Direct electrochemistry and electrocatalysis of hemoglobin entrapped in composite matrix based on chitosan and CaCO3 nanoparticles

The direct electron transfer between hemoglobin (Hb) and the underlying glassy carbon electrode (GCE) can be readily achieved via a high biocompatible composite system based on biopolymer chitosan (CHT) and inorganic CaCO₃ nanoparticles (nano-CaCO₃). Cyclic voltammetry of Hb-CHT/nano-CaCO₃/GCE showed a pair of stable and quasi-reversible peaks for HbFe(III)/Fe(II) redox couple in pH 7.0 buffer. The electrochemical reaction of Hb immobilized in CHT/nano-CaCO₃ composite matrix exhibited a surface-controlled process accompanied by electron and proton transfer. The electron transfer rate constant was estimated to be 1.8 s⁻¹. This modified electrode showed a high thermal stability up to 60 °C. The apparent Michaelis–Menten constant was calculated to be 7.5 × 10⁻⁴ M, indicating a high catalytic activity of the immobilized Hb toward H₂O₂. The interaction between Hb and this nano-hybrid material was also investigated using FT-IR and UV–vis spectroscopy, indicating that Hb retained its native structure in this hybrid matrix.     

Direct electrochemistry of hemoglobin and its electrocatalytic effect based on its direct immobilization on carbon ionic liquid electrode

Direct electrochemistry of hemoglobin (Hb) has been achieved by its direct immobilization on carbon ionic liquid electrode (CILE). CILE was immersed in a solution containing Hb and ionic liquid, octylpyridinium chloride ([OcPy][Cl]), to directly immobilize Hb on CILE. Cyclic voltammetry of modified electrode exhibited quasi-reversible peaks corresponding to Hb. The oxidation and reduction peak potentials of immobilized Hb in acetate buffer solution, pH 5.0 and at a scan rate of 0.1 V s⁻¹ were obtained at about –150 mV and –290 mV, respectively. The average surface coverage of the electroactive Hb adsorbed on the electrode surface was calculated as 8.4 × 10⁻¹¹ mol cm⁻². Hb retained its bioactivity on modified electrode and showed excellent electrocatalytic activity towards oxygen, hydrogen peroxide and nitrite. Hydrogen peroxide can be determined in the range of 1.0 × 10⁻⁴–5.0 × 10⁻³ M.     

Direct electrochemistry and electrocatalysis of hemoglobin in sodium alginate film on a BMIMPF6 modified carbon paste electrode

A room temperature ionic liquid (RTIL) modified carbon paste electrode was constructed based on the substitute of paraffin with 1-butyl-3-methyl-imidazolium hexafluorophosphate (BMIMPF₆) as binder for carbon paste. Direct electrochemistry and electrocatalytic behaviors of hemoglobin (Hb) entrapped in the sodium alginate (SA) hydrogel film on the surface of this carbon ionic liquid electrode (CILE) were investigated. The presence of IL in the CILE increased the electron transfer rate and provided a biocompatible interface. Hb remained its bioactivity on the surface of CILE and the SA/Hb modified electrode showed a pair of well-defined, quasi-reversible cyclic voltammetric peaks with the apparent standard potential (E^0′) at about −0.344 V (vs. SCE) in pH 7.0 Britton–Robinson (B–R) buffer solution, which was attributed to the Hb Fe(III)/Fe(II) redox couple. UV–Vis absorption spectra indicated that heme microenvironment of Hb in SA film was similar to its native status. Hb showed a thin-layer electrochemical behavior in the SA film with the direct electron transfer achieved on CILE without the help of electron mediator. Electrochemical investigation indicated that Hb took place one proton with one electron electrode process and the average surface coverage of Hb in the SA film was 3.2 × 10⁻¹⁰ mol/cm². The immobilized Hb showed excellent electrocatalytic responses to the reduction of H₂O₂ and nitrite.     

