Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO₂/Si₃N₄ electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10⁻⁴ to 10⁻¹ M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol⁻¹). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules.     

Amperometric xanthine biosensors based on electrodeposition of platinum on polyvinylferrocenium coated Pt electrode

Novel xanthine biosensors were successfully fabricated by immobilizing xanthine oxidase on polyvinylferrocenium perchlorate matrix (PVF⁺ClO₄⁻) and platinum electrodeposited polyvinylferrocenium perchlorate matrix. PVF⁺ClO₄⁻ film was coated on Pt electrode at +0.7 V vs. Ag/AgCl by electrooxidation of polyvinylferrocene (PVF). Platinum nanoparticles were deposited on PVF⁺ClO₄⁻ electrode by electrochemical deposition in 2.0 mM H₂PtCl₆ solution at −0.2 V. Xanthine oxidase was incorporated into the polymer matrix via ion exchange process by immersing modified Pt electrodes in the enzyme solution. The amperometric responses of the biosensors were measured via monitoring oxidation current of hydrogen peroxide at +0.5 V. Under the optimal conditions, the linear ranges of xanthine detection were determined as 1.73 × 10⁻³–1.74 mM for PVF⁺XO⁻ and 0.43 × 10⁻³–2.84 mM for PVF⁺XO⁻/Pt. The detection limits of xanthine were 5.20 × 10⁻⁴ mM for PVF⁺XO⁻ and 1.30 × 10⁻⁴ mM for PVF⁺XO⁻/Pt. Moreover, the effects of applied potential, electrodeposition potential, H₂PtCl₆ concentration, amount of electrodeposited Pt nanoparticles, thickness of polymeric film, temperature, immobilization time, xanthine and xanthine oxidase concentrations on the response currents of the biosensors were investigated in detail. The effects of interferents, the operational and storage stabilities of biosensors and the applicabilities to drug samples of the biosensors analysis were also evaluated.     

Zirconia-poly(propylene imine) dendrimer nanocomposite based electrochemical urea biosensor

In this article we report a selective urea electrochemical biosensor based on electro-co-deposited zirconia-polypropylene imine dendrimer (ZrO₂-PPI) nanocomposite modified screen printed carbon electrode (SPCE). ZrO₂ nanoparticles, prepared by modified sol–gel method were dispersed in PPI solution, and electro-co-deposited by cyclic voltammetry onto a SPCE surface. The material and the modified electrodes were characterised using FTIR, electron microscopy and electrochemistry. The synergistic effect of the high active surface area of both materials, i.e. PPI and ZrO₂ nanoparticles, gave rise to a remarkable improvement in the electrocatalytic properties of the biosensor and aided the immobilisation of the urease enzyme. The biosensor has an ampereometric response time of ∼4 s in urea concentration ranging from 0.01 mM to 2.99 mM with a correlation coefficient of 0.9985 and sensitivity of 3.89 μA mM⁻¹ cm⁻². The biosensor was selective in the presence of interferences. Photochemical study of the immobilised enzyme revealed high stability and reactivity.     

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

A biosensor based on gold nanoparticles,dihexadecylphosphate, and tyrosinase for the determination of catechol in natural water

In this work, a biosensor using a glassy carbon electrode modified with gold nanoparticles (AuNPs) and tyrosinase (Tyr) within a dihexadecylphosphate film is proposed. Cystamine and glutaraldehyde crosslinking agents were used as a support for Tyr immobilization. The proposed biosensor was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and cyclic voltammetry in the presence of catechol. The determination of catechol was carried out by amperometry and presented a linear concentration range from 2.5 × 10⁻⁶ to 9.5 × 10⁻⁵ mol L⁻¹ with a detection limit of 1.7 × 10⁻⁷ mol L⁻¹. The developed biosensor showed good repeatability and stability. Moreover, this novel amperometric method was successfully applied in the determination of catechol in natural water samples. The results were in agreement with a 95% confidence level for those obtained using the official spectrophotometric method.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV–vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of −0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10⁻¹⁰ mol cm⁻² and 3.36 s⁻¹, respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM – 2 mM with LOD of 4.1 μM, (2) 2 mM – 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H₂O₂. The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H₂O₂ in the linear range from 2.6 × 10^− 6 mol/L to 8.8 × 10^− 3 mol/L with a detection limit of 6.4 × 10^− 7 mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

Evaluation of the adverse effects of two commonly used fertilizers,DAP and urea,on motility and orientation of the green flagellate Euglena gracilis

