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Methyl paraoxon (MP) is a highly toxic, efficient and broad-spectrum organophosphorus pesticide, which poses significant risks to ecological environment and human health. Many detection methods for MP are based on the enzyme catalytic or inhibition effect. But natural biological enzymes are relatively expensive and easy to be inactivated with a short service life. As a unique tool of nanotechnology with enzyme-like characteristics, nanozyme has attracted increasing concern. However, a large proportion of nanozymes lack the intrinsic specificity, becoming a main barrier of constraining their use in biochemical analysis. Here, we use a one-pot reverse microemulsion polymerization combine the gold nanoclusters (AuNCs) with molecularly imprinted polymers (MIPs), polydopamine (PDA) and hollow CeO 2 nanospheres to synthesize the bright red-orange fluorescence probe (CeO 2 @PDA@AuNCs-MIPs) with high phosphatase-like activity for selective detection of MP. The hollow structure possesses a specific surface area and porous matrix, which not only increases the exposure of active sites but also enhances the efficiency of mass and electron transport. Consequently, this structure significantly enhances the catalytic activity by reducing transport distances. The introduced MIPs provide the specific recognition sites for MP. And Ce (III) can excite aggregation induced emission of AuNCs and enhance the fluorescent signal. The absolute fluorescence quantum yield (FLQY) of CeO 2 @PDA@AuNCs-MIPs (1.41 %) was 12.8-fold higher than that of the GSH-AuNCs (0.11 %). With the presence of MP, Ce (IV)/Ce (III) species serve as the active sites to polarize and hydrolyze phosphate bonds to generate p-nitrophenol (p-NP), which can quench the fluorescent signal through the inner-filter effect. The as-prepared CeO 2 @PDA@AuNCs-MIPs nanozyme-based fluorescence method for MP detection displayed superior analytical performances with wide linearities range of 0.45-125 nM and the detection limit of 0.15 nM. Furthermore, the designed method offers satisfactory practical application ability. The developed method is simple and effective for the in-field detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In view of the current serious situation of organophosphorus pesticides (OPs) residue contamination, developing rapid and accurate OPs sensors is a matter of urgency. Redox-nanozyme based colorimetric sensors have been widely researched and utilized in OPs residue determination, but overcoming the interference of external redox substances and the effect of single-signal modes on detection performance is still a challenge. Here we fabricated a Zr-based metal-organic framework (MOF) featuring specific phosphatase-like activity and strong aggregation-induced emission (AIE) fluorescence for redox interference-free bimodal pesticide sensing. In the MOF, the activity-tunable Zr 4+ node offered high hydrolytic activity and affinity toward P-O containing substrates, and the rigid framework structure effectively enhanced the fluorescence emission of the ligand 1,1,2,2-tetra(4-carboxylphenyl)ethylene. The developed AIEzyme could efficiently catalyze the hydrolysis of paraoxon to yellow p-nitrophenol, which further reduced the intrinsic AIE fluorescence of AIEzyme through internal filtration effect. Thereby, a natural enzyme-free dual-mode colorimetric/fluorescence approach was established for paraoxon detection with no interference from redox substances, and a smartphone-assisted portable platform was further developed to enable the facile, rapid, and high-performance sensing of the pesticide in complex practical matrices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Herein, we developed a method coupling TLC and enzyme inhibition principles to rapidly detect OPs (dichlorvos, paraoxon and parathion). After the removal of the organic solvent from the samples using TLC and paper-based chips, the enzyme was added to the detection system. The results showed that the current method effectively reduced the effects of solvents on enzyme behavior. Moreover, the pigments could be successfully retained on TLC with 40% ddH 2 O/ACN solution (v/v) as a developing solvent. Additionally, the detection limits (LODs) were 0.002 µg/mL for dichlorvos, 0.006 µg/mL for paraoxon, and 0.003 µg/mL for parathion. Finally, the method was applied to spiked cabbage, cucumber, and spinach and showed good average recoveries ranging between 70.22% and 119.79%. These results showed that this paper-based chip had high sensitivity, precleaning, and elimination of organic solvent properties. Furthermore, it provides a valuable idea for sample pretreatment and rapid determination of pesticide residues in food., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Monitoring of pesticide residues in food and environmental matrices is undoubtedly crucial to guarantee food safety and ecological health, yet how to realize their sensitive and convenient detection is still challenging. Herein, we propose an all-in-one test strip that elaborately integrates bioenzyme, nanozyme and chromogen together, and achieve the highly sensitive and convenient sensing of pesticide residues assisted by a smartphone. A sequential self-assembly strategy was first explored to acquire an integrative bioenzyme-nanozyme-chromogen assembly, and then the assembly was confined in a biocompatible hydrogel to construct the test strip. Thanks to both the proximity and confinement effects, a ∼1.2-fold improvement of the cascade catalytic efficiency was gained to benefit high-sensitivity detection. More importantly, since all the sensing elements, including target recognition units and signal amplification modules, were rationally integrated in the test strip, detection operation was significantly simplified, making it possible for in-field rapid analysis. Besides, the microenvironment provided by the alginate hydrogel carrier endowed the test strip with an excellent sensing stability. By taking paraoxon as a typical pesticide, high-performance detection of the target was accomplished via the smartphone-assisted all-in-one test strip. Moreover, the test strip was successfully applied for paraoxon detection in various real samples and exhibited good correlations with commercial kits, demonstrating its great prospect for practical applications. Our work not only offers a new tool for the high-sensitivity and convenient monitoring of pesticide residues, but will also inspire the development of efficient multi-enzyme sensing platforms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Exogenous pollution of Chinese medicinal materials by pesticide residues and heavy metal ions has attracted great attention. Relying on the rapid development of nanotechnology and multidisciplinary fields, fluorescent techniques have been widely applied in contaminant detection and pollution monitoring due to their advantages of simple preparation, low cost, high throughput and others. Most importantly, synchronous detection of multi-targets has always been pursued as one of the major goals in the design of fluorescent probes. Herein, we firstly develop a simultaneous sensing method for methyl-paraoxon (MP) and Nickel ion (Ni, Ⅱ) by using carbon based fluorescent nanocomposite with ratiometric signal readout and nanozyme. Notably, the designed system showed excellent effectiveness even when the two pollutants co-exist. Under the optimum conditions, this method provides low limits of detection of 1.25 µM for methyl-paraoxon and 0.01 µM for Ni (Ⅱ). To further verify the reliability, recovery studies of these two analytes were performed on ginseng radix et rhizoma, nelumbinis semen, and water samples. In addition, smartphone-based visual analysis has been introduced to expand its applicability in point of care detection. This work not only expands the application of the dual-mode approach to pollutant detection, but also provides insights into the analysis of multiple pollutants in a single assay., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Butyrylcholinesterase (BChE) is an important indicator for clinical diagnosis of liver dysfunction, organophosphate toxicity, and poststroke dementia. Point-of-care testing (POCT) of BChE activity is still a challenge, which is a critical requirement for the modern clinical diagnose. A portable photothermal BChE assay is proposed through modulating the photothermal effects of Cu 2 O nanoparticles. BChE can catalyze the decomposition of butyrylcholine, producing thiocholine, which further reduce and coordinate with CuO on surface of Cu 2 O nanoparticle. This leads to higher efficiency of formation of Cu 9 S 8 nanoparticles, through the reaction between Cu 2 O nanoparticle and NaHS, together with the promotion of photothermal conversion efficiency from 3.1 to 59.0%, under the excitation of 1064 nm laser radiation. An excellent linear relationship between the temperature change and the logarithm of BChE concentration is obtained in the range 1.0 to 7.5 U/mL, with a limit of detection of 0.076 U/mL. In addition, the portable photothermal assay shows strong detection robustness, which endows the accurate detection of BChE in human serum, together with the screening and quantification of organophosphorus pesticides. Such a simple, sensitive, and robust assay shows great potential for the applications to clinical BChE detection and brings a new horizon for the development of temperature based POCT., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Herein we report a multiplated and biopolymeric-based optical bioassay for organophosphate detection based on the use of acetylcholinesterase (AChE) as biocomponent and biopolymeric electrospun fibrous mats as eco-designed supports for AChE immobilisation. The principle of the detection relays on the decrease of enzymatic activity due to the capability of the organophosphorus pesticides to irreversibly inhibit AChE, which is optically detected using Ellman colorimetric method. The proposed bioassay consists in a novel, cost-effective, and multiplex-based 96-well system, in combination with customised biopolymeric membranes modified with AChE, with the aim to deliver a sustainable analytical tool. Indeed, the designed set-up should provide and guarantee several advantages, including: i) the re-use of plastic multi-plate with the only replacement of polymer dishes in the case of inhibition absence; ii) the exploiting of the properties of the immobilised enzyme, i.e. multiple analysis using the same amount of enzyme, reducing the AChE amount for analysis. In detail, three different biopolymers (i.e. polylactic acid (PLA), polycaprolactone (PCL), and poly-hydroxybutyrate-co-hydroxyvalerate (PHBV)) were investigated and morphologically characterised, as supports for enzyme immobilisation, to identify the optimal one. Among them, PHBV was selected as the best support to immobilise AChE by cross-linking method. The analytical features of the bioassay were then assessed by measuring standard solutions of paraoxon in a range of concentrations between 10 and 100 ppb, achieving a linear range up to 60 ppb and a detection limit of 10 ppb. Thus, the suitability of this sustainable bioassay to detect organophosphate at ppb level was demonstrated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Surface-enhanced Raman spectroscopy (SERS) is gaining importance as an ultrasensitive analytical tool for routine high-throughput analysis of a variety of molecular compounds. One of the main challenges is the development of robust, reproducible and cost-effective SERS substrates. In this work, we study the SERS activity of 3D silver mirror-like micro-pyramid structures extended in the z-direction up to 3.7 μm (G0 type substrate) or 7.7 μm (G1 type substrate), prepared by Si-based microfabrication technologies, for trace detection of organophosphorous pesticides, using paraoxon-methyl as probe molecule. The average relative standard deviation (RSD) for the SERS intensity of the peak displayed at 1338 cm -1 recorded over a centimetre scale area of the substrate is below 13% for pesticide concentrations in the range 10 -6 to 10 -15  mol L -1 . This data underlies the spatial uniformity of the SERS response provided by the microfabrication approach. According to finite-difference time-domain (FDTD) simulations, such remarkable feature is mainly due to the contribution on electromagnetic field enhancement of edge plasmon polaritons (EPPs), propagating along the pyramid edges where the pesticide molecules are preferentially adsorbed. Graphical abstract.