Direct electrochemistry of hemoglobin at vertically-aligned self-doping TiO2 nanotubes: A mediator-free and biomolecule-substantive electrochemical interface

The present work is dedicated to making the best of vertically-aligned TiO₂ nanotubes (TNTs) array to serve as a prospectively ideal “vessel” for protein immobilization and biosensor applications. The TNTs fabricated by electrochemical anodizing possess the advantageous of perpendicular alignment and tailored tubular architecture, as well as the good biocompatibility and hydrophilicity. But the electron-transfer resistance of the as-grown (AG-) TNTs is too large for the direct electron transfer and electrochemical biosensing. A simple strategy on controllable electrochemical reduction treatment of TNTs is adopted on it, leading TNTs in situ self-doped with Ti(III), which makes the Ti(III)–TNTs much better conductivity while the tubular and crystal structure of TNTs array still well maintained. Results show that the TNTs can be used as a super vessel for rapid and substantive immobilization of hemoglobin (Hb), with a large surface electroactive Hb coverage (Γ*) of 1.5 × 10^?9 mol cm^?2. The enhanced direct electron transfer of Hb is commendably observed on the Ti(III)–TNTs/Hb biosensor with a couple of well-defined redox peaks compared with the AG-TNTs/Hb. The biosensor further exhibits fast response, high sensitivity and stability for the amperometric biosensing of H₂O₂ with the detection limit of 1.5 × 10^?6 M, and the apparent Michaelis–Menten constant of 1.02 mM.     

Direct electron transfer of cytochrome c and its biosensor based on gold nanoparticles/room temperature ionic liquid/carbon nanotubes composite film

A robust and effective composite film based on gold nanoparticles (GNPs)/room temperature ionic liquid (RTIL)/multi-wall carbon nanotubes (MWNTs) modified glassy carbon (GC) electrode was prepared by a layer-by-layer self-assembly technique. Cytochrome c (Cyt c) was successfully immobilized on the RTIL-nanohybrid film modified GC electrode by electrostatic adsorption. Direct electrochemistry and electrocatalysis of Cyt c were investigated. The results suggested that Cyt c could be tightly adsorbed on the modified electrode. A pair of well-defined quasi-reversible redox peaks of Cyt c was obtained in 0.10 M, pH 7.0 phosphate buffer solution (PBS). RTIL-nanohybrid film showed an obvious promotion for the direct electron transfer between Cyt c and the underlying electrode. The immobilized Cyt c exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis currents increased linearly to the H₂O₂ concentration in a wide range of 5.0 × 10⁻⁵– 1.15 × 10⁻³ M. Based on the multilayer film, the third-generation biosensor could be constructed for the determination of H₂O₂.     

Gelatin-functionalized carbon nanotubes for the bioelectrochemistry of hemoglobin

In this paper, we compared the use of gelatin-functionalized carbon nanotubes (CNTs) as substrates for Hemoglobin (Hb) immobilization and as electrodes for electrochemical reduction of the absorbed Hb. The non-covalently gelatin-functionalized CNTs possessed an improved solubility in aqueous solution and may have an enhanced biocompatibility with Hb. The characteristics of Hb/gelatin-CNTs composite films were studied by using UV–vis spectroscopy, FTIR spectroscopy and electrochemical methods. The immobilized Hb showed a couple of quasi-reversible redox peaks with a formal potential of −0.35 V (vs. SCE) in 0.10 M pH 7.0 phosphate buffer solution (PBS). The surface concentration of electroactive Hb immobilized on gelatin-CNT/GC electrode was about 4.34 × 10⁻¹⁰ mol cm⁻².     

Sensing of 2,4-dichlorophenoxyacetic acid by surface-enhanced Raman scattering

Surface-enhanced Raman scattering (SERS) spectra of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was obtained by employing a bi-layer gold substrate, assembled by the reduction of Au(III) over gold-seeded nanoparticles immobilized on functionalized glass substrates. The SERS signal was linear with the logarithm of the solution concentrations between 1.0 × 10⁻⁷ mol L⁻¹ and 1.0 × 10⁻³ mol L⁻¹, indicating that the bi-layer gold substrate affords a significant dynamic range for SERS, providing an excellent analytical response within this concentration range, and revealing the high sensitivity of the gold surface towards such analyte. In addition, using the same gold substrate, a similar calibration curve was obtained for crystal-violet (CV), and it was possible to identify the concentration limit corresponding to the transition from the average SERS to the nonlinear SERS response.     

Cyclic voltammetric studies of carbohydrate–protein interactions on gold surface

A simple electrochemical method for the determination of association constants between carbohydrates and carbohydrate-binding proteins using cyclic voltammetry (CV) is described. The binding of concanavalin A (Con A) and cholera toxin (CT) to their specific α-mannose and β-galactose derivatives self-assembled on gold electrodes is electrochemically monitored with a redox probe of K₃Fe(CN)₆/K₄Fe(CN)₆. Upon binding of the proteins to the carbohydrate-modified electrodes, the redox current in CV decreases. The binding-induced change in electrochemical signal is thus used to construct Langmuir adsorption isotherm for the carbohydrate–protein interactions and to obtain the association constants. The association constants of carbohydrate–protein interactions determined by CV ((5.8 ± 1.2) × 10⁷ M^− 1 for mannose–Con A, (2.6 ± 0.5) × 10⁸ M^− 1 for galactose-CT) were in good agreement with those measured with electrochemical impedance spectroscopy and quartz crystal microbalance.     

