Amperometric Glucose Biosensors Based on Integration of Glucose Oxidase onto Prussian Blue/Carbon Nanotubes Nanocomposite Electrodes

Nanosized Prussian blue (PB) particles were synthesized with a chemical reduction method and then the PB nanoparticles were assembled on the surface of multiwall carbon nanotubes modified glassy carbon electrode (PB/MWNTs/GCE). The results showed that the PB/MWNTs nanocomposite exhibits a remarkably improved catalytic activity towards the reduction of hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the PB/MWNTs platform by an electrochemically polymerized o‐phenylenediamine (OPD) film to construct an amperometric glucose biosensor. The biosensor exhibited a wide linear response up to 8 mM with a low detection limit of 12.7 μM (S/N=3). The Michaelis–Menten constant K_m and the maximum current i_max of the biosensor were 18.0 mM and 4.68 μA, respectively. The selectivity and stability of the biosensor were also investigated.     

Development of a Xanthine Oxidase Modified Amperometric Electrode for the Determination of the Antioxidant Capacity

This paper describes the development of a xanthine oxidase/poly‐m‐phenylenediamine (XOD‐PPD)‐modified electrode. The biosensor was constructed by encapsulating XOD in a sol‐gel matrix deposited onto a platinum based screen‐printed electrode functionalized with a permselective PPD membrane. The hydrogen peroxide generated as a final product of the enzymatic reaction between the hypoxanthine and the XOD or by the spontaneous dismutation of superoxide radicals was selectively monitored at +700 mV. The use of a highly selective PPD layer blocked the nonspecific oxidation of other oxidizable molecules. Finally the biosensor was applied to the determination of the antioxidant capacity of acetylsalicylic acid.     

Electrochemical Fabrication of Prussian Blue Nanocube‐decorated Electroreduced Graphene Oxide for Amperometric Sensing of NADH

In this study, Prussian blue (PB) film on the electroreduced graphene oxide (ERGO)‐modified Au electrode surface (ERGO/PB) is easily prepared by means of cyclic voltammetric technique in the mixture of K₃Fe(CN)₆ and FeCl₃. Its electrochemical behaviors for NADH biosensor are studied. The structural and morphological characters of modified electrode material are analyzed with using of XPS, XRD, Raman, EDS, and SEM techniques. ERGO/PB hybrid nanocomposite for NADH biosensor is exhibited to the higher catalytic effect (linear range from 1.0 to 100 μM, detection limit of 0.23 μM at S/N=3) compared to naked Au, ERGO‐modified Au, and PB‐modified Au electrodes. In addition to, ERGO/PB electrode was used to voltammetric and amperometric detection of H₂O₂. ERGO/PB electrodes also showed the same behavior as the NADH sensor. This ERGO/PB‐modified electrode supplied a simple, new, and low‐cost route for amperometric sensing of both NADH and H₂O₂.     

Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

Prussian Blue Modified Carbon Ionic Liquid Electrode: Electrochemical Characterization and Its Application for Hydrogen Peroxide and Glucose Measurements

Prussian blue modified carbon ionic liquid electrodes (PB‐CILEs) were fabricated using chemical and electrochemical procedures. Chemically fabricated PB‐CILE exhibited an excellent sensitivity (0.0866 μA μM^?1), low detection limit (0.01 μM) and two linear ranges (0.01–1 and 1–600 μM) toward hydrogen peroxide. Then, glucose oxidase (GOx) was immobilized on the surface of PB‐CILE to fabricate glucose biosensor using three different procedures involving cross linking with glutaraldehyde (GLU) and bovine serum albumin (BSA), entrapment into the Nafion matrix and covering with a sol‐gel layer. Glucose biosensor fabricated using cross linking procedure showed the best sensitivity (0.0019 μA μM^?1) and operational stability for glucose.     