In this study, a quartz crystal microbalance (QCM) based sensor was developed that selectively recognizes and binds the paraoxon molecule. For this purpose, poly (styrene-maleic anhydride) (PSMA) polymer was synthesized to obtain nanofiber. A 30% (wt/wt) PSMA/DMSO solution was prepared for use in the electrospinning process, with operating conditions of 5 kV potential and 1 mL/h flow rate. After obtaining the nanofibers, AChE enzyme was immobilized to nanofiber. The QCM gold electrode surface was coated with AChE immobilized nanofiber. For this purpose, the QCM electrode was first functionalized with 4-aminothiophenol. The quartz electrode coated with the recognition layer was sequentially studied with aqueous solutions containing 50% (v/v) methanol, ranging from 0.1 ppm to 10 ppm of paraoxon. When the electrode interacts with paraoxon, the frequency value decreases. The obtained data were converted to graphs and a calibration graph was obtained. The LOD value was found to be 4.57 × 10 -8 and the LOQ value was found to be 1.52 × 10 -7  M. These results show us that the developed method can analyze very small quantities of paraoxon samples. The Langmuir adsorption equation was used to study the interaction of the bond between the electrode surface and the paraoxon. For the calculation of the coupling constant Ka, Δm/C (g/M) versus Δm (g) was plotted on the graph. The Ka binding constant of the obtained graphic was found to be 5 × 10 7  M -1 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A flexible acetylcholinesterase (AChE) film biosensor, based on a AuNPs-MoS 2 -reduced graphene oxide/polyimide flexible film (rGO/PI) electrode, has been synthesized for paraoxon detection. In this study, the rGO/PI film acts as the flexible substrate and AuNPs are reduced by monolayer MoS 2 under illumination. Transmission electron microscopy revealed that AuNPs are uniformly dispersed on the MoS 2 -rGO/PI electrode surface with a diameter ~10nm. X-ray photoelectron spectroscopy indicated that a strong binding force exists between reduced AuNPs and monolayer MoS 2 . The AChE modified AuNPs-MoS 2 -rGO/PI flexible film biosensor is used to hydrolyze acetylcholine chloride and obtain a large current response at 0.49V by differential pulse voltammetry, demonstrating successful immobilization of AChE. In view of the inhibition of paraoxon on the AChE, under optimal conditions, the AChE/AuNPs-MoS 2 -rGO/PI film biosensor shows a linear response over a concentration range 0.005-0.150μg/mL, a sensitivity of 4.44 uA/μg/mL, a detection limit of 0.0014μg/mL, acceptable reproducibility and stability to paraoxon. The flexible film biosensor has also proved used for detection of paraoxon in real samples., (Copyright © 2018. Published by Elsevier B.V.)

