Plant viruses are considered one of the main contributors to crop losses in the agricultural industry and thus induce high economic costs worldwide. The best strategy to slow down the spread of plant virus infections and prevent crop failures is to make an early and reliable diagnosis and provide prompt treatment. Electrolyte-insulator-semiconductor capacitors (EISCAP) represent an attractive transducer architecture for label-free electrostatic detection of virus particles by their intrinsic charge. In this work, a capacitive model of an EISCAP sensor loaded with negatively charged tobacco mosaic virus (TMV) particles is presented. In the developed model, the adsorbed TMV particles are assumed as local gates with dimensions in the nano- to sub-micrometer range. The impact of TMV surface coverage on the sensor characteristics of SiO2-gate EISCAPs was studied theoretically and experimentally. The observed EISCAP signal correlates well with the coverage of loaded TMV particles evaluated from scanning electron microscopy images of the SiO2-gate surface.

ABSTRACT Urea is formed from the metabolism of proteins and used as a biomarker for diagnosing and monitoring various medical conditions. In this work, a urea biosensor based on an electrolyte‐insulator‐semiconductor capacitor (EISCAP) modified with a stacked polyelectrolyte polyallylamine hydrochloride (PAH)/urease bilayer prepared by the layer‐by‐layer (LbL) technique is presented for the first time. The LbL formation of the PAH/urease bilayer was monitored with an underlying charge‐sensitive Al/p‐Si/SiO2/Ta2O5 EISCAP using convenient capacitive‐voltage and constant‐capacitance mode measurements. Urea‐sensitive EISCAP biosensors were electrochemically characterised in buffer solutions and artificial urine (AU) samples spiked with various concentrations of urea between 0.1 mM and 50 mM. The biosensors exhibited urea sensitivities of ca. 35.4 mV/dec and 32.1 mV/dec in buffer and AU solutions, respectively. Finally, local surface pH changes as a function of urea concentration have been evaluated. The obtained findings demonstrate the potential of PAH/urease‐modified EISCAPs for non‐invasive urea biomarker detection in urine samples at homecare or in‐field settings.

A user-friendly, portable, low-cost readout system for the on-site or point-of-care characterization of chemo- and biosensors based on an electrolyte–insulator–semiconductor capacitor (EISCAP) has been developed using a thumb-drive-sized commercial impedance analyzer. The system is controlled by a custom Python script and allows to characterize EISCAP sensors with different methods (impedance spectra, capacitance-voltage, and constant-capacitance modes), which are selected in a user interface. The performance of the portable readout system was evaluated by pH measurements and the detection of the antibiotic penicillin, hereby using EISCAPs consisting of Al/p-Si/SiO2/Ta2O5 structures and compared to the results obtained with a stationary commercial impedance analyzer. Both the portable and the commercial systems provide very similar results with almost perfectly overlapping recorded EISCAP signals. The new portable system can accelerate the transition of EISCAP sensors from research laboratories to commercial end-user devices.

Electrolyte-insulator-semiconductor capacitors (EISCAP) belong to field-effect sensors having an attractive transducer architecture for constructing various biochemical sensors. In this study, a capacitive model of enzyme-modified EISCAPs has been developed and the impact of the surface coverage of immobilized enzymes on its capacitance-voltage and constant-capacitance characteristics was studied theoretically and experimentally. The used multicell arrangement enables a multiplexed electrochemical characterization of up to sixteen EISCAPs. Different enzyme coverages have been achieved by means of parallel electrical connection of bare and enzyme-covered single EISCAPs in diverse combinations. As predicted by the model, with increasing the enzyme coverage, both the shift of capacitance-voltage curves and the amplitude of the constant-capacitance signal increase, resulting in an enhancement of analyte sensitivity of the EISCAP biosensor. In addition, the capability of the multicell arrangement with multi-enzyme covered EISCAPs for sequentially detecting multianalytes (penicillin and urea) utilizing the enzymes penicillinase and urease has been experimentally demonstrated and discussed.

