Emerging vistas on pesticides detection based on electrochemical biosensors - An update.

Organophosphates and carbamates pesticides are widely used to increase crop production globally causing a threat to human health and the environment. A variety of pesticides are applied during different stages of vegetable production. Therefore, monitoring the presence of pesticide residues in food and soil has great relevance to sensitive pesticide detection through distinct determination methods that are urgently required. Conventional techniques for the detection of pesticides have several limitations that can be overcome by the development of highly sensitive, fast, reliable and easy-to-use electrochemical biosensors. Herein, we describe the types of biosensors with the main focus on electrochemical biosensors fabricated for the detection of OPPs and carbamates pesticides. An overview of conventional techniques employed for pesticide detection is also discussed. This review aims to provide a glance of recently developed biosensors for some common pesticides like chlorpyrifos, malathion, parathion, paraoxon, and carbaryl which are present in food and environment samples.

A novel amperometric biosensor for rapid detection of ethanol utilizing gold nanoparticles and enzyme coupled PVC reaction cell.

This research was aimed at the fabrication of an improved enzyme-based amperometric biosensor for rapid quantification of ethanol. Alcohol oxidase (AOX) from Pichia pastoris was covalently immobilized on chemically treated polyvinylchloride (PVC) beaker and subsequently horseradish peroxidase (HRP), nafion (Nf), carboxylated multi-walled carbon nanotubes (c-MWCNTs), chitosan (CHIT) and gold nanoparticles (AuNPs) were immobilized onto Au electrode to fabricate a working electrode. The enzyme-coated PVC surface was analysed morphologically via scanning electron microscopy (SEM). At different stages of construction, the electrochemical properties of working electrode were deciphered by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor displayed optimal response in a short time span of 12 s at pH 7.5 and 35°C temperature. The working range exhibited by the proposed biosensor was 0.01-42 mM with a limit of detection (LOD) of 0.0001 µM and storage stability of 180 days at 4°C. When level of alcohol was evaluated in commercial samples via standard assay kit and existing biosensor, a good correlation (R2 = 0.98) was observed which authenticates its reliability.

Nano-interface driven electrochemical sensor for pesticides detection based on the acetylcholinesterase enzyme inhibition.

Forensic Science Laboratories usually receive numerous cases of suicidal, accidental, and homicidal poisoning most often involving organophosphorus (OP) pesticides. The toxicity of the OP pesticides arises due to their ability to inhibit the activity of acetylcholinesterase (AChE), a cholinergic enzyme that is essential for the proper functioning of the central nervous system. Conventional techniques which are currently in use for pesticide detection are time-consuming, need upskilled technicians as well as suffer from low sensitivity. Therefore, the more rapid and sensitive electrochemical biosensors based on the principle of AChE enzyme inhibition have emerged out to be a simple and promising alternative to the conventional techniques. Since, most of the time, the poison isolated from biological material in poisoning cases is in nM quantities, an attempt has been made for the development of biosensor with enhanced sensitivity in the nM range using reduced graphene oxide (rGO) and zinc oxide nanoflowers (ZnONFs). The rGO and ZnONFs were synthesized chemically and deposited electrochemically on the Au electrode. AChE was immobilized onto this prepared nano-interface (ZnONFs/rGO/Au) through chitosan and glutaraldehyde cross-linking. The fabricated sensor was characterized step by step with cyclic voltammogram and electrochemical impedance spectroscopy. This advanced nanomaterials based techniques has been explored for detecting pesticides in visceral samples. The limit of detection (LOD) for the present sensor was 0.01 nM for OP pesticides.

Novel approach using activated cellulose film for efficient immobilization of purified diamine oxidase to enhance enzyme performance and stability.

The presence of various contaminants in foodstuffs has led to serious public health concerns. Diamine oxidase (DAO) has attracted tremendous attention for guarding food safety as well as clinical and environmental industries. In this study, DAO from Pisum sativum (Pea) seedlings was extracted and purified by dialysis and gel filtration. Purified DAO was covalently immobilized onto the surface of nitrocellulose membrane using glutaraldehyde. The obtained bioaffinity support has efficiently shown high yield immobilization of DAO from pea seedlings. The optimal conditions of free and immobilized DAO activity were evaluated against the substrate, Putrescine dihydrochloride. The influence of pH, temperature, storage stability, and reusability of immobilized enzyme with comparison to the free enzyme was studied and the results showed that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 59% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized DAO carrier presents a new approach in large scale applications.