We propose a new capillary electrophoresis (CE)-based open-tubular immobilized enzyme microreactor (OT-IMER) and its application in acetylcholinesterase (AChE) assays. The IMER is fabricated at the capillary inlet (reactor length of ∼1 cm) with the inner surface modified by a micropore-structured layer (thickness of ∼220 nm, pore size of ∼15-20 nm). The use of IMER accomplishes the enzymatic reaction and separation/detection of the products in the same capillary within 3 min. The feasibility of the proposed method is evaluated via online analysis of the activity and inhibition of AChE enzymes. Such method exhibits good reproducibility with relative standard deviation (RSD) of less than 4% for 20 runs, and the enzyme remains over 82% of the initial activity after usage of 7 days. The IMERs are successfully applied to detect the organophosphorus pesticide, paraoxon, in three types of vegetable juice samples with a limit of detection of as low as 61 ng mL -1 . Results show that the spiked samples are in the range of 89.6-105.9% with RSD less than 2.7%, thereby indicating its satisfactory level of accurate and reliable analysis of real samples by using the proposed method. Our study indicates that, with combination of advantages of both porous-layer capillary and CE OT-IMER, the proposed method is capable to enhance enzymatic reactions and to achieve rapid analysis with simple instrumentation and operation, thus would pave the way for extensive application of CE-based IMERs in a variety of bioanalysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A practical electrochemical biosensor with high sensitivity was developed for detecting organophosphorus (OP). Initially, Ce metal was introduced into an UiO-66-template to form Ce/UiO-66. Later, graphene oxide (GO), carbon black (CB) and multi-walled carbon nanotubes (MWCNTs) were separately added to Ce/UiO-66 to compare the effect of different carbon-based material types on the performance of the biosensor. Exclusively, Ce/UiO-66/MWCNTs with a Ce (7%) and MWCNT (30%) matrix was found to not only load more acetylcholinesterase (AChE) onto vacant sites but also increase electron transfer and decrease the number of diffusion pathways between the thiocholine and electrode surface. Moreover, the appropriate oxophilicity of Ce coupled with the high surface area and good conductivity of MWCNTs in the UiO-66 structure revealed a high affinity to acetylthiocholine chloride (ATCl) and possible catalysis of the hydrolysis of ATCl with a Michaelis-Menten constant of 0.258 mM. This biosensor, under optimal conditions, demonstrated a rapid and sensitive detection of paraoxon over a wide linear range of 0.01-150 nM, with a low detection limit of 0.004 nM. As a result, the AChE/Ce/UiO-66/MWCNTs/GCE biosensor can be employed in laboratory and field experiments to determine paraoxon levels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel dual-mode analytical method by employing nanozyme was developed for the detection of organophosphorus pesticides (OPP) for the first time. The detection principle is that the pesticide could be hydrolyzed to para-nitrophenol (p-NP) in the presence of nanoceria as nanozyme. p-NP exhibits the bright yellow color, and its color intensity has a positive correlation with the pesticide concentration. Meanwhile, the characteristic absorption peak at 400 nm of p-NP increases gradually with the raised concentration of pesticide. Therefore, a dual-mode method including smartphone-based colorimetric and spectroscopic strategies was rationally developed. Herein, methyl-paraoxon was selected as the representative compound. Under the optimum conditions, the detection limits of both two strategies were calculated to be 0.42 μmol L -1 . Finally, the present method was successfully applied in three edible medicinal plants (Semen nelumbinis, Semen Armeniacae Amarum, Rhizoma Dioscoreae). The present work offers a reliable and convenient approach for routine detection of pesticide based on two different detection mechanisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Accurate analysis of organophosphate pesticides (OPs) with portable devices remain an elusive goal that have received widespread investigative attention in the areas of environmental contamination and disease prevention. Herein, using all-in-one enzyme-inorganic hybrid nanoflowers (ACC-HNFs) to fabricate high-performance artificial enzyme cascade system, we established a sensitive and affordable lab-on-paper biosensor. This biosensor incorporated disposable screen-printed carbon electrode (SPCE) and colorimetric test strips, which enabled the dual-modal readout (electrochemical and colorimetric signal) for on-site monitoring of OPs, achieving an "on-demand" tuning of the detection performance. Using paraoxon as a model analyte, the ACC-HNFs-based lab-on-paper platform could reach a limit of detection down to the femtogram/mL level (6 fg mL -1 ). Meticulous design of ACC-HNFs provided a versatile approach for constructing artificial enzyme as a recognizer and amplifier to fill the gap in constructing robust artificial enzyme systems which can be used for on-site contamination monitoring and biological diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Use of immobilized acetylcholine esterase (AChE) for detecting organophosphorus pesticides in water sources and body fluids can bring down the detection costs dramatically. In the present study, AChE was directly doped on multiwalled carbon nanotube (MWCNT) surface modified with carboxylic groups through amide bond and used for organophosphorus pesticide detection. Amide bond formation between MWCNTs and the enzyme molecules avoid use of any intermediate membranes, cross-linkers or binding materials. This strategy overcomes the hindrance to electron transfer posed by membranes or cross-linkers and increases the sensitivity of detection. MWCNTs carrying carboxyl groups were deposited on glassy carbon electrode and were subsequently immobilized with AChE. The activity of AChE was monitored by cyclic voltammetry after immobilization. Scanning electron microscopy and atomic force microscopy were used to characterize the electrode surface. FT-IR spectra were taken to characterize enzyme-MWCNT complex. Under optimized parameters, the electrode showed linear range between 10 and 50 nM, which is promising for detection of trace amounts of the pesticide. The lower and higher detection limits of the sensor are 0.1 nM and 500 nM respectively. The stability and reusability of the electrode were determined. Finally, successful detection of organophosphorus compounds in real samples established it as reliable for sensor applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biosensing applications require signal reporters to be sufficiently stable and biosafe as well as highly efficient. Aggregation-induced emission (AIE) nanoparticles have proven to be capable of cell-imaging and cancer therapy; however, realizing sensitive detection of biomolecules remains a great challenge because of their instability, biotoxicity, and lack of modifiable functional groups. Herein, we report a self-assembling strategy to fabricate AIE nanoparticles (PTDNPs) through the dispersion of amphiphilic polymers (PTDs) in phosphate-buffered saline. The PTDs were prepared through radical copolymerization of N -(1,2,2-triphenylvinyl)-4-acetylaniline and dimethyl diallyl ammonium chloride. We found that the particle size, morphology, functional groups, and fluorescence property of PTDNPs can be fine-tuned. Further, PTDNPs-0.10 were chosen as signal reporters to detect organophosphorus pesticides (OPs) with the aid of gold nanoparticles. Their sensing performance on OPs is superior to that using C-dot/quantum dot/rhodamine B as the signal reporter. This study not only provides new possibilities to fabricate novel AIE nanoparticles with exceptional properties, but also facilitates the AIE nanoparticle's application for target analyte biosensing.

