Capacitive field-effect biosensor modified with a stacked bilayer of weak polyelectrolyte and plant virus particles as enzyme nanocarriers.

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.

Essential Oils as Multicomponent Mixtures and Their Potential for Human Health and Well-Being.

Essential oils (EOs) and their individual volatile organic constituents have been an inherent part of our civilization for thousands of years. They are widely used as fragrances in perfumes and cosmetics and contribute to a healthy diet, but also act as active ingredients of pharmaceutical products. Their antibacterial, antiviral, and anti-inflammatory properties have qualified EOs early on for both, the causal and symptomatic therapy of a number of diseases, but also for prevention. Obtained from natural, mostly plant materials, EOs constitute a typical example of a multicomponent mixture (more than one constituent substances, MOCS) with up to several hundreds of individual compounds, which in a sophisticated composition make up the property of a particular complete EO. The integrative use of EOs as MOCS will play a major role in human and veterinary medicine now and in the future and is already widely used in some cases, e.g., in aromatherapy for the treatment of psychosomatic complaints, for inhalation in the treatment of respiratory diseases, or topically administered to manage adverse skin diseases. The diversity of molecules with different functionalities exhibits a broad range of multiple physical and chemical properties, which are the base of their multi-target activity as opposed to single isolated compounds. Whether and how such a broad-spectrum effect is reflected in natural mixtures and which kind of pharmacological potential they provide will be considered in the context of ONE Health in more detail in this review.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

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.

Detection of plant virus particles with a capacitive field-effect sensor.

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.

Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte's pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.

Capacitive Field-Effect Biosensor Studying Adsorption of Tobacco Mosaic Virus Particles.

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.

New planar assay for streamlined detection and quantification of ß-glucuronidase inhibitors applied to botanical extracts.

The inhibition of the ß-glucuronidase released from gut bacteria is associated with specific health-related benefits. Though a number of ß-glucuronidase inhibition assays are currently in use, none of them can directly measure the relevant activity of each single constituent in a complex mixture, without prior separation and tedious isolation of the pure compounds. Thus, the hyphenation of the high performance thin layer chromatography (HPTLC) technique with a ß-glucuronidase inhibition assay was investigated and successfully demonstrated for the first time. A colorimetric as well as fluorometric detection of the inhibitors was achieved using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide as a substrate. Hence, ß-glucuronidase inhibitors were detected as bright zones against an indigo blue or fluorescent background. The established method was optimized and validated employing the well-known inhibitor d-saccharic acid 1,4-lactone monohydrate. As proof of concept, the suitability of the new workflow was verified through analysis of two botanical extracts, Primula boveana and silymarin flavonolignans from Silybum marianum fruits. The found inhibitors were identified by spectroscopic methods; one of them, 3'-O-(ß-galactopyranosyl)-flavone, is here described as a newly isolated natural compound. The new hyphenation HPTLC-UV/Vis/FLD-ß-glucuronidase inhibition assay-HRMS covers four orthogonal dimensions, i.e. separation, spectral detection, biochemical activity and structural characterization, in a highly targeted time- and material-saving workflow for analysis of complex or costly mixtures.

Guided isolation of new iridoid glucosides from Anarrhinum pubescens by high-performance thin-layer chromatography-acetylcholinesterase assay.

Plants are an important source of natural iridoids. This study demonstrates for the first time the acetylcholinesterase (AChE) inhibitory activity of iridoids belonging to the class of antirrhinosides. As iridoids distinguish the chemical composition of most species of the Plantaginaceae family, the active AChE inhibitors were investigated in the hydro-alcoholic extract of Anarrhinum pubescens Fresen. High-performance thin-layer chromatography (HPTLC) in combination with the AChE inhibition assay is a time and material saving methodology, and thus was employed to directly point to the individual enzyme inhibitors occurring in the plant. The effect-directed screening successfully discovered three active metabolites. These were characterized as antirrhinoside-derived iridoids. Two of these are here reported as newly isolated natural compounds. Identification of the two new metabolites was based on analysis of their collected spectroscopic data (HRMS, 1D and 2D NMR). Their structures were elucidated to be 6-O-, 6'-O-di-trans-cinnamoyl-antirrhinoside (1) and 5-O-, 6-O-difoliamenthoyl-antirrhinoside (3), while the previously known compound 6-O-foliamenthoyl-(6'-O-cinnamoyl)-antirrhinoside (2) was assigned by extensive analysis of its HRMS and HRMS/MS data. The activity of the isolated compounds was referred to the known AChE inhibitor rivastigmine, i.e. their activity were calculated and expressed as values equivalently to rivastigmine. This neuroprotective potential of iridoids mediated through AChE inhibition promote them to compete as natural curatives for neurodegenerative disorders like Alzheimer's disease.

