Reagent-less detection of a competitive inhibitor of immobilized acetylcholinesterase

The interaction of monosulfonate tetraphenyl porphine (TPPS(1)) with immobilized acetylcholinesterase (AChE) yields a characteristic absorbance peak at 446 nm. Addition of acetylcholine iodide or the competitive inhibitor tetracaine to the immobilized TPPS(1)-AChE complex results in a decrease in absorbance intensity at 446 nm due to displacement of the porphyrin from the active site. The loss in intensity at 446 nm is linearly dependent on tetracaine concentration at levels below 100 ppb. Tetracaine concentrations as low as 300 ppt have been detected.     

Interaction of monosulfonate tetraphenyl porphyrin, a competitive inhibitor, with acetylcholinesterase

Monosulfonate tetraphenyl porphyrin (TPPS(1)) forms a 1:1 complex with electric eel acetylcholinesterase (AChE) inducing a loss in TPPS(1) absorbance at 402 nm and the appearance of a new absorbance centered at 442 nm. In the presence of AChE, the fluorescence of TPPS(1) at 652 nm is slightly narrowed, with the maximal 652 nm fluorescence shifted from 407 to 412 nm excitation wavelength. The fluorescence peak of TPPS(1) at 712 nm shifts to 716 nm in the presence of AChE. TPPS(1) is a competitive inhibitor of AChE. The addition of acetylcholine iodide (AChI) or the competitive inhibitor tetracaine to the preformed AChE-TPPS(1) complex results in a loss of the 442 nm absorbance band as the porphyrin is displaced from AChE. The absorbance peak does not decrease in the presence of procaine, a non-competitive inhibitor.     

Optical solid-state detection of organophosphates using organophosphorus hydrolase

We have developed a sensor surface for optical detection of organophosphates based on reversible inhibition of organophosphorus hydrolase (OPH) by copper complexed meso-tri(4-sulfonato phenyl) mono(4-carboxy phenyl) porphyrin (CuC1TPP). OPH immobilized onto glass microscope slides retains catalytic activity for more than 232 days. CuC1TPP is a reversible, competitive inhibitor of OPH, binding at the active site of the immobilized enzyme. The absorbance spectrum of the porphyrin-enzyme complex is measured via planar waveguide evanescent wave absorbance spectroscopy using a blue LED as a light source and an Ocean Optics USB2000 as the spectrophotometer. The characteristics of the absorbance spectrum of CuC1TPP are specific and different when the porphyrin is bound to the enzyme or is bound non-specifically to the surface of the slide. Addition of a substrate of OPH such as one of the organophosphates paraoxon, coumaphos, diazinon, or malathion displaces the porphyrin from the enzyme resulting in reduced absorbance intensity at 412 nm. Absorbance changes at 412 nm show log-linear dependence on substrate concentration. Paraoxon concentrations between 7 parts per trillion (ppt) and 14 parts per million (ppm) were investigated and a 3:1 S/N detection limit of 7 ppt was determined. Concentrations of 700 ppt to 40 ppm were investigated for diazinon, malathion, and coumaphos with detection limits of 800 ppt, 1 part per billion, and 250 ppt, respectively. This optical technique does not require the addition of reagents or solutions other than the sample and absorbance spectra can be collected in less than 6 s.     

Spectrophotometric detection of pentachlorophenol (PCP) in water using immobilized and water-soluble porphyrins

