A microconductometric biosensor based on lipase extracted from Candida rugosa for direct and rapid detection of organophosphate pesticides

Organophosphate pesticides (OPs) have been intensively used as insecticides in agriculture; after entering the aquatic environment, they may affect a wide range of organisms. A conductometric enzymatic biosensor based on lipase extracted from Candida rugosa (CRL) has therefore been developed for the direct and rapid quantitative detection of organophosphate pesticides: diazinon, methyl parathion and methyl paraoxon in water. The biosensor signal and response time were obtained under optimum conditions, the enzyme being immobilised in the presence of gold nanoparticles. Under these conditions, the enzymatic biosensor was able to measure concentrations as low as 60 µg/L of diazinon, 26 µg/L of methyl parathion and 25 µg/L of methyl paraoxon very rapidly (response time: 3 min). Moreover, this CRL biosensor was not sensitive to interferences such as carbamates. It presented good storage stability for 21 days when kept at 4°C and it was successfully applied to real samples.     

Electrochemical Biosensor for Organophosphate Pesticides and Huperzine‐A Detection Based on Pd Wormlike Nanochains/Graphitic Carbon Nitride Nanocomposites and Acetylcholinesterase

A new electrochemical biosensor was developed for organophosphorus pesticides (OPs) and huperzine‐A (hupA) detection based on Pd wormlike nanochains/graphitic carbon nitride (Pd WLNCs/g‐C₃N₄) nanocomposites and acetylcholinesterase (AChE). The morphologies and components of the nanocomposites were analyzed by transmission electron microscopy (TEM) and X‐ray photoelectron spectroscopy (XPS). The Pd WLNCs/g‐C₃N₄ nanocomposites could effectively immobilize enzymes and promote the signal amplification. Under the optimum condition, the proposed biosensor displayed well performance. The linear response ranges for the determination of OPs and hupA were 1.00 nM to 14.96 µM and 3.89 nM to 20.80 µM, respectively. The corresponding detection limits were 0.33 nM and 1.30 nM (S/N=3). Meanwhile, the biosensor owned good reproducibility and stability, and could also be applied to analyze practical samples, which would be a new hopeful method for pesticide analysis.     

Acetylcholinesterase based assay of eleven organophosphorus pesticides: finding of assay limitations

The study includes findings about limitations of acetylcholinesterase (AChE) based assay. Eleven organophosphorus pesticides: chlorpyrifos ethyl, chlorpyrifos methyl, DFP, dichlorvos, dimethoate, fenthion, paraoxon ethyl, paraoxon methyl, phosalone, pirimiphos methyl and pirimiphos ethyl were photometrically assayed using AChE as a recognition element. The study was carried out in order to find approachability of AChE based assay. In the first round, common organic solvents were tested for interfering in assay, since samples collection and extraction is a necessary part in samples processing. Isopropanol was found as the most convenient due to minimal inhibition not exceeding 5%. Though all analysed pesticides inhibit AChE in vivo, some of them are toxic after metabolisation. We found AChE based assay approachable for assay of DFP, paraoxons, and dichlorvos. These are oxoforms of organophosphorus pesticides. From thioforms of assayed pesticides, only fenthion was able significantly inhibit AChE in vitro. Electrochemical biosensor with AChE attached on platinum electrode was used for confirmation of interaction pesticide – AChE and complex stability estimation. DFP, paraoxons and dichlorvos were allowed to interact with AChE in biosensor. These pesticides were settled firmly in AChE active site as no spontaneous recovery of AChE activity was observed.     

A preliminary study on electrochemical biosensors for the determination of total cholinesterase inhibitors in strawberries

Organophosphorous (OP) insecticides reveal acute toxicity because of their capability to affect the nervous system through the inhibition of acetyl cholinesterase function in regulating the neurotransmitter acetylcholine. The present work shows an example of an easy to be handled inhibition electrochemical biosensor, based on thick film technology for low cost production of screen printed electrodes. Anti-cholinesterase activity in specific fruits was determined measuring the inhibition of acetyl cholinesterase enzyme owing to the presence of OP pesticides. Paraoxon was taken as reference pesticide for each measurement. The main fluidic critical parameters were investigated under flow injection analysis, through the comparison of different enzymatic immobilisation methods. Analytical features were evaluated as a function of experimental parameters. The analytical detection was developed in a three step procedure and the pesticides content was measured in strawberries samples taken from the local market. The separation between the acetyl cholinesterase inhibition and the electrochemical detection with the choline oxidase biosensor decreases the total analysis time, allowing improvements in reproducibility and stability of the system. A comparison with reference materials and standard analytical procedures for pesticides will be required in the future for evaluating the reliability of the method.     

