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.

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.

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.

Development of a borreliosis assay: Mannan coated polyethylene sinter bodies as a new platform technology.

Rapid diagnosis of Lyme borreliosis has been carried out on chemically modified porous polyethylene sinter bodies. Photografting of 2-propenol on sinter body's surface was performed as a first step, introducing active hydroxyl groups as a result of polyalcohol formation. The hydroxyl groups were used for further immobilization and could be linked via 3-aminopropyltriethoxysilane (APTES) to polysaccharides like mannan. Prone to coupling, mannan was activated using N, N'-disuccinimidyl carbonate (DSC) to allow smooth reaction with the primary amine groups of the silane layer. In a final preparation step, a recombinant fusion protein consisting of the mannan-binding domain of the lectin Concanavalin A (ConA) and a specific Borrelia surface antigen was immobilized by self-organization on the mannan surface. The fusion protein was used as biological interface structure. This strategy is highly efficient and resulted in a defined orientation of the antigen part of the fusion protein. Rapid and convenient differentiation could be then established between Borrelia-negative and a -positive serum even in 1000-fold diluted samples and detection of Lyme borreliosis in a rather early stage is likely. Furthermore, this generic strategy can be easily transferred to other bacterial or viral antigen structures.

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.

Amino acids in the cultivation of mammalian cells.

Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.

Study of interdigitated electrode arrays using experiments and finite element models for the evaluation of sterilization processes.

In this work, a sensor to evaluate sterilization processes with hydrogen peroxide vapor has been characterized. Experimental, analytical and numerical methods were applied to evaluate and study the sensor behavior. The sensor set-up is based on planar interdigitated electrodes. The interdigitated electrode structure consists of 614 electrode fingers spanning over a total sensing area of 20 mm². Sensor measurements were conducted with and without microbiological spores as well as after an industrial sterilization protocol. The measurements were verified using an analytical expression based on a first-order elliptical integral. A model based on the finite element method with periodic boundary conditions in two dimensions was developed and utilized to validate the experimental findings.

A novel method for the assessment of targeted PEI-based nanoparticle binding based on a static surface plasmon resonance system.

The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

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.

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.

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.

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.

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.

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.

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.