The spectrophotometric properties of porphyrins are altered upon interaction with chlorophenols and other organochlorine pollutants. Meso-tetra(4-sulfonatophenyl)porphyrin (TPPS), zinc meso-tetra(4-sulfonato phenyl)porphyrin (Zn-TPPS), monosulfonate-tetraphenylporphyrin (TPPS1), meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphyrin (C1TPP), meso-tetra(4-carboxyphenyl)porphyrin (C4TPP), and copper meso-tetra(4-carboxyphenyl)porphyrin (Cu-C4TPP) in solution exhibit a broad absorbance in the range 400-450 nm Soret region. The interaction of the above mentioned porphyrins in solution with pentachlorophenol (PCP) induces a red shift in the Soret spectrum with absorbance losses at 413, 418, 403, 405, 407, and 404 nm, respectively, and the appearance of new peaks at 421, 427, 431, 416, 417, and 416 nm, respectively. The intensity of the Soret spectral change is proportional to the pentachlorophenol concentration with a detection limit of 1, 0.5, 1.16, 1, 0.5, and 0.5 ppb, respectively. The interaction of (C4TPP) and (Cu-C4TPP) in solution with PCP shows to concentration dependent for concentrations less than 4 ppb the dependence was log-linear. However, for concentrations greater than 4 ppb the relation was linear. Monosulfonate-tetraphenylporphyrin immobilized as a monolayer on a Kimwipe tissue exhibits an absorbance peak in the Soret region at 422 nm. The interaction of the porphyrin with PCP induces a red shift in the Soret spectrum with absorbance loss at 419 nm and the appearance of new peaks at 446 nm. The intensity of the Soret spectral change is proportional to the log of PCP concentration. The detection limit with immobilized TPPS1 for PCP is 0.5 ppb. These results suggest the potential for development of spectrophotometric chemosensor for PCP residues in water with detection limits less than US EPA maximum contaminate level (MCL) of 1 ppb. The immobilized TPPS1 on the Kimwipe will make it possible to develop a wiping sensors to monitor the PCP or other pesticides residues on the vegetables or wood products.     

Immobilization of acetylcholinesterase in nanofibrous PVA/BSA membranes by electrospinning

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted continuous attention in the field of enzyme immobilization. In this study, acetylcholinesterase (AChE) has been successfully immobilized in PVA nanofibers via electrospinning of a mixture of AChE, BSA as an enzyme stabilizing additive and PVA. The maximum activity recovery of immobilized AChE was about 40%. In comparison with free enzyme, the immobilized AChE showed improved stability while retaining a considerable amount of activity at lower pH values. Moreover, the immobilized AChE retained >34% of its initial activity when stored at 30°C for 100 days and retained 70% of its initial activity after ten consecutive reactor batch cycles.     

Evaluation and optimization of factors affecting the performance of a flow-through system based on immobilized acetylcholineesterase as a biosensor

A flow-through system based on acetylcholineesterase (AChE) was studied. The system was prepared by mixing AChE and a multiwall carbon nanotube (MWCNT). Two important parameters, the ratios of AChE:MWCNT (X₁) and AChE-MWCNT:sol-gel (X₂) were optimized using response surface methodology. The results revealed that an enzyme immobilized within the MWCNT-sol-gel was more effective compared to one conducted with sol-gel. The optimum feed flow rate was 0.4 mL/min and ATChI concentration was found to be 1 mM. The optimum ratios of X₁ and X₂ for immobilization on ceramic packing were 1.07 and 0.43, respectively. The sensitivity of this flow-through system was 1.82 × 10⁻⁵/μM and long-term stability analyzed after 120 days was 74% of initial absorbance. With respect to an incubation time of 14 min, the detection limit for paraoxon was 7.3 × 10⁻¹² mol.     

Ectonucleotidase and acetylcholinesterase activities in silver catfish (Rhamdia quelen) exposed to different salinities

The purpose of this study was to investigate whether salinity adaptation can alter the purinergic (ecto-nucleoside triphosphate diphosphohydrolase; NTPDase and, 5′-nucleotidase) and cholinergic (acetylcholinesterase; AChE) systems in whole brain and blood tissue of the silver catfish, Rhamdia quelen. Silver catfish were gradually adapted to salinities of 0, 4 or 8 ppt and maintained at the experimental salinity for 10 days before brain and blood samples were collected. Blood AChE activity decreased significantly at 8 ppt and significant decreases in AChE activity were observed in the brain with salinity increases. ATP hydrolysis did not change between the groups. In contrast, ADP and AMP hydrolysis in silver catfish maintained at salinities of 4 and 8 ppt were significantly higher than those kept at 0 ppt. In conclusion, this study showed that there is an enhancement in the NTPDase (ADP hydrolysis) and 5′-nucleotidase activities in the brains of silver catfish exposed to increased salinity. Therefore, the activities of these enzymes can act as markers of salinity changes.     

Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer’s. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K_A) obtained from the fits were 3.8 × 10³ M⁻¹ for neostigmine and 1.7 × 10³ M⁻¹ for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor’s ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.     

Acetylcholinesterase-ISFET based system for the detection of acetylcholine and acetylcholinesterase inhibitors

A bioelectronic hybrid system for the detection of acetylcholine esterase (AChE) catalytic activity was assembled by way of immobilizing the enzyme to the gate surface of an ion-sensitive field-effect transistor (ISFET). Photometric methods used to characterize bonded enzyme and linker layers on silicon substrates confirm the existence of a stable amino-cyanurate containing AChE monolayer. The transduction of the enzyme-functionalized ISFET, in ionic solutions, is detected in response to application of acetylcholine (ACh). Recorded sensitivity of the modified ISFET to ACh has reached levels of up to 10(-5)M. The Michaelis-Menten constant of the immobilized AChE is only moderately altered. Nevertheless, the maximum reaction velocity is reduced by over an order of magnitude. The ISFET response time to bath or ionophoretic application of ACh from a micropipette was in the range of a second. The catalytic activity of the immobilized AChE is inhibited in a reversible manner by eserine, a competitive inhibitor of AChE. We conclude that the immobilized enzyme maintains its pharmacological properties, and thus the described bioelectronic hybrid can serve as a detector for reagents that inhibit AChE activity.     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.     

Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin

The photodynamic activity of 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on agar surfaces. The results obtained for the tricationic A3B3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin p-tosylate (TTAP4+), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence. In both cases, a higher photostability was found for A3B3+ than for TTAP4+. Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0-14 nmol) and irradiated with visible light for 3h. The light source used was either a projector or midday sun. The A3B3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through an optical fiber. Under these conditions, A3B3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photodynamic activity of A3B3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces complete inactivation of E. coli cells on the agar surface. Therefore, tricationic A3B3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection.     

Development of an oligo(ethylene glycol)-based SPR immunosensor for TNT detection

This paper describes the development of novel biosensor surfaces supported by robust self-assembled monolayers (SAMs) of aromatic alkanedithiol and oligo(ethylene glycol) (OEG) linker for highly sensitive surface plasmon resonance (SPR) detection of 2,4,6-trinitrotoluene (TNT). Aromatic alkanedithiol SAMs were firstly formed on Au sensor surface and TNT analogues were immobilized on it through OEG chain. Two kinds of OEG containing amine compounds, where H(2)N(C(2)H(4)O)(11)C(2)H(4)NHCOOC(CH(3))(3) served as a linker to react with carboxyl groups of TNT analogues while H(2)N(C(2)H(4)O)(3)C(2)H(4)OH served as a protein non-fouling background, were covalently bound to carboxyl terminal groups of SAMs with a certain ratio. Optimal ratio of them was also examined. Three kinds of TNT analogues, namely TNP-glycine, DNP-glycine, and DNP-acetic acid were used as immobilized ligands. Highly sensitive TNT detection by indirect competitive assay was conducted on the fabricated sensor surfaces; we examined how structural variations of them affect sensitivity in order to choose optimal hapten as well to improve sensitivity. The DNP-acetic acid immobilized surface, which had the lowest affinity to the TNT antibody among the three, showed the best limit of detection (LOD) value (ca. 80ppt (pgml(-1))). On the other hand, the TNP-glycine immobilized surface, which had the highest affinity, showed the worst LOD value (ca. 220ppt). The LOD got lower to ca. 50ppt by the use of the secondary antibody on the DNP-acetic acid immobilized surface. The sensor surfaces are durable for more than 100 times repeated use without any noticeable deterioration by their chemical stability and rather mild regeneration condition.     

