Acetylcholinesterase and butyrylcholinesterase co-immobilized on a copper containing Prussian blue modified electrode for the broad screening of insecticides.

We have developed a bienzymatic biosensor that contains acetylcholinesterase together with butyrylcholinesterase co-immobilized on the same electrode modified with a stabilized copper containing Prussian blue electrodeposited on electrodes coated with 4-aminothiophenol monolayer using diazonium chemistry and copper nanoparticles for improved sensitivity. There are organophosphorus and carbamate neurotoxic insecticides that inhibit only one of the two enzymes, e.g., pirimicarb inhibits butyrylcholinesterase at much lower concentrations than acetylcholinesterase while methomyl inhibits only acetylcholinesterase. Our system is simple and in a single measurement provides a sensitive signal for insecticides' presence based on the inhibition of the enzyme with the highest affinity for each toxic compound. The limits of detection are 50 ng/mL pirimicarb for the bienzymatic biosensor in comparison with 400 ng/mL pirimicarb for the acetylcholinesterase biosensor and 6 ng/mL methomyl for the bienzymatic biosensor, while inhibition is obtained for the butyrylcholinesterase biosensor at 700 ng/mL.

Cavitation-Effect-Based Treatments and Extractions for Superior Fruit and Milk Valorisation.

Ultrasound generates cavities in liquids with high-energy behaviour due to large pressure variations, leading to (bio)chemical effects and material modification. Numerous cavity-based treatments in food processes have been reported, but the transition from research to industrial applications is hampered by specific engineering factors, such as the combination of several ultrasound sources, more powerful wave generators or tank geometry. The challenges and development of cavity-based treatments developed for the food industry are reviewed with examples limited to two representative raw materials (fruit and milk) with significantly different properties. Both active compound extraction and food processing techniques based on ultrasound are taken into consideration.

Ternary water-organic solvent mixtures used for insecticide SPE extraction and analysis with acetylcholinesterase biosensor.

Few biosensors are reported for usage in combination with the organic solvent due to their negative impact on the enzymes. The usage of ternary water-organic solvent mixtures in combination with acetylcholinesterase biosensors allows to increase the useable total content of organic solvents with minimum negative effects to a higher content in comparison with a single organic solvent in water. The combination of acetonitrile/ethanol/water has a smaller negative effect on both enzyme activity and inhibition by insecticides in comparison with acetonitrile/methanol/water mixtures. The insecticides were eluted from solid-phase extraction (SPE) columns with a binary mixture of organic solvents acetonitrile/ethanol in 1/3 ratio and subsequently analysed with an acetylcholinesterase biosensor and the optimum total content of organic solvents of 12%. The analytical method allows the analysis of complex samples with improved selectivity and at improved limits of detection for chlorpyrifos-oxon and carbofuran analysis in river waters and soil samples. The usage of mixtures of organic solvents in combination with enzymes is an interesting approach that allows working with a higher total content of organic solvents than each individual solvent.

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives.

Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on possible solutions involving the use of nanomaterials to ensuring the biosensors' selectivity. Overall, the examples presented illustrate the various tools available when developing enzyme biosensors for new applications and stress the necessity to more comprehensively investigate their selectivity and validate the biosensors versus standard analytical methods.

Bienzymatic biosensor for rapid detection of aspartame by flow injection analysis.

A rapid, simple and stable biosensor for aspartame detection was developed. Alcohol oxidase (AOX), carboxyl esterase (CaE) and bovine serum albumin (BSA) were immobilised with glutaraldehyde (GA) onto screen-printed electrodes modified with cobalt-phthalocyanine (CoPC). The biosensor response was fast. The sample throughput using a flow injection analysis (FIA) system was 40 h⁻¹ with an RSD of 2.7%. The detection limits for both batch and FIA measurements were 0.1 µM for methanol and 0.2 µM for aspartame, respectively. The enzymatic biosensor was successfully applied for aspartame determination in different sample matrices/commercial products (liquid and solid samples) without any pre-treatment step prior to measurement.

