Development of light-addressable potentiometric sensor systems and their applications in biotechnological environments
The simultaneous analysis of multiple analytes and spatially resolved measurements of concentration distributions with a single sensor chip are an important task in the field of (bio-)chemical sensing. Together with the miniaturisation, this is a promising step forward for applications and processes...
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|Zusammenfassung:||The simultaneous analysis of multiple analytes and spatially resolved measurements of concentration distributions with a single sensor chip are an important task in the field of (bio-)chemical sensing. Together with the miniaturisation, this is a promising step forward for applications and processes that profit from (bio-)chemical sensors. In combination with biological recognition elements, like enzymes or cells, these biosensors are becoming an interesting tool for e.g., biotechnological, medical and pharmaceutical applications. One promising sensor principle is the light-addressable potentiometric sensor (LAPS). A LAPS is a semiconductor-based potentiometric sensor that allows determining analyte concentrations of aqueous solutions in a spatially resolved manner. Therefore, it is using a focused light source to address the area of interest. The light that illuminates the local area of the LAPS chip generates a photocurrent that correlates with the local analyte concentration on the sensor surface. Based on the "state of the art", further developments of LAPS set-ups were carried out within this PhD thesis. Furthermore, by utilising enzymes and whole cells, the benefits of these LAPS set-ups for biotechnological, medical and pharmaceutical applications are demonstrated. During the present thesis, three different LAPS set-ups were developed: The first LAPS set-up makes use of a field-programmable gate array (FPGA) to drive a 4x4 light-emitting diode (LED) array that defines 16 measurement spots on the sensor-chip surface. With the help of the FPGA, the driving parameters, like light brightness, modulation amplitude and frequency can be selected individually and all LEDs can be driven concurrently. Thus, a simultaneous readout of all measurement spots is possible and chemical images of the whole sensor surface can be achieved within 200 ms. The FPGA-based LAPS set-up is used to observe the frequency behaviour of LAPS chips. In a second LAPS set-up, a commercially available organic-LED (OLED) display is used as light source. The OLED panel consists of 96x64 pixels with a pixel size of 200x200 µm and thus, an over 16 times higher lateral resolution compared to the IR-LED array. It was demonstrated that chemical images of the whole sensor surface can be obtained in 2.5 min. Since the lateral resolution of LAPS is not only specified by the light source, but also by the LAPS chip itself, the lateral resolution of the LAPS structures is characterised. Therefore, the third LAPS set-up has been developed, which utilises a single laser diode that can be moved by an XY-stage. By scanning a specially patterned LAPS chip, a lateral resolution of the LAPS structures in the range of the pixel size of the OLED display is demonstrated. Label-free imaging of biological phenomena is investigated with the FPGA-based LAPS. With the help of an enzymatic layer with the enzyme acetylcholine esterase (AChE) the detection of the neuronal transmitter acetylcholine (ACh) is demonstrated. The dynamic and static response as well as the long-term stability is characterised and compared with another semiconductor-based chemical imaging sensor based on charge-coupled devices (CCD) using the same enzymatic layer. The usage of the FPGA-based LAPS as whole-cell-based biosensor is studied with the model organism Escherichia coli. Here, the metabolic activity of the E. coli cells is investigated by determining the extracellular acidification. An immobilisation technique for embedding the microorganisms in polyacrylamide gel on the sensor surface has been developed. The immobilisation is realised in an on-chip differential arrangement by making use of the addressability of LAPS. This way, external influences such as sensor drift, temperature changes and external pH changes can be compensated. In a comparative study of the extracellular acidification rate between immobilised E. coli and E. coli that are in suspension, acidification rates in the same order were determined, demonstrating that the immobilisation does not have any influence on the metabolic activity. Further measurements with this cell-based LAPS system underline the sensitivity towards different nutrient concentrations, namely glucose. The ability to observe the extracellular acidification of microorganisms and the sensitively towards nutrient concentrations enables to detect high-order effects, like toxicity or pharmacological activity in complex analytes.|