Physicochemical characterization of the layer-by-layer self-assembly of polyphenol oxidase and chitosan on glassy carbon electrode

This paper demonstrates the feasibility to build up layer-by-layer (L-b-L) self-assemblies of polyphenol oxidase (PPO) and quaternized chitosan (CHI⁺) on a glassy carbon (GC) rotating disk electrode. This work highlights the promising properties of this modified polysaccharide to immobilize PPO in self-assembled structures under conditions that preserve its catalytic activity. The film consists of a diffusional underlayer coupled with an outermost active layer of (PPO/CHI⁺)_n. UV-vis spectroscopy and quartz crystal microbalance (QCM) were used to follow the multilayer film formation. Referring to the low amount of enzyme immobilized in the multilayer structure, the amperometric response of the biosensor reached an excellent sensitivity of about 2000 A mol⁻¹ cm, proving that an important part of the entrapped enzyme is accessible to the substrate molecule while keeping a good level of activity. The experimental validation of the theoretical model that was carried out on the basis of the catechol and phenol-polyphenol oxidase model system shows that the amperometric responses are in excellent agreement with the model. From a comparison of the experimental results with theory, we were able to characterize the diffusion through the film which includes two different areas and extract its enzymatic activity related to catechol and phenol substrates.     

HRP biosensor based on sugar-lectin biospecific interactions for the determination of phenolic compounds

A layer-by-layer self-assembly of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) afforded multilayer thin films on the surface of a thiol-modifed gold electrode, through biospecific complexation of Con A and sugar residues in the glycoenzymes. The performance of the HRP biosensor is reported for the amperometric detection of phenolic compounds. The concentration of hydrogen peroxide and assembly conditions of the precursor film, such as pH, the ionic strength of the polyelectrolyte solutions and the number of assembled bilayers were investigated using catechol. With optimized conditions, the biosensor presented a linear response for catechol from 6.0 to 48.0 μmol l⁻¹, with a high sensitivity of 160 μmol⁻¹ l nA and a detection limit of 0.6 μmol l⁻¹. The response time of the biosensor for phenolic compounds was very short, reaching 95% of its maximum response in about 2 s. The differences in sensitivity observed for a series of phenolic substrates were discussed in terms of the stability of the oxidized phenolic compounds and the properties of substituents.     

Conversion of biomass model compound under hydrothermal conditions using batch reactor

Supercritical water has been focused on as an environmentally attractive reaction media where organic materials can be decomposed into smaller molecules. The reaction behavior of lignin model compound was studied in near- and supercritical water with a batch type reactor. Catechol was used as a model compound for aromatic rings in lignin. The reaction was carried out at temperatures of 643-693 K at various pressures under an argon atmosphere. The chemical species in the aqueous products were identified by gas chromatography mass spectrometry (GCMS) and quantified using high performance liquid chromatography (HPLC). The effect of pressure and reaction time on the conversion process of catechol was presented. The main products from the conversion of catechol was phenol and the value of global rate constant for catechol conversion (k) is 3.0 × 10⁻⁴-11.0 × 10⁻⁴ min⁻¹.     

Amperometric biosensor for ethanol based on co-immobilization of alcohol dehydrogenase and Meldola's Blue on multi-wall carbon nanotube

In this work, multi-wall carbon nanotube (MWCT) is evaluated as transducer, stabilizer and immobilization matrix for the construction of amperometric biosensor based on alcohol dehydrogenase (ADH) and Meldola's Blue (MB). The amperometric response was based on the electro catalytic properties of MB to oxidize NADH, which was generated in the enzymatic reaction of ethanol with NAD⁺ under catalysis of ADH. It is shown that the employed materials are promising as electrochemical mediators and enzyme stabilizers. The enzyme was immobilized onto the MWCT adsorbed with MB by cross-linking with glutaraldehyde. The dependence on the biosensor response for ethanol was investigated in terms of pH, supporting electrolyte, ADH and NAD⁺ amounts and working potential. The amperometric response for alcohol using this biosensor showed excellent sensitivity (4.75 μA cm⁻² mmol L⁻¹), operational stability (around 95% of the activity was maintained after 300 determinations) and wide linear response range (0.05-10 mmol L⁻¹). These favorable characteristics allowed its application for measurements of ethanol in a great variety of alcoholic beverages with a simple dilution. The precision and recovery data showed by the proposed biosensor may give reliable results for real complex matrices.     