Owing to their large specific surface, favorable electrical conductivity, and excellent electrocatalytic capabilities, two-dimensional transition metal dichalcogenides have received considerable attention in the field of biosensors. On the basis of these properties, we developed a portable and disposable enzyme-based biosensor for paraoxon detection using a metallic MoS 2 nanosheets modified screen-printed electrode (SPE). The exfoliated ultrathin metallic MoS 2 nanosheets can accelerate the electron transfer on electrode surface and contribute to the immobilization of acetylcholinesterase (AChE) via the cross-linking of glutaraldehyde. Electrodeposited gold nanoparticles (AuNPs) on SPE were used to immobilize MoS 2 nanosheets through the interaction between Au atoms on AuNPs and S atoms on MoS 2 . Using acetylcholine as the substrate, AChE can catalyze the formation of electroactive thiocholine and further generate the redox current. In the presence of paraoxon, the activity of AChE can be inhibited, making the related electrochemical signals weaken. Under the optimized conditions, this electrochemical biosensor exhibited a favorable linear relationship with the concentration of paraoxon from 1.0 to 1000 μg L -1 , with the detection limit of 0.013 μg L -1 . Furthermore, this developed biosensor was successfully applied to detect paraoxon in pretreated apple and pakchoi samples, which can provide a reliable method for the rapid analysis of organophosphorus pesticides in agricultural products.

A transparent, lateral-flow test strip coupled with a smartphone-based ambient light sensor was first proposed for detecting enzymatic inhibition and phosphorylation. The principle of the platform is based on the simultaneous measurement of the total amount of the enzyme and enzyme activity to biomonitor exposure to organophosphorus (OP) pesticides. In this study, butyrylcholinesterase (BChE) was adopted as the model enzyme and ethyl paraoxon was chosen as an analyte representing OP pesticides. The total amount of BChE was quantified by a sensitive colorimetric signal originating from a sandwich immunochromatographic assay utilizing PtPd nanoparticles as a colorimetric probe, which exhibited excellent catalytic activity for phenols. In the sandwich immunoassay, only one antibody against BChE was simultaneously utilized as the recognition antibody and the labeling antibody due to the tetrameric structure of native BChE. The BChE activity was estimated by another colorimetric signal using the Ellman assay. Both colorimetric signals on two separated test strips were detected by the smartphone-based ambient light sensor. The proposed sensor operated with an LED in a 3D-printed substrate, which emitted excitation light and transmitted it through a transparent, lateral-flow test strip. With the increase in the colorimetric signal in the test line of the test strip, the intensity of the transmitted light decreased. The smartphone-based sensor showed excellent linear responses for assaying the total amount of BChE and active BChE ranging from 0.05 to 6.4 nM and from 0.1 to 6.4 nM, respectively. A high portability and low detection limit were simultaneously realized in the common smartphone-based device. This low-cost, portable, easy-operation, and sensitive immunoassay strategy shows great potential for online detection of OP exposure and monitoring other disease biomarkers.

A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with "egg-box" structure is triggered inside a capillary with releasing of Ca 2+ by changing the pH of the sol solution, which is accomplished in situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for online CE assays with immobilized enzymes, which can be widely applied in bioanalysis.

The aim of this study was to determine optimal conditions for in vitro skin decontamination using water and detergents as decontamination agents and to test the cleansing efficiency of selected detergents. Experiments were performed using a peristaltic pump for showering of pig skin in modified static diffusion cells. Several conditions were tested including different flow rates (from 5 to 33 ml s -1 ), quantity of rinsing fluid (from 40 to 400 ml) and concentration of detergents (2; 5; 10%). Further, several types of detergents/commercial decontamination agents were evaluated under the selected conditions to find the most effective means of decontamination. The amount of paraoxon removed from the skin surface following wet-type decontamination was detected in the rinsing fluid spectrophotometrically after hydrolysis of paraoxon - a model contaminant. The efficacy of rinsing by water/Spolapon AES 253 increased with flow rate up to 25 ml s -1 and a rinsing volume of 200 ml. Lutensol AT 25 achieved maximum efficacy at the lowest tested concentration (2%). A flow rate of 16 ml s -1 , rinsing volume of 100 ml (values from the middle part of the sigmoid curve) and 5% concentration of decontaminant solution were used for further evaluation of detergents as cleansing agents under the selected conditions. Cetylpyridinium bromide (cationic surfactant), carbethopendecinii bromidum (cationic surfactant) and polyoxyethylene-10-tridecyl ether (non-ionic surfactant), SDS (anionic surfactant), althosan MB (cationic surfactant), sodium dodecylbenzene sulphonate (anionic surfactant), neodekont (mixture), tergitol NPX (non-ionic surfactant), Korynt P (non-ionic surfactant) were found to be the most effective. These decontaminants were able to wash away more than 92% of paraoxon from the contaminated skin.

Carbon dots (CDs) combined with a nanomaterial-based quencher has created an innovative way for designing promising sensors. Herein, a novel fluorescent-sensing platform was designed for sensitive detection of organophosphorus pesticides (OPs). The preparation of CDs was based on one-step hydrothermal reaction of 3-aminobenzeneboronic acid. The fluorescence of CDs can be quenched by manganese dioxide (MnO 2 ) nanosheets via the Förster resonance energy transfer (FRET). In the presence of butyrylcholinesterase (BChE) and acetylthiocholine, the enzymatic hydrolysate (thiocholine) can efficiently trigger the decomposition of MnO 2 nanosheets, resulting in the recovery of CDs fluorescence. OPs as inhibitors for BChE activity can prevent the generation of thiocholine and decomposition of MnO 2 nanosheets, accompanying the fluorescence "turn-off" of the system. So the BChE-ATCh-MnO 2 -CDs system can be utilized to detect OPs quantitatively based on the fluorescence turn "on-off". Under the optimum conditions, the present FRET-based approach can detect paraoxon ranging from 0.05 to 5 ng mL -1 with a detection limit of 0.015 ng mL -1 . Meanwhile, the present strategy also showed a visual color change in a concentration-dependent manner. Thus, the proposed assay can potentially be a candidate for OPs detection.

