Amperometric Biosensor Based on Immobilization Acetylcholinesterase on Manganese Porphyrin Nanoparticles for Detection of Trichlorfon with Flow‐Injection Analysis System

A highly sensitive amperometric biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso‐tetraphenylporphyrin (MnTPP) nanoparticles (NPs)‐modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE‐CHIT/MnTPPNP/GC biosensor integrated with a flow‐injection analysis (FIA) system was used to monitor trichlorfon (typical OP). A wide linear inhibition response for trichlorfon is observed in the range of 1.0 nM–1.0 mM, corresponding to 10–83% inhibition for AChE with a detection limit of 0.5 nM.     

Design of a new hypoxanthine biosensor: xanthine oxidase modified carbon film and multi-walled carbon nanotube/carbon film electrodes

A new and simple-to-prepare hypoxanthine biosensor has been developed using xanthine oxidase (XOD) immobilised on carbon electrode surfaces. XOD was immobilised by glutaraldehyde cross-linking on carbon film (CF) electrodes and on carbon nanotube (CNT) modified CF (CNT/CF). A comparison of the performance of the two configurations was carried out by the current response using amperometry at fixed potential; the best characteristics being exhibited by XOD/CNT/CF modified electrodes. The effects of electrolyte pH and applied potential were evaluated, and a proposal is made for the enzyme mechanism of action involving competition between regeneration of flavin adenine dinucleotide and reduction of hydrogen peroxide. Under optimised conditions, the determination of hypoxanthine was carried out at ?0.2 V vs. a saturated calomel electrode (SCE) with a detection limit of 0.75 μM on electrodes with CNT and at ?0.3 V vs. SCE with a detection limit of 0.77 μM on electrodes without CNT. The applicability of the biosensor was verified by performing an interference study, reproducibility and stability were investigated, and hypoxanthine was successfully determined in sardine and shrimp samples.     

Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples

A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD⁺) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 μL of a 1 % (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 μM NAD⁺), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 μM. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5 % in the 1–10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95–110 % range.

Amperometric Ethanol Biosensor Based on Carbon Nanotubes Dispersed in Sol–Gel‐Derived Titania–Nafion Composite Film

Composite solution of sol–gel‐derived titania and perfluorosulfonated ionomer (Nafion) was used as a solubilizing agent for multiwalled carbon nanotubes (CNT) as well as an encapsulation matrix for alcohol dehydrogenase (ADH) for the fabrication of a highly sensitive and stable amperometric ethanol biosensor. ADH was immobilized within a thin film of CNT–titania–Nafion composite film coated on a glassy carbon electrode. Because of the mesoporous nature of the CNT–titania–Nafion composite film, the present biosensor exhibited remarkably fast response time within 2 s. The presence of CNT in the composite film increases not only the sensitivity of the ethanol biosensor but also the long‐term stability of the biosensor. The present biosensor responds linearly to ethanol in the wide concentration ranges from 1.0×10^?5 M to 3.0×10^?3 M with the sensitivity of 51.6 mA M^?1cm^?2. The present biosensor showed good long‐term stability with 75% of its activity retained after 4 weeks of storage in 50 mM phosphate buffer at pH 7.0.     

聚中性红修饰玻碳电极葡萄糖生物传感器

以中性红为电子媒介体,电聚合于Nafion修饰的玻碳电极表面,以戊二醛作交联剂固定葡萄糖氧化酶,最后覆盖一层Nafion膜防止酶流失,构建一种新型葡萄糖生物传感器.详细探讨了传感器的电化学性能及对葡萄糖的最佳响应条件.结果表明,30℃时,传感器在pH 7.0的PBS中对葡萄糖的线性响应范围为1.0×10-5~5.0×10-3mol.L-1.该传感器制作简单、性能优良,有潜在应用前景.     

