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Nanotubular tobacco mosaic virus (TMV) particles and RNA-free lower-order coat protein (CP) aggregates have been employed as enzyme carriers in different diagnostic layouts and compared for their influence on biosensor performance. In the following, we describe a label-free electrochemical biosensor for improved glucose detection by use of TMV adapters and the enzyme glucose oxidase (GOD). A specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CP aggregates was achieved via bioaffinity binding. Glucose sensors with adsorptively immobilized [SA]-GOD, and with [SA]-GOD cross-linked with glutardialdehyde, respectively, were tested in parallel on the same sensor chip. Comparison of these sensors revealed that TMV adapters enhanced the amperometric glucose detection remarkably, conveying highest sensitivity, an extended linear detection range and fastest response times. These results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for applications in biosensorics and biochips. Here, we describe the fabrication and use of amperometric sensor chips combining an array of circular Pt electrodes, their loading with GOD-modified TMV nanotubes (and other GOD immobilization methods), and the subsequent investigations of the sensor performance.
The conjunction of (bio-)chemical recognition elements with nanoscale biological building blocks such as virus particles is considered as a very promising strategy for the creation of biohybrids opening novel opportunities for label-free biosensing. This work presents a new approach for the development of biosensors using tobacco mosaic virus (TMV) nanotubes or coat proteins (CPs) as enzyme nanocarriers. Sensor chips combining an array of Pt electrodes loaded with glucose oxidase (GOD)-modified TMV nanotubes or CP aggregates were used for amperometric detection of glucose as a model system for the first time. The presence of TMV nanotubes or CPs on the sensor surface allows binding of a high amount of precisely positioned enzymes without substantial loss of their activity, and may also ensure accessibility of their active centers for analyte molecules. Specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CPs was achieved via bioaffinity binding. These layouts were tested in parallel with glucose sensors with adsorptively immobilized [SA]-GOD, as well as [SA]-GOD crosslinked with glutardialdehyde, and came out to exhibit superior sensor performance. The achieved results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for future applications in biosensorics and biochips.
Monitoring of organic acids (OA) and volatile fatty acids (VFA) is crucial for the control of anaerobic digestion. In case of unstable process conditions, an accumulation of these intermediates occurs. In the present work, two different enzyme-based biosensor arrays are combined and presented for facile electrochemical determination of several process-relevant analytes. Each biosensor utilizes a platinum sensor chip (14 × 14 mm²) with five individual working electrodes. The OA biosensor enables simultaneous measurement of ethanol, formate, d- and l-lactate, based on a bi-enzymatic detection principle. The second VFA biosensor provides an amperometric platform for quantification of acetate and propionate, mediated by oxidation of hydrogen peroxide. The cross-sensitivity of both biosensors toward potential interferents, typically present in fermentation samples, was investigated. The potential for practical application in complex media was successfully demonstrated in spiked sludge samples collected from three different biogas plants. Thereby, the results obtained by both of the biosensors were in good agreement to the applied reference measurements by photometry and gas chromatography, respectively. The proposed hybrid biosensor system was also used for long-term monitoring of a lab-scale biogas reactor (0.01 m³) for a period of 2 months. In combination with typically monitored parameters, such as gas quality, pH and FOS/TAC (volatile organic acids/total anorganic carbonate), the amperometric measurements of OA and VFA concentration could enhance the understanding of ongoing fermentation processes.
Sterilisation processes are compulsory in medicine, pharmacy, and food industries to prevent infections of consumers and microbiological contaminations of products. Monitoring the sterilisation by conventional microbiological methods is time- and lab-consuming. To overcome this problem, in this work a novel biosensor has been proposed. The sensor enables a fast method to evaluate sterilisation processes. By means of thin-film technology the sensor's transducer structures in form of IDEs (interdigitated electrodes) have been fabricated on a silicon substrate. Physical characterisation of the developed sensor was done by AFM, SEM, and profilometry. Impedance analyses were conducted for the electrical characterisation. As microbiological layer spores of B. atrophaeus have been immobilised on the sensing structure; spores of this type are a well-known sterilisation test organism. Impedance measurements at a fixed frequency over time were performed to monitor the immobilisation process. A sterilisation process according to aseptic filling machines was applied to demonstrate the sensor functionality. After both, immobilisation and sterilisation, a change in impedance could successfully be detected.