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- FG Nanobiotechnologie (2) (remove)
The present work aimed to develop a new biochemical sensor based on a gold surface with well-defined structural nano-motifs.
The new anchor molecule 1,3-dimercaptopropan-2-ol was synthesized for this purpose. Due to its symmetrical structure with two thiol groups, it forms a very stable self-assembled monolayer (SAM) on the gold surface. The monolayers of 1,3-dimercaptopropan-2-ol were studied by cyclic voltammetry, impedance spectrometry, X-ray photoelectron spectroscopy (XPS), kinetics of capacitance and contact angle measurements. The structure of the SAM is determined by the adsorption conditions. The comparative study of the desorption of SAM shows that the stability of SAM increases when the molecules are mostly bound through both thiol groups.
The electrochemically active p-benzoquinone was immobilized on this monolayer. The 1,3- dimercaptopropan-2-ol served as an anchor molecule, 3-mercaptopropionic acid acted as a spacer, and 1,4-benzoquinone as a head group. The surface concentration of p-benzoquinone was 2.5 ± 0.2×10-10 mol•cm⁻². This corresponds to a functionalization of 65 ± 5% SAM molecules. The assembled layer can be used for electrically addressable immobilization of biomolecules or development of electrocatalytic sensors.
The SAM with terminal benzoquinone was used as a sensor for the quantitative detection of hydrogen peroxide. The sensor characterization was performed with cyclic voltammetry in the potential range from -0.6 V to +0.9 V as well as in the anodic or cathodic range only. The results indicate an oxidative electrochemical decomposition of the hydrogen peroxide at a potential of about +0.4 V with oxygen formation, while at cathodic potentials a reduction of the formed oxygen as well as of the hydrogen peroxide takes place. The reduction of the oxidation potential for hydrogen peroxide at the benzoquinone-coated gold electrode compared with the identical layer structure without benzoquinone indicates an electrocatalytic effect of this molecule in the oxidative decomposition of hydrogen peroxide.
The analytical evaluation of the sensor performance was performed in voltammetric as well as in amperometric mode. In the concentration range from 0.1 to 2.5 mM of hydrogen peroxide, the sensor response is linear, with a lower limit of detection of approximately 4 µM. The amperometric chemosensor shows good selectivity in the presence of typical interfering substances such as ascorbic acid, uric acid or glucose. The high sensitivity suggests that this system should not only be used as a hydrogen peroxide sensor, but also as a transducer for biosensors with immobilized oxidoreductases, such as a glucose biosensor with glucose oxidase.
Towards ultrasensitive SPR-based sensing: self-referencing and detection of single nanoparticles
(2018)
Surface plasmon resonance (SPR) and its extensions, surface plasmon resonance imaging (SPRi) and surface plasmon resonance microscopy (SPRM) both enabling visualization of the sensor surface, belong to classical, indispensable highly sensitive and robust optical (bio)analytical techniques to study affinity processes on a surface. Nevertheless, SPR and SPRi/SPRM undergo a continuous development in regard to the improvement of sensitivity. The main challenge in this direction is attributed to the separation of signals due to the binding of analytes and those due to the bulk effect. The main task of the present thesis was to apply different strategies to improve the performance of SPR sensing. Within this scope, two main objectives were pursued: (1) implementation and realization of a so-called internal referencing towards suppression of the bulk effect leading to an improvement and optimization of the signal-to-noise ratio (SNR) and (2) application of wide-field (WF)-SPRM to detect, to visualize and to characterize single nanoparticles adsorbed to modified surfaces. The first objective of this thesis comprises the realization of three different internal-referencing approaches. In the first approach, a self-referencing effect based on arbitrarily distributed micro-patterned self-assembled monolayer (SAM) containing sensing and referencing spots was realized. Measurements of classical antigen-antibody-interaction resulted in a 10-fold improvement of the SNR by suppression of the bulk effect and the corresponding microfluctuations of the bulk temperature. The application of the second internal-referencing-approach, ionic referencing, acting as an assessment of patterned SAM was realized using electrolytes with a high molar refraction of either anions or cations to micro-patterned SAM combined with WF-SPRM as detecting technology. As a result, successful, unobtrusive visualization and spatial distinction of micro-patterned surfaces was shown. Unlike visualization of micro-scaled surface areas, the application of spatio-temporal referencing in WF-SPRM, as a third type of internal referencing, enables to detect, moreover to visualize and localize, smallest changes in refractive index near/on the sensor surface. In that sense, the second objective of this thesis was dedicated to the application of the WF-SPRM technology combined with spatio-temporal referencing to detect, to visualize, to quantify and to characterize single nanoparticles adsorbed to the sensor surface; here, nanoparticles act as analyte species. Based on a sophisticated image analysis, successful detection and characterization of single nanoparticles in complex media such as wine, juice and sun cream was performed. Besides being a powerful solution for nanoparticles analytics, the WF-SPRM technology represents a base to develop novel, ultra-sensitive and fast (bio)sensing platforms. Within this scope, enzyme-assisted generation of nanoparticles was studied.