Urea Biosensor Based on Electrochromic Properties of Prussian Blue

Prussian blue (PB) is an electrochromic material, which can be used as a signal transducer in the formation of optical urea biosensors. The previous researches in electrochromic properties of PB demonstrated the optical PB response to ammonium ions, which occurs when ammonium ions are interacting with PB layer at a constant 0.2 V vs Ag|AgCl|KCl_sat potential. In this work PB optical dependence on ammonium ions concentration was applied in the formation of electrochromic urea biosensor. Biosensor was formed by modifying the optically transparent indium tin oxide (ITO) coated glass electrode (glass/ITO) with Prussian blue layer and immobilizing urease (glass/ITO/PB‐urease). Calibration curve showed the linear dependency (R²=0.995) between the change of maximal absorbance (ΔA) and urea concentration in concentration range varying from 3 mM to 30 mM. The highest sensitivity (4 ΔA M^?1) of glass/ITO/PB‐urease biosensor is in the concentration range from 7 mM to 30 mM. It was determined that working principle of the glass/ITO/PB‐urease biosensor is not related to pH changes occurring during enzymatic hydrolysis of urea.     

Amperometric Immunosensor for the Determination of α‐1‐Fetoprotein Based on Core‐Shell‐Shell Prussian Blue‐BSA‐Nanogold Functionalized Interface

In the present work, a newly functional nanoparticle has been prepared to immobilize the protein for the detection of α‐1‐fetoprotein (AFP). Prussian blue (PB) nanoparticle was initially synthesized under ultrasonic condition, then bovine serum albumin (BSA) was used to coat the PB nanoparticle to improve the stability of the PB nanoparticle as well as functionalize the surface of PB nanoparticle, and then gold colloids were loaded on the BSA‐coated PB nanoparticle to construct a core‐shell‐shell nanostructure via the conjunction of thiolate linkages or alkylamines of the BSA. Finally, a convenient, effective and sensitivity amperometric immunosensor for the detection of α‐1‐fetoprotein (AFP) was constructed by the employment of these functional core‐shell‐shell microspheres. The preparation of the nanoparticle (Au‐BSA‐PB NPs) was characterized by transmission electron microscopy (TEM), and the assembly of the biosensor was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The dynamic range of the resulted immunosensor for the detection of AFP is from 0.02 ng/mL to 200.0 ng/mL with a detection limit of 0.006 ng/mL (S/N=3). Moreover, this biosensor displays good selectivity, stability and reproducibility.     

New Nanocomposite Based on Prussian Blue Nanoparticles/Carbon Nanotubes/Chitosan and Its Application for Assembling of Amperometric Glucose Biosensor

Abstract

A new nanocomposite was developed by combination of prussian blue (PB) nanoparticles and multiwalled carbon nanotubes (MWNTs) in the matrix of biopolymer chitosan (CHIT). The PB and MWNTs had a synergistic electrocatalytic effect toward the reduction of hydrogen peroxide. The CHIT/MWNTs/PB nanocomposite‐modified glassy carbon (GC) electrode could amplify the reduction current of hydrogen peroxide by ～35 times compared with that of CHIT/MWNTs/GC electrode and reduce the response time from ～60 s for CHIT/PB/GC to 3 s. Besides, the CHIT/MWNTs/PB nanocomposite‐modified GC electrode could reduce hydrogen peroxide at a much lower applied potential and inhibit the responses of interferents such as ascorbic acid (AA) uric acid (UA) and acetaminophen (AC). With glucose oxidase (GOx) as an enzyme model, a new glucose biosensor was fabricated. The biosensor exhibited excellent sensitivity (the detection limit is down to 2.5 µM), fast response time (less than 5 s), wide linear range (from 4 µM to 2 mM), and good selection.     

Chloroperoxidase Modified Electrode for Amperometric Determination of 2,4,6‐Trichlorophenol

A carbon paste‐poly(o‐phenylendiamine)‐modified electrode to be used as amperometric biosensor for 2,4,6‐trichlorophenol (TCP) is described. The enzyme chloroperoxidase (EC 1.11.1.10) from Caldariomyces fumago is immobilized through dispersion in a graphite paraffin oil carbon paste covered by an electrogenerated poly(o‐phenylendiamine) (PPD) layer. The main enzymatic dehalogenation product, 2,6‐dichloro‐1,4‐benzoquinone (DCQ) is characterized by liquid chromatography‐mass spectrometry and cyclic voltammetry. This product is electrochemically active and can be detected amperometrically at +150 mV vs. Ag|AgCl|KCl (3 M). The biosensor exhibits a response time of 4 min, a detection limit of 10^?7 M, and a dynamic linear range between 10^?7 and 10^?6 M. Selectivity as well as operating and storage stability were evaluated.     

