Amperometric Tyrosinase Biosensor Based on Carbon Nanotube–Titania–Nafion Composite Film

A highly sensitive and stable amperometric tyrosinase biosensor has been developed based on multiwalled carbon nanotube (MWCNT) dispersed in mesoporous composite films of sol–gel‐derived titania and perfluorosulfonated ionomer (Nafion). Tyrosinase was immobilized within a thin film of MWCNT–titania–Nafion composite film coated on a glassy carbon electrode. Phenolic compounds were determined by the direct reduction of biocatalytically‐liberated quinone species at ?100 mV versus Ag/AgCl (3 M NaCl) without a mediator. The present tyrosinase biosensor showed good analytical performances in terms of response time, sensitivity, and stability compared to those obtained with other biosensors based on different sol–gel matrices. Due to the large pore size of the MWCNT–titania–Nafion composite, the present biosensor showed remarkably fast response time with less than 3 s. The present biosensor responds linearly to phenol from 1.0×10^?7 M to 5.0×10^?5 M with an excellent sensitivity of 417 mA/M and a detection limit of 9.5×10^?8 M (S/N=3). The enzyme electrode retained 89% of its initial activity after 2 weeks of storage in 50 mM phosphate buffer at pH 7.0.     

Fabrication of Tyrosinase Biosensor Based on Multiwalled Carbon Nanotubes‐Chitosan Composite and Its Application to Rapid Determination of Coliforms

A tyrosinase (Tyr) biosensor was fabricated by immobilizing Tyr on the surface of multiwalled carbon nanotubes (MWNTs)‐chitosan (Chit) composite modified glassy carbon electrode (GCE). The MWNTs‐Chit composite film provided a biocompatible platform for the Tyr to retain the bioactivity and the MWNTs possessed excellent inherent conductivity to enhance the electron transfer rate. The Tyr/MWNTs‐Chit/GCE biosensor showed high sensitivity (412 mA/M), broad linear response (1.0×10^?8–2.8×10^?5 M), low detection limit (5.0 nM) and good stability (remained 93% after 10 days) for determination of phenol. The biosensor was further applied to rapid detection of the coliforms, represented by Escherichia coli (E. coli) in this work. The current responses were proportional to the quantity of coliforms in the range of 10⁴–10⁶ cfu/mL. After 5.0 h of incubation, E. coli could be detected as low as 10 cfu/mL.     

Layered Zirconium(IV) Aminoethylphosphonate Intercalated with Horseradish Peroxidase for an Amperometric Biosensor

Layered zirconium(IV) aminoethylphosphonate (ZrAEP) have been used as matrices for immobilization of horseradish peroxidase (HRP) to fabricate enzyme electrode for an amperometric biosensor. The biocompatible HRP–ZrAEP films were fabricated on gold electrode surface by electro‐co‐deposition method. The morphology of the HRP–ZrAEP composite was characterized by scanning electron microscopy (SEM). UV–vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the ZrAEP. The immobilized HRP at the HRP–ZrAEP films exhibited good electro catalytic responses to the reduction of hydrogen peroxide. The response time of the biosensor was less than 3 s, and the linear range is from 2.5 × 10^?6 to 3.22 × 10^?3 M, with a detection limit of 7.0 × 10^?7 M (S/N = 3). The Michaelis–Menten constant (K^app_M) value is estimated to be 2.21 mM. In addition, the obtained biosensor possesses high sensitivity, good stability and reproducibility.     

Determination of Phenolic Compounds Based on Co‐Immobilization of Methylene Blue and HRP on Multi‐Wall Carbon Nanotubes

This work evaluated an amperometric biosensor based on multi‐wall carbon nanotubes (MWCNT), chemically modified with methylene blue (Met) and horseradish peroxidase (HRP), for detection of phenolic compounds. The dependences of the biosensor response due to the enzyme immobilization procedure, HRP amounts, pH and working potential were investigated. The amperometric response for catechol using the proposed biosensor showed a very wide linear response range (1 to 150 μmol L^?1), good sensitivity (50 nA cm^?2 μmol^?1 L), excellent operational stability (after 300 determinations the response remained at 97%) and very good storage stability (lifetime>3 months). Based on all these characteristics, it is possible to affirm that the material is promising for phenol detection due to its good electrochemical response and enzyme stabilization. The biosensor response for various phenolic compounds was investigated.     