Acetylcholinesterase immobilised eggshell membrane-based optical biosensor for organophosphate detection

The development of an optical biosensor for the determination of malathion based on acetylcholinesterase (AChE) inhibition using Ellman’s reagent is reported. The AChE has been immobilised onto the eggshell membrane (ESM) using glutaraldehyde as a cross-linking agent. Scanning Electron Microscopic (SEM) studies and Fourier Transformed Infra-Red (FTIR) characterisations have been carried out to affirm the successful immobilisation of AChE onto the ESM. Under optimum conditions, the developed biosensor estimated the pesticide concentration in the range of 0.1–50 ng/mL with a limit of detection (LOD) of 0.1 ng/mL within 30 min. Parameters affecting the biosensor response such as concentration of enzyme, substrate and inhibition time were optimised. The stability and reusability of the AChE/ESM sensor have been observed as 31 days at 4°C and two times, respectively.     

Glucose biosensor based on a platinum electrode modified with rhodium nanoparticles and with glucose oxidase immobilized on gold nanoparticles

We have developed an enzymatic glucose biosensor that is based on a flat platinum electrode which was covered with electrophoretically deposited rhodium (Rh) nanoparticles and then sintered to form a large surface area. The biosensor was obtained by depositing glucose oxidase (GOx), Nafion, and gold nanoparticles (AuNPs) on the Rh electrode. The electrical potential and the fractions of Nafion and GOx were optimized. The resulting biosensor has a very high sensitivity (68.1 μA mM^?1 cm^?2) and good linearity in the range from 0.05 to 15 mM (r?=?0.989). The limit of detection is as low as 0.03 mM (at an SNR of 3). The glucose biosensor also is quite selective and is not interfered by electroactive substances including ascorbic acid, uric acid and acetaminophen. The lifespan is up to 90 days. It was applied to the determination of glucose in blood serum, and the results compare very well with those obtained with a clinical analyzer.

Selective Determination of Quercetin Using Carbon Nanotube‐Modified Electrodes

Electrochemical detection of quercetin has been carried out on glassy carbon electrodes modified with carbon nanotubes and Nafion (GC/Nafion‐CNT). GC/Nafion‐CNT electrodes did not show passivation effect that occurs on the unmodified electrodes and displayed better stability and reproducibility. Quercetin oxidation was most favorable in acidic conditions and current gradually decreased as the solution pH increased. No oxidation was observed when two ? OH groups in a catechol moiety were fully deprotonated. These electrodes enabled selective determination of quercetin in the presence of interfering species such as ascorbic acid, uric acid, glucose, and catechol in large excess. Quantification of quercetin in a yellow onion has been made and favorably compared with reported values. Good selectivity and high sensitivity obtained by Osteryoung sSquare‐wave voltammetry can open new possibilities of direct quercetin determination in vegetables with a minimal sample treatment.     

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

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

Ionophore Based Bismuth Film Electrode for Lead

Abstract

A Nafion/ionophore, 4-tert-butylcalix[4]arene-tetrakis(N,N-dimethylthioacetamide) composite coated and bismuth film modified glassy carbon electrode. (GC/NA-IONO/BiFE) was described to determine trace lead sensitively and selectively. The characteristics of such modified GC/NA-IONO/BiFE were studied by scanning electron microscopy and cyclic voltammetry. The influence of various experimental parameters upon the stripping lead signal at the GC/NA-IONO/BiFE was explored. Under the optimized conditions, the differential pulse voltammetric stripping response is highly linear over the 0.1–8.0 nM lead range examined (180 s preconcentration at ?1.2 V), with a detection limit of 0.044 nM and good precision (RSD = 5.4% at 0.5 nM). Also applicability to seawater samples was demonstrated at such modified electrode. The high selectivity of ionophore coupled with the excellent electrochemical characteristics of bismuth endow the GC/NA-IONO/BiFE a promising and robust tool for monitoring of trace lead rapidly and precisely.     

