Construction of an improved amperometric acrylamide biosensor based on hemoglobin immobilized onto carboxylated multi-walled carbon nanotubes/iron oxide nanoparticles/chitosan composite film

A method is described for construction of an improved amperometric acrylamide biosensor based on covalent immobilization of hemoglobin (Hb) onto nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT) and iron oxide nanoparticles (Fe₃O₄NPs) electrodeposited onto Au electrode through chitosan (CHIT) film. The Hb/cMWCNT-Fe₃O₄NP/CHIT/Au electrode was characterized by scanning electron microscopy, Fourier transform infra-red spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry at different stages of its construction. The biosensor was based on interaction between acrylamide and Hb, which led to decrease in the electroactivity of Hb, i.e., current generated during its reversible conversion [Fe(II)/Fe(III)]. The biosensor showed optimum response within 8 s at pH 5.0 and 30 °C. The linear working range for acrylamide was 3–90 nM, with a detection limit of 0.02 nM and sensitivity of 36.9 μA/nM/cm². The biosensor was evaluated and employed for determination of acrylamide in potato crisps.     

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

Abstract

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

Thermostable alkaline protease from Thermomyces lanuginosus: optimization,purification and characterization

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-₁₀₀ and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K _m), maximal reaction velocity (V _max) and turnover number (K _cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s^?1, respectively. The best enzyme activators were K⁺, Ca²⁺ and Mn², respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.     

SENSITIVE NITRATE DETERMINATION IN WATER AND MEAT SAMPLES BY AMPEROMETRIC BIOSENSOR

This study describes a novel biosensor method for specific determination of nitrate in food and water samples by using nitrate reductase (NR) (EC 1.9.6.1) biosensor based on the detection of oxidation peak current of redox mediator, methyl viologen, related to nitrate concentration. The method was shown to be selective and sensitive to determine the nitrate levels of water samples and processed meat samples. Immobilization procedure and also working conditions of the biosensor were optimized. Dynamic range attained with this method was established as (5.0–90.0 × 10^?9 M) for nitrate concentration with a 10 s response time. Limit of detection (LOD) and quantification (LOQ) of the biosensor were calculated as 2.2 × 10^?9 M and 5.79 × 10^?9 M, respectively. Reproducibility experiments was established on repetitive measurements by using a freshly prepared biosensor for avoiding the memory effect. The RSD was calculated as 1.22% at a nitrate concentration of 4.7 × 10^?8 M (n = 7).     

Construction of an amperometric TG biosensor based on AuPPy nanocomposite and poly (indole-5-carboxylic acid) modified Au electrode

A method is described for construction of an amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase and glycerol-3-phosphate oxidase onto gold polypyrrole nanocomposite decorated poly indole-5-carboxylic acid electrodeposited on the surface of a gold electrode. The enzyme electrode was characterized by transmission electron microscopy, scanning electron microscopy, electrochemical impedance studies, Fourier transform infrared spectroscopy and cyclic voltammetry. Biosensor showed optimum response within 4 s at pH 6.5 and 35 °C, when polarized at +0.1 V against Ag/AgCl. There was a linear relationship between sensor response and triolein concentration in the range 50–700 mg/dl. Biosensor was employed for determination of TG in serum. Detection limit of the biosensor was 20 mg/dl. Biosensor was evaluated with 91–95 % recovery of added triolein in sera and 4.14 and 5.85 % within and between batch coefficients of variation, respectively. There was a good correlation (r = 0.99) between sera TG values by standard method (Enzymic colorimetric) and the present method. The biosensor was unaffected by a number of serum substances at their physiological concentration. Biosensor lost 50 % of its initial activity after its 100 uses over 7 months, when stored at 4 °C.     

Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

Characterization of microbial membranes used for the estimation of biochemical oxygen demand with a biosensor

Summary A bacterial mixed culture was immobilized in Millipore filters to construct microbial-membranes for BOD determination using an oxygen electrode. The biosensor response was best when 0.5 mg cells were immobilized per cm² of membrane, at 30°C, pH 7 and 0.05 M phosphate buffer. Reproducible microbial-membranes can be constructed and they can be stored for up to 20 days without appreciable loss of their response characteristics.     

