Advanced scanning electrochemical probe microscopy : from sensing to electrochemical force spectroscopy

Abstract

Scanning probe microscopy (SPM) techniques, which provide information on topography, conductivity, chemical flux and interface reactivity at the micro- to nanometer scale, are becoming increasingly important not only in energy-related topics such as battery research and catalysis but also in biomedical research. For example, the formation of biofilms and adhesion of microorganisms that attach and grow on solid surfaces, causing about 80% of infections in humans is still not fully understood. Quantitative detection of biomedical signaling molecules such as adenosine triphosphate (ATP) and hydrogen peroxide (H2O2), a stable representative of the reactive oxygen species (ROS) at the cell level may contribute to gain fundamental knowledge for pulmonary function under physiological and pathophysiological conditions. Among the multitude of SPM techniques, atomic force microscopy (AFM) providing high-resolution topographical information as well as physical properties of the sample and scanning electrochemical microscopy (SECM), which gives access to reactivity mapping and localized sensing of molecules are highly attractive for biomedical studies. In recent years, strong efforts were dedicated towards the development of hybrid SPM techniques, such as scanning ion conductance microscopy (SICM)-SECM and AFM-SECM, as they combine the benefits of the individual SPM technique, providing simultaneously multidimensional information, which is essential for samples that may alter over a short span of time. This cumulative thesis is based on five articles published in international peer-reviewed journals, presenting microsensor developments and their applications in biomedical research and advanced AFM-SECM probe design, fabrication and applications. The research comprises the development of modified ultramicroelectrodes (UME) for the detection of biomedically relevant analytes, and colloidal AFM-SECM probes and their functionalization for e.g., electrochemical force spectroscopy measurements. In respect to sensing, UMEs were modified with recognition layers or electrocatalytically active layers for the sensitive and selective detection of signaling molecules. UMEs were also used for structured deposition of functional polymer spots. The detection of the signaling molecules such as ATP, catecholamines and ROS using modified UME was one focus of this dissertation. For the detection of H2O2, the electrode surface was modified with Prussian Blue (PB, KFeIII[FeII(CN)6]), which allows the reduction of H2O2 at a potential of -0.05 V vs. Ag/AgCl. Prussian White (PW, K2FeII[FeII(CN)6]) is oxidized to PB, while H2O2 is reduced to hydroxide. Such low potential for H2O2 detection eliminates the co-detection of other electroactive species (e.g. ascorbic acid), usually present in biological/biomedical samples, in contrast to e.g. bare platinum electrodes where H2O2 is oxidized at a potential of 0.6 V vs. Ag/AgCl. Due to the limited stability of PB-modified electrodes at pH values > 7.0, platinum black (Pt-black) was also investigated as electrocatalytic layer, reducing the oxidation potential of H2O2 from 0.6 V (bare Pt-UME) to 0.3 V vs. Ag/AgCl. Detection limits were achieved in the nanomolar range enabling the detection of extracellular H2O2 concentrations from blood cells of trauma-induced swine. For Pt-black modified electrodes the required potential of 0.3 V vs. Ag/AgCl for H2O2 oxidation cannot assure that interfering analytes (ephedrine and norephedrine) possibly present in the sample are not co-determined. Because of that, a method for the simultaneous measurement of catecholamines as possible interfering species using carbon fiber electrodes (oxidation of catecholamines at 0.2 V vs. Ag/AgCl) has been established. ATP as one of the most important signaling molecule in eukaryotic cells and plays a significant role, i.e. ATP serves as energy resource in all living organisms. Therefore, its regeneration by ATP-synthases is of particular importance. ATP can be detected electrochemically using biosensors, which has the advantage that ATP release can be monitored over time. Amperometric microsensors with increased stability and sensitivity based on poly(benzoxazines) as immobilization matrix were developed, characterized and used to determine ATP concentrations of inverted vesicles from E. coli and P. putida. Such experiments were performed using dual microelectrodes, with one electrode modified with glucoseoxidase (GOx) and hexokinase (HEX) and the other electrode for positioning of the dual electrode assembly in combination with SECM. In addition to sensing of ATP and ROS, tip-integrated chemical sensing of dopamine and ascorbic acid with colloidal BDD-AFM-SECM probes were also developed in the course of this thesis. For this purpose and for the first time, a colloidal AFM-SECM probe was developed, which comprises a spherical boron-doped diamond (BDD) electrode attached to a – besides the contacting area - insulated soft silicon nitride cantilever. This combined SPM probe has several advantages, such as a broad potential window for electrochemical measurements, physical and chemical inertness of the spherical probe but at the same time a relatively low force constant of the cantilever for force spectroscopy. Applying a potential to the AFM-SECM probe enables force spectroscopy measurements, while at the same time electrochemical properties of the sample can be probed. Recording force distance curves simultaneously with current-distance curves will be termed ‘electrochemical force spectroscopy’ within this thesis. These probes represent an innovative addition to scanning electrochemical probe microscopy (SEPM), as they are suitable for a variety of different complementary experiments. For force spectroscopic measurements in air, altering the surface termination of BDD spheres e.g., from hydrogen terminated to oxygen-terminated changes the chemical properties from hydrophobic to hydrophilic and hence, changes significantly the force interaction with the sample surface. Moreover, in electrolyte solution, a potential can be applied to the colloidal AFM-SECM probe and electrochemical force spectroscopy can be carried out. Such probes are also suitable to perform conductive current sensing AFM (CS-AFM) studies, due to the physical robustness and conductivity of the probe. A series of different SPM measurement can be performed using the same probe switching from measurements in air to immersing the sample in solution. Given the excellent electrochemical properties, these probes can be also used in sensing applications as exemplarily shown with the quantification of dopamine in the presence of ascorbic acid. A contribution, which was just published presents the modification of conductive colloidal probes with functional polymer films such as polydopamine (PDA) via electropolymerization. PDA is highly attractive as the surface functionality can be switched via oxidation or reduction of the film. Within this contribution, adhesion measurements in dependence of the applied potential were performed e.g., at bacterial cells. By applying a potential, the functional groups on the surface can be switched from oxidized state to reduced state (phenolic and quinoid groups), which has a strong effect on adhesion measurements. The versatility of this combined probe for force spectroscopic applications at electrified interfaces was demonstrated at model substrates. Hydrophilic gold surfaces and self-assembling monolayers with different wettability were used to investigate the influence on the adhesion and first measurements on the P. fluorescens bacteria were shown. The adhesion is dependent on the applied potential of the PDA-modified colloidal AFM-SECM probes and thus ultimately dependent on the functional surface groups of the PDA film. Moreover, it could be demonstrated that the deposited PDA has polymeric character based on the measured contour lengths, which describes the length of a polymer chain at maximum physically possible extension. PDA is a mussel inspired polymer derived from naturally occurring melanin, formed by the oxidation of dopamine. Typically, PDA films are obtained via a simple dip coating process, but films can also be obtained via electropolymerization. A method for the generation of PDA films through pulsed electrochemical deposition was developed. Aim of this study was to correlate the deposition parameters with the obtained morphology, film thickness and electron transfer properties. In order to perform screening experiments, PDA microspots were deposited via SECM direct mode. Similar to the conventional electrochemical deposition using cyclic voltammetry, the number of applied pulses enables control of the PDA film thickness and morphology. The newly developed pulsed method was used in studies to modify ultramicroelectrodes and colloidal AFM-SECM probes.