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Pesticide toxicity assessment using an electrochemical biosensor with <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and a bioluminescence inhibition assay with <Emphasis Type="Italic">Vibrio fischeri</Emphasis>

Two different toxicity tests, an electrochemical biosensor Cellsense and a bioluminescence inhibition assay ToxAlert were performed in order to establish and compare the acute toxicity responses of different types of raw and spiked water for a selected group of pesticides. The selected compounds were endosulfan, chlorfenvinphos, dimethoate, fenamiphos, ametryn, deltamethrin and alpha-cypermethrin; all of them are used in large quantities for agricultural purposes. In the first step, the study of the toxicity responses for each individual pesticide with Milli-Q water was carried out. Next, the toxic responses of different mixtures of these pesticides in different water matrices, i.e., Milli-Q water, surface water, groundwater and wastewater were studied in order to evaluate (i) device advantages and limitations for the toxicity evaluation of real environmental samples, (ii) antagonistic or synergistic effects and (iii) the influence of the water matrices. The survey of pesticides in real samples was carried out using a combined method involving both chemical analysis and toxicity bioassays. Chemical analysis involved the use of solid-phase micro-extraction (SPME) followed by gas chromatography with electron capture detection (GC/ECD) or thermoionic specific detection (GC/TSD) with mass spectrometric confirmation (GC/MS).     

Development of an EnFET for the detection of organophosphorous and carbamate insecticides

A biosensor for the detection of insecticides based on an ion-sensitive field-effect transistor (ISFET) was developed. The resulting device combines the simplicity of potentiometric sensors and the use of associated electronic systems as powerful tools for the acquisition and the processing of data. The enzyme acetylcholinesterase (AChE) was entrapped in a membrane placed on the gate of the ISFET forming an enzyme field-effect transistor (EnFET). The biosensor is applied to the determination of pesticides in spiked real samples. Organophosphorous and carbamate insecticides were measured with a detection limit of 10(-8) mol L(-1). The measurement is based on the production of hydrogen ions due to the hydrolysis of acetylthiocholine by the enzyme. The resulting local pH change is picked up by the underlying pH-sensitive ISFET and transduced as potential variations. The preparation of the membrane is simple and reproducible. The analysis in spiked real samples was performed in tap water and showed detection limits comparable to those obtained by other researchers.     

Amperometric screen-printed biosensor arrays with co-immobilised oxidoreductases and cholinesterases

Amperometric screen-printed biosensor arrays for detection of pesticides (organophosphates and carbamates) and phenols have been developed. Cholinesterases (AChE and BChE), tyrosinase (TYR), peroxidases (SBP, soybean and HRP, horseradish) and cellobiose dehydrogenase (CDH) were combined on the same array consisting of one Ag/AgCl reference electrode surrounded by eight radially distributed working electrodes of either carbon or platinum. Mainly cross-linking with glutaraldehyde was employed for enzyme immobilisation. The substrates for the enzymes were acetylthiocholine for cholinesterases (ChEs), cellobiose for CDH and hydrogen peroxide for peroxidases. Hydrogen peroxide was generated in the presence of glucose by co-immobilised glucose oxidase (GOx). All measurements were performed in an electrochemical steady state system specially constructed for eight channel screen-printed electrode arrays. The achieved relative standard deviation values calculated for different enzyme substrates (10 measurements) were typically below 7% and one assay was completed within less than 10 min. The detection limits for pesticides and phenols were in the nanomolar and micromolar ranges, respectively. The developed biosensor array was evaluated on wastewater samples. To simplify interpretation of results, the measured data were treated with multivariate analysis-principal component analysis (PCA).     

The mitochondria from beef heart as biosensors for the selective monitoring of phenols

We propose a simple and rapid procedure which allows for the selective monitoring in solution of toxic compounds which behave as uncouplers of the oxidative phosphorylation. Since all phenols, are uncouplers of the oxidative phosphorylation, the procedure allows for the selective monitoring in solution of phenols even in presence of other toxic compounds. The biological sensor are the mitochondria from beef heart. This biosensor is easily available without a stabular and therefore the biosensor and the whole procedure is very simple and not expensive. By linear regression analysis, it results that the procedure well predicts the response of the standard fish method to phenols. Therefore the procedure can be utilized as prescreening analysis for the monitoring the phenols in aqueous samples.     