Electrophoretic transfer protein zymography

A mixture of commercial creatinine amidohydrolase (CA), creatine amidinohydrolase (CI), and sarcosine oxidase (SO) was coimmobilized covalently via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto carboxylated multiwalled carbon nanotube (c-MWCNT)/polyaniline (PANI) nanocomposite film electrodeposited over the surface of a platinum (Pt) electrode. A creatinine biosensor was fabricated using enzyme/c-MWCNT/PANI/Pt as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor detected creatinine levels as low as 0.1 μM, estimated at a signal-to-noise ratio of 3, within 5 s at pH 7.5 and 35 °C. The optimized biosensor showed a linear response range of 10 to 750 μM creatinine with sensitivity of 40 μA/mM/cm². The fabricated biosensor was successfully employed for determination of creatinine in human serum. The biosensor showed only 15% loss in its initial response after 180 days when stored at 4 °C.     

PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROM Scopulariopsis spp.

A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0–12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co²⁺ and Mn²⁺ at 10 mM but was unaffected by Ba²⁺, Mg²⁺, Cu²⁺, Na⁺, K⁺, and Fe²⁺. Ca²⁺ and Fe³⁺ moderately reduced the activity (～18%); however, a reduction of about 40% was seen for Zn²⁺ and Hg²⁺. The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H₂O₂ and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (?20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel–enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.     

A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

Changes in methane oxidation activity and methanotrophic community composition in saline alkaline soils

The soil of the former Lake Texcoco is a saline alkaline environment where anthropogenic drainage in some areas has reduced salt content and pH. Potential methane (CH₄) consumption rates were measured in three soils of the former Lake Texcoco with different electrolytic conductivity (EC) and pH, i.e. Tex-S1 a >18 years drained soil (EC 0.7 dS m^?1, pH 8.5), Tex-S2 drained for ~10 years (EC 9.0 dS m^?1, pH 10.3) and the undrained Tex-S3 (EC 84.8 dS m^?1, pH 10.3). An arable soil from Alcholoya (EC 0.7 dS m^?1, pH 6.7), located nearby Lake Texcoco was used as control. Methane oxidation in the soil Tex-S1 (lowest EC and pH) was similar to that in the arable soil from Alcholoya (32.5 and 34.7 mg CH₄ kg^?1 dry soil day^?1, respectively). Meanwhile, in soils Tex-S2 and Tex-S3, the potential CH₄ oxidation rates were only 15.0 and 12.8 mg CH₄ kg^?1 dry soil day^?1, respectively. Differences in CH₄ oxidation were also related to changes in the methane-oxidizing communities in these soils. Sequence analysis of pmoA gene showed that soils differed in the identity and number of methanotrophic phylotypes. The Alcholoya soil and Tex-S1 contained phylotypes grouped within the upland soil cluster gamma and the Jasper Ridge, California JR-2 clade. In soil Tex-S3, a phylotype related to Methylomicrobium alcaliphilum was detected.     

Three-dimensional macro-scale assessment of regional and temporal wall shear stress characteristics on aortic valve leaflets