Electrode materials play a key role in the development of electrochemical sensors, particularly enzyme-based biosensors. Here, a novel NiCo 2 S 4 with reticulated hollow spheres assembled from rod-like structures was prepared by a one-pot solvothermal method and its formation mechanism was discussed. Moreover, comparison of NiCo 2 S 4 materials from different experiment conditions as biosensors was investigated by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV), and the best one that was reticulated hollow spheres assembled from rod-like structures NiCo 2 S 4 has been successfully employed as a matrix of AChE immobilization for the special structure, superior conductivity and rich reaction active sites. When using common two kinds of organophosphate pesticides (OPs) as model analyte, the biosensors demonstrated a wide linear range of 1.0×10 -12 -1.0×10 -8 gmL -1 with the detection limit of 4.2×10 -13 gmL -1 for methyl parathion, and 1.0×10 -13 -1.0×10 -10 gmL -1 with the detection limit of 3.5×10 -14 gmL -1 for paraoxon, respectively. The proposed biosensors exhibited many advantages such as acceptable stability and low cost, providing a promising tool for analysis of OPs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, a new colorimetric biosensor for the assay of paraoxon was developed via the conventional iodine-starch color reaction and multi-enzyme cascade catalytic reactions. In the presence of acetylcholine chloride, acetylcholinesterase (AChE) and choline oxidase (ChO) catalyzed the formation of H 2 O 2 , which then activated horseradish peroxidase (HRP) to catalyze the oxidation of KI to produce an iodine-starch color reaction. Upon exposure to paraoxon, the catalytic activity of AChE was inhibited and less H 2 O 2 generated, resulting in a decrease in the production of I 2 and a drop in the intensity of solution color. This colorimetric biosensor showed high sensitivity for the assay of paraoxon with a limit of detection 4.7 ppb and was applied for the assay of paraoxon in spiked real samples. By employing the conventional iodine-starch color reaction, this biosensor has the potential of on-site assay of OPs residues in environmental samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Assays for organophosphorus pesticides (OPs) with high sensitivity as well as on-site screening have been urgently required to protect ecosystem and prevent disease. Herein, a novel fluorimetric sensing platform was constructed for quantitative detection of OPs via tyrosinase (TYR) enzyme-controlled quenching of gold nanoclusters (AuNCs). One-step green synthetic approach was developed for the synthesis of AuNCs by using chicken egg white (CEW) as template and stabilizer. Initially, TYR can catalyze the oxidation of dopamine to dopaminechrome, which can efficiently quench the fluorescence intensity of AuNCs at 630nm based on dynamic quenching process. However, with the presence of OPs, the activity of TYR was inhibited, resulting in the fluorescence recovery of AuNCs. This proposed fluorescence platform was demonstrated to enable rapid detection for OPs (paraoxon as model) and to provide excellent sensitivity with a detection limit of 0.1ngmL -1 . Significantly, the fluorescence probe was used to prepare paper-based test strips for visual detection of OPs, which validated the excellent potential for real-time and on-site application., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Organophosphorus compounds (OP), which mainly penetrate via the percutaneous pathway, represent a threat for both military and civilians. Body surface decontamination is vital to prevent victims poisoning. The development of a cost-effective formulation, which could be efficient and easy to handle in case of mass contamination, is therefore crucial. Metal oxides nanoparticles, due their large surface areas and the large amount of highly reactive sites, present high reactivity towards OP. First, this study aimed at evaluating the reaction of CeO 2 nanoparticles, synthetized by microwave path and calcined at 500 or 600 °C, with Paraoxon (POX) in aqueous solution. Results showed that both nanoparticles degraded 60%-70% of POX. CeO 2 calcined at 500 °C, owing to its larger specific area, was the most effective. Moreover, the degradation was significantly increased under Ultra-Violet irradiation (initial degradation rate doubled). Then, skin decontamination was studied in vitro using the Franz cell method with pig-ear skin samples. CeO 2 powder and an aqueous suspension of CeO 2 (CeO 2 -W) were applied 1 h after POX exposure. The efficiency of decontamination, including removal and/or degradation of POX, was compared to Fuller's earth (FE) and RSDL lotion which are, currently, the most efficient systems for skin decontamination. CeO 2 -W and RSDL were the most efficient to remove POX from the skin surface and decrease skin absorption by 6.4 compared to the control not decontaminated. FE reduced significantly (twice) the absorbed fraction of POX, contrarily to CeO 2 powder. Considering only the degradation rate of POX, the products ranged in the order CeO 2  > RSDL > CeO 2 -W > FE (no degradation). This study showed that CeO 2 nanoparticles are a promising material for skin decontamination of OP if formulated as a dispersion able to remove POX like CeO 2 -W and to degrade it as CeO 2 powder., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

A biocompatible nanocomposite including bovine serum albumin (BSA) template Cu nanoclusters (CuNCs@BSA) and single-walled carbon nanotubes (SWCNT) was synthesized to fabricate a highly sensitive electrochemical biosensor for paraoxon as a model of organophosphates. The UV-vis, fluorescence and Fourier transform infrared (FTIR) demonstrated that BSA entrapped in the nanocomposite film have been changed in its secondary structure so that it provided an enzyme like activity attributing to the high electrical conductivity of the entrapped copper nanoclusters. Also, the morphology and structure of prepared nanocomposites were investigated by transmission electronic microscopy (TEM) and scanning electron microscopy (SEM). In the prepared nanocomposite, the CuNCs@BSA found to play as a conductive holder as well as an accumulator of redox active centers on the surface of the electrode, and SWCNT improves the electrocatalytic activity along with conductivity of glassy carbon electrode (GCE) surface. The fabricated biosensor exhibited excellent sensitivity, acceptable stability, fast response, and high electrocatalytic activity toward the reduction of paraoxon. The reduction peak current vs paraoxon concentration was linear over the range 50nM to 0.5μM and 0.5-35μM, with a limit of detection of 12.8nM. Notable electrocatalytic properties of the developed electrode toward paraoxon indicated that the nanocomposite possesses a promising potential to fabricate the third generation enzyme-free electrochemical biosensors, bioelectronics and state-of-the-art biomedical devices in the future., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the present study, a novel colorimetric sensing platform was constructed for quantitative detection of acetylcholinesterase (AChE) activity and its inhibitor. Manganese dioxide (MnO 2 ) nanosheets as an oxidase-mimicking nanomaterial could directly oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into oxTMB without the need for horseradish peroxidase and H 2 O 2 . When AChE was introduced, acetylthiocholine could be catalytically hydrolyzed to produce thiocholine, which easily triggers the decomposition of MnO 2 nanosheets, causing the decrease of solution absorbance. Owing to the inhibition effect of organophosphorus pesticides, the enzymatic activity was suppressed, preventing the decomposition of MnO 2 and resulting in the increase of absorbance. Under optimal conditions, the colorimetric platform shows sensitive responses to AChE and paraoxon in the range of 0.1-15 mU mL -1 and 0.001-0.1 μg mL -1 , respectively. The detection limits of AChE and paraoxon reached 35 μU mL -1 and 1.0 ng mL -1 , respectively. Furthermore, the MnO 2 -TMB platform has been used to fabricate test strips for rapid and convenient visual detection of AChE and its inhibitor with highly promising performance.

