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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.
A New Class of Biosensors Based on Tobacco Mosaic Virus and Coat Proteins as Enzyme Nanocarrier
(2016)
A new approach for the development of electrochemical biosensors using tobacco mosaic virus (TMV) nanotubes and coat protein (CP) aggregates as enzyme nanocarriers is presented. The generic approach has been demonstrated by realizing amperometric glucose sensors combining an array of Pt electrodes loaded with glucose oxidase (GOD)-modified TMV nanotubes and CPs as a model system. The presence of TMV nanotubes or CPs on the sensor surface allows binding of a high amount of enzymes without substantial loss of their activity. The obtained results underline the potential of integrating biomolecule/virus hybrids with electronic transducers for applications in biosensorics.
Designing novel or optimizing existing biodegradable polymers for biomedical applications requires numerous tests on the effect of substances on the degradation process. In the present work, polymer-modified electrolyte–insulator–semiconductor (PMEIS) sensors have been applied for monitoring an enzymatically catalyzed degradation of polymers for the first time. The thin films of biodegradable polymer poly(d,l-lactic acid) and enzyme lipase were used as a model system. During degradation, the sensors were read-out by means of impedance spectroscopy. In order to interpret the data obtained from impedance measurements, an electrical equivalent circuit model was developed. In addition, morphological investigations of the polymer surface have been performed by means of in situ atomic force microscopy. The sensor signal change, which reflects the progress of degradation, indicates an accelerated degradation in the presence of the enzyme compared to hydrolysis in neutral pH buffer media. The degradation rate increases with increasing enzyme concentration. The obtained results demonstrate the potential of PMEIS sensors as a very promising tool for in situ and real-time monitoring of degradation of polymers.
In vitro studies of the degradation kinetic of biopolymers are essential for the design and optimization of implantable biomedical devices. In the presented work, a field-effect capacitive sensor has been applied for the real-time and in situ monitoring of degradation processes of biopolymers for the first time. The polymer-covered field-effect sensor is, in principle, capable to detect any changes in bulk, surface and interface properties of the polymer induced by degradation processes. The feasibility of this approach has been experimentally proven by using the commercially available biomedical polymer poly(D,L-lactic acid) (PDLLA) as a model system. PDLLA films of different thicknesses were deposited on the Ta₂O₅-gate surface of the field-effect structure from a polymer solution by means of spin-coating method. The polymer-modified field-effect sensors have been characterized by means of capacitance–voltage and impedance-spectroscopy method. The degradation of the PDLLA was accelerated by changing the degradation medium from neutral (pH 7.2) to alkaline (pH 9) condition, resulting in drastic changes in the capacitance and impedance spectra of the polymer-modified field-effect sensor.
A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 μAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.