The aortic valve (AV) achieves unidirectional blood flow between the left ventricle and the aorta. Although hemodynamic stresses have been shown to regulate valvular biology, the native wall shear stress (WSS) experienced by AV leaflets remains largely unknown. The objective of this study was to quantify computationally the macro-scale leaflet WSS environment using fluid–structure interaction modeling. An arbitrary Lagrangian–Eulerian approach was implemented to predict valvular flow and leaflet dynamics in a three-dimensional AV geometry subjected to physiologic transvalvular pressure. Local WSS characteristics were quantified in terms of temporal shear magnitude (TSM), oscillatory shear index (OSI) and temporal shear gradient (TSG). The dominant radial WSS predicted on the leaflets exhibited high amplitude and unidirectionality on the ventricularis (TSM>7.50 dyn/cm², OSI < 0.17, TSG>325.54 dyn/cm² s) but low amplitude and bidirectionality on the fibrosa (TSM < 2.73 dyn/cm², OSI>0.38, TSG < 191.17 dyn/cm² s). The radial WSS component computed in the leaflet base, belly and tip demonstrated strong regional variability (ventricularis TSM: 7.50–22.32 dyn/cm², fibrosa TSM: 1.26–2.73 dyn/cm²). While the circumferential WSS exhibited similar spatially dependent magnitude (ventricularis TSM: 1.41–3.40 dyn/cm², fibrosa TSM: 0.42–0.76 dyn/cm²) and side-specific amplitude (ventricularis TSG: 101.73–184.43 dyn/cm² s, fibrosa TSG: 41.92–54.10 dyn/cm² s), its temporal variations were consistently bidirectional (OSI>0.25). This study provides new insights into the role played by leaflet–blood flow interactions in valvular function and critical hemodynamic stress data for the assessment of the hemodynamic theory of AV disease.     

Streptomyces sp. LK3 mediated synthesis of silver nanoparticles and its biomedical application

In the present study, the marine actinobacteria mediated biosynthesis of silver nanoparticles (AgNps) was achieved using Streptomyces sp LK3. The synthesized AgNps showed the characteristic absorption spectra in UV–vis at 420 nm, which confirmed the presence of nanoparticles. XRD analysis showed intense peaks at 2θ values of 27.51°, 31.87°, 45.57°, 56.56°, 66.26°, and 75.25° corresponding to (210), (113), (124), (240), (226), and (300) Bragg’s reflection based on the fcc structure of AgNps. The FTIR spectra exhibited prominent peaks at 3,417 cm^?1 (OH stretching due to alcoholic group) and 1,578 cm^?1 (C=C ring stretching). TEM micrograph showed that the synthesized AgNps were spherical in shape with an average size of 5 nm. Surface morphology and topographical structure of the synthesized AgNps were dignified by AFM. The synthesized AgNps showed significant acaricidal activity against Rhipicephalus microplus and Haemaphysalis bispinosa with LC₅₀ values of 16.10 and 16.45 mg/L, respectively. Our results clearly indicate that AgNps could provide a safer alternative to conventional acaricidal agents in the form of a topical antiparasitic formulation. The present study aimed to develop a novel, cost-effective, eco-friendly actinobacteria mediated synthesis of AgNps and its antiparasitic activity.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.     

Fabrication of an amperometric d-amino acid biosensor based on nickel hexacyanoferrate polypyrrole hybrid film deposited on glassy carbon electrode

A nickel hexacyanoferrate polypyrrole film was synthesized through an electrochemical two-step methodology leading to a very stable and homogenous robust hybrid film. A highly sensitive, specific and rapid amperometric d-amino acid biosensor was constructed by immobilizing d-amino acid oxidase on this film deposited over the surface of a glassy carbon electrode. The modified electrode was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and Fourier transform infrared spectrophotometry. The biosensor showed optimum response within 1 s, when operated at 50 mV s^?1 in 0.01 M Tris HCl buffer, pH 7.0 at 30 °C. The biosensor exhibited excellent sensitivity with a detection limit of 1.5 µM (S/N = 3) for d-amino acids and wider linear range, 20–500 µM. Analytical recovery of added d-alanine (5 and 10 mM) in serum samples was 98.00 and 98.80 %, respectively. Within-batch and between-batch coefficients of variation in serum samples were 1.36 and 2.77 %, respectively. The enzyme electrode was used more than 50 times over 2 months, when stored at 4 °C. The proposed modified electrode exhibited sufficient mechanical and electrochemical stability and high sensitivity compared to earlier electrochemical d-amino acid biosensors. Interference by ascorbic acid and uric acid, the main interfering species in the biological samples, was negligible.     