Screen-Printed Carbon Electrodes Modified with Graphene Oxide for the Design of a Reagent-Free NAD+-Dependent Biosensor Array.

A facile approach for the construction of reagent-free electrochemical dehydrogenase-based biosensors is presented. Enzymes and cofactors (NAD+ and Fe(CN)63-) were immobilized by modification of screen-printed carbon electrodes with graphene oxide (GO) and an additional layer of cellulose acetate. The sensor system was exemplarily optimized for an l-lactate electrode in terms of GO concentration, working potential, and pH value. The biosensor exhibited best characteristics at pH 7.5 in 100 mM potassium phosphate buffer at an applied potential of +0.250 V versus an internal pseudo Ag reference electrode. Thereby, sensor performance was characterized by a linear working range from 0.25 to 4 mM and a sensitivity of 0.14 µA mM-1. The detection principle was additionally evaluated with three other dehydrogenases (d-lactate dehydrogenase, alcohol dehydrogenase, and formate dehydrogenase, respectively). The developed reagentless biosensor array enabled simultaneous and cross-talk free determination of l-lactate, d-lactate, ethanol, and formate.

Combined calorimetric gas- and spore-based biosensor array for online monitoring and sterility assurance of gaseous hydrogen peroxide in aseptic filling machines.

A combined calorimetric gas- and spore-based biosensor array is presented in this work to monitor and evaluate the sterilization efficacy of gaseous hydrogen peroxide in aseptic filling machines. H2O2 has been successfully measured under industrial conditions. Furthermore, the effect of H2O2 on three different spore strains , namely Bacillus atrophaeus, Bacillus subtilis and Geobacillus stearothermophilus, has been investigated by means of SEM, AFM and impedimetric measurements. In addition, the sterilization efficacy of a spore-based biosensor and the functioning principle are addressed and discussed: the sensor array is convenient to be used in aseptic food industry to guarantee sterile packages.

Effect-directed analysis by high-performance thin-layer chromatography for bioactive metabolites tracking in Primula veris flower and Primula boveana leaf extracts.

The genus Primula (Primulaceae) comprises species with high medicinal as well as ornamental values. Plants belonging to this genus are well recognized for their richness in bioactive constituents. The huge variety of secondary metabolites make their complete analysis impossible. In order to cope with this challenge, effect-directed analysis (EDA) via HPTLC coupled to structure elucidation techniques was applied on Primula species for the first time. As straightforward non-target bioanalytical technique, HPTLC-UV/Vis/FLD-EDA-ESI-HRMS hyphenates three different orthogonal dimensions, i.e. chromatography with spectrometric detection, biological/enzymatic assays and HRMS. The bioactive metabolites were determined in the middle polar extracts of two Primula species, P. veris (flower) and P. boveana (leaf). The bioactivity profiling comprised the antibacterial activity against Aliivibrio fischeri and Bacillus subtilis bacterial strains and acetyl-/butyrylcholinesterase (AChE/BChE) inhibition assays. The compounds were characterized and identified via their recorded spectral data (HRMS and 1H NMR). The results showed that linoleic and linolenic acids were the principle bioactive compounds present in the studied P. veris flower extract. In the P. boveana leaf extract, flavone, 2'-methoxy-, 2'-hydroxy- and 5,6,2',6'-tetramethoxyflavone (zapotin) were determined as active metabolites. The identification of zapotin, which was previously undescribed in the investigated plant, was considered as the strength of the straightforward non-target bioanalytical technique. Flavone turned out to be the highest potent metabolite, and at the same time, a multipotent compound referring to its various bioactivities discovered. An equivalency calculation of the HPTLC-AChE inhibition by flavone was performed with reference to the well-known inhibitor rivastigmine. As a result, the amount of flavone contained in 10.0 µg dry powder of P. boveana (corresponding to 0.1 µL extract) inhibited as strong as 4.5 µg rivastigmine. In other words, the flavone contained in P. boveana leaf extract powder turned out to be half as strong as the well-known AChE inhibitor rivastigmine.

Surface regeneration and reusability of label-free DNA biosensors based on weak polyelectrolyte-modified capacitive field-effect structures.