Critical evaluation of acetylthiocholine iodide and acetylthiocholine chloride as substrates for amperometric biosensors based on acetylcholinesterase.

Numerous amperometric biosensors have been developed for the fast analysis of neurotoxic insecticides based on inhibition of cholinesterase (AChE). The analytical signal is quantified by the oxidation of the thiocholine that is produced enzymatically by the hydrolysis of the acetylthiocholine pseudosubstrate. The pseudosubstrate is a cation and it is associated with chloride or iodide as corresponding anion to form a salt. The iodide salt is cheaper, but it is electrochemically active and consequently more difficult to use in electrochemical analytical devices. We investigate the possibility of using acetylthiocholine iodide as pseudosubstrate for amperometric detection. Our investigation demonstrates that operational conditions for any amperometric biosensor that use acetylthiocholine iodide must be thoroughly optimized to avoid false analytical signals or a reduced sensitivity. The working overpotential determined for different screen-printed electrodes was: carbon-nanotubes (360 mV), platinum (560 mV), gold (370 mV, based on a catalytic effect of iodide) or cobalt phthalocyanine (110 mV, but with a significant reduced sensitivity in the presence of iodide anions).

Creativity and Generation of Ideas in the Design of Children's Toys.

Creativity offers new, interesting, and valuable things that can be intangible (ideas, a theory, songs, etc.) or physical objects (a painting, invention, machine). Creativity implies a lot of qualities of the creator such as imagination, creative work, and innovation and it also improves learning and memory. Many of history's most important discoveries are the results of creative activity. Repetition leads to mastery of a concept through understanding and produces increased self-confidence. Confidence increases the willingness to act on creativity-to explore, discover, and learn. This positive cycle of learning is fueled by the curiosity and enjoyment that comes from discovery and understanding. We are social creatures, so the greatest reward and pleasure comes from the admiration and support received from loved and respected people. Stimulating children's interest through play also defines solving through exploration regarding the accumulation of new essential information for knowing values and other useful information, by stimulating curiosity and creativity as well as discovering new resources that generate creative ideas, allowing the acquisition of practical skills. All these aspects are oriented and define the premises for the harmonious development of children towards a new existential stage. Thus, taking these aspects into account will have future effects on self-confidence, work strategies, school results, as well as the desire to study and the ability to store and organize accumulated information. The approach of the case study presents through the game, a motivational alternative, staged regarding the generation of creative ideas in the development and materialization of the concept. It is well known that during childhood, many things are acquired by children through selective association and depending on the sensory perception of objects, namely preferred colors, functions, and predefined shapes, proportional to the anthropometric dimensions specific to preschool age. The article proposes the creative approach and generation of ideas on the design of children's toys; namely, a case study is presented: children's toy set-teacup.

Technological Barriers in the Use of Electrochemical Microsensors and Microbiosensors for in vivo Analysis of Neurological Relevant Substances.

In this paper is presented an overview of the technological barriers faced by the in vivo brain analysis with microelectrodes. Numerous microsensors and enzymatic microbiosensors have been developed for the real time monitoring of neurotransmitters, neuromodulators, drugs and diverse other biological relevant substances. A clear understanding of the working principle, advantages and limitations is essential for the acquisition of valid data in neurological investigations. Some of the aspects presented here refer to: microelectrode insertion and positioning related to possibilities to minimize tissue damage, spatial and temporal resolution of the measurements, actual controversies in data interpretation and sensor calibration, simultaneous detection of multiple analytes, interferences and state of the art in the development of wireless devices.

Aspects Regarding the Innovative Conceptual Design for Children's Recreational Areas.

Conceptual design approaches the definition of an innovative concept design applied to a product intended for children'-=s recreational area, corresponding to a personal profile located around the age of childhood, namely aged 2 to 5. The case study presented highlights the development of an innovative concept for children's recreational areas, to be precise the design, and the conceptual design applied to a roto-pendular carousel for children's recreational areas. The working method aims at identifying the chronological stages of development and the conceptual design, starting from the idea, prospectographic research, analysis of constructive solutions, and finally the materialization of the assisted design in detail, applied to the new concept. Based on the prospectographic study, several constructive variants are highlighted, at the draft level, and after the analysis, the optimized variant of the carousel concept is established that should match the functions and characteristics pursued and imposed on the new concept. Following the justification of the choice of the optimal variant of the concept, the model is designed in an assisted environment in order to follow aspects related to technology associated with each component of the whole assembly, as well as aspects related to ergonomics, safety in exploration and proportion associated to the typology and to the age criteria of children.

Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor using a 50 MHz electronic oscillator circuit.

This work deals with the design of a high sensitivity DNA sequence detector using a 50 MHz quartz crystal microbalance (QCM) electronic oscillator circuit. The oscillator circuitry is based on Miller topology, which is able to work in damping media. Calibration and experimental study of frequency noise are carried out, finding that the designed sensor has a resolution of 7.1 ng/cm(2) in dynamic conditions (with circulation of liquid). Then the oscillator is proved as DNA biosensor. Results show that the system is able to detect the presence of complementary target DNAs in a solution with high selectivity and sensitivity. DNA target concentrations higher of 50 ng/mL can be detected.

Determination of silver(I) by differential pulse voltammetry using a glassy carbon electrode modified with synthesized N-(2-aminoethyl)-4,4'-bipyridine.

A new modified glassy carbon electrode (GCE) based on a synthesized N-(2-aminoethyl)-4,4'-bipyridine (ABP) was developed for the determination of Ag(I) by differential pulse voltammetry (DPV). ABP was covalently immobilized on GC electrodes surface using 4-nitrobenzendiazonium (4-NBD) and glutaraldehyde (GA). The Ag(I) ions were preconcentrated by chemical interaction with bipyridine under a negative potential (-0.6 V); then the reduced ions were oxidized by differential pulse voltammetry and a peak was observed at 0.34 V. The calibration curve was linear in the concentration range from 0.05 µM to 1 µM Ag(I) with a detection limit of 0.025 µM and RSD=3.6%, for 0.4 µM Ag(I). The presence of several common ions in more than 125-fold excess had no effect on the determination of Ag(I). The developed sensor was applied to the determination of Ag(I) in water samples using a standard addition method.

Ester flavorants detection in foods with a bienzymatic biosensor based on a stable Prussian blue-copper electrodeposited on carbon paper electrode.

A stable and reproducible layer of Prussian blue (PB) modified with copper was electrodeposited on carbon paper electrodes for the multiple detection of ester flavorants with a bienzymatic biosensor. Carbon fiber composite paper was investigated as high-surface, low-cost substrate for biosensor development. The pre-activation of the electrode surface by cyclic voltammetry was necessary to improve the electrochemical properties before the electrochemical deposition of Prussian blue-copper film (PB-Cu). The stability and the reproducibility of the obtained PB-Cu carbon paper electrode was demonstrated at pH 7.4, optimum for biosensor development. The developed biosensor is based on the immobilization of two enzymes (carboxyl esterase and alcohol oxidase) by cross-linking with glutaraldehyde onto PB-Cu carbon paper electrode. A mixture of key aroma ester compounds (methyl butyrate, ethyl butyrate, methyl cinnamate and ethyl cinnamate) was detected in several food samples with low interferences.

Simple, selective and fast detection of acrylamide based on glutathione S-transferase.

Acrylamide (AA) is a toxic compound formed in thermally prepared foods by Maillard reaction. Besides foods, AA may be found in cosmetic products as an impurity of the widely-used non-toxic polyacrylamide. We present a novel, fast and selective detection method based on the amperometric monitoring of the coupling reaction between reduced glutathione (GSH) and AA catalyzed by glutathione S-transferase (GST) to produce an electrochemically inactive compound. We have used electrodes modified with cobalt-phthalocyanine to monitor the decrease of GHS concentration at +300 mV. Our system is simple, does not require supplementary substrates such as 1-chloro-2,4-dinitrobenzene (CDNB) nor have disadvantageous competitive kinetics characteristic to inhibition like signals. Using the optimum concentration of 100 µM GSH we have obtained a linear calibration graph from 7 to 50 µM AA and a limit of detection of 5 µM AA. The method is not affected by interfering compounds usually found in foods and was applied for real sample analysis.