Enzymatic biosensors for the quantification of biogenic amines: a literature update.

The present review emphasizes on the quantification of biogenic amines (BAs) which are regarded as a quality indicator of food freshness or spoilage and for evaluating microbial action while food processing. BAs have various potential adverse effects on human health and they are widely found in varying concentrations in different food stuffs. In the quest for a reliable method for their precise detection, BA biosensors have emerged as an efficient tool which enables rapid and accurate assessment in miniature form. Various combinations of amine oxidase enzymes have been used for the fabrication of biosensors in order to enhance specific biorecognition and signal transduction. This article also summarizes the widely employed components used in the construction of a pertinent biosensor and the research results conducted previously. The meticulous description regarding the choice of transducers and the significant role of mediators in a high response biosensor has been reviewed. Moreover, it also encompasses the utilization of highly attractive electrolytic characteristics of nanoparticles to enhance the specificity and accuracy of BA biosensors.

Enzyme-Based Ultrasensitive Electrochemical Biosensors for Rapid Assessment of Nitrite Toxicity: Recent Advances and Perspectives.

In the present era of rapid international globalization and industrialization, intensive use of nitrite as a fertilizing agent in agriculture, preservative, dyeing agent, food additive and as corrosion inhibitor in industrial sectors is adversely effecting environment, natural habitats and human health. The issue of toxicity and carcinogenicity due to excessive ingestion of nitrites via the dietary intake has led to an imminent need for its efficient real-time monitoring in situ. Nitrite detection employing electrochemical biosensors has been gaining high credibility in the field of clinical research. Nitrite biosensors have emerged as an outstanding choice for portable point of care testing of nitrite quantification owing to the excellent properties, such as rapidity, miniaturization, ultra-low limits of detection, multiplexing and enhanced detection sensitivity. The article is enclosed with an interesting outlook on latest emerging trends in the development of nitrite biosensors utilizing nanomaterials, such as metal nanoparticles, carbon nanotubes, metal oxide nanoparticles, nanocomposites, polymers and biomaterials. The present review embarks on the highlights relevant to the nitrite quantification in real samples, then proceeds with a meticulous description of the most pertinent electrochemical nitrite biosensors, which have been proposed by adopting diverse materials and strategies of fabrication and finally end with the achievements and future outlook signifying the application of these nanoengineered biosensors for environmental surveillance and human safety.

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol.

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8 s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM-50 mM and 0.0001 µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190 days) at 4°C. The sensitivity of the sensor was found to be 155 µA mM-1 cm-2. A good correlation (R2 = 0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.

Recent trends and perspectives in enzyme based biosensor development for the screening of triglycerides: a comprehensive review.

Clinical manifestations of the elevated plasma triacylglycerol (TG) include a greater prevalence of atherosclerotic heart disease, acute pancreatitis, diabetes mellitus, hypertension, and ischemic vascular disease. Hence, these significant health troubles have attracted scientific attention for the precise detection of TG in biological samples. Numerous techniques have been employed to quantify TG over many decades, but biosensors hold the leading position owing to their superior traits such as highly specific recognition for target molecules, accuracy, minituarization, small sample requirement and rapid response. Enzyme-based electrochemical biosensors represent an instantaneous resolution for the foremost bottlenecks constraining laboratory prototypes to reach real time bedside applications. We highlight the choice of transducers and constructive strategies to design high-performance biosensor for the quantification of triglycerides in sera and early diagnosis of health problems related to it. In the present review, a small effort has been made to emphasize the significant role of enzymes, nanostructured metal oxides, graphene, conducting polypyrrole, nanoparticles, porous silicon, EISCAP and ENFET in enabling TG biosensors more proficient and taking a revolutionary step forward.

Identification of coding sequence and its use for functional and structural characterization of catalase from Phyllanthus emblica.

Catalase is an essential antioxidant enzyme that is well characterized from microbial and animal sources. The structure of plant catalase is unknown. Therefore, it is of interest to understand the functional and structural characteristics of catalase from an Indian gooseberry, Phyllanthus emblica (or Emblica officinalis). Hence, catalase from P. emblica was cloned in pUC18 plasmid, sequenced and submitted to GenBank with the accession numbers "MF979112" and "ATO98311.1". InterProScan showed that the coding sequence is monofunctional and haem-dependent catalase-like superfamily. Multiple sequence alignment (MSA) followed by phylogenetic analysis showed that the P. emblica catalase groups with soybean catalase. We further report the characteristics of structural model of the enzyme for functional characterization.