The effect of two commonly used fertilizers, DAP (diammonium phosphate) and urea was studied on the freshwater flagellate Euglena gracilis using the automatic biotest ECOTOX. NOEC and EC₅₀ values for various parameters like motility, velocity, cell shape and gravitaxis were calculated. The NOEC and EC₅₀ values obtained for DAP were much lower than those for urea; i.e. DAP showed a stronger inhibitory effect as compared to urea. The inhibition caused by DAP increased with increasing exposure time over 24 h but urea showed no augmentation with increasing exposure time. Application of DAP resulted in an increased pH and high concentrations of ammonia but urea did neither affect the pH nor affect the ammonia concentration. Recovery experiments in deionized water after urea application showed a reconstitution of motility after 72 h. After an application of 1.35 g L⁻¹ (24 h EC₅₀ for motility) DAP motility recovered after 72 h but motility did not recover when the concentration was doubled (2.7 g L⁻¹). The EC₅₀ values obtained were compared with the EC₅₀/LC₅₀ values reported for other aquatic organisms and were found to be comparable with the reported values.     

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.     

Nitrogen uptake kinetics of Prymnesium parvum (Haptophyte)

The uptake rates of different nitrogen (N) forms (NO₃⁻, urea, and the amino acids glycine and glutamic acid) by N-deficient, laboratory-grown cells of the mixotrophic haptophyte, Prymnesium parvum, were measured and the preference by the cells for the different forms determined. Cellular N uptake rates (ρ_cell, fmol N cell⁻¹ h⁻¹) were measured using ¹⁵N-labeled N substrates. P. parvum showed high preference for the tested amino acids, in particular glutamic acid, over urea and NO₃⁻ under the culture nutrient conditions. However, extrapolating these rates to Baltic Seawater summer conditions, P. parvum would be expected to show higher uptake rates of NO₃⁻ and the amino acids relative to urea because of the difference in average concentrations of these substrates. A high uptake rate of glutamic acid at low substrate concentrations suggests that this substrate is likely used through extracellular enzymes. Nitrate, urea and glycine, on the other hand, showed a non-saturating uptake over the tested substrate concentration (1–40 μM-N for NO₃⁻ and urea, 0.5–10 μM-N for glycine), indicating slower membrane-transport rates for these substrates.     

Synthesis,biological evaluation and molecular docking of N-phenyl thiosemicarbazones as urease inhibitors

Urease is an important enzyme which breaks urea into ammonia and carbon dioxide during metabolic processes. However, an elevated activity of urease causes various complications of clinical importance. The inhibition of urease activity with small molecules as inhibitors is an effective strategy for therapeutic intervention. Herein, we have synthesized a series of 19 benzofurane linked N-phenyl semithiocarbazones (3a–3s). All the compounds were screened for enzyme inhibitor activity against Jack bean urease. The synthesized N-phenyl thiosemicarbazones had varying activity levels with IC₅₀ values between 0.077 ± 0.001 and 24.04 ± 0.14 μM compared to standard inhibitor, thiourea (IC₅₀ = 21 ± 0.11 μM). The activities of these compounds may be due to their close resemblance of thiourea. A docking study with Jack bean urease (PDB ID: 4H9M) revealed possible binding modes of N-phenyl thiosemicarbazones.     

Gastro-intestinal handling of water and solutes in three species of elasmobranch fish,the white-spotted bamboo shark,Chiloscyllium plagiosum,little skate,Leucoraja erinacea and the clear nose skate Raja eglanteria

The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca²⁺ (19.67 ± 3.65 mM) and Mg²⁺ (43.99 ± 5.11 mM) were high in L. erinacea and Mg²⁺ was high (130.0 ± 39.8 mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO₃^? values were low (8.19 ± 2.42 and 8.63 ± 1.48 mM) in both skates but very high in C. plagiosum (73.3 ± 16.3 mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0 ± 8.1 to 0 mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.     

Two ways to achieve an anammox influent from real reject water treatment at lab-scale: Partial SBR nitrification and SHARON process

A comparative study to produce the correct influent for Anammox process from anaerobic sludge reject water (700–800 mg NH₄⁺-N L⁻¹) was considered here. The influent for the Anammox process must be composed of NH₄⁺-N and NO₂⁻-N in a ratio 1:1 and therefore only a partial nitrification of ammonium to nitrite is required. The modifications of parameters (temperature, ammonium concentration, pH and solid retention time) allows to achieve this partial nitrification with a final effluent only composed by NH₄⁺-N and NO₂⁻-N at the right stoichiometric ratio. The equal ratio of HCO₃⁻/NH₄⁺ in reject water results in a natural pH decrease when approximately 50% of NH₄⁺ is oxidised. A Sequencing batch reactor (SBR) and a chemostat type of reactor (single-reactor high activity ammonia removal over nitrite (SHARON) process) were studied to obtain the required Anammox influent. At steady state conditions, both systems had a specific conversion rate around 40 mg NH₄⁺-N g⁻¹ volatile suspended solids (VSS) h⁻¹, but in terms of absolute nitrogen removal the SBR conversion was 1.1 kg N day⁻¹ m⁻³, whereas in the SHARON chemostat was 0.35 kg N day⁻¹ m⁻³ due to the different hydraulic retention time (HRT) used. Both systems are compared from operational (including starvation experiments) and kinetic point of view and their advantages/disadvantages are discussed.     