Advances in Enzyme-Based Biosensors for Pesticide Detection.

The intensive use of toxic and remanent pesticides in agriculture has prompted research into novel performant, yet cost-effective and fast analytical tools to control the pesticide residue levels in the environment and food. In this context, biosensors based on enzyme inhibition have been proposed as adequate analytical devices with the added advantage of using the toxicity of pesticides for detection purposes, being more "biologically relevant" than standard chromatographic methods. This review proposes an overview of recent advances in the development of biosensors exploiting the inhibition of cholinesterases, photosynthetic system II, alkaline phosphatase, cytochrome P450A1, peroxidase, tyrosinase, laccase, urease, and aldehyde dehydrogenase. While various strategies have been employed to detect pesticides from different classes (organophosphates, carbamates, dithiocarbamates, triazines, phenylureas, diazines, or phenols), the number of practical applications and the variety of environmental and food samples tested remains limited. Recent advances focus on enhancing the sensitivity and selectivity by using nanomaterials in the sensor assembly and novel mutant enzymes in array-type sensor formats in combination with chemometric methods for data analysis. The progress in the development of solar cells enriched the possibilities for efficient wiring of photosynthetic enzymes on different surfaces, opening new avenues for development of biosensors for photosynthesis-inhibiting herbicides.

Electrodeposited Organic Layers Formed from Aryl Diazonium Salts for Inhibition of Copper Corrosion.

Copper substrates deposed on a gold screen-printed electrode were covered with different aryl diazonium salts by electrodeposition at 0.25 mA for 30 or 300 s. Seven compounds were investigated: 4-aminophenylacetic acid, 4-aminophenethyl alcohol, 4-fluoroaniline, 4-(heptadecafluorooctyl)aniline, 4-aminoantipyrine, 4-(4-aminophenyl)butyric acid and 3,4,5-trimethoxyaniline. Quantitative monitoring of the electrodeposition process was carried out by electrogravimetry using quartz crystal microbalance (QCM). The electrodeposited mass varies between 26 ng/cm² for 4-fluoroaniline formed during 30 s to 442 ng/cm² for 4-phenylbutyric acid formed during 300 s. The corrosion inhibition properties of aryl-modified layers have been studied in buffer citrate with pH = 3 or 3.5% NaCl solutions using electrochemical noise (ECN) and Tafel potentiodynamic polarization measurements. A corrosion inhibiting efficiency up to 90% was found. The highest corrosion inhibition was obtained for 4-(4-aminophenyl)butyric acid and the lowest for 4-fluoroaniline. A relation between the inhibition efficiency and the chemical nature of the substituents in the protective layer was found.

Strategies to develop malic acid biosensors based on malate quinone oxidoreductase (MQO).

An amperometric biosensor based on malate quinone oxidoreductase (MQO) was developed for monitoring of the malolactic fermentation of wines. Screen-printed electrodes coupled with appropriate mediators were used as transducers for this novel biosensor. MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) on the surface of the working electrode. Several electrochemical mediators were studied in order to lower the applied potential and minimise the matrix effects. Among them, 2,6-dichlorophenol indophenol (DPIP) and phenazine methosulfate (PMS) were chosen for further development. The working conditions (mediator concentration, applied potential and pH) were optimised for both DPIP and PMS. Detection limits for both types of biosensors were of 5 microM malic acid. Sensitivities obtained for the linear part of the calibration curve were 0.85 and 1.7 mA/M for the biosensors based on DPIP and PMS, respectively. Interferences due to non-specific oxidations were shown to be negligible when using PMS as mediator.

Low-interferences Determination of the Antioxidant Capacity in Fruits Juices Based on Xanthine Oxidase and Mediated Amperometric Measurements in the Reduction Mode.