Development of an Amperometric Enzymatic Biosensor Based on Gold Modified Magnetic Nanoporous Microparticles

Gold modified nanoporous silica based magnetic microparticles have been prepared as support for the immobilization of the enzyme horseradish peroxidase (HRP). The enzyme modified gold microparticles were retained onto the surface of a solid carbon paste electrode with the help of a permanent magnet. The analytical performances of the resulting biosensor were characterized by studying hydroquinone (HQ) and hydrogen peroxide. The former was monitored by the direct electroreduction of the biocatalytically generated quinone. Several experimental parameters influencing the biosensor response were investigated. A linear response to HQ was obtained in the concentration range comprised between 5×10^?7 and 4.5×10^?6 M with a detection limit of 4×10^?7 M. The enzyme electrode provided a linear response to hydrogen peroxide over a concentration range comprised between 5×10^?7?1.3×10^?4 M with a detection limit of 4×10^?7 M. The inhibition of the biosensor response in the presence of thiols e.g. cysteine, captopril, glutathione and Nacystelyn (NAL) has been pointed out.     

Synthesis of multi-branched gold nanoparticles by reduction of tetrachloroauric acid with Tris base, and their application to SERS and cellular imaging

A biosensor for hydrogen peroxide was constructed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe₂O₄ nanoparticles on a glassy carbon electrode (GCE). The electron mediator carboxyferrocene was also immobilized on the surface of the GCE. UV?Cvis spectra, Fourier transform IR spectra, scanning electron microscopy, and electrochemical impedance spectra were acquired to characterize the biosensor. The experimental conditions were studied and optimized. The biosensor responds linearly to H₂O₂ in the range from 1.0?×?10^?5 to 2.0?×?10^?3?M and with a detection limit of 2.0?×?10^?6?M (at S/N?=?3).

Sensitive phenol determination based on co-modifying tyrosinase and palygorskite on glassy carbon electrode

A sensitive tyrosinase biosensor, based on co-modifying tyrosinase and palygorskite on glassy carbon electrode, was developed for phenol analysis. Palygorskite, a kind of natural one-dimensional clay with good biocompatibility, high specific surface area and porous morphology, works as a perfect matrix of enzyme. Tyrosinase retains its inherent bioactivity when immobilized in palygorskite, which leads to a high sensitivity of 1.897 A mol^?1 L. The sensor response achieves 95% of steady-state-current in no more than 3 s, and the linear range of the bioelectrode spans the concentration of phenol from 5?×?10^?8 to 1?×?10^?4 mol L^?1 with a correlation coefficient of 0.9992. The results show no apparent decrease in the response over 2 weeks, and about 80% of the response was retained after 2 months when the electrode was stored at 4–5 °C.     

An Amperometric Biosensor for trans‐Resveratrol Determination in Aqueous Solutions by Means of Carbon Paste Electrodes Modified with Peroxidase Basic Isoenzymes from Brassica Napus