Spectroscopic determination of acetylcholine esterase–inhibitor complex: determination of conformational shifts of proteins

Changes in the absorbance spectrum of tetraphenylporphyrin sulfonate (TPPS) are observed that are unique for the proteins lysozyme, luciferase, apomyoglobin, myoglobin, gamma globulin, insulin, RNAase, phosphotransacetylase, papain, ovalbumin, bovine serum albumin (BSA), protamine sulfate, and polylysine. The absorbance spectrum of porphyrins is different for native compared with heat denatured RNAase. A unique absorbance wavelength red shift is observed with trypsin when trypsin inhibitor is present, indicating that porphyrins incorporated with proteins can detect conformational changes in the protein. The absorbance spectrum of the Soret band of TPPS undergoes bathochromic shifts upon addition of local anesthetics to acetylcholine esterase (AChE), suggesting that the absorbance spectrum of porphyrins can be used as a reporter of the presence of inhibitors of AChE by indicating conformational changes on binding of the inhibitor.     

Genetically engineered acetylcholinesterase-based biosensor for attomolar detection of dichlorvos

The design of a biosensor for the detection of dichlorvos at attomolar levels is described based on a highly sensitive double mutant (E69Y Y71D) of the Drosophila melanogaster acetylcholinesterase (Dm. AChE). This enzyme has a k(i) for dichlorvos equal to 487 microM(-1)min(-1), which is 300 and 20,000 times higher than that of the wild type Dm. AChE and the Electrophorus electricus AChE (E.el. AChE), respectively. The enzyme is immobilized into microporous-activated conductive carbon, and is used as such for the development of an inhibitor electrochemical biosensor. This E69Y Y71D mutant enables the decrease in the detection limit of the biosensor down to 10(-17) M, which is five orders of magnitude lower compared to the Electropharus electricus-based biosensor and eight orders of magnitude lower than the biosensors described so far.     

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid‐based nanofiber membrane

In this study, polyacrylic acid‐based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid‐based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid‐based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Ca²⁺ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE‐based biosensors.     

Acetylcholinesterase with mesoporous silica: Covalent immobilization,physiochemical characterization,and its application in food for pesticide detection

Toxic contamination of commonly consumed food products and water due to food chain vulnerability via agricultural products and commodities is a serious health hazard. This study reports on Santa Barbara Amorphous (SBA-15), a type of mesoporous silica nanoparticles, for efficient and stable acetylcholinesterase (AChE) adhesion toward detection of toxic pesticides. AChE was immobilized to the inert framework of mesoporous materials viz. SBA-15 with a proficient hydrolytic response toward acetylthiocholine. The immobilized system acts as a biosensor for the detection of pesticides, which are organophosphorus compounds in food. Both the SBA-15 and immobilized SBA-15 were characterized to give an insight on the physiochemical and morphological modification properties. The enzyme activity was accessed by Ellman’s spectrophotometric bioassay for bare and enzyme-immobilized SBA-15 that resulted in promising enzymatic activity with the counterpart. Enzyme stability was also studied, which exhibited that immobilized AChE retained its catalytic activity up to 60 days and retained 80% of the hydrolytic activity even at 37°C. On the basis of the success of immobilized enzyme (covalent) being inhibited by acetylthiocholine, the sensor was administered for the inhibition by monocrotophos and dimethoate that are used widely as pesticides in agricultural. The inhibitory concentration (IC₅₀) value was found to be 2.5 ppb for monocrotophos and 1.5 ppb for dimethoate inhibiting immobilized AChE. This was verified using cyclic voltammetry, an electrochemical analysis thus proving that the SBA-15@AChE complex could be used as a sensitive and highly stable sensor for detecting the concentration of hazardous pesticide compounds.     

