A capacitive electrolyte-insulator-semiconductor (EISCAP) biosensor modified with Tobacco mosaic virus (TMV) particles for the detection of acetoin is presented. The enzyme acetoin reductase (AR) was immobilized on the surface of the EISCAP using TMV particles as nanoscaffolds. The study focused on the optimization of the TMV-assisted AR immobilization on the Ta2O5-gate EISCAP surface. The TMV-assisted acetoin EISCAPs were electrochemically characterized by means of leakage-current, capacitance-voltage, and constant-capacitance measurements. The TMV-modified transducer surface was studied via scanning electron microscopy.

Acetoin and diacetyl have a major impact on the flavor of alcoholic beverages such as wine or beer. Therefore, their measurement is important during the fermentation process. Until now, gas chromatographic techniques have typically been applied; however, these require expensive laboratory equipment and trained staff, and do not allow for online monitoring. In this work, a capacitive electrolyte–insulator–semiconductor sensor modified with tobacco mosaic virus (TMV) particles as enzyme nanocarriers for the detection of acetoin and diacetyl is presented. The enzyme acetoin reductase from Alkalihalobacillus clausii DSM 8716T is immobilized via biotin–streptavidin affinity, binding to the surface of the TMV particles. The TMV-assisted biosensor is electrochemically characterized by means of leakage–current, capacitance–voltage, and constant capacitance measurements. In this paper, the novel biosensor is studied regarding its sensitivity and long-term stability in buffer solution. Moreover, the TMV-assisted capacitive field-effect sensor is applied for the detection of diacetyl for the first time. The measurement of acetoin and diacetyl with the same sensor setup is demonstrated. Finally, the successive detection of acetoin and diacetyl in buffer and in diluted beer is studied by tuning the sensitivity of the biosensor using the pH value of the measurement solution.

Introduction Viruses are not only infectious agents, they are also known as promising functional building blocks for application in nano- and biotechnologies. The tobacco mosaic virus (TMV) was the first studied plant virus and is widely distributed; it infects vegetables, like tomato, bell pepper, beans and other members of the family Solanaceae, while it is totally harmless for mammals [1]. It is one of the most studied plant viruses and its genome is completely sequenced, whereby genetical and chemical modification is easy. TMV has a nanotube-like shape with a length of 300 nm, an outer diameter of 18 nm and an inner diameter of 4 nm. Since it possesses a high chemical and physical robustness, it can be integrated with different electronic transducers for bio- and chemical sensing applications [2]. Recently, we presented a TMV-based amperometric glucose biosensor [3] and a potentiometric penicillin biosensor [4]. TMV was used as enzyme nanocarrier for the enzyme glucose oxidase and penicillinase, respectively [3,4]. The sensitivity and detection limit of these biosensors, among others, depend on the density of TMVs on the sensor surface. The surface density of the immobilized TMVs is strongly influenced by the electrostatic interactions between the charged TMVs and sensor surface as well as by the inter-TMV-nanotubes repulsion, which could be changed by varying the pH value and the ionic strength of the TMV solution. In this study, we investigated an impact of the pH value and ionic strength of the TMV solution on the surface density of TMV nanotubes immobilized onto Ta2O5-gate capacitive field-effect electrolyte-insulator-semiconductor (EIS) sensors. Materials and Methods TMV particles modified with biotin-linker molecules (TMVBio), which serve as binding sites for streptavidin-conjugated enzymes, have been immobilized onto capacitive field-effect EIS sensors with Ta2O5 as transducer layer, as shown in Fig. 1. Immobilization was performed from TMVBiosolutions with different values of pH between pH 3.0 and pH 9.5 and ionic strength between 0.1 mM and 750 mM. The sensors have been electrochemically characterized before and after TMVBio immobilization by capacitance-voltage- and constant-capacitance methods, respectively. In addition, the density of the immobilized TMVs and morphology of the sensor surface has been investigated by means of scanning electron microscopy (SEM). Results and Conclusions The TMVBio density on the Ta2O5 sensor surface was influenced by varying the pH value (as exemplarily shown in Fig. 1) and ionic strength (not shown) of the TMVBio solution. The amplitude of the field-effect sensor signal correlates well with the density of the immobilized TMVBionanotubes. Thus, optimized conditions for the high-density immobilization of TMVBio nanotubes onto the Ta2O5 surface and thereby enhanced biosensing have been found. Details of the experiments and the obtained results will be presented and discussed. Acknowledgements The authors like to thank Dr. Claudia Koch and Rebecca Hummel, Stuttgart, for scientific and technical support. References [1] X. Z. Fan, E. Pomerantseva, M. Gnerlich, Tobacco mosaic virus: A biological building block for micro/nano/bio systems, Journal of Vacuum Science & Technology A. 31 (2013) 050815. doi: 10.1116/1.4816584. [2] M. Knez, M. Sumer, A. Bittner, C. Wege, H. Jeske, D. Hoffmann, K. Kuhnke, K. Kern, Binding the tobacco mosaic virus to inorganic surfaces, Langmuir 20 (2004) 441–447. doi: 10.1021/la035425o. [3] M. Bäcker, C. Koch, F. Geiger, F. Eber, H. Gliemann, A. Poghossian, C. Wege, M. J. Schöning, Tobacco mosaic virus as enzyme nanocarrier for electrochemical biosensors, Sensors and Actuators B: Chemical 238 (2017) 716– 722. doi: 10.1016/j.snb.2016.07.096. [4] A. Poghossian, M. Jablonski, C. Koch, T. S. Bronder, D. Rolker, C. Wege, M. J. Schöning, Field-effect biosensor using virus particles as scaffolds for enzyme immobilization, Biosensors and Bioelectronics 110 (2018) 168–174. doi: 10.1016/j.bios.2018.03.036 Figure 1: Measurement set-up with schematic layer structure of the capacitive EIS sensor modified with negatively charged TMVBio particles (a). SEM images of the Ta2O5-sensor surface modified with TMVBio nanotubes at pH 4.5 (b) and pH 7.0 (c), respectively. Figure 1