Isolation of antioxidant catechins from green tea and its decaffeination

Ethyl acetate, n-butanol and n-hexane were used as solvents to separate catechins from green tea extract solution and the catechins were decaffeinated using citric acid solution. Ethyl acetate was confirmed to be an appropriate solvent for isolating catechins from tea extract, based on yield of catechins and concentrations of caffeine. The optimum extraction conditions were that 100 g tea was extracted in 3 L water at 80 °C for 40 min and the catechins in the extracted solution were isolated using 1.5 L ethyl acetate for three times. The extracted catechins in the ethyl acetate phase was then decaffeinated by washing the organic phase with 1.5 L of 10 g L⁻¹ citric acid solution for three times. The obtained product contained 694.47 mg g⁻¹ catehchins and 37.89 mg g⁻¹ caffeine, with 78.8% caffeine being removed. The method is considered to be an alternative to replace traditional chloroform decaffeination.     

Microsomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 2,4-dichlorophenol—An endocrine disruptor compound

Cytochrome P450-3A4 (CYP3A4) is a monooxygenase enzyme that plays a major role in the detoxification of bioactive compounds and hydrophobic xenobiotics (e.g. medicines, drugs, environmental pollutants, food supplements and steroids). Physiologically the monooxygenation reactions of this class II, microsomal, b-type heme enzyme, usually requires cytochrome P450 reductase, NADPH. A novel CYP3A4 biosensor system that essentially simplified the enzymatic redox processes by allowing electron transfer between the electrode and the enzyme redox centre to occur, without any need for the physiological redox partners, was developed for the detection of 2,4-dichlorophenol (2,4-DCP), a priority environmental pollutant and an endocrine disruptor. The biosensor, GC/Naf-Co(Sep)³⁺/CYP3A4/Naf, was constructed by encapsulating CYP3A4 in a Nafion-cobalt (III) sepulchrate (Naf-Co(Sep)³⁺) composite film on a glassy carbon (GC) electrode. The responses of the biosensor to 2,4-dichlorophenol, erythromycin (CYP3A4 native substrate) and ketoconazole (CYP 3A4 natural inhibitor) were studied by cyclic and square wave voltammetric techniques. The detection limit (DL) of the biosensor for 2,4-dichlorophenol was 0.043 μg L⁻¹, which is by an order of magnitude lower than the EU limit (0.3 μg L⁻¹) for any pesticide compound in ground water. The biosensor's DL is lower than the U.S. Environmental Protection Agency's drinking water equivalent level (DWEL) value for 2,4-DCP, which is 2 μg L⁻¹.     

Hybrid electrochemical biosensor for organophosphorus pesticides quantification

An electrochemical biosensor for organophosphorus (OP) pesticides trace level concentrations determination was developed and characterized. It integrates a hybrid biorecognition element consisting of immobilized Arthrobacter globiformis and free acetylcholinesterase (ACh) with a Clark type oxygen probe transducer. The bacteria convert the ACh-generated choline to betaine with oxygen consumption measured as a Clark probe current change. This change representing the sensor response correlates to the concentration of the OP pesticides inhibiting the Ach-catalyzed acetylcholine hydrolysis to choline. The conditions for maximal sensor response to choline were optimized according to the methodology of design of experiments. The analytical performances of the enzyme substrate determination in a wide concentration range (0.1-20 μmol dm⁻³ of acetylcholine) and different ACh activities were established. It was demonstrated that the biosensor ensures reproducible, accurate and reliable chlorophos quantification reaching a limit of detection (LOD) of 1 nmol dm⁻³ and a sensitivity of 0.0252 μA/p(mol dm⁻³) under optimal experimental conditions. The biosensor response time is 200 s and the storage stability is t_L50 = 49 days for the bacterial membrane at ambient temperature. The device is reusable, the bacterial membrane being not affected by OP. The biosensor was applied to chlorophos determination in contaminated milk.     