The catalytic properties of peroxidase basic isoenzymes (PBI's) from Brassica napus towards trans‐resveratrol (t‐Res) oxidation were demonstrated by the first time by conventional UV‐visible spectroscopic measurements. The enzymatic reaction rate was studied under different experimental conditions and the kinetics parameters were determined. An amperometric biosensor based on Brassica napus PBI's to determine t‐Res is also proposed by the first time. The method employs a dialysis membrane covered, PBI's entrapped and ferrocene (Fc)‐embedded carbon paste electrode (PBI's‐Fc‐CP) and is based on the fact that the decreased amount of H₂O₂ produced by the action of PBI's is proportional to the oxidised amount of t‐Res in the solution. Comparative amperometric experiments showed that, in spite of PBI's activity was much lower than commercial horseradish peroxidase (HRP) activity, t‐Res was a much better substrate for PBI's biosensors than those biosensors constructed by using HRP. The PBI's‐Fc‐CP biosensors showed a very good stability during at least twenty days. The reproducibility and the repeatability were 4.5% and 8.3%, respectively, showing a good biosensor performance. The calibration curve was linear in the t‐Res concentration (c_t‐Res) range from 1×10^?6 to 2.5×10^?5 M, with a sensibility of (2.31±0.05)×10⁶ nA M^?1. The lowest c_t‐Res value measured experimentally for a signal to noise ratio of 3 : 1 was 0.83 μM.     

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H₂O₂) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM^?1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

Dendritic Silver/Silicon Dioxide Nanocomposite Modified Electrodes for Electrochemical Sensing of Hydrogen Peroxide

A novel biosensor for hydrogen peroxide was prepared by immobilizing horseradish peroxidase (HPR) on newly synthesized dendritic silver/silicon dioxide nanocomposites, which were coated on a glassy carbon electrode. The modified electrode was characterized with XPS, SEM, and electrochemical methods. This biosensor showed a very fast amperometric response to hydrogen peroxide with a linear range from 0.7 to 120 μM, a limit of detection of 0.05 μM and a sensitivity of 1.02 mA mM^?1 cm^?2. The Michaelis‐Menten constant of the immobilized HRP was estimated to be 0.21 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity. The preparation of the proposed biosensor was convenient, and it showed high sensitivity and good stability.     

Disposable Amperometric Biosensor Based on Poly‐L‐lysine and Fe3O4 NPs‐chitosan Composite for the Detection of Tyramine in Cheese

An amperometric tyramine biosensor based on poly‐L‐lysine (PLL) and Fe₃O₄ nanoparticles (Fe₃O₄NP) modified screen printed carbon electrode (SPCE) was developed. PLL was formed on the SPCE by the electropolymerization of L‐lysine. Subsequently, Fe₃O₄NP suspension prepared in chitosan (CH) solution was casted onto the PLL/SPCE. Tyrosinase (Ty) enzyme was immobilized onto the modified Fe₃O₄?CH/PLL/SPCE and the electrode was coated with Nafion to fabricate the Ty/Fe₃O₄?CH/PLL/SPCE. Different techniques including scanning electron microscopy, chronoamperometry (i–t curve), cyclic voltammetry and electrochemical impedance spectroscopy were utilized to study the fabrication processes, electrochemical characteristics and performance parameters of the biosensor. The analytical performance of the tyramine biosensor was evaluated with respect to linear range, sensitivity, limit of detection, repeatability and reproducibility. The response of the biosensor to tyramine was linear between 4.9×10^?7–6.3×10^?5 M with a detection limit of 7.5×10^?8 M and sensitivity of 71.36 μA mM^?1 (595 μA mM^?1 cm^?2). The application of the developed biosensor for the determination of tyramine was successfully tested in cheese sample and mean analytical recovery of added tyramine in cheese extract was calculated as 101.2±2.1 %. The presented tyramine biosensor is a promising approach for tyramine analysis in real samples due to its high sensitivity, rapid response and easy fabrication.     