Purification and Analytical Application of Vigna mungo Chitinase for Determination of Total Fungal Load of Stored Cereals.

A novel chitinase from urd bean (Vigna mungo) seeds was purified up to homogeneity and optimized with respect to its optimum working conditions of pH, temperature, and substrate concentration. Overall, 145-fold purification with 70% yield of the purified chitinase was achieved. The notable features of the purified enzyme were its appreciative substrate affinity as well as catalytic efficiency, high thermo stability (70% retention of initial activity at 70 °C after 60 min of continuous exposure), and pretty good storage stability (half-life of 45 days at 5 °C). The enzyme was used for determination of total chitin contents of the stored cereals that in the absence of any insect infestation, were considered to be directly proportional to the total fungal load of the tested samples. The method was linear up to 7.0 mM with 0.04 mM as the limit of detection. Percent recoveries of added chitin were < 90.0% and within-day and between-day coefficients of variations were > 3.0% for all the samples. Chitin values in stored cereal samples obtained by the present method and the popular DNS method showed good correlation, the value for coefficient of determination (R2) being < 0.98. Overall, the method yielded acceptable sensitivity, reproducibility, and accuracy. Graphical Abstract ᅟ.

Improved protein determination assays obtained after substitution of copper sulfate by copper oxide nanoparticles.

Copper oxide nanoparticles (nano CuO) provide Cu2+ ions which can be easily harnessed for protein determination as an alternative to the use of copper sulfate (CuSO4). In the present work, nano CuO of size <25 nm were substituted for CuSO4 in two of the well-known protein assays viz. Lowry method and bicinchoninic acid (BCA) method. Use of nano CuO in the Lowry's assay had no effect on the assay time (30 min) but significantly lowered the limit of detection (LOD) from 0.01 to 0.001 µg/ml, while the BCA method when performed using nano CuO resulted in notable reduction of not only the assay time from 30 to 20 min but also the LOD from 0.1 to 0.001 µg/ml. Nano CuO based protein determination in the human serum and urd bean seeds extract produced reliable, reproducible and consistent results. Nano CuO also alleviated the inhibition of both the methods by common interfering substances such as ammonium sulfate, glucose, EDTA, SDS, Triton X-100, dithiothreitol and 2-mercaptoethanol. Hence, successful modification and improvement of Lowry and BCA methods by substitution of CuSO4 with nano CuO for protein determination has been demonstrated.

A novel polyurethane/nano ZnO matrix for immobilization of chitinolytic enzymes and optical sensing of chitin.

Purified chitinase from Vigna mungo and N-acetyl ß glucosaminidase (NAGase) from Canavalia ensiformis were immobilized on to the novel polyurethane (PU)/zinc oxide nanoparticles (nano ZnO) composite matrix with a conjugation yield of 0.785±0.01mg/cm2 and 96.19±0.85% retention of specific activity. Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) also confirmed the presence of nano ZnO and enzymes on the PU support. Thus synthesized PU/nano ZnO/chitinase/NAGase conjugates were optimized with respect to pH, temperature and substrate concentration and successfully employed for development of an absorbance based optical biosensor for chitin determination in stored wheat grains. The limit of detection was 0.01mM with linearity from 0.1 to 10.0mM. The% recoveries of added chitin (0.1 and 0.2mM) were >95.0% and >96.5% respectively and within-day and between-day coefficients of variations were 1.03% and 1.78% respectively. The method showed good correlation (R2=0.996) with the popular 3,5-dinitrosalicylic acid method. PU/nano ZnO bound chitinase/NAGase showed good thermal and storage stabilities and could be reused 10 times without any appreciable loss of activity.

Enzymes loaded chitosan/coconut fibre/zinc oxide nanoparticles strip for polyamine determination.

Most often, the immobilized enzyme based quantification is an attractive alternative to other chromatographic, electrochemical and mass spectrometry based methods due to its specificity and simplicity. In the present study, polyamine oxidase specific for spermine and spermidine and diamine oxidase specific for putrescine, were co-immobilized onto a novel chitosan/coconut fibre/zinc oxide nanoparticles (CS/CF/nZnO) hybrid support to yield a polyamine sensing strip. The strip worked optimally at pH 7.0, temperature 25°C and 6min of incubation time. Pretty good values for kinetic constants Kmapp (6.60mM), Vmax (17.69µmol/min mg protein) and kcatapp (1987.64s-1) as well as for thermal (<50 % activity retained at 40°C), storage (half life-40days) and operational stabilities (<90 % activity retained after 20 reuses) were obtained. The strip was employed for polyamine determination in some of the locally grown fruit and vegetables and the results were found to be comparable, reliable and reproducible.