Low potential detection of glucose at carbon nanotube modified glassy carbon electrode with electropolymerized poly(toluidine blue O) film

A mediator glucose biosensor has been constructed by immobilizing glucose oxidase at electropolymerized poly(toluidine blue O) film on carbon nanotube modified glass carbon electrode. The toluidine blue O moieties served as redox mediators for enzymatic glucose oxidation and as polymeric network to maintain the biosensor activity. Great enhancement in current response was observed for the glucose biosensor. The detection potential could be decreased to −0.1 V (versus Ag|AgCl), where common interferences such as ascorbic acid, uric acid and acetamidophenol were not oxidized to cause interferences. The amperometric glucose biosensor offered a sensitivity of 14.5 mA M⁻¹ cm⁻² for the linear range of 1-7 mM.     

Electrochemical study on cobalt film modified glassy carbon electrode and its application

A novel electroactive material for ascorbic acid (AA) determination was successfully prepared by plating/potential cycling method. The cobalt film was first deposited on the surface of glassy carbon electrode (GCE) in CoSO₄ solution by potential cycling, and then a cobalt film on the surface of GCE was activated by potential cycling in 0.1 mol L⁻¹ NaOH. The electrochemical performance of the resulted film (Co/GCE) and factors affecting its electrochemical activity were investigated by cyclic voltammetry and amperometry. This film electrode exhibited good electrocatalytic activity to the oxidation of AA. This biosensor had a fast response of AA less than 3 s and excellent linear relationships were obtained in the concentration range of 3 × 10⁻⁷ to 1 × 10⁻⁴ mol L⁻¹ with a detection limit of 2 × 10⁻⁷ mol L⁻¹ (S/N = 3) under the optimum conditions. Moreover, the selectivity, stability and reproducibility of this biosensor were evaluated with satisfactory results.     

Alcohol dehydrogenase amperometric biosensor based on a colloidal gold-carbon nanotubes composite electrode

A novel ethanol biosensor based on the bulk incorporation of alcohol dehydrogenase (ADH) into a colloidal gold (Au_coll)-multiwalled carbon nanotubes (MWCNTs) composite electrode using Teflon as binding material is reported. The composite Au_coll-MWCNTs-Teflon electrode exhibited significantly improved electrooxidation of NADH when compared with other carbon composite electrodes, including those based on carbon nanotubes. Amperometric measurements for NADH at +0.3 V showed significant differences in sensitivity between Au_coll-MWCNTs-Teflon and MWCNTs-Teflon composite electrodes. Incorporation of ADH into the bulk electrode material allowed the construction of a mediatorless ethanol biosensor. Both the enzyme loading and the NAD⁺ concentration in solution were optimized. The ADH-Au_coll-MWCNTs-Teflon biosensor allowed a limit of detection for ethanol of 4.7 μmol l⁻¹, which is remarkably better than those reported for other CNTs-based ADH biosensors. The apparent Michaelis-Menten constant was 4.95 mmol l⁻¹, which is much lower than that reported by immobilization of ADH onto a gold electrode. Both repeatability of the ethanol amperometric measurements, reproducibility with different biosensors, lifetime and storage ability can be, in general, advantageously compared with other ADH-CNTs biosensors. The biosensor was applied for the rapid determination of ethanol in commercial and certified beer samples.     