Catalytic activity of acetylcholinesterase immobilized on mesoporous molecular sieves

MCM-41 and FSM-16 were used for enzyme immobilization on account of their good physical and chemical properties. In this work, the catalytic activity of acetylcholinesterase (AChE) immobilized on these materials was investigated, using neostigmina as AChE inhibitor. The results show that AChE was adsorbed on MCM-41 and on FSM-16-TIPB. AChE immobilized on the latter material maintained 70% of its activity and the material did not hydrolyze ACh (as MCM-41) by itself. Therefore, FSM-16-TIPB was the best material, considering also that when neostigmine was applied to AChE immobilized on FSM-16-TIPB, the activity of AChE decreased as occurs in its free from. Hence, this model could be useful in the evaluation of different kinds of AChE inhibitors, allowing the recycling of enzymes and making possible several assays and thereby, lowering cost.     

Determination of organophosphorous pesticides by a novel biosensor based on localized surface plasmon resonance

Liquid and gas chromatography are commonly used to measure organophosphorus pesticides. However, these methods are relatively time consuming and require a tedious sample pretreatment. Here, we applied the localized surface plasmon resonance (LSPR) of gold nanoparticles covalently coupled with acetylcholinesterase (AChE) to create a biosensor for detecting an example of serial signals responding to paraoxon in the range of 1-100 ppb by an AChE modified LSPR sensor immersing in a 0.05 mM ACh solution. The underlying mechanism is that paraoxon prevents acetylcholine chloride (ACh) reacting with AChE by destroying the OH bond of serine in AChE. We found that the AChE modified LSPR sensors prepared by incubation with 12.5 mU/mL of AChE in phosphate buffer solution at pH 8.5 room temperature for 14 h have the best linear inhibition response with a 0.234 ppb limit of paraoxon detection. A 14% of inhibition on the sensor corresponds to the change of paraoxon concentration from 1 to 100 ppb. The sensor remained 94% of its original activity after six cycles of inhibition with 500 ppb paraoxon followed with reactivation of AChE by 0.5 mM 2-pyriding-aldoxime methoiodide (2-PAM). In addition, the sensor retains activity and gives reproducible results after storage in dry state at 4 degrees C for 60 days. In conclusion, we demonstrated that the AChE modified LSPR sensors can be used to determine the concentration of paraoxon biosensor with high sensitive and stable characteristics.     

Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates <Emphasis Type="Italic">in vitro</Emphasis> blood coagulation similarly to thromboplastin on fibroblasts

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.     

Immobilization of rat brain acetylcholinesterase on porous gold-nanoparticle-CaCO₃ hybrid material modified Au electrode for detection of organophosphorous insecticides

An acetylcholinesterase (AChE) purified from rat brain was immobilized onto gold nanoparticles (AuNPs) assembled on the surface of porous calcium carbonate (CaCO₃) microsphere. The resulting AChE-AuNPs-CaCO₃ bioconjugate was mounted on the surface of Au electrode with the help of silica sol-gel matrix to prepare the working electrode. This electrode was connected to Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode through a potentiostat to construct an organophosphorus (OP) biosensor. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The biosensor showed optimum response at pH 7.0, 30 °C, when polarized at +0.2 V. Two OP compounds, malathion and chlorpyrifos could be detected in the range of 0.1-100 nM and 0.1-70 nM, respectively at 2.0-3.0% inhibition level of AChE. The sensor was reactivated by immersing it in 0.1 mM 2-pyridine aldoxime for 10 min. The detection limit of the sensor was 0.1 nM for both malathion and chlorpyrifos. The biosensor exhibited good reusability (50 times without considerable loss) and storage stability (50% within 60 days, when stored at 4 °C).