It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 μg L(-1), and a low detection limit equal to 0.05 μg L(-1) (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels. Graphical Abstract Biosensors based on screen-printed electrodes modified with Acetylcholinesterase, Carbon Black, and Chitosan for organophosphorus pesticide detection.

Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates that could be used in designing enzyme-based sensors for detecting OP compounds. In this work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme-chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well, to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate, leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased Km value by more than 50%, and increased Vmax and Kcat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2-12) and temperature (25-90°C) was also achieved. The method worked in the 0 to 1050 nM concentration ranges, and had a detection limit as low as 5 × 10(-11) M.

A sensitive amperometric acetylcholinesterase (AChE) biosensor, based on gold nanorods (AuNRs), was developed for the detection of organophosphate pesticide. Compared with Au@Ag heterogeneous NRs, AuNRs exhibited excellent electrocatalytic properties, which can electrocatalytically oxidize thiocholine, the hydrolysate of acetylthiocholine chloride (ATCl) by AChE at +0.55V (vs. SCE). The AChE/AuNRs/GCE biosensor was fabricated on basis of the inhibition of AChE activity by organophosphate pesticide. The biosensor could detect paraoxon in the linear range from 1nM to 5μM and dimethoate in the linear range from 5nM to 1μM, respectively. The detection limits of paraoxon and dimethoate were 0.7nM and 3.9nM, which were lower than the reported AChE biosensor. The proposed biosensor could restore to over 95% of its original current, which demonstrated the good reactivation. Moreover, the biosensor can be applicable to real water sample measurement. Thus, the biosensor exhibited low applied potential, high sensitivity and good stability, providing a promising tool for analysis of pesticides., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A nano-silver electrode immobilizing acetylcholinesterase (AChE) for the detection of organophosphorus (OPPs) pesticides is reported. Scanning electron microscopy (SEM) was used to characterize the surface structure of two kinds of electrodes fabricated with different sizes of silver powders and the interface between chitosan layer and nano-silver powder layer. Cyclic voltammetry was carried out to characterize the response of silver/chitosan electrode in the absence and in the presence of thiocholine (TCh). It was also used to evaluate the insulativity of the chitosan layer. An amperometric method was performed to measure the response of the electrode to TCh, which is the product of the enzymatic reaction for detecting organophosphorus pesticides indirectly. Although there are many kinds of nanoparticles, silver was chosen for its internal advantage in detecting TCh at low potential without further modification. The result shows nano-silver powder has better performance than usual silver powder, and the limit of detection of paraoxon is 4 ppb under optimized conditions. One percent (w/v) chitosan solution was used as binder for the immobilization of nano-silver powder and AChE, which made it possible for independent electrode fabrication at room temperature, whereas 3% (w/v) chitosan solution was used as insulating compound for controlling the electrode area. Unlike traditional organic insulating ink, chitosan is safe and environmentally friendly, and it is used as insulating material for the first time. The flexible nano-silver/AChE/chitosan electrode was evaluated in Chinese chives and cabbage, and the recoveries of standard addition were 105.11 and 96.41%, respectively. Owing to the antibacterial property of nano-silver and the biocompatibility, safety, and biodegradability of chitosan, the proposed method is safe, facile, environmentally friendly, and has great potential in organophosphorus pesticide detection for food safety. Graphical Abstract Current response of nano-silver electrode (a) and silver electrode (b) to thiocholine in 0.02 M PBS + KCl at 0.15 V; addition of thiocholine (0.09 mM) every 50 s (↓); inset: calibration curve of nano-silver (▲) and silver (◆) electrode.

Solvent tolerance of immobilized enzymes is important for many biosensing and biotechnological applications. In this paper we report an acetylcholinesterase (AChE) biosensor based on chitosan that exhibits high solvent resistance and enables sensitive detection of pesticides in presence of a high content of organic solvents. The solvent effect was established comparatively for the enzyme immobilized in chitosan and covalently cross-linked with glutaraldehyde. The activity of the immobilized AChE was dependent on the immobilization method and solvent type. The enzyme entrapped in chitosan fully conserved its activity in up to 25% methanol, 15% acetonitrile and 100% cyclohexane while the enzyme cross-linked with glutaraldehyde gradually lost its activity starting at 5% acetonitrile and methanol, and showed variable levels in cyclohexane. The detection limits of the biosensor for paraoxon were: 7.5 nM in 25% methanol, 100 nM in 15% acetonitrile and 2.5 μM in 100% cyclohexane. This study demonstrates that chitosan provides an excellent immobilization environment for AChE biosensors designed to operate in environments containing high amounts of organic solvents. It also highlights the effect of the immobilization material and solvent type on enzyme stability. These findings can enable future selection of the immobilization matrix and solvent type for the development of organic phase enzyme based systems., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

New acetylcholinesterase (AChE) biosensor based on unsubstituted pillar[5]arene (P[5]A) as electron mediator was developed and successfully used for highly sensitive detection of organophosphate and carbamate pesticides. The AChE from electric eel was immobilized by carbodiimide binding on carbon black (CB) placed on glassy carbon electrode. The working potential of 200mV was obtained in chronoamperometric mode with the measurement time of 180 s providing best inter-biosensors precision of the results. The AChE biosensor developed made it possible to detect 1×10(-11)-1×10(-6) M of malaoxon, 1×10(-8)-7×10(-6) M of methyl-paraoxon, 1×10(-10)-2×10(-6) M of carbofuran and 7×10(-9)-1×10(-5) M of aldicarb with 10 min incubation. The limits of detection were 4×10(-12), 5×10(-9), 2×10(-11) and 6×10(-10) M, respectively. The AChE biosensor was tested in the analysis of pesticide residuals in spiked samples of peanut and beetroot. The protecting effect of P[5]A derivative bearing quaternary ammonia groups on malaoxon inhibition was shown., (Copyright © 2015 Elsevier B.V. All rights reserved.)