Direct electron transfer of horseradish peroxidase on Nafion-cysteine modified gold electrode

Direct electron transfer of horseradish peroxidase, immobilized on a functional membrane-modified gold electrode, was studied. The electrode showed a quasi-reversible electrochemical redox behavior with a formal potential of 60 mV (versus Ag/AgCl) in 20 mM potassium phosphate buffer solution at pH 7.0 and temperature 25 °C. The cathodic transfer coefficient was 0.42 and electron transfer rate constant was evaluated to be 1.6 s⁻¹. Furthermore, the modified electrode was used as a biosensor and exhibited a satisfactory stability and sensitivity to H₂O₂. The linear range of this biosensor for H₂O₂ determination was from 5.0 × 10⁻⁶ to 1.5 × 10⁻⁴ M while its detection limit, based on a signal-to-noise ratio of 3, was 1.3 × 10⁻⁶ M. The apparent Michaelis-Menten constant () for immobilized HRP was calculated to be 1.6 × 10⁻⁴ M.     

Cd transfer across water/1,2-dichloroethane microinterfaces facilitated by complex formation with 1,10-Phenanthroline

The transfer of Cd²⁺ facilitated by 1,10-Phenanthroline (phen) was investigated at the microinterface of two immiscible electrolyte solutions, hosted by a 25 μm diameter orifice of a micropipette. Cyclic voltammetry (CV) was employed to examine the transfer in the conditions of the ligand (organic phase) in excess and Cd²⁺ (aqueous phase) in excess. In these conditions, asymmetric (peak-shaped in the forward scan and steady state in the backward scan), and reversible steady state (for the two scan directions) voltammograms were observed. The dependence of half-wave potential on the ligand concentration suggested that the equilibrium was effectively displaced towards a 1:3 (Cd²⁺:ligand) stoichiometry, with a formation constant, β₃ = 3.9 × 10²⁹. The diffusion coefficients of Cd²⁺ in the aqueous solution and those of phen, Cd(phen)₃²⁺ in organic phase were evaluated to be 6.5 × 10⁻⁶, 5.8 × 10⁻⁶, and 5.1 × 10⁻⁶ cm² s⁻¹ respectively, using CV.     

A novel hydrogen peroxide biosensor based on Au-graphene-HRP-chitosan biocomposites

Graphene was prepared successfully by introducing -SO₃⁻ to separate the individual sheets. TEM, EDS and Raman spectroscopy were utilized to characterize the morphology and composition of graphene oxide and graphene. To construct the H₂O₂ biosensor, graphene and horseradish peroxidase (HRP) were co-immobilized into biocompatible polymer chitosan (CS), then a glassy carbon electrode (GCE) was modified by the biocomposite, followed by electrodeposition of Au nanoparticles on the surface to fabricate Au/graphene/HRP/CS/GCE. Cyclic voltammetry demonstrated that the direct electron transfer of HRP was realized, and the biosensor had an excellent performance in terms of electrocatalytic reduction towards H₂O₂. The biosensor showed high sensitivity and fast response upon the addition of H₂O₂, under the conditions of pH 6.5, potential −0.3 V. The time to reach the stable-state current was less than 3 s, and the linear range to H₂O₂ was from 5 × 10⁻⁶ M to 5.13 × 10⁻³ M with a detection limit of 1.7 × 10⁻⁶ M (S/N = 3). Moreover, the biosensor exhibited good reproducibility and long-term stability.     

A third-generation hydrogen peroxide biosensor based on Horseradish Peroxidase (HRP) enzyme immobilized in a Nafion-Sonogel-Carbon composite

