Reconfigurable Implication and Inhibition Boolean logic gates based on NAD+-dependent enzymes: Application to signal-controlled biofuel cells and molecule release

AbstractThe Implication and Inhibition Boolean logic gates were realized using NAD+/NADH‐dependent dehydrogenases combined with hexokinase competing for biomolecule input signals. Both logic gates operated with the same enzyme composition and their reconfiguration was achieved simply by redefining the input signals. The output signals produced by the logic gates were analyzed optically and electrochemically, particularly using enzyme‐modified electrodes. The logically processed input signals were used to switch operation of a biofuel cell and activate a molecule release process

Integration of biomolecular logic principles with electronic transducers on a chip

Boolean operations applied in biology and integrated with electronic transducers allow the development of a new class of digital biosensors for the detection of multiple input signals simultaneously and in real-time. With the help of Boolean functions (AND, OR, etc.), an electrical output signal will be directly delivered, representing a ”1” or “0” binary notation, corresponding to a “true” or “false” statement, respectively. Such digital biosensors have the future potential to create medical devices and systems for intelligent or smart diagnostics. The present thesis describes the realization of different enzyme-based biomolecular logic gates combined with electronic transducers for the possible application in medicine or food industry. In a first concept, a so called BioLogicChip is developed combining a “sense-act-treat” function integrated on one chip. The present system exemplarily mimics an “artificial pancreas” designed as a closed-loop drug-release system. A glucose sensor is constructed as enzyme-based AND logic gate, a temperature-depending hydrogel imitates the actuator function switching ON and OFF with its shrinking or swelling property, and an additional insulin sensor is developed to monitor and control the release of the drug (here: insulin) from the actuator. In this study, the results of the individual components such as the amperometric glucose sensor, the temperature-dependent hydrogel and the amperometric insulin sensor are presented, which are necessary to create such BioLogicChip. Moreover, a digital adrenaline biosensor is developed to proof the catheter position during adrenal vein sampling. The sensor consists of an oxygen electrode modified by a bi-enzyme system with the enzymes laccase and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) to realize substrate-recycling principle to detect low adrenaline concentrations (in the nanomolar concentration range). The sensor`s behavior at different pH values and at different temperatures is studied. Measurements in Ringer`s solution are performed. In addition, the sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine is investigated. Furthermore, the adrenaline biosensor is successfully examined in human blood plasma. Finally, “proof-of-principle” experiments have been performed by combining the adrenaline biosensor with Boolean operations to get a rapid qualitative statement of the presence or absence of adrenaline, thus validating the correct position of the catheter in a YES/NO form. This adrenaline biosensor is further miniaturized as a thin-film platinum adrenaline biosensor. Here, the bioelectrocatalytical measurement principle is applied by immobilization of the enzyme PQQ-GDH to detect adrenaline in the nanomolar concentration range, too. The measurement conditions such as pH value, glucose concentration in the analyte solution and temperature are optimized with regard to a high sensitivity and low detection limit. Also, this sensor has been verified towards other catecholamines (noradrenaline, dopamine and dobutamine). The platinum thin-film adrenaline biosensor is successfully applied in blood plasma for the detection of different spiked adrenaline concentrations. Furthermore, the developed adrenalin biosensor is able to detect the concentration difference between adrenal blood and peripheral blood. In contrast to the above-mentioned amperometric biosensor examples for biomolecular gates, also a field-effect-based platform is given attention in this thesis. The field-effect electrolyte-insulator-semiconductor (EIS) sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5 and is used to create an acetoin biosensor for the first time to control different fermentation processes. The sensor chip is modified by the enzyme acetoin reductase from B. clausii DSM 8716T for the catalytical reaction of (R)-acetoin to (R,R)-butanediol and meso-butanediol, respectively, in the presence of NADH. The linear measurement range, the optimal immobilization strategy (cross-linking by using glutaraldehyde and adsorptive binding) as well as the optimal working pH value and long-term stability are investigated by means of constant-capacitance measurements. Finally, the acetoin sensor was successfully applied in wine probes to detect different spiked acetoin concentrations. The sensor shows opportunities to be further developed as digital acetoin biosensor

Control of Noise in Chemical and Biochemical Information Processing

We review models and approaches for error-control in order to prevent the buildup of noise when gates for digital chemical and biomolecular computing based on (bio)chemical reaction processes are utilized to realize stable, scalable networks for information processing. Solvable rate-equation models illustrate several recently developed methodologies for gate-function optimization. We also survey future challenges and possible new research avenues.Comment: 39 pages, 8 figures, PD

Light addressable gold electrodes

The main objective carried out in this dissertation was to fabricate Light Amplified Potentiometric sensors (LAPS) based upon the semiconductor nanoparticles (quantum dots) instead of its bulk form. Quantum dots (QDs) were opted for this device fabrication because of their superior fluorescent, electric and catalytic properties. Also in comparison to their bulk counterparts they will make device small, light weighted and power consumption is much lower. QDs were immobilized on a Au substrate via 1,4 benzene dithiol (BDT) molecule. Initially a self-assembled monolayer (SAM) of BDT was established on Au substrate. Because of SAM, the conductivity of Au substrate decreased dramatically. Furthermore QDs were anchored with the help of BDT molecule on Au substrate. When QDs immobilized on Au substrate (QD/Au) via BDT molecule were irradiated with UV-visible light, electron-hole pairs were generated in QDs. The surface defect states in QDs trapped the excited electrons and long lived electron-hole pairs were formed. By the application of an appropriate bias potential on Au substrate the electrons could be supplied or extracted from the QDs via tunneling through BDT. Thus a cathodic or anodic current could be observed depending upon bias potential under illumination. However without light illumination the QD/Au electrode remained an insulator. To improve the device different modifications were made, including different substrates (Au evaporated on glass, Au evaporated on mica sheets and Au sputtered on SiO2/Si) and different dithiol molecules (capped and uncapped biphenyl 4,4 dithiol and capped and uncapped 4,4 dimercaptostilbenes) were tried. Also different QD immobilization techniques (normal incubation, spin coating, layer by layer assembly (LbL) of polyelectrolytes and heat immobilization) were employed. This device was able to detect electrochemically different analytes depending upon the QDs incorporated. For example CdS QDs were able to detect 4-Aminophenol, a product of an enzymatic reaction of Alkaline Phosphatase with p-Aminophenyl Phosphate. Subsequently this reaction was observed at CdS/Au electrode, by enzyme-substrate reaction within the electrolyte solution, and also by immobilizing the enzyme on top of QDs via LbL assembly of polyelectrolytes. With another kind of CdS-FePt dimer QDs, detection of hydrogen peroxide (H2O2) was demonstrated. Only at CdS/Au electrode there was no impact made by H2O2 but with the presence of Pt within QDs H2O2 was detected via reduction even at a bias potential of -100mV