Bilayer lipid membrane biosensor with enhanced stability for amperometric determination of hydrogen peroxide

In this paper, a polydopamine (PDA) film is electropolymerized on the surface of bilayer lipid membrane (BLM) which is immobilized with horseradish peroxidase (HRP). The coverage of the PDA film on HRP/BLM electrode is monitored by electrochemical impedance spectroscopy (EIS). The electrocatalytic reduction of H₂O₂ at the PDA/HRP/BLM electrode is studied by means of cyclic voltammetry (CV). The biosensor has a fast response to H₂O₂ of less than 5 s and an excellent linear relationship is obtained in the concentration range from 2.5 × 10⁻⁷ to 3.1 × 10⁻³ mol L⁻¹, with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The response current of BLM/HRP/PDA biosensor retains 84% of its original response after being stored in 0.1 mol L⁻¹ pH 7.0 PBS at 4 °C for 3 weeks. The selectivity, repeatability, and storage stability of PDA/HRP/BLM biosensor are greatly enhanced by the coverage of polydopamine film on BLM.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

Amperometric biosensor for hydrogen peroxide based on coimmobilized horseradish peroxidase and methylene green in ormosils matrix with multiwalled carbon nanotubes

A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV-vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H₂O₂ at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H₂O₂, less than 10 s, and excellent linear range of concentration from 5 × 10⁻⁷ to 2 × 10⁻⁵ M with the detection limit of 0.5 μM (S/N = 3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4 °C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability.     

Sensitive DNA biosensor improved by Luteolin copper(II) as indicator based on silver nanoparticles and carbon nanotubes modified electrode

A novel and sensitive electrochemical DNA biosensor has been developed for the detection of DNA hybridization. The biosensor was proposed by using copper(II) complex of Luteolin C₃₀H₁₈CuO₁₂ (CuL₂) as an electroactive indicator based on silver nanoparticles and multi-walled carbon nanotubes (Ag/MWCNTs) modified glassy carbon electrode (GCE). In this method, the 4-aminobenzoic acid (4-ABA) and Ag nanoparticles were covalently grafted on MWCNTs to form Ag/4-ABA/MWCNTs. The proposed method dramatically increased DNA attachment quantity and complementary ssDNA detection sensitivity for its large surface area and good charge-transport characteristics. DNA hybridization detection was performed using CuL₂ as an electroactive indicator. The CuL₂ was synthesized and characterized using elemental analysis (EA) and IR spectroscopy. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction between CuL₂ and ds-oligonucleotides (dsDNA). It was revealed that CuL₂ presented high electrochemical activity on GCE, and it could be intercalated into the double helices of dsDNA. The target ssDNA of the human hepatitis B virus (HBV) was quantified in a linear range from 3.23 × 10⁻¹² to 5.31 × 10⁻⁹ M (r = 0.9983). A detection limit of 6.46 × 10⁻¹³ M (3σ, n = 11) was achieved.     

Hydrogen peroxide and peracetic acid determination in waste water using a reversible reagentless biosensor

During the reversible reaction between peroxidase (HRP) and peroxides, several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for their determination. On this basis, a reversible reagentless optical biosensor based on HRP for hydrogen peroxide and peracetic acid determinations has been developed. The biosensor (which can be used for at least 3 months and/or more than 200 measurements) is prepared by HRP entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the analyte concentration is also presented. Both peroxides show similar responses in the sensor film. Under the recommended working conditions, the biosensor shows linear response ranges from 6 × 10⁻⁷ to 1.0 × 10⁻⁴ M using FIA mode, and from 2 × 10⁻⁷ to 1.5 × 10⁻⁵ M using continuous mode for both peroxides; the precision, expressed as R.S.D., is about 4%. This biosensor has been applied for peroxide determination in waste water samples previously treated with peroxides.     

Amperometric biosensor for hydrogen peroxide based on hemoglobin entrapped in titania sol-gel film

Hemoglobin (Hb) was entrapped in a titania sol-gel matrix and used as a mimetic peroxidase to construct a novel amperometric biosensor for hydrogen peroxide. The Hb entrapped titania sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process for protein immobilization. The morphologies of both titania sol-gel and the Hb films were characterized using scanning electron microscopy (SEM) and proved to be chemically clean, porous, homogeneous. This matrix provided a biocompatible microenvironment for retaining the native structure and activity of the entrapped Hb and a very low mass transport barrier to the substrates. H₂O₂ could be reduced by the catalysis of the entrapped hemoglobin at −300 mV without any mediator. The reagentless H₂O₂ sensor exhibited a fast response (less than 5 s) and sensitivity as high as 1.29 mA mM⁻¹ cm⁻². The linear range for H₂O₂ determination was from 5.0×10⁻⁷ to 5.4×10⁻⁵ M with a detection limit of 1.2×10⁻⁷ M. The apparent Michaelis-Menten constant of the encapsulated hemoglobin was calculated to be 0.18±0.02 mM. The stability of the biosensor was also evaluated.     