A third-generation biosensor based on HRP and a Sonogel-Carbon electrode has been fabricated with the aim of monitoring hydrogen peroxide in aqueous media via a direct electron transfer process. The redox activity of native HRP, typical of thin-layer electrochemistry, was observed. The charge coefficient transfer, α, and the heterogeneous electron transfer rate constant, k_s, were calculated to be 0.51 ± 0.04 and 1.29 ± 0.04 s⁻¹, respectively. Topographic study by atomic force microscopy (AFM) shows that the enzyme may have been introduced inside the ionic cluster of the Nafion. The immobilized HRP exhibited excellent electrocatalytical response to the reduction of H₂O₂ and preserved its native state after the immobilization stage. Several important experimental variables were optimized. The resulting biosensor showed a linear response to H₂O₂ over a concentration range from 4 to 100 μM, with a sensitivity of 12.8 nA/μM cm⁻² and a detection limit of 1.6 μM, calculated as (3 S.D./sensitivity). The apparent Michaelis-Menten constant was calculated to be 0.295 ± 0.020 mM. The biosensor showed high sensitivity as well as good stability and reproducibility. The performance of the biosensor was evaluated with respect to four possible interferences.     

Synthesis of a poly(vinylcatechol-co-divinylbenzene) resin and accessibility to catechol units

Poly(vinylcatechol-co-divinylbenzene) resins have been prepared by suspension copolymerization of 3,4-dimethoxystyrene (DMS) with divinylbenzene (DVB) using toluene as the porogen and followed by deprotection of the catechol functions. The ratio of DMS versus DVB was modified and led to a maximum catechol content (determined by elemental analysis) varying from 0.82 to 3.16 mmol g⁻¹. Large surface areas were found for copolymers prepared with a divinylbenzene content larger than 35% v/v: from 464 to 658 m² g⁻¹. Accessibility (determined by back titration of the hydroxyl groups) was improved by prior contact with ethanol but remained inferior to 20%. This can be the result of the morphology of the resin beads and of the bottle-shape form of the pores, or/and of the embedding of the catechol units inside the polymer network. The sorption capacities were determined for Cu(II) and Cd(II) and reached 101.5 and 81.0 μmol g⁻¹ for copper and cadmium, respectively. Moreover, a significant variation of coloration has been observed upon complexation of copper by the resins.     

Amperometric glucose biosensor prepared with biocompatible material and carbon nanotube by layer-by-layer self-assembly technique

Prussian Blue (PB) based glucose biosensor was prepared by immobilizing glucose oxidase (GOD) in layer-by-layer (LBL) films with chitosan (Chi) and multi-walled carbon nanotubes (MWNTs). With the increasing of Chi/MWNTs/GOD layers, the response current to glucose was changed regularly and reached a maximum value when the number of layer was six. At the optimized condition, the biosensor exhibits excellent response performance to glucose with a linear range from 1 to 7 mM and a low detection limit of 0.05 mM. The biosensor also shows a high sensitivity of 8.017 μA mM⁻¹ cm⁻², which is attributed to the biocompatible nature of the LBL films. Furthermore, the biosensor shows rapid response, good reproducibility, long-term stability and freedom of interference from other co-existing electroactive species such as ascorbic acid and acetaminophen.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-coated carbon nanotubes nanocomposite for rapid detection of coliforms

A tyrosinase (Tyr) biosensor was developed based on Fe₃O₄ magnetic nanoparticles (MNPs)-coated carbon nanotubes (CNTs) nanocomposite and further applied to detect the concentration of coliforms with flow injection assay (FIA) system. Negatively charged MNPs were absorbed onto the surface of CNTs which were wrapped with cationic polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA). The Fe₃O₄ MNPs-coated CNTs nanocomposite was modified on the surface of the glassy carbon electrode (GCE), and Tyr was loaded on the modified electrode by glutaraldehyde. The immobilization matrix provided a good microenvironment for retaining the bioactivity of Tyr, and CNTs incorporated into the nanocomposite led to the improved electrochemical detection of phenol. The Tyr biosensor showed broad linear response of 1.0 × 10⁻⁸-3.9 × 10⁻⁵ M, low detection limit of 5.0 × 10⁻⁹ M and high sensitivity of 516 mA/M for the determination of phenol. Moreover, the biosensor integrated with a FIA system was used to monitor coliforms, represented by Escherichia coli (E. coli). The detection principle was based on determination of phenol which was produced by enzymatic reaction in the E. coli solution. Under the optimal conditions, the current responses obtained in the FIA system were proportional to the concentration of bacteria ranging from 20 to 1 × 10⁵ cfu/mL with detection limit of 10 cfu/mL and the overall assay time of about 4 h. The developed biosensor with the FIA system was well suited for quick and automatic clinical diagnostics and water quality analysis.     

