Development of an automatized biosensor production and visualization of enzyme activity using an in-house scanning electrochemical microscope

Abstract

The aim of this thesis was the development of a tool for producing high quality, enzyme-based third generation lactose biosensors with a high throughput rate, as well as a tool for quality control of those biosensors by visualization of local distribution of enzyme catalytic activity which was realized with an in-house-made Scanning Electrochemical Microscope (SECM). By using the implementation of automatized production of biosensors, higher production rate, sensitivity and reproducibility was achieved. For the detection of lactose, the enzyme cellobiose dehydrogenase (CDH) was obtained from a wood degrading rot fungi and applied on a transducer as the sensitive biological element. CDH was deposited on screen printed carbon electrode (SPCE) strips via a dispenser which dispenses drops in nano-liter range to impregnate the hydrophobic surface of the electrode which acts as the transducer. Two linear piezo positioners for x and y axis allow positioning of the working electrode under the dispenser with precision in the lower micrometer range. Communication and coordination between the dispenser and the positioners of the electrodes was achieved using self-made LabVIEW software, which reads the coordinates for the dispensing area from a standard Excel-file. A direct relation between current and area covered with enzyme was shown. Furthermore the mix containing the enzyme was optimized in regard of variation the amount of cross-linker. Effects of surface pretreatment by applying cross-linker before enzyme modification on the working electrode were investigated in order to see a better fixation of the enzyme to the carbon and therefore a more stable signal over time. The local enzyme activity on the surface of the produced sensors was detected now through the SECM with a resolution in micro-meter-range and therefore knowledge about the local behavior can be gained. A platinum disk with a diameter of 25 µm sealed in a glass capillary was used as ultra-microelectrode. Again, LabVIEW was used to control the hardware, in this case the linear positioners - which were already used during production - and a potentiostat. The acquired coordinates and recorded currents were stored in an Excel-file for easy processing and manipulating. After successfully performing linear scans also the whole area of enzyme spots were visualized through the catalytic current and compared to the images obtained from scanning electron microscope (SEM). To show that SECM is not only used for visualization but also for evaluation of various performances of mutants, comparisons between the CDH wild-type and mutants were carried out. Therefore variants of the enzyme were deposited in lines next to the wild-type and then a linear scan crossing both domains was performed. Providing different substrates, a direct conclusion of possible advantageous or disadvantageous behavior between mutants and WT can be fast and easy drawn with a simple linear scan.