Development of a stable biosensor based on a SiO2 nanosheet–Nafion–modified glassy carbon electrode for sensitive detection of pesticides

SiO₂ nanosheets (SNS) have been prepared by a chemical method using montmorillonite as raw material and were characterized by scanning electron microscopy and X-ray diffraction. SiO₂ nanosheet–Nafion nanocomposites with excellent conductivity, catalytic activity, and biocompatibility provided an extremely hydrophilic surface for biomolecule adhesion. Chitosan was used as a cross-linker to immobilize acetylcholinesterase (AChE), and Nafion was used as a protective membrane to efficiently improve the stability of the AChE biosensor. The AChE biosensor showed favorable affinity for acetylthiocholine chloride and catalyzed the hydrolysis of acetylthiocholine chloride with an apparent Michaelis–Menten constant of 134 μM to form thiocholine, which was then oxidized to produce a detectable and fast response. Based on the inhibition by pesticides of the enzymatic activity of AChE, detection of the amperometric response from thiocholine on the biosensor is a simple and effective way to biomonitor exposure to pesticides. Under optimum conditions, the biosensor detected methyl parathion, chlorpyrifos, and carbofuran at concentrations ranging from 1.0?×?10^?12 to 1?×?10^?10?M and from 1.0?×?10^?10 to 1?×?10^?8?M. The detection limits for methyl parathion, chlorpyrifos, and carbofuran were 5?×?10^?13?M. The biosensor developed exhibited good sensitivity, stability, reproducibility, and low cost, thus providing a new promising tool for analysis of enzyme inhibitors.

Chemometric interpretation of pesticide occurence in soil samples from an intensive horticulture area in north Portugal

An extensive monitoring programme of pesticides was carried out in soil samples from an intensive horticulture area in north of Portugal, putting into practice the needs for increased control of soil quality as far as organic pollution is concerned. The area under investigation was additionally defined as vulnerable to nitrates due to local soil and aquifer characteristics, which might be extended to pesticides contamination. Five sampling sites were selected and soils analysed at three depths in eight sampling campaigns, for the period of 2 years. A stepwise multivariate statistical approach was selected to uncover most relevant patterns inside a complex environmental data matrix. Cluster analysis was applied both to group pesticides and samples, giving a primary and unsupervised overlook of privileged relationships. Clusters of persistent pesticides and selected herbicides were identified, whereas sample classes were also formed and disposed geographically. Thirty eight percent of analysed soils samples fell into one class characterized by low contamination (class 1 in cluster analysis), which is entirely representative of the sampling site no. 1. Afterwards, linear discriminant analysis was applied to identify those pesticides, which had a higher impact in the definition of classes. Finally, factor analysis using a five component model was implemented in order to bring to light the constitution and data variance explained by each of the five main principal components, as well as, their relation to pest management practices. A factor was identified (PC1 – 22% variance) composed of chlorinated pesticides, which was representative of one of the investigated sites indicating its high contamination status. Qualitative main findings and class average concentration values were obtained through this multivariate statistical approach.     

Development of a biosensor for caffeine

We have utilized a microbe, which can degrade caffeine to develop an Amperometric biosensor for determination of caffeine in solutions. Whole cells of Pseudomonas alcaligenes MTCC 5264 having the capability to degrade caffeine were immobilized on a cellophane membrane with a molecular weight cut off (MWCO) of 3000-6000 by covalent crosslinking method using glutaraledhyde as the bifunctional crosslinking agent and gelatin as the protein based stabilizing agent (PBSA). The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg mL⁻¹. With read-times as short as 3 min, this caffeine biosensor acts as a rapid analysis system for caffeine in solutions. Interestingly, successful isolation and immobilization of caffeine degrading bacteria for the analysis of caffeine described here was enabled by a novel selection strategy that incorporated isolation of caffeine degrading bacteria capable of utilizing caffeine as the sole source of carbon and nitrogen from soils and induction of caffeine degrading capacity in bacteria for the development of the biosensor. This biosensor is highly specific for caffeine and response to interfering compounds such as theophylline, theobromine, paraxanthine, other methyl xanthines and sugars was found to be negligible.Although a few biosensing methods for caffeine are reported, they have limitations in application for commercial samples. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. The optimum pH and temperature of measurement were 6.8 and 30 ± 2 °C, respectively. Interference in analysis of caffeine due to different substrates was observed but was not considerable. Caffeine content of commercial samples of instant tea and coffee was analyzed by the biosensor and the results compared well with HPLC analysis.     