Functionalization of Single‐Walled Carbon Nanotubes with Cubic Prussian Blue and Its Application for Amperometric Sensing

In this work, we synthesized electroactive cubic Prussian blue (PB) modified single‐walled carbon nanotubes (SWNTs) nanocomposites using the mixture solution of ferric‐(III) chloride and potassium ferricyanide under ambient conditions. The successful fabrication of the PB‐SWNTs nanocomposites was confirmed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV‐vis absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and cyclic voltammetry (CV). PB nanocrystallites are observed to be finely attached on the SWNTs sidewalls in which the SWNTs not only act as a carrier of PB nanocrystallites but also as Fe(III)‐reducer. The electrochemical properties of PB‐SWNTs nanocomposites were also investigated. Using the electrodeposition technique, a thin film of PB‐SWNTs/chitosan nanocomposites was prepared onto glassy carbon electrode (GCE) for the construction of a H₂O₂ sensor. PB‐SWNTs/chitosan nanocomposites film shows enhanced electrocatalytic activity towards the reduction of H₂O₂ and the amperometric responses show a linear dependence on the concentration of H₂O₂ in a range of 0.5–27.5 mM and a low detection limit of 10 nM at the signal‐to‐noise ratio of 3. The time required to reach the 95% steady state response was less than 2 s. CV studies demonstrate that the modified electrode has outstanding stability. In addition, a glucose biosensor is further developed through the simple one‐step electrodeposition method. The observed wide concentration range, high stability and high reproducibility of the PB‐SWNTs/chitosan nanocomposites film make them promising for the reliable and durable detection of H₂O₂ and glucose.     

Sensitive and Selective Electrochemical Analysis of Methyl‐parathion (MPT) and 4‐Nitrophenol (PNP) by a New Type p‐NiTSPc/p‐PPD Coated Carbon Fiber Microelectrode (CFME)

A novel modified carbon fiber microelectrode (CFME) was obtained by combination of tetrasulfonated nickel phtalocyanine (p‐NiTSPc) electroformed film associated to para‐phenylenediamine (p‐PPD) electropolymerized outer‐coating. The modified CFMEs where denoted C/p‐NiTSPc and C/p‐NiTSPc/p‐PPD, respectively. These electrodes are dedicated to the organophosphates compounds (OPs) methyl‐parathion (MPT) and para‐nitrophenol (PNP). Our contribution shows that both OPs can be determined simultaneously on the unmodified and modified C/p‐NiTSPc CFMEs. A clear electrocatalytic activity towards both MPT and PNP redox process was observed, for the first time, in presence of p‐NiTSPc. The obtained sensitivity for the C/p‐NiTSPc CFME was 80 nA L mg^?1 in the concentration range 0.01 to 10 mg/L with a detection limit of 40 μg/L. Also the combination of p‐NiTSPc and p‐PPD electrodeposited films show, for the first time, the possibility to discriminate on the C/p‐NiTSPc/p‐PPD CFME between MPT and PNP. Stability experiments were also conducted for 3 weeks in acetate buffer showing a good reproductibility of the sensitivity to PNP vs. time in presence of MPT with a little loss of sensitivity (5%) after 3 weeks.     

Graphene/Poly(vinylferrocene) Composite Based Amperometric Biosensor for L‐lysine Determination

A novel and sensitive amperometric biosensor for L‐lysine determination based on a glassy carbon electrode (GCE) modified with graphene (GR) and redox polymer poly(vinylferrocene) (PVF) was constructed. L‐lysine‐α‐oxidase was immobilized onto the modified GCE by a glutaraldehyde/bovine serum albumin cross‐linking procedure. SEM, CV and EIS were used for the characterization of the surface morphology and stepwise fabrication processes of PVF/GR composite. Optimal composition of the biosensor and experimental conditions that affect the performance of the biosensor are discussed. The effect of buffer pH on biosensor response was studied in detail over a wide pH range. L‐lysine biosensor displayed a linear range of 9.9×10⁻⁷ ‐ 3.1×10⁻⁴ M with a low detection limit of 2.3×10⁻⁷ M and K_M ^app value of 0.4 mM. The L‐lysine biosensor was tested using pharmaceutical sample and cheese with satisfactory results.     