A mediator-free amperometric hydrogen peroxide biosensor based on HRP immobilized on a nano-Au/poly 2,6-pyridinediamine-coated electrode

A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine (pPA). The modified process was characterized by electrochemical impedance spectroscopy (EIS), and the electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent electrocatalytical response to reduction of H₂O₂ without the aid of an electron mediator, the linear range was 4.2 × 10⁻⁷–1.5 × 10⁻³ mol/L (r = 0.9977), with a detection limit of 1.4 × 10⁻⁷ mol/L. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor exhibited permselectivity, good stability and good fabrication reproducibility.     

Omp85 genosensor for detection of human brain bacterial meningitis

The 5′-thiolated DNA probe based on specific virulence gene, Omp85, was immobilized onto a screen-printed gold electrode followed by hybridization with 6–100 ng/6 μl (5.9 × 10⁵–9.3 × 10⁶ c.f.u.) of Neisseria meningitidis single stranded genomic DNA (ssG-DNA) for 10 min at 25 °C from the cerebrospinal fluid (CSF) of a meningitis patient. The Omp85 genosensor can detect as little as 6 ng ssG-DNA in 6 μl CSF of a human brain meningitis patient in 30 min including a response time of 1 min by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance. The sensitivity of the genosensor electrode was 2.6(μA/cm²)/ng using DPV with regression coefficient (R²) 0.954. The genosensor was characterized using Fourier transform infrared spectroscopy and atomic force microscopy. Omp85 genosensor was stable for 12 months at 4 °C with 12 % loss in DPV current.     

Toxicity of A Novel Neonicotinoid Insecticide Paichongding to Earthworm Eisenia fetida

Traditional neonicotinoid insecticides are used worldwide. Paichongding (IPP), as a novel neonicotinoid pesticide, has been widely used in China. However, the ecotoxicity of IPP to non-target invertebrates in soil ecosystem has not been reported yet. In this study, acute toxicity of IPP to earthworm Eisenia fetida, as well as the antioxidant response after IPP exposure, was evaluated. In the filter paper contact test, the LC₅₀ at 24 hr and 48 hr for IPP were 14.98 μg/cm² and 7.59 μg/cm², respectively. In artificial soil test, the LC₅₀ (lethal concentration) at 14 days and 28 days for IPP were 541.07 mg/kg and 238.51 mg/kg, respectively. The LC₅₀ of IPP is much higher than that of traditional neonicotinoid insecticides. However, earthworm body weight assessment demonstrated that the growth of earthworm was inhibited by extended exposure to IPP at sublethal doses. The activities of antioxidative enzymes superoxide dismutase and catalase in earthworms were significantly induced after IPP exposure. Malondialdehyde, a biomarker of lipid peroxidation, was also increased after IPP exposure. Although the results indicated that IPP had potentially adverse effect on earthworms, its toxicity was much lower than traditional neonicotinoids.     

An electrochemical biosensor for 3-hydroxybutyrate detection based on screen-printed electrode modified by coenzyme functionalized carbon nanotubes

3-Hydroxybutyrate, one of the main blood ketone bodies, has been considered as a critical indicator for diagnosis of diabetic ketoacidosis. Biosensors designed for detection of 3-hydroxybutyrate with advantages of precision, easiness and speedy performance have attracted increasing attention. This study attempted to develop a 3-hydroxybutyrate dehydrogenase-based biosensor in which single-walled carbon nanotubes (SWCNT) was used in order to immobilize the cofactor, NAD⁺, on the surface of screen-printed electrode. The formation of NAD⁺–SWCNT conjugates was assessed by electrochemistry and electron microscopy. Cyclic voltammetry was used to analyze the performance of this biosensor electrochemically. The considerable shelf life and reliability of the proposed biosensor to analyze real sample was confirmed by this method. The reduction in the over potential of electrochemical oxidation of NADH to ?0.15 V can be mentioned as a prominent feature of this biosensor. This biosensor can detect 3-hydroxybutyrate in the linear range of 0.01–0.1 mM with the low detection limit of 0.009 mM. Simultaneous application of screen-printed electrode and SWCNT has made the biosensor distinguished which can open new prospects for detection of other clinically significant metabolites.