Determination of pesticides in surface and ground waters by liquid chromatography-electrospray-tandem mass spectrometry

The sensitive multiresidual analytical method for simultaneous analysis of 14 most commonly used agricultural pesticides in Serbia was developed and optimized. The selected insecticides, fungicides and herbicides belong to seven chemical classes (organophosphates, neonicotinoids, carbamates, diacylhydrazines, benzimidazoles, triazines and phenylureas). The method was based on solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. The following parameters that may affect the SPE procedure efficiency were optimized: the sorbent type in combination with different elution solvents, the sample pH and the sample volume. For each pesticide, MSⁿ analysis was performed and distinctive ions and transitions were selected for identification and quantification, as well as for confirmation purposes. External matrix-matched calibration method was used to eliminate variable matrix effect and ensure precise quantification. Good recoveries (72-129%), and low limits of detection (0.4-5.5 ng L⁻¹) and quantification (1.1-18.2 ng L⁻¹) were achieved for all selected pesticides. The developed and optimized method was successfully applied in the analysis of several river waters, as well as ground waters in Serbia, influenced by agriculture. The most frequently detected pesticide was carbendazim. Dimethoate, carbofuran and propazine were also found in the investigated samples.     

Simultaneous multi-residue pesticide analysis in soil samples with ultra-high-performance liquid chromatography–tandem mass spectrometry using QuEChERS and pressurised liquid extraction methods

To assess soil contamination, it is important to be able to measure different classes of pesticides simultaneously. For this reason we developed a sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of 25 pesticides in soil samples. Multi-class pesticides (triazines, phenylureas, phenoxy acid pesticides etc.) were analysed using a single mass spectrometry method with a fast polarity switching option, allowing the analysis of 19 compounds in the positive ionisation mode and six compounds in the negative ionisation mode. Extraction of pesticides from soil samples was performed employing a pressurised liquid extraction (PLE) and a quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure, recently developed for the extraction of multi-residue pesticides from food matrices. The extraction efficiency, performance and recoveries of these two procedures were evaluated and compared. In addition, we studied the effect of matrix on signal suppression or enhancement. Isotope-labelled internal standards (ILIS) were used to compensate the suppression or enhancement of signal intensities in the extracted samples. The method was validated using reference soil material (EUROSOIL 7) spiked with 50 μg/kg of each pesticide. The average recovery by PLE varied between 65.1% and 122.2% with RSDs of 1.7–23.4%. QuEChERS provided better recoveries for most of the pesticides, the extraction recovery ranging from 79.4% to 113.3% with RSDs of 1.0–12.2%. Limits of quantification for all target compounds were within a range of 0.1–2.9 µg/kg.     

Biosensor for direct determination of fenitrothion and EPN using recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode

A whole cell-based amperometric biosensor for highly selective, sensitive, rapid, and cost-effective determination of the organophosphate pesticides fenitrothion and ethyl p-nitrophenol thiobenzenephosphonate (EPN) is discussed. The biosensor comprised genetically engineered p-nitrophenol (PNP)-degrading bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorous hydrolase (OPH) on its cell surface as biological sensing element and carbon paste electrode as the amperometric transducer. Surface-expressed OPH catalyzed the hydrolysis of organophosphorous pesticides such as fenitrothion and EPN to release PNP and 3-methyl-4-nitrophenol, respectively, which were subsequently degraded by the enzymatic machinery of P. putida JS444 through electrochemically active intermediates to the TCA cycle. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. Operating at optimum conditions, 0.086 mg dry wt of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citratephosphate buffer, pH 7.5, with 50 μM CoCl₂ at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN. There was no interference from phenolic compounds, carbamate pesticides, triazine herbicides, or organophosphate pesticides without nitrophenyl substituent. The service life of the biosensor and the applicability to lake water were also demonstrated.     