An o-aminobenzoic acid film-based immunoelectrode for detection of the cardiac troponin T in human serum

An amperometric immunosensor for cardiac troponin T detection in human serum troponin T, a marker considered as “gold standard” for acute myocardial infarction diagnosis, is described. A stable carboxylic film to covalently bind antibodies against cTnT onto electrode surface was achieved with electropolymerization of the o-aminobenzoic acid. A fractional factorial study was performed to optimize the electropolymerization parameters. Cyclic voltammetry assays were carried out for characterize steps of the modified electrode surface. The obtained calibration curve at −0.05 V by amperometry presented a good linear response range from 0.05 to 5.0 ng mL⁻¹ cTnT with a correlation coefficient of 0.992 (n = 6) and 0.016 ng mL⁻¹ detection limit. The electrodes showed a good stability upon the analytical responses retaining 91.6% of its initial response after 18 days. This sensor showed outgoing results regarding sensitivity allowing reliable measurements of the cTnT at levels of clinical significance for acute myocardial infarctions diagnosis.     

Gallic acid interference on polyphenolic amperometric biosensing using Trametes versicolor laccase

The present work reports the gallic acid (GA) interference on polyphenolic amperometric biosensing using Trametes versicolor laccase (TvLac). GA′ inhibitory effect on TvLac activity was investigated on the oxidation of caffeic acid (CA) by free TvLac and its immobilised form on modified polyethersulfone membrane (PES/TvLac), using spectrophotometric and amperometric biosensor detection methods. The results have indicated that GA presents inhibitory behaviour on TvLac activity in a concentration-dependent manner. The GA concentration leading to 50% activity lost, IC₅₀, was determined to be 19.15 ± 0.11 μM and 5.11 ± 0.19 μM for free and immobilised enzyme, respectively. The results have also shown that GA exhibited a competitive and a mixed inhibition types on the TvLac activity for spectrophotometric and amperometric biosensor methods, respectively. Further GA′ and CA′ cyclic voltammetry studies have demonstrated that GA′ oxidation products interfered with CA′ redox reaction products. In fact, a decrease of the reduction current was observed at cyclic voltammograms of CA, when mixed with GA. Therefore, the GA′ interference on polyphenolic amperometric biosensing is the result of the combination of two factors: on one hand, we have the inhibitory enzymatic effect, and on the other, the reaction of GA′ oxidation products with the o-quinones obtained by the enzymatic oxidation of CA. Both gave rise to the amperometric signal decreasing effect.     

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC₅₀ = 0.5 nM) with a strong binding to sEH (K_i = 3.1 nM) and a moderately long residence time on the enzyme (k_off = 1.05 × 10⁻³ s⁻¹; t_1/2 = 11 min).     

Process intensification for an enhanced replication of a newly adapted RM-65 sheep pox virus strain in Vero cells grown in stirred bioreactor

Sheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 10^5.875 TCID₅₀ (median tissue culture infective dose) ml⁻¹.To optimize the production process, the effects of MOI (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MOI of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supplemented with 5 mM glucose, 5 mM fructose and 25 mM sucrose. Spinner culture confirms these data; virus titer was 10^7.375 TCID₅₀ ml⁻¹.In addition Vero cells were cultivated in a 7-l bioreactor in batch mode on 3 g l⁻¹ Cytodex1, and infected at cell seeding at a MOI of 0.005. Maximal virus titer was 10^7.275 TCID₅₀ ml⁻¹. This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM + 2% FCS.     

Development of electrochemical sulfite biosensor based on SOX/PBNPs/PPY modified Au electrode

A sulfite oxidase (SO_X) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto Prussian blue nanoparticles/polypyrrole (PBNPs/PPY) nanocomposite film electrodeposited onto the surface of gold (Au) electrode. An electrochemical sulfite biosensor was fabricated using SO_X/PBNPs/PPY/Au electrode as working electrode, Ag/AgCl as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The working electrode was characterized by Fourier Transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor showed optimum response within 2 s, when operated at 20 mV s⁻¹ in 0.1 M Tris–HCl buffer, pH 8.0 and at 30 °C. Linear range and minimum detection limit were 0.5–1000 μM and 0.1 μM (S/N = 3) respectively. The sensor was evaluated with 95.0% recovery of added sulfite in red wine samples and 1.9% and 3.3% within and between batch coefficients of variation respectively. There was a good correlation (r = 0.96) between red wine samples sulfite value by standard DTNB method and the present method. The sensor was employed for determination of sulfite level in red, white and rose wine samples. The enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C.