Amperometric Study of Au‐Colloid Function on Xanthine Biosensor Based on Xanthine Oxidase Immobilized in Polypyrrole Layer

Four kinds of xanthine oxidase (XOD) based amperometric biosensors were fabricated and their analytical performances were compared. Polypyrrole (PPY)/XOD biosensor was constructed by electrochemical oxidation of pyrrole in the solution containing xanthine oxidase and pyrrole in this paper. Colloidal Au was then immobilized on the biosensor. On the other hand, electron mediator, Prussian Blue (PB), was deposited on the electrode before the immobilization of PPY/XOD to enhance electron‐transfer rate and current response. The results showed that PPY/XOD, PPY/XOD/Au‐colloid, PB/PPY/XOD and PB/PPY/XOD/Au‐colloid biosensors exhibit good response to xanthine in 1×10^?6 M and 2×10^?5 M and Michaelis‐Menten constants (K_m) of these biosensors were 242.2, 113.4, 144.5, 43.2 μmol?L^?1, respectively. The dependence of current responses with applied voltages was discussed, and different mechanisms of these biosensors were discussed. It has been found that colloidal Au can enhance the current response at the same concentration of xanthine solution and decrease the energy‐barrier of electron‐transfer reaction on the electrode.     

Preparation of GOD/Sol‐Gel Silica Film on Prussian Blue Modified Electrode for Glucose Biosensor Application

A novel amperometric glucose biosensor was fabricated by in situ incorporating glucose oxidase (GOD) within the sol‐gel silica film on a Prussian blue (PB) modified electrode. The method is simple and controllable, which combined the merits of in situ immobilizing biomolecules in sol‐gel silica film by electrochemical method and the synergic catalysis effects of PB and GOD molecules. Scanning electron microscopy (SEM) showed that the GOD/sol‐gel silica film was homogeneous with a large number of three‐dimensional nanopores, which not only enhanced mass transport, but also maintained the active configuration of the enzyme molecule and prevented the leakage of enzyme, therefore improved the stability and sensitivity of the biosensor. The fabricated biosensor showed fast response time (10 s), high sensitivity (26.6 mA cm^?2 M^?1), long‐term stability, good suppression of interference, and linear range of 0.01 mM–5.8 mM with a low detection limit of 0.94 μM for the detection of glucose. In addition, the biosensor was successfully applied to determine glucose in human serum samples.     

An Amperometric Hydrogen Peroxide Biosensor Based on a Hemoglobin‐Immobilized Dopamine‐Oxidation Polymer/Prussian Blue/Au Electrode

A novel method to immobilize hemoglobin (Hb) in a polymer grown from dopamine (DA) oxidation was proposed. The growth of the polymeric films during DA oxidation at the Prussian blue (PB) modified Au electrode in weak alkaline phosphate buffer (pH 9.18) and the immobilization of Hb into the polymeric films during their growth were traced by the electrochemical quartz crystal impedance analysis (EQCIA) method. A hydrogen peroxide (H₂O₂) biosensor was thus constructed, and effects of experimental parameters on the sensor performance, including the applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of ?0.25 V vs. SCE, the current response of the biosensor in the selected phosphate buffer (pH 5.29) was linear with the concentration of H₂O₂ from 0.01 to 4.5 mM, with a lower limit of detection of 0.5 μM (S/N=3), short response time (within 10 s) and good anti‐interferent ability. The Michaelis constant (K_m^app) was estimated to be 3.80 mM. Compared with the separate film of PB or Hb, the composite film of Hb and PB exhibited a higher catalytic activity toward the reduction of H₂O₂, as a result of the additive effect of the chemical and biological catalyses.     