Analytical Biosensing of Hydrogen Peroxide on Brilliant Cresyl Blue/Multiwalled Carbon Nanotubes Modified Glassy Carbon Electrode

A cationic quinine‐imide dye brilliant cresyl blue (BCB) and horseradish peroxidase (HRP) were co‐immobilized within ormosil on multiwalled carbon nanotubes modified glassy carbon electrode for the fabrication of highly sensitive and selective hydrogen peroxide biosensor. The presence of epoxy group in ormosil as organic moiety improves the mechanical strength and transparency of the film and amino group provides biocompatible microenvironment for the immobilization of enzyme. The presence of MWCNTs improved the conductivity of the nanocomposite film. The surface characterization of MWCNT modified ormosil nanocomposite film was performed with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cyclic voltammetry and amperometry measurements were used to study and optimize the performance of the resulting peroxide biosensor. The apparent Michaelis–Menten constant was determined to be 1.5 mM. The proposed H₂O₂ biosensor exhibited wide linear range from 3×10^?7 to 1×10^?4 M, and low detection limit 1×10^?7 M (S/N=3) with fast response time <5 s. The probable interferences in bio‐matrix were selected to test the selectivity and no significant response was observed in the biosensor. This biosensor possessed good analytical performance and long term storage stability.     

Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on Fe3O4/Chitosan Modified Glassy Carbon Electrode

A new type of amperometric hydrogen peroxide biosensor was constructed based on horseradish peroxidase (HRP) immobilized on Fe₃O₄/chitosan modified glassy carbon electrode. The effects of some experimental variables such as the concentration of supporting electrolyte, pH, enzyme loading, the concentration of the mediator of methylene blue (MB) and the applied potential were investigated. The linear range of the calibration curve for H₂O₂ was 2.0×10^?4–1.2×10^?2 M with a detection limit of 1.0×10^?4 M (S/N=3). The response time was less than 12 s. The apparent Michaelis‐Menten constant K_m was 21.4 mM and it illustrated the excellent biological activity of the fixed enzyme. In addition, the biosensor had long‐time stability and good reproducibility. And this method has been used to determine H₂O₂ concentration in the real sample.     

Use of capillary electrophoresis with chemiluminescence detection for sensitive determination of homocysteine

A sensitive capillary electrophoresis (CE) method with chemiluminescence (CL) detection was developed for the determination of homocysteine (HCys) in human plasma. In this work, N‐(4‐aminobutyl)‐N‐ethylisoluminol was used as tagging reagent to label the analyte for achieving high assay sensitivity. N‐(4‐Aminobutyl)‐N‐ethylisoluminol‐tagged HCys after CE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase, producing CL emission. Experimental conditions for labeling analyte, CE separation, and CL detection were studied. The CL intensity was proportional to the concentration of HCys in the range of 2.5×10^?8 to 5.0×10^?6 M. Detection limit (S/N=3) was 7.6×10^?9 M. Human plasma samples from healthy donors were analyzed by the presented method. HCys levels were found to be in the range of 9.50–15.3 μM.     

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

CuO‐Doped Mesoporous Silica Hybrid for Rapid and Sensitive Amperometric Detection of Phenolic Compounds

This work constructed an amperometric biosensing platform using CuO doped mesoporous silica hybrid (CuO/SBA‐15) as a carrier. The CuO/SBA‐15 showed a pair of redox peaks of Cu^2+/0. Upon immobilization of tyrosinase on the hybrid, the resulting biosensor exhibited a rapid (<0.5 s) and sensitive amperometric response to phenolic compounds under the optimized conditions. The linear response to catechol ranged from 1.2×10^?9 to 3.0×10^?5 M. The activation energy for enzymatic reaction was calculated to be 26.6 kJ mol^?1. The apparent Michaelis–Menten constants of the enzyme electrode were estimated to be 54.6, 145, 17.0, 74.8 and 633 µM for catechol, phenol, p‐cresol, m‐cresol and dopamine hydrochloride, respectively. The metal oxide doped mesoporous silica hybrid exhibited excellent performance for construction of new biosensors.     