Uric acid determination using uricase and the autotransducer molecular absorption properties of peroxidase

Uric acid (UA) is determined using the UV-vis molecular absorption properties of peroxidase (HRP). The method as a whole involves UA oxidation in the presence of uricase (UOx), giving H₂O_2. The H₂O₂ then reacts with HRP forming the compound I species which returns to its initial form by reaction with UA and intramolecular reduction. The molecular absorption changes of HRP at 420 nm during the reaction enable the UA to be determined. A mathematical model relating the analytical signal to UA, UOx and HRP has been developed and experimentally validated. The possibility of carrying out both enzymatic reactions sequentially or simultaneously is discussed, the latter option producing better analytical performances. The method permits UA determination in the range 1.5 × 10⁻⁶-4.0 × 10⁻⁵ M, with an R.S.D. of about 3% (n = 5, 1.5 × 10⁻⁶ M UA). It has been applied to analyte determination in synthetic serum samples.     

Electrochemistry and scanning electron microscopy of polyaniline/peroxidase-based biosensor

An amperometric biosensor was prepared by in situ deposition of horseradish peroxidase (HRP) enzyme on a polyaniline (PANI)-doped platinum disk electrode. The PANI film was electrochemically deposited on the electrode at 100 mV s⁻¹/Ag-AgCl. Cyclic voltammetric characterization of the PANI film in 1 M HCl showed two distinct redox peaks, which prove that the PANI film was electroactive and exhibited fast reversible electrochemistry. The surface concentration and film thickness of the adsorbed electroactive species was estimated to be 1.85×10⁻⁷ mol cm⁻² and approximately 16 nm, respectively. HRP was electrostatically immobilized onto the surface of the PANI film, and voltammetry was used to monitor the electrocatalytic reduction of hydrogen peroxide under diffusion-controlled conditions. Linear responses over the concentration range 2.5×10⁻⁴ to 5×10⁻³ M were observed. Spectroelectrochemistry was used to monitor the changes in UV-vis properties of HRP, before and after the catalysis of H₂O₂. The biosensor surface morphology was characterized by scanning electron microscopy (SEM) using PANI-doped screen-printed carbon electrodes (SPCEs) in the presence and absence of (i) peroxidase and (ii) peroxide. The SEM images showed clear modifications of the conducting film surface structure when doped with HRP, as well as the effect of hydrogen peroxide on the morphology of biosensor.     

A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO₃⁻) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO₃⁻ layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO₃⁻-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO₃⁻. The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH₃)₆³⁺ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10¹³ strands cm⁻² and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)₃^3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)₃^3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10⁻¹³ M to 1.0 × 10⁻⁸ M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2 × 10⁻¹⁴ M based on 3σ.     

Preparation of cobalt-tetraphenylporphyrin/reduced graphene oxide nanocomposite and its application on hydrogen peroxide biosensor

A novel cobalt-tetraphenylporphyrin/reduced graphene oxide (CoTPP/RGO) nanocomposite was prepared by a π–π stacking interaction and characterized by ultraviolet–visible absorption spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). The CoTPP/RGO nanocomposite exhibited high electrocatalytic activity both for oxidation and reduction of H₂O₂. The current response was linear to H₂O₂ concentration with the concentration range from 1.0 × 10⁻⁷ to 2.4 × 10⁻³ mol L⁻¹ (R = 0.998) at the reductive potential of −0.20 V and from 1.0 × 10⁻⁷ to 4.6 × 10⁻⁴ mol L⁻¹ (R = 0.996) at the oxidative potential of +0.50 V. The H₂O₂ biosensor showed good anti-interfering ability towards oxidative interferences at the oxidative potential of +0.50 V and good anti-interfering ability towards reductive interferences at the reductive potential of −0.20 V.     

Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Electrogenerated chemiluminescence biosensor based on Ru(bpy)3(2+) and dehydrogenase immobilized in sol-gel/chitosan/poly(sodium 4-styrene sulfonate) composite material

A new electrogenerated chemiluminescence biosensor was fabricated by immobilizing ECL reagent Ru(bpy)₃²⁺ and alcohol dehydrogenase in sol-gel/chitosan/poly(sodium 4-styrene sulfonate) (PSS) organically modified composite material. The component PSS was used to immobilize ECL reagent Ru(bpy)₃²⁺ by ion-exchange, while the addition of chitosan was to prevent the cracking of conventional sol-gel-derived glasses and provide biocompatible microenvironment for alcohol dehydrogenase. Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection for quantification of enzyme substrate and it was much simpler than previous double-layer design. The detection limit was 9.3 × 10⁻⁶ M for alcohol (S/N = 3) with a linear range from 2.79 × 10⁻⁵ to 5.78 × 10⁻² M. With ECL detection, the biosensor exhibited wide linear range, high sensitivity and good stability.     

