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An amperometric bi-enzyme biosensor based on substrate recycling principle for the amplification of the sensor signal has been developed for the detection of adrenaline in blood. Adrenaline can be used as biomarker verifying successful adrenal venous sampling procedure. The adrenaline biosensor has been realized via modification of a galvanic oxygen sensor with a bi-enzyme membrane combining a genetically modified laccase and a pyrroloquinoline quinone-dependent glucose dehydrogenase. The measurement conditions such as pH value and temperature were optimized to enhance the sensor performance. A high sensitivity and a low detection limit of about 0.5–1 nM adrenaline have been achieved in phosphate buffer at pH 7.4, relevant for measurements in blood samples. The sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine has been studied. Finally, the sensor has been successfully applied for the detection of adrenaline in human blood plasma.
A field-effect biosensor employing tobacco mosaic virus (TMV) particles as scaffolds for enzyme immobilization is presented. Nanotubular TMV scaffolds allow a dense immobilization of precisely positioned enzymes with retained activity. To demonstrate feasibility of this new strategy, a penicillin sensor has been developed by coupling a penicillinase with virus particles as a model system. The developed field-effect penicillin biosensor consists of an Al-p-Si-SiO₂-Ta₂O₅-TMV structure and has been electrochemically characterized in buffer solutions containing different concentrations of penicillin G. In addition, the morphology of the biosensor surface with virus particles was characterized by scanning electron microscopy and atomic force microscopy methods. The sensors possessed a high penicillin sensitivity of ~ 92 mV/dec in a nearly-linear range from 0.1 mM to 10 mM, and a low detection limit of about 50 µM. The long-term stability of the penicillin biosensor was periodically tested over a time period of about one year without any significant loss of sensitivity. The biosensor has also been successfully applied for penicillin detection in bovine milk samples.
Phase Repeatable Synthesizers as a New Harmonic Phase Standard for Nonlinear Network Analysis
(2018)
Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.
Magnetic detection structure for Lab-on-Chip applications based on the frequency mixing technique
(2018)
A magnetic frequency mixing technique with a set of miniaturized planar coils was investigated for use with a completely integrated Lab-on-Chip (LoC) pathogen sensing system. The system allows the detection and quantification of superparamagnetic beads. Additionally, in terms of magnetic nanoparticle characterization ability, the system can be used for immunoassays using the beads as markers. Analytical calculations and simulations for both excitation and pick-up coils are presented; the goal was to investigate the miniaturization of simple and cost-effective planar spiral coils. Following these calculations, a Printed Circuit Board (PCB) prototype was designed, manufactured, and tested for limit of detection, linear response, and validation of theoretical concepts. Using the magnetic frequency mixing technique, a limit of detection of 15 µg/mL of 20 nm core-sized nanoparticles was achieved without any shielding.