For the sensitive and convenient detection of pesticides, several sensing methods and materials have been widely explored. However, it is still a challenge to obtain sensitive, simple detection techniques for pesticides. Here, the simple and sensitive Time-resolved SERS mapping (T-SERS) and Spatial-resolved SERS mapping (S-SERS) are presented for detection of pesticides by using Au@Ag NPs as SERS substrate. The Time-resolved SERS mapping (T-SERS) is based on state translation nanoparticles from the wet state to the dry state to realize SERS measurements. During the SERS measurement, adhesive force drives the particles closer together and then average interparticle gap becomes smaller. Following, air then begins to intersperse into the liquid network and the particles are held together by adhesive forces at the solid-liquid-air interface. In the late stage of water evaporation, all particles are uniformly distributed. Thus, so called hotspots matrix that can hold hotspots between every two adjacent particles in efficient space with minimal polydispersity of particle size are achieved, accompanying the red-shift of surface plasmon peak and appearance of an optimal SPR resonated sharply with excitation wavelength. Here, we found that the T-SERS method exhibits the detection limits of 1-2 orders of magnitude higher than that of S-SERS. On the other hand, the T-SERS is very simple method with high detection sensitivity, better reproducibility (RSD=10.8%) and is beneficial to construction of a calibration curve in comparison with that of Spatial-resolved SERS mapping (S-SERS). Most importantly, as a result of its remarkable sensitivity, T-SERS mapping strategies have been applied to detection of several pesticides and the detect limit can down to 1nM for paraoxon, 0.5nM for sumithion. In short, T-SERS mapping measurement promises to open a market for SERS practical detection with prominent advantages., (Copyright © 2015. Published by Elsevier B.V.)

This work introduced an efficient immobilization of acetylcholinesterase (AChE) onto amino functionalized carbon nanotubes (CNT-NH2), in order to fabricate high sensitive and practical organophosphorus pesticide (OPs) biosensors. Compared with the pristine, -COOH and -OH decorated CNTs, there were larger amount of enzymes adsorbed on the surface of CNT-NH2 with a favorable orientation and the best amperometric response was obtained on the AChE/CNT-NH2/GC electrode. Furthermore, the biosensor modified with CNT-NH2 showed a high affinity to acetylthiocholine chloride (ATCh) and could catalyze the hydrolysis of ATCh with an apparent Michaelis-Menten constant (Km) value of 67.4 µM. Using paraoxon as a model compound, wide linear ranges from 0.2 nM to 1 nM and 1 nM to 30 nM, and a low detection limit of 0.08 nM were obtained with satisfactory reproducibility and stability. Moreover, the biosensor had also been successfully employed for the determination of low concentrations of pesticides in real vegetable samples. This method could be extended to other functionalized nano-materials for their application in constructing biosensors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

Rapid detection of organophosphorous (OP) compounds such as paraoxon would allow taking immediate decision on efficient decontamination procedures and could prevent further damage and potential casualties. In the present study, a biosensor based on nanomagnet-silica core-shell conjugated to organophosphorous hydrolase (OPH) enzyme was designed for detection of paraoxon. Coumarin1, a competitive inhibitor of the OPH enzyme, was used as a fluorescence-generating molecule. Upon excitation of cumarin1 located at the active site of the enzyme, i.e., OPH, the emitted radiations were intensified due to the mirroring effect of the nanomagnet-silica core-shell conjugated to the enzyme. In presence of paraoxon and consequent competition with the fluorophore in occupying enzyme's active site, a significant reduction in emitted radiations was observed. This reduction was proportional to paraoxon concentration in the sample. The method worked in the 10- to 250-nM concentration range had a low standard deviation (with a coefficient of variation (CV) of 6-10%), and the detection limit was as low as 5 × 10(-6) μM.

A sensitive photoelectrochemical (PEC) biosensor for detection of organophosphorus pesticides (OPs) using the nanocomposite of CdSe@ZnS quantum dots (QDs) and graphene deposited on the ITO coated glass electrode as a photoactive electrode is presented. The integration of CdSe@ZnS/graphene nanocomposite with biomolecules acetylcholinesterase (AChE) as a biorecognition element yields a novel biosensing platform. Under visible light irradiation, the AChE-CdSe@ZnS/graphene nanocomposite can generate a stable photocurrent and the photocurrent is found to be inversely dependent on the concentration of OPs. Under the optimal experimental conditions, the photocurrents were proportional to the logarithm of paraoxon and dichlorvos within the concentration range of 10(-12)-10(-6) M. The detection limits (LOD) of the proposed biosensor for paraoxon and dichlorvos are as low as 10(-14) M and 10(-12) M. The photoelectrochemical biosensor shows good sensitivity, reproducibility, stability, and could be successfully applied to detection of OPs in real fruit samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Many important considerations in the design of practical Surface enhanced Raman spectroscopy (SERS) substrates are necessary, such as the low cost, simple preparation, mass production and high efficiency of sample collection, which the conventional rigid substrates are lack of. In this work, practical SERS substrates based on deposition of silver nanoparticles (Ag NPs) on commercially available low-cost filter paper were prepared by simple silver mirror reaction in a large scale, and utilized for rapid, portable and accurate identification and detection of pesticide residues at various peels. Compared with conventional substrates, this novel SERS substrate dramatically enhanced the sample collection efficiency by simply swabbing paper-based device across different surfaces without destroying the sample, meanwhile avoiding the substrate signal of real-world samples. Considering their low cost, portability, simplicity and high sample collection efficiency, Ag NP-decorated filter paper, as practical SERS substrate, are used in solving critical problems for detection of pesticide residues at various peels. SERS experiments were carried out on Ag NP-decorated filter paper combined with 'dynamic SERS' (D-SERS) due to its high detection sensitivity. The excellent detection performance of the Ag NP-based filter paper was demonstrated by detection thiram and paraoxon residues at various peels. Besides, the stability and reproducibility of the practical substrates were also involved., (Copyright © 2014 Elsevier B.V. All rights reserved.)