Analysis of the potato glycoalkaloids by using of enzyme biosensor based on pH-ISFETs

The applicability of an enzyme biosensor based on pH-ISFETs for direct determination of total glycoalkaloids content in real potato samples, without any pre-treatment, is shown. The results of determination of the total glycoalkaloids concentrations in potato samples from different experimental varieties obtained by the biosensor are well correlated with the analogous data obtained by the HPLC method with standard complex sample pre-treatment procedure. The detection of total glycoalkaloids content by biosensors is reproducible, the relative standard deviation was around 3%. The dependence of total glycoalkaloids content on various parts of the potato tuber and their size, different growing area has been shown using the biosensor developed.The method based on biosensors is cheap, easy to operate and requires a shorter analysis time than the one needed using traditional methods for glycoalkaloids determination. The biosensor can operate directly on potato juice, or even directly on a suspension of potato or plant material. It can provide a way to save time and costs, with a possibility of taking rapid assessment of total glycoalkaloids content in a wide variety of potato cultivars. Furthermore the operational and storage stability of this biosensor are quite good with a drift lower than 1% per day and response being stable for more than 3 months.     

Immobilised tyrosinase-based biosensor for the detection of tea polyphenols

An amperometric principle-based biosensor containing immobilized enzyme tyrosinase has been used for detection of polyphenols in tea. The immobilized tyrosinase-based biosensor could detect tea polyphenols in the concentration range 10–80 mmol L⁻¹. Immobilization of the enzyme by the crosslinking method gave good stable response to tea polyphenols. The biosensor response reached the steady state within 5 min. The voltage response was found to have a direct linear relationship with the concentration of polyphenols in black tea samples. Enzyme membrane fouling was observed with number of analyses with a single immobilised enzyme membrane. The tyrosinase-based biosensor gave maximum response to tea polyphenols at 30°C. The optimum pH was 7.0. This biosensor system can be applied for analysis of tea polyphenols. Variation in the biosensor response to black tea infusions gave an indication of the different amounts of theaflavins in the samples, which is an important parameter in evaluating tea quality. A comparative study of the quality attributes of a variety of commercially available brands of tea were performed using the biosensor and conventional analytical techniques such as spectrophotometry.     

Elaboration of Urease Adsorption on Silicalite for Biosensor Creation

A possibility of efficient urease adsorption on silicalite for the purpose of biosensor creation was investigated. The procedure of urease adsorption on silicalite is notable for such advantages as simple and fast performance and non‐use of toxic or auxiliary compounds. Optimal conditions for modifying transducer surfaces with silicalite and subsequent urease adsorption on these surfaces were selected. The working parameters of the created biosensor were optimized. The developed biosensor with adsorbed urease was characterized by good intra‐reproducibility (RSD – 4.5 %), improved inter‐reproducibility (RSD of urea determination is 9 %) and operational stability (less than 10 % loss of activity after 10 days). Besides, the developed method for enzyme adsorption on silicalite was compared with the traditional methods of urease immobilization in biosensorics. Working conditions of the produced biosensor (pH and ionic strength) were shown to be close to those of the biosensor based on urease immobilized in GA vapor. For these reasons, it was concluded that the method of enzyme adsorption on silicalite is well‐suited for biosensor standardization aimed at its further manufacture.     

Determination of selected pesticides in environmental water by employing liquid-phase microextraction and liquid chromatography–tandem mass spectrometry

An optimised extraction and cleanup method for the analysis of pesticide in natural water samples is presented. Sixteen pesticides of different polarity and from the different chemical classes (organophosphates, triazines, benzimidazoles, carbamates, carbamides, neonicotinoides, methylureas, phenylureas and benzohydrazides), most frequently used in Serbia, were selected for the analysis. Liquid-phase microextraction in a single hollow fibre (HF-LPME) has been applied for sample preparation. The concentrations of pesticides were determined using HPLC-MS/MS method with electrospray ionisation. The extraction behaviour and selection of the experimental conditions was predicted based on log D and pK _a values of targeted pesticides, which were calculated applying the computer software ACD/Labs PhysChem Suite v12. The influence of the donor pH and concentration of pesticides, organic phase composition as well as the extraction time on the extraction efficiency was investigated. Optimum extraction conditions were evaluated with respect to the investigated parameters of the extraction. The extraction method was validated for 10 out of 16 studied pesticides. Linear range of the pesticides was 0.1–5 μg L⁻¹ with the correlation coefficient from 0.991 to 0.9998, and the relative standard deviation for three standard measurements was between 0.2 and 11.8%. The limits of detections ranged from 0.026 to 0.237 μg L⁻¹ and the limits of quantifications from 0.094 to 0.793 μg L⁻¹. The optimised two-phase HF-LPME method was successfully applied for determination of moderately polar as well low-polar pesticides in the environmental water samples.