Integrated multienzyme electrochemical biosensors for the determination of glycerol in wines

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150 mV (vs. Ag/AgCl), and the reduction of TTF⁺ at 0 mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0 × 10⁻⁶ to 2.0 × 10⁻⁵ or 1.0 × 10⁻⁶ to 1.0 × 10⁻⁵ M glycerol and sensitivities of 1214 ± 21 or 1460 ± 34 μA M⁻¹ were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0 × 10⁻⁷ and 3.1 × 10⁻⁷ M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines

Integrated amperometric biosensors for the determination of l-malic and l-lactic acids were developed by coimmobilization of the enzymes l-malate dehydrogenase (MDH) and diaphorase (DP), or l-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100 mV (vs. Ag/AgCl), and the reduction of TTF⁺ at −50 mV were used for the monitoring of the enzyme reactions involved in l-malic and l-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for l-malic and l-lactic were obtained with linear ranges of 5.2 × 10⁻⁷ to 2.0 × 10⁻⁵ and 4.2 × 10⁻⁷ to 2.0 × 10⁻⁵ M, respectively. The calculated detection limits were 5.2 × 10⁻⁷ and 4.2 × 10⁻⁷ M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748^T into a synthetic wine. Samples collected during MLF were assayed for l-malic and l-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits.     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

Enthalpy change and mechanism of oxidation of o-phenylenediamine by hydrogen peroxide catalyzed by horseradish peroxidase

The hydrogen peroxide-oxidation of o-phenylenediamine (OPD) catalyzed by horseradish peroxidase (HRP) at 37 °C in 50 mM phosphate buffer (pH 7.0) was studied by calorimetry. The apparent molar reaction enthalpy with respect to OPD and hydrogen peroxide were −447 ± 8 kJ mol⁻¹ and −298 ± 9 kJ mol⁻¹, respectively. Oxidation of OPD by H₂O₂ catalyzed by HRP (1.25 nM) at pH 7.0 and 37 °C follows a ping-pong mechanism. The maximum rate V_max (0.91 ± 0.05 μM s⁻¹), Michaelis constant for OPD K_m,S (51 ± 3 μM), Michaelis constant for hydrogen peroxide Km,H₂O₂ (136 ± 8 μM), the catalytic constant k_cat (364 ± 18 s⁻¹) and the second-order rate constants k₊₁ = (2.7 ± 0.3) × 10⁶ M⁻¹ s⁻¹ and k₊₅ = (7.1 ± 0.8) × 10⁶ M⁻¹ s⁻¹ were obtained by the initial rate method.     

Tyrosinase immobilized magnetic nanobeads for the amperometric assay of enzyme inhibitors: application to the skin whitening agents

The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC₅₀, under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC₅₀ = 3.7 × 10⁻⁶ M, K_i = 8.6 × 10⁻⁷ M), ascorbic acid (IC₅₀ = 1.2 × 10⁻⁵ M), benzoic acid (IC₅₀ = 7.2 × 10⁻⁵ M, K_i = 2.0 × 10⁻⁵ M) and azelaic acid (IC₅₀ = 1.3 × 10⁻⁴ M, K_i = 4.2 × 10⁻⁵ M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.     

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10⁻⁷ M), short response time (within 10 s), high sensitivity (88.6 μA mM⁻¹ cm⁻²) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H₂O₂ by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083 ± 0.001) ×10⁻³ M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.     

Examination of performance of glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection

A composite electrode was prepared by modifying glassy carbon microparticles with gold nanoparticles (Au-nps) and xanthine oxidase enzyme (XOD) for xanthine (X) and hypoxanthine (Hx) detection. After the optimization of the system for X, the biosensor was characterized for X and Hx. A linearity was obtained in the concentration range between 5.00 × 10⁻⁷ and 1.00 × 10⁻⁵ M for X with equation of y = 0.24x + 0.712 and 5.00 × 10⁻⁶ to 1.50 × 10⁻⁴ M for Hx, with equation of y = 0.014x + 0.575, respectively. Obtained results were compared to X and/or Hx biosensors including/not including Au-np in the structure. The developed system was also applied for detection of Hx in canned tuna fish sample and very promising results were obtained.