applications

Combined atomic force microscopy-scanning electrochemical microscopy (AFM-SECM) is a powerful emerging technology capable of providing simultaneous topographical and electrochemical imaging at the sample surface. Specifically, AFM-SECM based on tip-integrated electrodes that are recessed from the apex of the AFM tip provides miniaturized electrodes that can be positioned at a constant distance to the sample surface. Surface modification of the tip-integrated electrode area (e.g., with biosensors) further enhances the versatility of such bifunctional probes. The integration of amperometric biosensors into AFM-SECM probes facilitates obtaining enhanced information during measurements of relevant molecular processes at live biological specimen. Of particular interest to this work was the detection of adenosine triphosphate (ATP) at a cellular level, as ATP is involved in many biologically relevant processes. However, there are several challenges concerning the integration of biosensors into bifunctional AFM-SECM probes. This thesis focuses on addressing and advancing several of these limitations.;The first part of the thesis describes novel thin film insulation materials for combined AFM-SECM probes. Insulation materials for microelectrochemical experiments are of crucial importance, since they need to be temporally stable, pinhole free, and sufficiently thin. The latter aspect is of particular importance for AFM-SECM based applications to decrease possible interactions of the probes during scanning of sample surfaces with the sample topology along with improved SECM performance. Plasma-polymerization is introduced as an attractive alternative to current state-of-the-art insulation techniques. Insulation layers with a thickness of <300 nm were found to exhibit excellent insulating properties and satisfying temporal stability for successful application in AFM-SECM approach/imaging experiments.;The second focus of this work was the implementation of novel approaches for increasing the AFM tip-integrated electrode area. Particularly in conjunction with biosensing experiments, the electrode areas in conventionally focused ion beam (FIB) fabricated AFM-SECM probes are too small for generating a detectable current response during scanning experiments. However, while increasing the tip-integrated electrode area, sufficient electrochemical resolution during the SECM experiment should be maintained. Ion beam induced deposition (IBID) was used to generate platinum carbon (PtC) composite materials at AFM-SECM probes, thereby successfully increasing the tip-integrated electrode area, as determined by cyclic voltammetry. Moreover, PtC materials fabricated via IBID were thoroughly characterized in terms of their physical and electrochemical properties. Studies at PtC-based ultramicroelectrodes (UMEs) revealed that the carbon fraction in the composite was inhibiting the charge transfer kinetics at the electrode surface for certain analytes. Therefore, several pre-treatment strategies were investigated including annealing, UV/ozone treatment, and post-deposition FIB milling. It was found that annealing lead to the desired electrode properties, as obtained from PtC UMEs, however, was of limited applicability to AFM cantilevers. FIB milling proved to be the most promising alternative treatment procedure improving charge transfer properties at the electrode along with fabrication compatibility at AFM-SECM probes. The third part of this thesis aimed at providing fundamental studies on AFM-SECM application at live epithelial cell monolayers. Due to the soft and dynamic nature of the samples along with the variability of the cell surface, thorough characterization of the cell surface was mandatory prior to AFM-SECM experiments. Therefore, AFM was used in different imaging modes to characterize the surface structures of epithelial cells. It was found that epithelial cell monolayers are amenable to extended AFM imaging; however, the force applied to the sample surfaces has to be carefully optimized, which was accentuated by results obtained during AFM-SECM based feedback mode experiments. Prior to the incorporation of ATP biosensors into batch-fabricated AFM-SECM probes, SECM-based experiments were performed, and have confirmed the presence of ATP at the surface of live epithelial cell monolayers. Moreover, imaging experiments conducted by AFM-SECM have enabled laterally resolved detection of ATP at live epithelial cell monolayers for the first time. Additionally, PtC composite materials introduced in the second part of this thesis were evaluated for applicability as transducer platforms for enzymatic biosensors. It was shown that pristine PtC did not exhibit adequate charge transfer characteristics for the electrooxidation of H2O2, whereas post-treated composites revealed strongly increased oxidation currents approaching the behavior of pure platinum electrodes. Glucose biosensors were deposited at PtC-based UMEs, and satisfying sensitivities and saturation currents were observed. The response time at this point was insufficient for imaging applications consequently further improvement of the biosensor immobilization procedure at PtC materials is required. It is anticipated that combination of all advancements obtained throughout this thesis along with an enhanced immobilization procedure will lead to optimized and miniaturized tip-integrated ATP biosensors for the localized detection of ATP at the surface of live epithelial cell monolayers.