A low-interferences enzymatic sensor for evaluating the antioxidant capacity was developed. Xanthine oxidase was used to produce superoxide radicals that spontaneously dismutate to hydrogen peroxide. Low xanthine concentrations were used to minimize the rapid dismutation of the superoxide radical before its fast reaction with antioxidants. The sensor operates in the reduction mode, and evaluations with low interferences of the antioxidant capacity are based on the detection of remaining hydrogen peroxide using Prussian blue electrodes at low potentials. The linear calibration graph is between 2 - 10 µM ascorbic acid. No interferences were observed from easily oxidisable substances including uric acid, which is produced in the enzymatic reaction or other substances usually found in foods. The method was used to evaluate the antioxidant capacity in different real juice samples.

Biosensor based on inhibition of monoamine oxidases A and B for detection of ß-carbolines.

ß-Carbolines are inhibitors of monoamine oxidases (MAO-A and MAO-B) and can be found in foods, hallucinogenic plant or various drugs. We have developed a fast analysis method for ß-carbolines based on the inhibition of MAO. The enzymes were immobilized on screen-printed electrodes modified with a stabilized film of Prussian blue that contain also copper. We have used benzylamine as substrate for the enzymatic reaction and the hydrogen peroxide was measured amperometrically at -50 mV. The detection limits obtained were 5.0 µM for harmane and 2.5 µM for both harmaline and norharmane. The MAO-A is inhibited by all three tested ß-carbolines (harmane, norharmane, and harmaline) while MAO-B is inhibited only by norharmane. The presence of norharmane in mixtures of ß-carbolines can be identified based on the difference between the cumulative inhibition of MAO-A by all ß-carbolines and MAO-B inhibition. The developed biosensors were used for food analysis.

Versatile method of cholinesterase immobilisation via affinity bonds using Concanavalin A applied to the construction of a screen-printed biosensor.

Development of new and more reliable methods to immobilise biomolecules has emerged rapidly due to a continuous need for more stable, sensitive and reliable biosensors. This paper reports a new method of acetylcholine-esterase (AChE) immobilisation based on the high affinity interaction between the glycoproteic enzyme and Concanavalin A (Con A). In order to establish the nature of the link formed between the glycoenzyme, lectin and support, three different configurations are presented. The optimum immobilisation procedure was further used for biosensor manufacturing. The non-specific adsorption is around 3% and the chemical cross-linking of the proteins is avoided. The optimised method allows loading of the working electrode surface with different amounts of enzyme ranging from 0.3 to 3.3 mIU with a good operational stability. The sensor showed a linear response range to acetylthiocholine substrate between 10 and 110 micromol l(-1) with a sensitivity of 3.6 mA l mol(-1). The applicability of the method to the detection of organophosphorus insecticides resulted in a detection limit of 10(-8) mol l(-1) for chlorpyriphos.

Organophosphorus insecticides extraction and heterogeneous oxidation on column for analysis with an acetylcholinesterase (AChE) biosensor.

This paper presents an analysis method for organophosphorus insecticides based on AChE biosensors coupled with a preconcentration and oxidation on a solid phase column. Three organic solvents, acetonitrile (ACN), ethanol and methanol were tested for their influence on AChE activity, insecticide inhibition and their ability to elute the adsorbed insecticides. Our results showed that ACN in a concentration of 5% (v/v) had the less negative effect on biosensor analysis and was the most appropriate organic solvent for the column elution. The presence of the organic solvent in the incubation media of the biosensor was found to induce a reduction of the inhibition percentages. The inhibition of the biosensors was performed in phosphate buffer with 5% (v/v) ACN, while the initial and remaining response of the biosensors were measured in PBS. In these conditions, the LODs of paraoxon and dichlorvos were measured with or without a preconcentration step. The LODs of the AChE biosensor without sample preconcentration were 8 x 10(-8) M for paraoxon and 1 x 10(-7) M dichlorvos and the LOD obtained after the preconcentration step were 2.5 x 10(-8) M for paraoxon and 2.5 x 10(-8) M for dichlorvos. Moreover, the use of the column allowed the heterogeneous oxidation of organophosphorus insecticides for improved LOD.