Immobilized Laccase on Activated Poly(Vinyl Alcohol) Microspheres For Enzyme Thermistor Application

Poly(vinyl alcohol) (PVA) microspheres were prepared by inverse suspension crosslinked method, with glutaraldehyde as a crosslinking agent. PVA microspheres activated with aldehyde groups were employed for Trametes versicolor laccase immobilization. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the activated PVA microspheres and PVA microspheres with immobilized laccase (Lac/PVA microspheres), which show that laccase was successfully immobilized on the PVA microspheres. The optimum pH and temperature coupling conditions for the immobilized laccase were determined to be 3.3 and 30 °C, respectively. Residual activity was also investigated by soaking the immobilized laccase in organic solvents at different concentrations, proving it chemically stable. Immobilized laccase exhibited good storage stability at 4 °C. The enzyme biosensor showed good performance in 2,2-azinobis(3-ethylthiazoline-6-sulfonate) and bisphenol A, with concentration ranges of 2 to 8 mM and 0.05 to 0.25 mM, respectively. Therefore, PVA microspheres may have high potential as support for enzyme thermistor applications.     

Electrogenerated Chemiluminescence Ethanol Biosensor Based on Carbon Nanotube‐Titania‐Nafion Composite Film

Mesoporous titania‐Nafion composite doped with carbon nanotube (CNT) has been used for the immobilization of tris(2,2′‐bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) and alcohol dehydrogenase on an electrode surface to yield a highly sensitive and stable electrogenerated chemiluminescence (ECL) ethanol biosensor. The presence of CNT in the composite film increases not only the sensitivity of the ECL biosensor but also the long‐term stability of the biosensor. The present biosensor responds linearly to ethanol in the wide concentration ranges from 1.0×10^?5 M to 1.0×10^?1 M with a detection limit of 5.0×10^?6 M (S/N=3). The present ECL ethanol biosensor exhibited higher ECL response compared to that obtained with the ECL biosensor based on the corresponding composite without CNT. The present CNT‐based ECL biosensor showed good long‐term stability with 75% of its initial activity retained after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0.     

A Highly Sensitive and Selective Multiwall Carbon Nanotubes/Nafion/Au Microarrays Electrode for Dopamine Determination

This work presents the fabrication of Nafion (Nf) or Nafion/Multiwalled Carbon Nanotubes (Nf/MWCNTs) modified gold microarray (Au‐µA) and macro‐(Au‐M)electrode biosensors. The surface morphologies of the above electrodes were examined using SEM. The catalytic properties of the above electrodes towards dopamine were tested using square wave voltammetric technique. The Nf/MWCNT/Au‐µA electrode exhibited a wide range (0.1–1000 nM) of linearity among the other electrodes. The LOD of Nf/MWCNT/Au‐µA electrode was 50 pM for dopamine in the presence of 5000 µM ascorbic acid. Therefore, the Nf/MWCNT/Au‐µA biosensor was applied for the determination of dopamine in human serum.     

AChE biosensor based on zinc oxide sol-gel for the detection of pesticides

Zinc oxide has been used as a matrix for immobilization of acetylcholinesterase (AChE) and detection of the pesticide paraoxon. The immobilized enzyme retained its enzymatic activity up to three months when stored in phosphate buffered saline (pH 7.4) at 4 °C. An amperometric biosensor for the detection of paraoxon was designed. The biosensor detected paraoxon in the range 0.035-1.38 ppm and can be used to detect other AChE inhibiting organophosphate pesticides.     

Organophosphorus pesticides detection using acetylcholinesterase biosensor based on gold nanoparticles constructed by electroless plating on vertical nitrogen-doped single-walled carbon nanotubes

An acetylcholinesterase (AChE) biosensor was constructed based on gold nanoparticles (AuNPs) using electroless plating on vertical nitrogen-doped single-walled carbon nanotubes (VNSWCNTs) for detecting organophosphorus pesticides (OPs). AChE was immobilised on AuNPs via Au–S bonding, and VNSWCNTs were produced by spontaneous chemical adsorption of NSWCNTs on gold electrode, also via Au–S bonding. This modified electrode exhibited excellent electron transfer capacity due to the synergy between AuNPs and VNSWCNTs. The developed biosensor showed good linear relations at concentrations of 10^?5 – 1 ppb, and the detection limits were 3.04 × 10^?6 ppb for methyl parathion, 1.96 × 10^?6 ppb for malathion and 2.06 × 10^?6 ppb for chlorpyrifos, respectively. The AChE biosensor revealed satisfactory stability, excellent sensitivity and good repeatability. These results suggest that this biosensor has good application prospects and can function as a sensitive device in OPs analysis.     