New acetylcholinesterase (AChE) sensor based on Ag nanoparticles decorated with macrocyclic ligand has been developed and successfully used for highly sensitive detection of organophosphate and carbamate pesticides. AChE was immobilized by carbodiimide binding on carbon black (CB) layer deposited on a glassy carbon electrode. The addition of Ag nanoparticles decreased the working potential of the biosensor from 350 to 50 mV. The AChE sensor made it possible to detect 0.4 nM-0.2 μM of malaoxon, 0.2 nM-0.2 μM of paraoxon, 0.2 nM-2.0 μM of carbofuran and 10 nM-0.20 μM of aldicarb (limits of detection 0.1, 0.05, 0.1 and 10 nM, respectively) with 10 min incubation. The AChE sensor was tested for the detection of residual amounts of pesticides in spiked samples of peanut and grape juice. The protecting effect of new macrocyclic compounds bearing quaternary ammonia fragments was shown on the example of malaoxon inhibition., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report the use of Polystyrene/Ag (PS/Ag) nanoparticles as dynamic surface-enhanced Raman spectroscopy (dynamic-SERS) substrates for sensitive detection of low levels of organophosphorus pesticides. The PS particles clearly observed using Raman microscopy provide the masterplate for in situ growth of Ag NPs, leading to multiple active sites for SERS measurements. Besides obtaining the fingerprints of target molecules and recording time-resolved Raman spectra, this dynamic-SERS method can be used as an ultra-sensitive analytical technique which can enhance 1-2 orders of magnitude the signals of analytes in comparison to that of the traditional methods. On the other hand, importantly, it shows much better correlations between concentration and intensity than does the conventional SERS technique so that it can build the foundation for quantitative analysis of analytes. The as-prepared individual PS/Ag nanoparticle has been demonstrated for the sensitive detection of organophosphorus paraoxon and sumithion. SERS spectra are acquired at different concentrations of each pesticide and linear calibration curves are obtained by monitoring the strongest intensity value of bands arising from stronger stretching mode as a function of analyte concentration. The limits of detection and limits of quantitation are reported for two pesticides. The limit of detection for paraoxon is 96 nM (0.026 ppm) and for sumithion is 34 nM (0.011 ppm). The limits of quantitation are 152 nM (0.042 ppm) and 57 nM (0.016 ppm) for paraoxon and sumithion, respectively. It can be seen that these two organophosphorus pesticides can be detected in the low nM range based on this dynamic-SERS analytical method. Also, in the real sample experiments of paraoxon and sumithion, the results confirm that this dynamic-SERS technique would have potential applicability for quantitative analysis with slight interference., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Changes in ionic current flowing through nanopores due to binding or translocation of single biopolymer molecules enable their detection and characterization. It is, however, much more challenging to detect small molecules due to their rapid and small signal signature. Here we demonstrate the use of de novo designed peptides for functionalization of nanopores that enable the detection of a small analytes at the single molecule level. The detection relies on cooperative peptide conformational change that is induced by the binding of the small molecule to a receptor domain on the peptide. This change results in alteration of the nanopore effective diameter and hence induces current perturbation signal. On the basis of this approach, we demonstrate here the detection of diethyl 4-nitrophenyl phosphate (paraoxon), a poisonous organophosphate molecule. Paraoxon binding is induced by the incorporation of the catalytic triad of acetylcholine esterase in the hydrophilic domain of a short amphiphilic peptide and promotes β-sheet assembly of the peptide both in solution and for peptide molecules immobilized on solid surfaces. Nanopores coated with this peptide allowed the detection of paraoxon at the single molecule level revealing two binding arrangements. This unique approach, hence, provides the ability to study interactions of small molecules with the corresponding engineered receptors at the single molecule level. Furthermore, the suggested versatile platform may be used for the development of highly sensitive small analytes sensors.

In this work we demonstrate the efficient immobilization of histidine 6-tagged organophosphate hydrolase (His6-OPH), an organophosphate-degrading enzyme, on mesoporous titania thin films. This permits the use of the biocatalyst films as efficient tools in the detection/detoxification of paraoxon. His6-OPH was immobilized on mesoporous thin films with uniform (9 nm) and bimodal (13-38 nm) pore size distribution, through covalent attachment and physical adsorption. The biocatalyst films show good activity, and enhanced stability with respect to the free enzyme at extreme conditions of pH and temperature, especially around neutral pH and room temperature. In addition, the bioactive films can be easily separated from the reaction media and reused multiple times without significant loss of activity.

There is an increasing need to develop biosensors for the detection of harmful pesticide residues in food and water. Here, we report on a versatile strategy to synthesize functionalized graphene oxide nanomaterials with abundant affinity groups that can capture histidine (His)-tagged acetylcholinesterase (AChE) for the fabrication of paraoxon biosensors. Initially, exfoliated graphene oxide (GO) was functionalized by a diazonium reaction to introduce abundant carboxyl groups. Then, Nα,Nα-bis(carboxymethyl)-l-lysine hydrate (NTA-NH2) and Ni(2+) were anchored onto the GO based materials step by step. AChE was immobilized on the functionalized graphene oxide (FGO) through the specific binding between Ni-NTA and His-tag. A low anodic oxidation potential was observed due to an enhanced electrocatalytic activity and a large surface area brought about by the use of FGO. Furthermore, a sensitivity of 2.23 μA mM(-1) to the acetylthiocholine chloride (ATChCl) substrate was found for our composite covered electrodes. The electrodes also showed a wide linear response range from 10 μM to 1mM (R(2)=0.996), with an estimated detection limit of 3 μM based on an S/N=3. The stable chelation between Ni-NTA and His-tagged AChE endowed our electrodes with great short-term and long-term stability. In addition, a linear correlation was found between paraoxon concentration and the inhibition response of the electrodes to paraoxon, with a detection limit of 6.5×10(-10) M. This versatile strategy provides a platform to fabricate graphene oxide based nanomaterials for biosensor applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

p-Nitrophenyl organophosphates (OPs) including paraoxon, parathion and methyl parathion, etc, are highly poisonous OPs, for which sensitive and rapid detection method is most needed. In this work, an ultrasensitive electrochemical sensor for the determination of p-nitrophenyl OPs was developed based on ordered mesoporous carbons (OMCs) modified glassy carbon electrode (GCE) (OMCs/GCE). The electrochemical behavior and reaction mechanism of p-nitrophenyl OPs at OMCs/GCE was elaborated by taking paraoxon as an example. Experimental conditions such as buffer pH, preconcentration potential and time were optimized. By using differential pulse voltammetry, the current response of the sensor at -0.085 V was linear with concentration within 0.01-1.00 μM and 1.00-20 μM paraoxon. Similar linear ranges of 0.015-0.5 μM and 0.5-10 μM were found for parathion, and 0.01-0.5 μM and 0.5-10 μM for methyl parathion. The low limits of detection were evaluated to be 1.9nM for paraoxon, 3.4 nM for parathion and 2.1 nM for methyl parathion (S/N=3). Common interfering species had no interference to the detection of p-nitrophenyl OPs. The sensor can be applicable to real samples measurement. Therefore, a simple, sensitive, reproducible and cost-effective electrochemical sensor was proposed for the fast direct determination of trace p-nitrophenyl OPs at low potential without deoxygenization., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate-the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.

Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410 nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16 U/OD₆₀₀ using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55 °C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5-150 μM paraoxon, 1-200 μM parathion and 2.5-200 μM methyl parathion, and limits of detection were 0.2 μM, 0.4 μM and 1 μM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications., (Copyright © 2013 Elsevier Inc. All rights reserved.)