This chapter describes the use of enzymes in biosensors. Enzyme-based biosensors have enjoyed a prosperous growth market for more than five decades and are increasingly being used in biotechnological processes. Typically, they comprise a biological recognition element together with a physicochemical transducer. The main advantages of biosensors are their easy-to-use operation, cost-effective manufacturing, and their ability for sensitive and highly accurate detection of specific analytes. A brief overview on "biosensor technology" is given, including relevant sensor parameters, followed by major developments in electrochemical enzyme biosensors with a more detailed focus on possible applications in the field of biotechnology. Looking beyond the "end of one's nose" will offer insight into alternative transducer principles and biomolecules.

In comparison to single‐analyte devices, multiplexed systems for a multianalyte detection offer a reduced assay time and sample volume, low cost, and high throughput. Herein, a multiplexing platform for an automated quasi‐simultaneous characterization of multiple (up to 16) capacitive field‐effect sensors by the capacitive–voltage ( C – V ) and the constant‐capacitance (ConCap) mode is presented. The sensors are mounted in a newly designed multicell arrangement with one common reference electrode and are electrically connected to the impedance analyzer via the base station. A Python script for the automated characterization of the sensors executes the user‐defined measurement protocol. The developed multiplexing system is tested for pH measurements and the label‐free detection of ligand‐stabilized, charged gold nanoparticles.

This work presents a new approach for the development of field-effect biosensors based on an electrolyte-insulator-semiconductor capacitor (EISCAP) modified with a stacked bilayer of weak polyelectrolyte and tobacco mosaic virus (TMV) particles as enzyme nanocarriers. With the aim to increase the surface density of virus particles and thus, to achieve a dense immobilization of enzymes, the negatively charged TMV particles were loaded onto the EISCAP surface modified with a positively charged poly(allylamine hydrochloride) (PAH) layer. The PAH/TMV bilayer was prepared on the Ta2O5-gate surface by means of layer-by-layer technique. The bare and differently modified EISCAP surfaces were physically characterized by fluorescence microscopy, zeta-potential measurements, atomic force microscopy and scanning electron microscopy. Transmission electron microscopy was used to scrutinize the PAH effect on TMV adsorption in a second system. Finally, a highly sensitive TMV-assisted EISCAP antibiotics biosensor was realized by immobilizing the enzyme penicillinase onto the TMV surface. This PAH/TMV bilayer-modified EISCAP biosensor was electrochemically characterized in solutions with different penicillin concentrations via capacitance-voltage and constant-capacitance methods. The biosensor possessed a mean penicillin sensitivity of 113 mV/dec in a concentration range from 0.1 mM to 5 mM.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.

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.

Miniaturized electrolyte–insulator–semiconductor capacitors (EISCAPs) with ultrathin gate insulators have been studied in terms of their pH‐sensitive sensor characteristics: three different EISCAP systems consisting of Al–p‐Si–Ta 2 O 5 (5 nm), Al–p‐Si–Si 3 N 4 (1 or 2 nm)–Ta 2 O 5 (5 nm), and Al–p‐Si–SiO 2 (3.6 nm)–Ta 2 O 5 (5 nm) layer structures are characterized in buffer solution with different pH values by means of capacitance–voltage and constant capacitance method. The SiO 2 and Si 3 N 4 gate insulators are deposited by rapid thermal oxidation and rapid thermal nitridation, respectively, whereas the Ta 2 O 5 film is prepared by atomic layer deposition. All EISCAP systems have a clear pH response, favoring the stacked gate insulators SiO 2 –Ta 2 O 5 when considering the overall sensor characteristics, while the Si 3 N 4 (1 nm)–Ta 2 O 5 stack delivers the largest accumulation capacitance (due to the lower equivalent oxide thickness) and a higher steepness in the slope of the capacitance–voltage curve among the studied stacked gate insulator systems.

Nanoparticles are recognized as highly attractive tunable materials for designing field-effect biosensors with enhanced performance. In this work, we present a theoretical model for electrolyte-insulator-semiconductor capacitors (EISCAP) decorated with ligand-stabilized charged gold nanoparticles. The charged AuNPs are taken into account as additional, nanometer-sized local gates. The capacitance-voltage (C–V) curves and constant-capacitance (ConCap) signals of the AuNP-decorated EISCAPs have been simulated. The impact of the AuNP coverage on the shift of the C–V curves and the ConCap signals was also studied experimentally on Al–p-Si–SiO2 EISCAPs decorated with positively charged aminooctanethiol-capped AuNPs. In addition, the surface of the EISCAPs, modified with AuNPs, was characterized by scanning electron microscopy for different immobilization times of the nanoparticles.