Activity and stability of horseradish peroxidase in hydrophilic room temperature ionic liquid and its application in non-aqueous biosensing

The activity and stability of horseradish peroxidase (HRP) were investigated in a hydrophilic room temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluroborate ([bmim][BF₄]) by electrochemical methods. Although no detectable activity exhibited in anhydrous [bmim][BF₄], HRP was active in the presence of a small amount of water (4.53%, v/v). And its activity can be improved by immobilization in agarose hydrogel. The immobilized HRP possesses excellent activity at 65 °C. It remained 80.2% of its initial activity after being immersed for 10.5 h in an aqueous mixture of [bmim][BF₄] with some hydrogen peroxide (H₂O₂) under room temperature, implying extremely high stability. Moreover, the immobilized HRP was found to be very sensitive and stable in H₂O-containing [bmim][BF₄] for the detection of H₂O₂, with a wide linear range of 6.10 × 10⁻⁷ to 1.32 × 10⁻⁴ mol l⁻¹ and low detection limit of 1.0 × 10⁻⁷ mol l⁻¹.     

Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol–gel silica thin films on glassy carbon

This work points out that electrogeneration of silica gel (SG) films on glassy carbon electrodes (GCEs) can be applied to immobilize biomolecules – hemoglobin (Hb) or glucose oxidase (GOD) or both of them in mixture – without preventing their activity. These proteins were physically entrapped in the sol–gel material in the course of the electro-assisted deposition process applied to form the thin films onto the electrode surface. SG films were prepared from a precursor solution by applying a suitable cathodic potential likely to induce a local pH increase at the electrode/solution interface, accelerating thereby polycondensation of the silica precursors with concomitant film formation. Successful immobilization of proteins was checked by various physico-chemical techniques. Both Hb and GOD were found to undergo direct electron transfer, as demonstrated by cyclic voltammetry. GCE–SG–Hb gave rise to well-defined peaks at potentials E_c = −0.29 V and E_a = −0.17 V in acetate buffer, corresponding to the Fe^III/Fe^II redox system of heme group of the protein, while GCE–SG–GOD was characterized by the typical signals of FAD group at E_c = −0.41 V and E_a = −0.33 V in phosphate buffer. These two redox processes were also evidenced on a single voltammogram when both Hb and GOD were present together in the same SG film. Hb entrapped in the silica thin film displayed an electrocatalytic behavior towards O₂ and H₂O₂ in solution, respectively in the mM and μM concentration ranges. Immobilized GOD kept its biocatalytic properties towards glucose. Combined use of these two proteins in mixture has proven to be promising for detection of glucose in solution via the electrochemical monitoring of oxygen consumption (decrease of the oxygen electrocatalytic signal).     

One-step fabrication of chitosan–hematite nanotubes composite film and its biosensing for hydrogen peroxide

This work reports on a novel chitosan–hematite nanotubes composite film on a gold foil by a simple one-step electrodeposition method. The hybrid chitosan–hematite nanotubes (Chi–HeNTs) film exhibits strong electrocatalytic reduction activity for H₂O₂. Interestingly, two electrocatalytic reduction peaks are observed at −0.24 and −0.56 V (vs SCE), respectively, one controlled by surface wave and the other controlled by diffusion process. The Chi–HeNTs/Au electrode shows a linear response to H₂O₂ concentration ranging from 1 × 10⁻⁶ to 1.6 × 10⁻⁵ mol L⁻¹ with a detection limit of 5 × 10⁻⁸ mol L⁻¹ and a sensitivity as high as 1859 μA μM⁻¹ cm⁻².     

Electrogenerated chemiluminescence aptamer-based biosensor for the determination of cocaine

A novel electrogenerated chemiluminescence aptamer-based (ECL-AB) biosensor for the determination of a small molecule drug is designed employing cocaine-binding aptamer as molecular recognition element for cocaine as a model analyte and ruthenium complex served as an ECL label. A 5′-terminal cocaine-binding aptamer with the ECL label at 3′-terminal of the aptamer was utilized as an ECL probe. The ECL-AB biosensors were fabricated by immobilizing the ECL probe onto a gold electrode surface via thiol-Au interactions. An enhanced ECL signal is generated upon recognition of the target cocaine, attributed to a change in the conformation of the ECL probe from random coil-like configuration on the probe-modified film to three-way junction structure, in close proximity to the sensor interface. The integrated ECL intensity versus the concentration of cocaine was linear in the range from 5.0 × 10⁻⁹ to 3.0 × 10⁻⁷ M. The detection limit was 1.0 × 10⁻⁹ M. This work demonstrates that the combination of a highly binding aptamer to analyte with a highly sensitive ECL technique to design ECL-AB biosensor is a great promising approach for the determination of small molecule drugs.     