Enhanced Hydrogen Peroxide Sensing Based on Tetraruthenated Porphyrins/Nafion/Glassy Carbon‐modified Electrodes via Incorporating of Carbon Nanotubes

The incorporation of carbon nanotubes to a Nafion/tetraruthenated cobalt porphyrin/ glassy carbon electrode (GC/Nf/CoTRP vs GC/Nf/CNTCoTRP) enhanced the amperometric determination of hydrogen peroxide. Both electrodes produced a decrease in the overpotential required for the hydrogen peroxide oxidation in about 100 mV compared to glassy carbon under the same experimental conditions. Nevertheless, for GC/Nf/CNT/CoTRP, the increase in the current is remarkable. The GC/Nf/CoTRP modified electrode gave no significant analitycal signal for hydrogen peroxide reduction. Moreover, a great increase in current is observed with GC/Nf/CNT/CoTRP at ?150mV which suggests a significant increase in the sensitivity of the modified electrode. Scanning electrochemical microscopy (SECM) revealed an enhancement in the electroactivity of the GC/Nf/CNT/CoTRP modified electrode. This fact has been explained in terms of enhanced homogeneity of the electrodic surface as a consecuence of better dispersibility of CNT‐CoTRP produced by a Nafion polyelectrolyte.     

Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor

A dimeric organophosphorus hydrolase (OPH; EC 3.1.8.1; 72 kDa) was isolated from wild-type bacteria, analyzed for its 16s rRNA sequence, purified, and immobilized on gold nanoparticles (AuNPs) to form the transducer part of a biosensor. The isolated strain was identified as Pseudomonas aeruginosa. The AuNPs were characterized by transmission electron microscopy and localized surface plasmon resonance. Covalent binding of OPH to the AuNPs was confirmed by spectrophotometry, enzymatic activity assays, and FTIR spectroscopy. Coumarin 1, a competitive inhibitor of OPH, was used as a fluorogenic probe. The bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence that is directly proportional to the concentration of paraoxon. The gold-OPH conjugates were then used to determine paraoxon in serum samples spiked with varying levels of paraoxon. The method works in the 50 to 1,050 nM concentration range, has a low standard deviation (with a CV of 5.7–11 %), and a detection limit as low as 5?×?10^?11 M.

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

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

Functionalized graphene oxide for the fabrication of paraoxon biosensors

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, N_α,N_α-bis(carboxymethyl)-l-lysine hydrate (NTA-NH₂) and Ni²⁺ 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 1 mM (R² = 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.     

Amperometric glucose biosensor based on electrodeposition of platinum nanoparticles onto covalently immobilized carbon nanotube electrode

A new amperometric biosensor for glucose was developed based on adsorption of glucose oxidase (GOx) at the gold and platinum nanoparticles-modified carbon nanotube (CNT) electrode. CNTs were covalently immobilized on gold electrode via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM). The fabricated GOx/Au_nano/Pt_nano/CNT electrode was covered with a thin layer of Nafion to avoid the loss of GOx in determination and to improve the anti-interferent ability. The immobilization of CNTs on the gold electrode was characterized by quartz crystal microbalance technique. The morphologies of the CNT/gold and Pt_nano/CNT/gold electrodes have been investigated by scanning electron microscopy (SEM), and the electrochemical performance of the gold, CNT/gold, Pt_nano/gold and Pt_nano/CNT/gold electrodes has also been studied by amperometric method. In addition, effects of electrodeposition time of Pt nanoparticles, pH value, applied potential and electroactive interferents on the amperometric response of the sensor were discussed.

The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for glucose in the absence of a mediator. The linear range was from 0.5 to 17.5 mM with correction coefficient of 0.996. The biosensor had good reproducibility and stability for the determination of glucose.