The coupling of ligand-stabilized gold nanoparticles with field-effect devices offers new possibilities for label-free biosensing. In this work, we study the immobilization of aminooctanethiol-stabilized gold nanoparticles (AuAOTs) on the silicon dioxide surface of a capacitive field-effect sensor. The terminal amino group of the AuAOT is well suited for the functionalization with biomolecules. The attachment of the positively-charged AuAOTs on a capacitive field-effect sensor was detected by direct electrical readout using capacitance-voltage and constant capacitance measurements. With a higher particle density on the sensor surface, the measured signal change was correspondingly more pronounced. The results demonstrate the ability of capacitive field-effect sensors for the non-destructive quantitative validation of nanoparticle immobilization. In addition, the electrostatic binding of the polyanion polystyrene sulfonate to the AuAOT-modified sensor surface was studied as a model system for the label-free detection of charged macromolecules. Most likely, this approach can be transferred to the label-free detection of other charged molecules such as enzymes or antibodies.

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.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

A multiplexer platform for the automatic electrochemical characterization of up to 16 individual capacitive electrolyte-insulator-semiconductor field-effect sensors is presented. CAD (computer aided design) models of the top and bottom part of the multi-cell are compared to their 3D-printed counterparts. The functionality of the 3D-printed multi-cell and written Python script is demonstrated by the quasi-simultaneous pH characterization of 8 different sensors by means of capacitance-voltage and constant-capacitance measurements.

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.

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

The on-chip integration of multiple biochemical sensors based on field-effect electrolyte-insulator-semiconductor capacitors (EISCAP) is challenging due to technological difficulties in realization of electrically isolated EISCAPs on the same Si chip. In this work, we present a new simple design for an array of on-chip integrated, individually electrically addressable EISCAPs with an additional control gate (CG-EISCAP). The existence of the CG enables an addressable activation or deactivation of on-chip integrated individual CG-EISCAPs by simple electrical switching the CG of each sensor in various setups, and makes the new design capable for multianalyte detection without cross-talk effects between the sensors in the array. The new designed CG-EISCAP chip was modelled in so-called floating/short-circuited and floating/capacitively-coupled setups, and the corresponding electrical equivalent circuits were developed. In addition, the capacitance-voltage curves of the CG-EISCAP chip in different setups were simulated and compared with that of a single EISCAP sensor. Moreover, the sensitivity of the CG-EISCAP chip to surface potential changes induced by biochemical reactions was simulated and an impact of different parameters, such as gate voltage, insulator thickness and doping concentration in Si, on the sensitivity has been discussed.

Gold-Nanopartikel (AuNP) sind durch ihre Biokompatibilität, Stabilität, vielseitige Oberflächenchemie, sowie ihr hohes Verhältnis von Oberfläche zu Volumen für viele biomedizinische Anwendungen attraktiv [1]. Ein aktuelles Beispiel dafür ist ihr Einsatz als optische Marker in Covid-19 AntikörperSchnelltests [2]. Negativ geladene Citrat-stabilisierte AuNP auf der Oberfläche von kapazitiven FeldeffektStrukturen wurden u.a. bereits für die markierungsfreie Detektion von positiv geladenen Makromolekülen, als Nanoträger für Desoxyribonukleinsäure (DNA), die schichtweise Abscheidung von Polymeren und die Immobilisierung von Enzymen verwendet [3]. In dieser Arbeit wird die Immobilisierung von positiv geladenen Aminooctanthiol-stabilisierten GoldNanopartikeln (AuAOT) auf der Oberfläche von kapazitiven Feldeffekt-Sensoren vorgestellt [4]. Die terminale Aminogruppe der eingesetzten Partikel ist sehr gut für die spätere Funktionalisierung mit Biomolekülen geeignet. Die Anbindung der positiv geladenen AuAOTs auf der Sensoroberfläche konnte durch eine signifikante Verschiebung des Sensorsignals nachgewiesen werden. Bei einer höheren Partikeldichte auf der Sensoroberfläche war die gemessene Signaländerung entsprechend ausgeprägter. Zusätzlich konnte die elektrostatische Anbindung des Polyanions Polystyrolsulfonat an der AuAOT-modifizierten Sensoroberfläche gemessen werden.

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.