Recent approaches to ameliorate selectivity and sensitivity of enzyme based cholesterol biosensors: a review.

The healthcare area is often reluctant to execute new technology unless they are proven to be safe, constructive and secure. Eventually, an aspiration stands for providing point-of-care testing service to allow a better estimation of the biochemical levels of a patient that entails an insistent remedial action. With increasing mortality rate due to cardiovascular diseases (CVDs) in present scenario, it has become the need of hour to develop more advance methods for their diagnosis, so that it can be determined at sensitive levels and can be prevented from being fatal. Elevated level of cholesterol in blood stream is one of the utmost risk factors which lead to CVDs. Discernible from the vast research in this field, worth of cholesterol biosensors is already recognized and flourished in the clinical analysis of brain and cardiac vascular diseases. It necessitates unremitting progress in the development of biosensing technology towards fabrication, miniaturization and multiplexing ability of cholesterol quantification devices so that they can endow with lab-on-chip-analysis systems to the medical field. Different strategies have been meticulously explored for the engineering of cholesterol biosensors utilizing nanocomposites, conducting polymers, nanotubes and nanoparticles. Foremost, this article reviews the contemporary evolution in cholesterol biosensors, which encompass various strategies for immobilization of enzymes and roles of various matrices and artificial mediators used for the biosensor fabrication. Still there remains an enormous challenge to congregate the demands of performance and yield in a cost effective manner for its application in successful treatments of CVDs.

Alcohol quantification: recent insights into amperometric enzyme biosensors.

Biosensors are the switching channels that make sense. The biosensors have found an empirical role in health applications (e.g. clinical diagnostics) as they represent the technological counterpart of living senses. On a global scale, alcohol analysis is indispensable for criminal justice systems, monitoring medical conditions of HIV patients & pregnant women as well as public safety issues regarding pilots, metro drivers, doctors etc. For addressing the clinical and toxicological problems, much advancement in the improvement of biosensors have been witnessed in recent decades. Currently, electrochemical biosensors dominate the field which harnesses the synergistic action of enzymes and nanomaterials for the analysis of ethanol. The enzymatic biosensors are the most explored biosensing devices among all the types of biosensors, and employment of nanomaterials has paved a way to the further improvements in this gem of a discovery. The relative comparison to precise the alcohol biosensors has been aptly discussed in the review on the basis of several analytical properties including fabrication, linearity, sensitivity, response time, detection limit as well as storage stability. Finally, the recent trends and emerging future prospects of alcohol biosensors have been reviewed.

Bilirubin enzyme biosensor: potentiality and recent advances towards clinical bioanalysis.

Bilirubin detection plays a major role in healthcare. Its high concentration in human serum is lethal and must be determined accurately. Clinically, it is vital for assessing patients with deleterious health conditions such as jaundice or icterus, hepatitis, mental disorders, cerebral palsy and brain damage especially in the case of neonates. In evaluating the drawbacks regarding the conventional methodology of bilirubin detection, there is need for a superior analytical tool. Bilirubin oxidase (BOx)-based sensors have been designed for the ultrasensitive analysis of bilirubin and quality deliverance of treatment and this review highlights the different mechanisms of bilirubin detection using different modified electrodes. Further, it also addresses the exploitation of highly attractive electrocatalytic properties of elite nanoparticles such as gold and zirconia- coated silica nanoparticles in enhancing the reproducibility and specificity of bilirubin biosensors.

Increasing the efficiency of immobilization and chitin determination using copper oxide nanoparticles.