Plant virus-like particles, and in particular, tobacco mosaic virus (TMV) particles, are increasingly being used in nano- and biotechnology as well as for biochemical sensing purposes as nanoscaffolds for the high-density immobilization of receptor molecules. The sensitive parameters of TMV-assisted biosensors depend, among others, on the density of adsorbed TMV particles on the sensor surface, which is affected by both the adsorption conditions and surface properties of the sensor. In this work, Ta2O5-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption. The impact of the TMV concentration on both the sensor signal and the density of TMV particles adsorbed onto the Ta2O5-gate surface has been studied systematically by means of field-effect and scanning electron microscopy methods. In addition, the surface density of TMV particles loaded under different incubation times has been investigated. Finally, the field-effect sensor also demonstrates the label-free detection of penicillinase immobilization as model bioreceptor on TMV particles.

Introduction Viruses are not only infectious agents, they are also known as promising functional building blocks for application in nano- and biotechnologies. The tobacco mosaic virus (TMV) was the first studied plant virus and is widely distributed; it infects vegetables, like tomato, bell pepper, beans and other members of the family Solanaceae, while it is totally harmless for mammals [1]. It is one of the most studied plant viruses and its genome is completely sequenced, whereby genetical and chemical modification is easy. TMV has a nanotube-like shape with a length of 300 nm, an outer diameter of 18 nm and an inner diameter of 4 nm. Since it possesses a high chemical and physical robustness, it can be integrated with different electronic transducers for bio- and chemical sensing applications [2]. Recently, we presented a TMV-based amperometric glucose biosensor [3] and a potentiometric penicillin biosensor [4]. TMV was used as enzyme nanocarrier for the enzyme glucose oxidase and penicillinase, respectively [3,4]. The sensitivity and detection limit of these biosensors, among others, depend on the density of TMVs on the sensor surface. The surface density of the immobilized TMVs is strongly influenced by the electrostatic interactions between the charged TMVs and sensor surface as well as by the inter-TMV-nanotubes repulsion, which could be changed by varying the pH value and the ionic strength of the TMV solution. In this study, we investigated an impact of the pH value and ionic strength of the TMV solution on the surface density of TMV nanotubes immobilized onto Ta2O5-gate capacitive field-effect electrolyte-insulator-semiconductor (EIS) sensors. Materials and Methods TMV particles modified with biotin-linker molecules (TMVBio), which serve as binding sites for streptavidin-conjugated enzymes, have been immobilized onto capacitive field-effect EIS sensors with Ta2O5 as transducer layer, as shown in Fig. 1. Immobilization was performed from TMVBiosolutions with different values of pH between pH 3.0 and pH 9.5 and ionic strength between 0.1 mM and 750 mM. The sensors have been electrochemically characterized before and after TMVBio immobilization by capacitance-voltage- and constant-capacitance methods, respectively. In addition, the density of the immobilized TMVs and morphology of the sensor surface has been investigated by means of scanning electron microscopy (SEM). Results and Conclusions The TMVBio density on the Ta2O5 sensor surface was influenced by varying the pH value (as exemplarily shown in Fig. 1) and ionic strength (not shown) of the TMVBio solution. The amplitude of the field-effect sensor signal correlates well with the density of the immobilized TMVBionanotubes. Thus, optimized conditions for the high-density immobilization of TMVBio nanotubes onto the Ta2O5 surface and thereby enhanced biosensing have been found. Details of the experiments and the obtained results will be presented and discussed. Figure 1: Measurement set-up with schematic layer structure of the capacitive EIS sensor modified with negatively charged TMVBio particles (a). SEM images of the Ta2O5-sensor surface modified with TMVBio nanotubes at pH 4.5 (b) and pH 7.0 (c), respectively. Acknowledgements The authors like to thank Dr. Claudia Koch and Rebecca Hummel, Stuttgart, for scientific and technical support. References [1] X. Z. Fan, E. Pomerantseva, M. Gnerlich, Tobacco mosaic virus: A biological building block for micro/nano/bio systems, Journal of Vacuum Science & Technology A. 31 (2013) 050815. doi: 10.1116/1.4816584. [2] M. Knez, M. Sumer, A. Bittner, C. Wege, H. Jeske, D. Hoffmann, K. Kuhnke, K. Kern, Binding the tobacco mosaic virus to inorganic surfaces, Langmuir 20 (2004) 441–447. doi: 10.1021/la035425o. [3] M. Bäcker, C. Koch, F. Geiger, F. Eber, H. Gliemann, A. Poghossian, C. Wege, M. J. Schöning, Tobacco mosaic virus as enzyme nanocarrier for electrochemical biosensors, Sensors and Actuators B: Chemical 238 (2017) 716– 722. doi: 10.1016/j.snb.2016.07.096. [4] A. Poghossian, M. Jablonski, C. Koch, T. S. Bronder, D. Rolker, C. Wege, M. J. Schöning, Field-effect biosensor using virus particles as scaffolds for enzyme immobilization, Biosensors and Bioelectronics 110 (2018) 168–174. doi: 10.1016/j.bios.2018.03.036. Figure 1