A Biosensor for Genetic Modified Soybean DNA Determination via Adsorption of Anthraquinone‐2‐sulphonic Acid in Reduced Graphene Oxide

An electrochemical DNA biosensor for DNA determination of genetically modified (GM) soybean (CaMV 35S target genes) was developed utilizing a new detection concept based on the adsoption of anthraquinone‐2‐sulphonic acid (AQMS) on the reduced graphene oxide nano‐particles (rGO) during DNA hybridization events. The aminated DNA probe for CaMV 35S was immobilized onto poly(n‐butyl acrylate) film modified with succinimide functional groups [poly(nBA‐NAS)] via peptide covalent bond. Nanosheets of rGO were entrapped in the poly(nBA‐NAS) film to form a conducting [poly(nBA‐NAS)‐rGO] film of the DNA biosensor. Besides facilitating the electron transfer reactions, the rGO also functioned as an adsorbent for AQMS. The sensing mechanism of the proposed DNA biosensor involved measuring the oxidation current of the AQMS adsorbed on the electrode surface at −0.50 V using differential pulse voltammetry (DPV) before and after a DNA hybridization event. Under optimum conditions, the DNA biosensor demonstrated a linear proportionality between AQMS oxidation signal and logarithm cDNA concentration from 1.0×10⁻¹⁵ M to 1.0×10⁻⁸ M target DNA with a detection limit of 6.3×10⁻¹⁶ M. The electrochemical DNA biosensor possessed good selectivity and a shelf life of about 40 days with relative standard deviation of reproducibility obtained in the range of 3.7–4.6% (n=5). Evaluation of the DNA biosensor using GM soybean DNA extracts showed excellent recovery percentages of 97.2–104.0.     

Prussian Blue‐Modified Titanate Nanotubes: A Novel Nanostructured Catalyst for Electrochemical Reduction of Hydrogen Peroxide

Prussian blue (PB) modified titanate nanotubes (PB‐TiNT) have been synthesized by the reaction of Fe²⁺‐modified TiNT with hexacyanoferrate(III) ions. The rate constant for heterogeneous catalytic reaction between PB‐TiNT and H₂O₂ was found to be k=2×10⁴ dm³ mol^?1 s^?1, which is an order of magnitude higher than the values of k reported for conventionally prepared, electrochemically deposited PB films. On the PB‐TiNT modified electrode with subnanomolar surface concentration of PB (Γ(PB)=2.8×10^?11 mol/cm²), a stable, reproducible and linear response towards H₂O₂ was obtained in the concentration range 0.02–4 mM, with the sensitivity of 0.10 AM^?1 cm^?2 at ?150 mV.     

A Selective Cholesterol Biosensor Based on Composite Film Modified Electrode for Amperometric Detection

An amperometric cholesterol biosensor based on immobilization of cholesterol oxidase in a Prussian blue (PB)/polypyrrole (PPy) composite film on the surface of a glassy carbon electrode was fabricated. Hydrogen peroxide produced by the enzymatic reaction was catalytically reduced on the PB film electrode at 0 V with a sensitivity of 39 μA (mol/L)^?1. Cholesterol in the concentration range of 10^?5 ? 10^?4 mol/L was determined with a detection limit of 6 × 10^?7 mol/L by amperometric method. Normal coexisting compounds in the bio‐samples such as ascorbic acid and uric acid do not interfere with the determination. The excellent properties of the sensor in sensitivity and selectivity are attributed to the PB/PPy layer modified on the sensor.     

Behavior of PQQ Glucose Dehydrogenase on Prussian Blue‐Modified Carbon Electrode

Glucose sensitive biosensor containing pyrroloquinoline quinone (PQQ)‐dependent glucose dehydrogenase immobilized on Prussian blue (PB)‐modified graphite electrode was designed. Properties of the biosensor were investigated in the cathodic and anodic response detection regions. It was shown, that anodic response of the biosensor is sum of two signals: direct electron transport from reduced PQQ to the electrode and by formation of the PQQ‐oxygen‐PB‐carbon ternary complex. Cathodic response of the biosensor is based on the oxidation of the reduced PQQ by PB‐oxygen‐PB complex. Electrochemical regeneration of the enzyme does not produce free hydrogen peroxide.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.