Electrochemical biosensor based on hairpin DNA probe using 2-nitroacridone as electrochemical indicator for detection of DNA species related to Chronic Myelogenous Leukemia

In this article, a new kind of hairpin DNA Electrochemical biosensor using nitroacridone as electrochemical indicator was first designed, and used to detect BCR/ABL fusion gene in Chronic Myelogenous Leukemia (CML). The results indicated that in pH 7.0 Tris–HCl buffer solution, the oxidation peak current was linear with the concentration of complementary strand in the range of 6.2 × 10⁻⁸–3.1 × 10⁻⁷ mol/l with a detection limit of 5.3 × 10⁻⁹ mol/l. This new hairpin DNA electrochemical biosensor demonstrates its excellent specificity for single-base mismatch and complementary (dsDNA) after hybridization, and this probe has been used for assay of PCR product of a real sample with satisfactory result.     

Ferrocene-modified Fe3O4@SiO2 magnetic nanoparticles as building blocks for construction of reagentless enzyme-based biosensors

A novel amperometric glucose biosensor was developed by entrapping glucose oxidase (GOD) in chitosan (CS) composite doped with ferrocene monocarboxylic acid-modified magnetic core-shell Fe₃O₄@SiO₂ nanoparticles (FMC-AFSNPs). It is shown that the obtained magnetic bio-nanoparticles attached to the surface of a carbon paste electrode (CPE) with the employment of a permanent magnet showed excellent electrochemical characteristics and at the same time acted as mediator to transfer electrons between the enzyme and the electrode. Under optimal conditions, this biosensor was able to detect glucose in the linear range from 1.0 × 10⁻⁵ to 4.0 × 10⁻³ M with a detection limit of 3.2 μM (S/N = 3). This immobilization approach effectively improved the stability of the electron transfer mediator and is promising for construction of biosensor and bioelectronic devices.     

New methodology for anodic stripping voltammetric determination of methylmercury

A novel procedure for trace methylmercury determinations by anodic stripping voltammetry at a gold film electrode is presented. Measurements were performed in a flow system. For selective determination of methylmercury, the Hg²⁺ ions were masked by complexation with DTPA. Hg-DTPA complex is not reduced at the gold film electrode at the potential of methylmercury reduction to the metallic state. The calibration graph was linear from 5 × 10⁻⁹ to 1 × 10⁻⁷ mol L⁻¹ for an accumulation time 600 s. A detection limit (based on 3σ criterion) for methylmercury was 2.3 × 10⁻⁹ mol L⁻¹. The validation of the proposed procedure was made by analyses of human hair certified reference material.     

Sorption of iron(II) and ruthenium(III)-triazine complexes on silica gel and its analytical applicability

2,4,6-Tri(2′-pyridyl)-s-triazine (TPTZ) complexes with iron(II) and ruthenium(III) were prepared. Their sorption and desorption features on silica gel have been investigated. Both complexes were strongly adsorbed. This has been utilized for separating and preconcentrating iron(II) and ruthenium(III) using TPTZ-impregnated silica gel. The chromatographic behavior of TPTZ on silica gel column was examined and found to be effective modifier for silica gel surface. The sorption capacity of silica gel for those metal-triazine complexes has been determined under static conditions and was found to be 5.28 × 10^–3 mM (Fe(TPTZ)₂²⁺) and 2.9 × 10^–3 mM (Ru(TPTZ)₂³⁺). Saturated methanolic solutions of KI or 25% NaClO₄ solutions desorbed both complexes quantitatively from the silica gel surface.     

