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The integration of biomolecular logic principles with electronic transducers allows designing novel digital biosensors with direct electrical output, logically triggered drug-release, and closed-loop sense/act/treat systems. This opens new opportunities for advanced personalized medicine in the context of theranostics. In the present work, we will discuss selected examples of recent developments in the field of interfacing enzyme logic gates with electrodes and semiconductor field-effect devices. Special attention is given to an enzyme OR/Reset logic gate based on a capacitive field-effect electrolyte-insulator-semiconductor sensor modified with a multi-enzyme membrane. Further examples are a digital adrenaline biosensor based on an AND logic gate with binary YES/NO output and an integrated closed-loop sense/act/treat system comprising an amperometric glucose sensor, a hydrogel actuator, and an insulin (drug) sensor.
The incorporation of nanomaterials that are biocompatible with different types of biological compounds has allowed the development of a new generation of biosensors applied especially in the biomedical field. In particular, the integration of film-based nanomaterials employed in field-effect devices can be interesting to develop biosensors with enhanced properties. In this paper, we studied the fabrication of sensitive nanofilms combining ZnO nanocrystals and carbon nanotubes (CNTs), prepared by means of the layer-by-layer (LbL) technique, in a capacitive electrolyte-insulator-semiconductor (EIS) structure for detecting glucose and urea. The ZnO nanocrystals were incorporated in a polymeric matrix of poly(allylamine) hydrochloride (PAH), and arranged with multi-walled CNTs in a LbL PAH-ZnO/CNTs film architecture onto EIS chips. The electrochemical characterizations were performed by capacitance–voltage and constant capacitance measurements, while the morphology of the films was characterized by atomic force microscopy. The enzymes glucose oxidase and urease were immobilized on film’s surface for detection of glucose and urea, respectively. In order to obtain glucose and urea biosensors with optimized amount of sensitive films, we investigated the ideal number of bilayers for each detection system. The glucose biosensor showed better sensitivity and output signal for an LbL PAH-ZnO/CNTs nanofilm with 10 bilayers. On the other hand, the urea biosensor presented enhanced properties even for the first bilayer, exhibiting high sensitivity and output signal. The presence of the LbL PAH-ZnO/CNTs films led to biosensors with better sensitivity and enhanced response signal, demonstrating that the adequate use of nanostructured films is feasible for proof-of-concept biosensors with improved properties that may be employed for biomedical applications.
The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.
The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided.
The coupling of charged molecules, nanoparticles, and more generally, inorganic/organic nanohybrids with semiconductor field-effect devices based on an electrolyte–insulator–semiconductor (EIS) system represents a very promising strategy for the active tuning of electrochemical properties of these devices and, thus, opening new opportunities for label-free biosensing by the intrinsic charge of molecules. The simplest field-effect sensor is a capacitive EIS sensor, which represents a (bio-)chemically sensitive capacitor. In this chapter, selected examples of recent developments in the field of label-free biosensing using nanomaterial-modified capacitive EIS sensors are summarized. In the first part, we present applications of EIS sensors modified with negatively charged gold nanoparticles for the label-free electrostatic detection of positively charged small proteins and macromolecules, for monitoring the layer-by-layer formation of oppositely charged polyelectrolyte (PE) multilayers as well as for the development of an enzyme-based biomolecular logic gate. In the second part, examples of a label-free detection by means of EIS sensors modified with a positively charged weak PE layer are demonstrated. These include electrical detection of on-chip and in-solution hybridized DNA (deoxyribonucleic acid) as well as an EIS sensor with pH-responsive weak PE/enzyme multilayers for enhanced field-effect biosensing.
The LAPS (light-addressable potentiometric sensor) platform is one of the most attractive approaches for chemical and biological sensing with many applications ranging from pH and ion/analyte concentration measurements up to cell metabolism detection and chemical imaging. However, although it is generally accepted that LAPS measurements are spatially resolved, the light-addressability feature of LAPS devices has not been discussed in detail so far. In this work, an extended electrical equivalent-circuit model of the LAPS has been presented, which takes into account possible cross-talk effects due to the capacitive coupling of the non-illuminated region. A shunting effect of the non-illuminated area on the measured photocurrent and addressability of LAPS devices has been studied. It has been shown, that the measured photocurrent will be determined not only by the local interfacial potential in the illuminated region but also by possible interfacial potential changes in the non-illuminated region, yielding cross-talk effects. These findings were supported by the experimental investigations of a penicillin-sensitive multi-spot LAPS and a metal-insulator-semiconductor LAPS as model systems.
Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (L-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.
In this paper, carbon nanotubes (CNTs) were incorporated in penicillinase-phospholipid Langmuir and Langmuir–Blodgett (LB) films to enhance the enzyme catalytic properties. Adsorption of the penicillinase and CNTs at dimyristoylphosphatidic acid (DMPA) monolayers at the air–water interface was investigated by surface pressure–area isotherms, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to solid supports through the LB technique, forming mixed DMPA-CNTs-PEN films, which were investigated by quartz crystal microbalance, vibrational spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV–vis spectroscopy and the feasibility of the supramolecular device nanostructured as ultrathin films were essayed in a capacitive electrolyte–insulator–semiconductor (EIS) sensor device. The presence of CNTs in the enzyme–lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks, showing increased values of activity. Viability as penicillin sensor was demonstrated with capacitance/voltage and constant capacitance measurements, exhibiting regular and distinctive output signals over all concentrations used in this work. These results may be related not only to the nanostructured system provided by the film, but also to the synergism between the compounds on the active layer, leading to a surface morphology that allowed a fast analyte diffusion because of an adequate molecular accommodation, which also preserved the penicillinase activity. This work therefore demonstrates the feasibility of employing LB films composed of lipids, CNTs, and enzymes as EIS devices for biosensing applications.
Research collaborations provide opportunities for both practitioners and researchers: practitioners need solutions for difficult business challenges and researchers are looking for hard problems to solve and publish. Nevertheless, research collaborations carry the risk that practitioners focus on quick solutions too much and that researchers tackle theoretical problems, resulting in products which do not fulfill the project requirements.
In this paper we introduce an approach extending the ideas of agile and lean software development. It helps practitioners and researchers keep track of their common research collaboration goal: a scientifically enriched software product which fulfills the needs of the practitioner’s business model.
This approach gives first-class status to application-oriented metrics that measure progress and success of a research collaboration continuously. Those metrics are derived from the collaboration requirements and help to focus on a commonly defined goal.
An appropriate tool set evaluates and visualizes those metrics with minimal effort, and all participants will be pushed to focus on their tasks with appropriate effort. Thus project status, challenges and progress are transparent to all research collaboration members at any time.
Field-effect EIS (electrolyte-insulator-semiconductor) sensors modified with a positively charged weak polyelectrolyte layer have been applied for the electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge. The EIS sensors are able to detect the existence of target DNA amplicons in PCR (polymerase chain reaction) samples and thus, can be used as tool for a quick verification of DNA amplification and the successful PCR process. Due to their miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge, those sensors can serve as possible platform for the development of label-free DNA chips. Possible application fields as well as challenges and limitations will be discussed.
The capacitive electrolyte–insulator–semiconductor (EIS) structure is a typical device based on a field-effect sensor platform. With a simple silicon-based structure, EIS have been useful for several sensing applications, especially with incorporation of nanostructured films to modulate the ionic transport and the flat-band potential. In this paper, we report on ion transport and changes in flat-band potential in EIS sensors made with layer-by-layer films containing poly(amidoamine) (PAMAM) dendrimer and single-walled carbon nanotubes (SWNTs) adsorbed on p-Si/SiO 2 /Ta 2 O 5 chips with an Al ohmic contact. The impedance spectra were fitted using an equivalent circuit model, from which we could determine parameters such as the double-layer capacitance. This capacitance decreased with the number of bilayers owing to space charge accumulated at the electrolyte–insulator interface, up to three PAMAM/SWNTs bilayers, after which it stabilized. The charge-transfer resistance was also minimum for three bilayers, thus indicating that this is the ideal architecture for an optimized EIS performance. The understanding of the influence of nanostructures and the fine control of operation parameters pave the way for optimizing the design and performance of new EIS sensors.