Amperometric glutamate biosensor based on chitosan enzyme film

A simple method for enzyme immobilization in electrochemical biosensors for monosodium glutamate (MSG) was developed. The method relied on the precipitation of complexes of polyanionic enzyme l-glutamate oxidase (GmOx) with polycationic chains of chitosan (CHIT) on the surface of platinum electrode. Such ionotropic gelation allowed the CHIT matrix to retain ∼57% of applied GmOx (0.30-3.0 units). The CHIT + GmOx based biosensor displayed a low detection limit of 1.0 × 10⁻⁷ M MSG (S/N = 3, E = 0.400 V), linear range up to 2 × 10⁻⁴ M (R² = 0.991), sensitivity of 85 mA M⁻¹ cm⁻², and a short response time (t_90% = 2 s). The biosensors maintained ∼80% of the MSG signal even after 11 h of continuous use, which indicated good operational stability. Stability studies revealed that a majority of signal loss was due to a slow transformation of glutamate in a solution into the redox inactive pyroglutamate. After 4 months of storage in water at 4 °C, the CHIT + GmOx films retained ∼80-90% of their original activity toward the MSG. The CHIT + GmOx films are promising candidates for the development of simple and reliable chromatographic detectors for glutamate.     

Nucleic acid biosensor for DNA hybridization detection using rutin-Cu as an electrochemical indicator

The complex of rutin-Cu (C₈₁H₈₆Cu₂O₄₈, abbreviated by Cu₂R₃, R = rutin) was synthesized and characterized by elemental analysis and IR spectra. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Cu₂R₃ with salmon sperm DNA. It was revealed that Cu₂R₃ could interact with double-stranded DNA (dsDNA) by a major intercalation role. Using Cu₂R₃ as a novel electroactive indicator, an electrochemical DNA biosensor for the detection of specific DNA fragment was developed and its selectivity for the recognition with different target DNA was assessed by differential pulse voltammetry (DPV). The target DNA related to coliform virus gene could be quantified ranged from 1.62 × 10⁻⁸ mol L⁻¹ to 8.10 × 10⁻⁷ mol L⁻¹ with a good linearity (r = 0.9989) and a detection limit of 2.3 × 10⁻⁹ mol L⁻¹ (3σ, n = 7) was achieved by the constructed electrochemical DNA biosensor.     

Suppression of floatability of pyrite in coal processing by carrier microencapsulation

Carrier microencapsulation, CME, is a technique to form a thin layer of metal oxide or hydroxide on pyrite surface using a water soluble organic carrier combined with metal ions. The present study investigated the effect of CME using a tris-catecholato complex of Si⁴⁺, Si(cat)₃²⁻ on pyrite-coal separation by dynamic bubble pick-up experiments and Hallimond tube flotation experiments using coal, pyrite, and a coal-pyrite mixture. The mineral samples were treated in 0-5 mol m^− 3 Si(cat)₃²⁻ solutions at pH 4-9 at treatment times of 1-24 h. Dynamic bubble pick-up experiments showed that CME treatment converted the pyrite surface from hydrophobic to hydrophilic but did not affect coal's hydrophobic surface. The results of the Hallimond tube flotation experiments of a coal-pyrite mixture at pH 7-9 in the presence of kerosene as a collector showed that pyrite floatability was selectively suppressed after 1 h CME treatment with 0.5 mol^− 3 Si(cat)₃²⁻ while both coal and pyrite were floated without the treatment. This indicates that CME treatment is effective in suppressing pyrite floatability in coal-pyrite flotation.