The reusability of capacitive field-effect electrolyte-insulator-semiconductor (EIS) sensors modified with a cationic weak polyelectrolyte (poly(allylamine hydrochloride) (PAH)) for the label-free electrical detection of single-stranded DNA (ssDNA), in-solution- and on-chip-hybridized double-stranded DNA (dsDNA) has been studied. It has been demonstrated that via simply regeneration of the gate surface of the EIS sensor by means of an electrostatic adsorption of a new PAH layer, the same biosensor can be reused for at least five DNA-detection measurements. Because of the reversal of the charge sign of the outermost layer after each surface modification with the cationic PAH or negatively charged DNA molecules, the EIS-biosensor signal exhibits a zigzag-like behavior. The amplitude of the signal changes has a tendency to decrease with increasing number of macromolecular layers. The direction of the EIS-signal shifts can serve as an indicator for a successful DNA-immobilization or -hybridization process. In addition, we observed that the EIS-signal changes induced by each surface-modification step (PAH adsorption, immobilization of ssDNA or dsDNA molecules and on-chip hybridization of complementary target cDNA) is decreased with increasing the ionic strength of the measurement solution, due to the more efficient macromolecular charge-screening by counter ions. The results of field-effect experiments were supported by fluorescence-intensity measurements of the PAH- or DNA-modified EIS surface using various fluorescence dyes.

Toward a Hybrid Biosensor System for Analysis of Organic and Volatile Fatty Acids in Fermentation Processes.

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.

Application of a Portable Multi-Analyte Biosensor for Organic Acid Determination in Silage.

Multi-analyte biosensors may offer the opportunity to perform cost-effective and rapid analysis with reduced sample volume, as compared to electrochemical biosensing of each analyte individually. This work describes the development of an enzyme-based biosensor system for multi-parametric determination of four different organic acids. The biosensor array comprises five working electrodes for simultaneous sensing of ethanol, formate, d-lactate, and l-lactate, and an integrated counter electrode. Storage stability of the biosensor was evaluated under different conditions (stored at +4 °C in buffer solution and dry at −21 °C, +4 °C, and room temperature) over a period of 140 days. After repeated and regular application, the individual sensing electrodes exhibited the best stability when stored at −21 °C. Furthermore, measurements in silage samples (maize and sugarcane silage) were conducted with the portable biosensor system. Comparison with a conventional photometric technique demonstrated successful employment for rapid monitoring of complex media.

Development and characterization of a field-effect biosensor for the detection of acetoin.

A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.

Detection of PCR-Amplified Tuberculosis DNA Fragments with Polyelectrolyte-Modified Field-Effect Sensors.

Field-effect-based electrolyte-insulator-semiconductor (EIS) sensors were modified with a bilayer of positively charged weak polyelectrolyte (poly(allylamine hydrochloride) (PAH)) and probe single-stranded DNA (ssDNA) and are used for the detection of complementary single-stranded target DNA (cDNA) in different test solutions. The sensing mechanism is based on the detection of the intrinsic molecular charge of target cDNA molecules after the hybridization event between cDNA and immobilized probe ssDNA. The test solutions contain synthetic cDNA oligonucleotides (with a sequence of tuberculosis mycobacteria genome) or PCR-amplified DNA (which origins from a template DNA strand that has been extracted from Mycobacterium avium paratuberculosis-spiked human sputum samples), respectively. Sensor responses up to 41 mV have been measured for the test solutions with DNA, while only small signals of ∼5 mV were detected for solutions without DNA. The lower detection limit of the EIS sensors was ∼0.3 nM, and the sensitivity was ∼7.2 mV/decade. Fluorescence experiments using SybrGreen I fluorescence dye support the electrochemical results.

Spore-based biosensor to monitor the microbicidal efficacy of gaseous hydrogen peroxide sterilization processes.

In this work, a spore-based biosensor is evaluated to monitor the microbicidal efficacy of sterilization processes applying gaseous hydrogen peroxide (H2O2). The sensor is based on interdigitated electrode structures (IDEs) that have been fabricated by means of thin-film technologies. Impedimetric measurements are applied to study the effect of sterilization process on spores of Bacillus atrophaeus. This resilient microorganism is commonly used in industry to proof the sterilization efficiency. The sensor measurements are accompanied by conventional microbiological challenge tests, as well as morphological characterizations with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The sensor measurements are correlated with the microbiological test routines. In both methods, namely the sensor-based and microbiological one, a tailing effect has been observed. The results are evaluated and discussed in a three-dimensional calibration plot demonstrating the sensor's suitability to enable a rapid process decision in terms of a successfully performed sterilization.