One-step synthesis of poly(thionine)-Au nano-network and nanowires and its application for non-enzyme biosensing of hydrogen peroxide

We describe the preparation of novel poly(thionine)-Au materials, where the poly(thionine)-Au nano-network and nanowires have been synthesized in aqueous solution via the polymerization of thionine using HAuCl₄ as the oxidant in a single reaction setup. The synthesis process does not require templates, nor does it require large amounts of organic solvents or electrochemical methods. The morphology of the nanocomposites can be controlled by varying the thionine/HAuCl₄ ratio. The resulting poly(thionine)-Au network was used to fabricate a novel non-enzyme hydrogen peroxide (H₂O₂) biosensor. In pH 7.0 phosphate buffer, almost interference-free determination of H₂O₂ was realized at − 0.1 V versus Ag/AgCl with a linear of 1 × 10^− 4 to 5 × 10^− 2 M, a correlation coefficient of 0.998 and a response time of < 2 s. The developed biosensor showed a detection limit of 0.2 μM (S/N = 3) with very good stability, reproducibility and high selectivity.     

Using flowerlike polymer–copper nanostructure composite and novel organic–inorganic hybrid material to construct an amperometric biosensor for hydrogen peroxide

A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping horseradish peroxidase (HRP) in the organic–inorganic hybrid material composed of zirconia–chitosan sol–gel and Au nanoparticles (ZrO₂–CS–AuNPs). The sensitivity of the biosensor was enhanced by a flowerlike polymer–copper nanostructure composite (pPA–FCu) which was prepared from co-electrodeposition of CuSO₄ solution and 2,6-pyridinediamine solution. Several techniques, including UV–vis absorption spectroscopy, scanning electron microscopy, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy were employed to characterize the assembly process and performance of the biosensor. The results showed that this pPA–FCu nanostructure not only had excellent redox electrochemical activity, but also had good catalytic efficiency for hydrogen peroxide. Also the ZrO₂–CS–AuNPs had good film forming ability, high stability and good retention of bioactivity of the immobilized enzyme. The resulting biosensors showed a linear range from 7.80 × 10^?7 to 3.7 × 10^?3 mol L^?1, with a detection limit of 3.2 × 10^?7 mol L^?1 (S/N = 3) under optimized experimental conditions. The apparent Michaelis–Menten constant was determined to be 0.32 mM, showing good affinity. In addition, the biosensor which exhibits good analytical performance, acceptable stability and good selectivity, has potential for practical applications.     

Kinetics of the diazotization and azo coupling reactions of procaine in the micellar media

The kinetics of the diazotization reaction of procaine in the presence of anionic micelles of sodium dodecyl sulfate (SDS) and cationic micelles of cetyltrimethyl ammonium bromide (CTAB), dodecyltrimethyl ammonium bromide (DDTAB) and tetradecyltrimethyl ammonium bromide (TDTAB) were carried out spectrophotometrically at λ_max = 289 nm. The values of the pseudo first order rate constant were found to be linearly dependent upon the [NaNO₂] in the concentration range of 1.0 × 10⁻³ mol dm⁻³ to 12.0 × 10⁻³ mol dm⁻³ in the presence of 2.0 × 10⁻² mol dm⁻³ acetic acid. The concentration of procaine was kept constant at 6.50 × 10⁻⁵ mol dm⁻³. The addition of the cationic surfactants increased the reaction rate and gave plateau like curve. The addition of SDS micelles to the reactants initially increased the rate of reaction and gave maximum like curve. The maximum value of the rate constant was found to be 9.44 × 10⁻³ s⁻¹ at 2.00 × 10⁻³ mol dm⁻³ SDS concentration. The azo coupling of diazonium ion with β-naphthol (at λ_max = 488) nm was found to linearly dependent upon [ProcN₂⁺] in the presence of both the cationic micelles (CTAB, DDTAB and TDTAB) and anionic micelles (SDS). Both the cationic and anionic micelles inhibited the rate of reactions. The kinetic results in the presence of micelles are explained using the Berezin pseudophase model. This model was also used to determine the kinetic parameters e.g. k_m, K_s from the observed results of the variation of rate constant at different [surfactants].     

Electrochemiluminescent biosensor based on multi-wall carbon nanotube/nano-Au modified electrode

The electrochemiluminescent (ECL) behavior of lucigenin on a multi-wall carbon nanotube/nano-Au modified glassy carbon electrode (MWNT/nano-Au/GCE) was studied in this paper. Compared with the bare GCE, the ECL intensity of lucigenin can be greatly enhanced at MWNT/nano-Au/GCE. Based on the fact that superoxide dimutase (SOD) could obviously inhibit the ECL of lucigenin at MWNT/nano-Au/GCE, a sensitive ECL biosensor for determination of SOD was developed with a wide linear range of 5.0 × 10⁻⁸–5.0 × 10⁻⁶ mol/L with detection limit of 2.5 × 10⁻⁸ mol/L.