Amperometric Determination of Hydrogen Peroxide and its Mathematical Simulation for Horseradish Peroxidase Immobilized on a Sonogel Carbon Electrode

A mathematical model of a horseradish peroxidase biosensor was applied to simulate the amperometric response for the detection of hydrogen peroxide. The development of the mathematical model was based on the Michaelis–Menten equation and Fick’s Second Law. The theoretical study is based on the determination of physico-chemical and geometric parameters of a horseradish peroxidase biosensor as well as the kinetic parameters of reaction mechanism such as diffusion coefficients of hydrogen peroxide, the thickness of enzymatic layer, and the Michaelis–Menten kinetic constant. The theoretical analysis provides an accurate estimate of parameters affecting the biosensor performance such as the diffusion coefficient of hydrogen peroxide in the biomembrane that was estimated to be 56?×?10^?12 m²/s. The thickness of diffusion layer was estimated to be 80–100?µm and the biomembrane 7.5?µm. The experimental and numerical values of kinetic parameters were 0.92 and 0.98?µM for the Michaelis–Menten constants and 0.010 and 0.012?µM/s for the catalytic activity rates. The model was validated for hydrogen peroxide detection and exhibited a good agreement with the experimental measurements.     

An H2O2 Biosensor Based on Immobilization of Horseradish Peroxidase Labeled Nano‐Au in Silica Sol‐Gel/Alginate Composite Film

Abstract

A novel approach to assemble an H₂O₂ amperometric biosensor was introduced. The biosensor was constructed by entrapping horseradish peroxidase (HRP) labeled nano‐scaled particulate gold (nano‐Au) (HRP‐nano‐Au electrostatic composite) in a new silica sol‐gel/alginate hybrid film using glassy carbon electrode as based electrode. This suggested strategy fully merged the merits of sol‐gel derived inorganic‐organic composite film and the nano‐Au intermediator. The silica sol‐gel/alginate hybrid material can improve the properties of conventional sol‐gel material and effectively prevent cracking of film. The entrapment of HRP in the form of HRP‐nano‐Au can not only factually prevent the leaking of enzyme out of the film but also provide a favorable microenvironment for HRP. With hydroquinone as an electron mediator, the proposed HRP electrode exhibited good catalytic activity for the reduction of H₂O₂. The parameters affecting both the qualities of sol‐gel/alginate hybrid film and the biosensor response were optimized. The biosensor exhibited high sensitivity of 0.40 Al mol^?1 cm^?2 for H₂O₂ over a wide linear range of concentration from 1.22×10^?5 to 1.46×10^?3 mol L^?1, rapid response of <5 s and a detection limit of 0.61×10^?6 mol L^?1. The enzyme electrode has remarkable stability and retained 86% of its initial activity after 45 days of storage in 0.1 mol L^?1 Tris‐HCl buffer solutions at pH 7.     

Biosensor for H2O2 Response Based on Horseradish Peroxidase: Effect of Different Mediators Adsorbed on Silica Gel Modified with Niobium Oxide

Reagentless biosensors sensitive to hydrogen peroxide have been developed and compared. These biosensors are comprised of a carbon paste electrode modified with horseradish peroxidase (HRP) and one phenothiazine (methylene blue), one phenoxazine (meldola's blue) or one phenazine (phenazine methosulfate) dye adsorbed on silica gel modified with niobium oxide (SN). The enzyme was immobilized onto the graphite powder by cross‐linking with glutaraldehyde and mixing with one of the electron transfer mediators (dyes) adsorbed on SN. The amperometric response was based on the electrocatalytic properties of the dye to mediate electrons, which were generated in the enzymatic reaction of hydrogen peroxide under catalysis of HRP. The dependence on the biosensor response in terms of pH, buffer, HRP amounts and applied potential was investigated. The best results were found with a biosensor containing methylene blue dye showing an excellent operational stability (around 92% of the activity was maintained after 300 determinations). The proposed biosensor also presented good sensitivity (32.87 nA cm^{?2 μ}mol^?1 L) allowing hydrogen peroxide quantification at levels down to 0.52×10^?6 mol L^?1 an optimum response at pH 6.8 and at a potential of ?50 mV (vs. SCE) and showing a wide linear response range (from 1 to 700 μmol L^?1 for hydrogen peroxide).     

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.