A novel polyurethane (PU) support with and without copper oxide nanoparticles (nCuO) was employed for immobilization of two chitinolytic enzymes, chitinase and N-acetyl ß glucosaminidase (NAGase) to yield PU/nCuO/chitinase/NAGase and PU/chitinase/NAGase conjugates respectively. The surface morphologies, topologies and bonded interactions between different components of the immobilized enzyme conjugates were characterized using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Fourier Transform Infrared Spectroscopy (FTIR) respectively. High conjugation yield of 0.811±0.10mg/cm2 with 97.66±0.50% retention of specific activity of enzymes on PU/nCuO support vis-a-vis a conjugation yield of 0.531±0.50mg/cm2 with 75.23±0.60% retention activity on PU support was achieved. Additionally, increased pH and temperature tolerance, better kinetic parameters and enhanced stabilities of PU/nCuO/chitinase/NAGase conjugates established their superiority over the PU/chitinase/NAGase conjugates. However, both the conjugates were successfully employed for the determination of chitin contents in the apparently healthy stored rice grains. The methods were tested on analytical parameters such as linearity, limit of detection, the coefficient of variation and reproducibility of the externally added chitin, which were again found to be superior for PU/nCuO/chitinase/NAGase conjugates.

Effect of changing the nanoscale environment on activity and stability of nitrate reductase.

Nitrate reductase (NR) is employed for fabrication of nitrate sensing devices in which the enzyme in immobilized form is used to catalyze the conversion of nitrate to nitrite in the presence of a suitable cofactor. So far, instability of immobilized NR due to the use of inappropriate immobilization matrices has limited the practical applications of these devices. Present study is an attempt to improve the kinetic properties and stability of NR using nanoscale iron oxide (nFe3O4) and zinc oxide (nZnO) particles. The desired nanoparticles were synthesized, surface functionalized, characterized and affixed onto the epoxy resin to yield two nanocomposite supports (epoxy/nFe3O4 and epoxy/nZnO) for immobilizing NR. Epoxy/nFe3O4 and epoxy/nZnO support could load as much as 35.8±0.01 and 33.20±0.01µg/cm(2) of NR with retention of about 93.72±0.50 and 84.81±0.80% of its initial activity respectively. Changes in surface morphology and chemical bonding structure of both the nanocomposite supports after addition of NR were confirmed by scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Optimum working conditions of pH, temperature and substrate concentration were ascertained for free as well as immobilized NR preparations. Further, storage stability at 4°C and thermal stability between 25-50°C were determined for all the NR preparations. Analytical applications of immobilized NR for determination of soil and water nitrates along with reusability data has been included to make sure the usefulness of the procedure.

Immobilization of nitrate reductase onto epoxy affixed silver nanoparticles for determination of soil nitrates.

Epoxy glued silver nanoparticles were used as immobilization support for nitrate reductase (NR). The resulting epoxy/AgNPs/NR conjugates were characterized at successive stages of fabrication by scanning electron microscopy and fourier transform infrared spectroscopy. The immobilized enzyme system exhibited reasonably high conjugation yield (37.6±0.01 µg/cm(2)), with 93.54±0.88% retention of specific activity. Most favorable working conditions of pH, temperature and substrate concentration were ascertained to optimize the performance of epoxy/AgNPs/NR conjugates for soil nitrate quantification. The analytical results for soil nitrate determination were consistent, reliable and reproducible. Minimum detection limit of the method was 0.05 mM with linearity from 0.1 to 11.0 mM. The % recoveries of added nitrates (0.1 and 0.2 mM) were<95.0% and within-day and between-day coefficients of variations were 0.556% and 1.63% respectively. The method showed good correlation (R(2)=0.998) with the popular Griess reaction method. Epoxy/AgNPs bound NR had a half-life of 18 days at 4 °C and retained 50% activity after 15 reuses.

A new immobilization and sensing platform for nitrate quantification.

Nitrate reductase (NR) from Aspergillus niger was covalently coupled to the epoxy affixed gold nanoparticles (epoxy/AuNPs) with a conjugation yield of 35.40±0.01 µg/cm(2) and 93.90±0.85% retention of specific activity. The bare and NR bound epoxy/AuNPs support was characterized using scanning electron microscopy and Fourier Transform Infrared Spectroscopy. The immobilized enzyme system was optimized with respect to pH, temperature and substrate concentrations and successfully employed for determination of nitrate contents in ground water. The minimum detection limit of the method was 0.05 mM with linearity from 0.1 to 10.0 mM. The % recoveries of added nitrates (0.1 and 0.2 mM) were >95.0% and within-day and between-day coefficients of variations were 1.012% and 3.125% respectively. The method showed good correlation (R(2)=0.998) with the popular Griess reaction method. Epoxy/AuNPs bound NR showed good thermal and storage stabilities and retained 50% activity after 16 reuses.