Direct Electrochemistry and Electrocatalysis of Hemoglobin in Lipid Film Incorporated with Room‐Temperature Ionic Liquid

A facile phospholipid/room‐temperature ionic liquid (RTIL) composite material based on dimyristoylphosphatidylcholine (DMPC) and 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([bmim]PF₆) was exploited as a new matrix for immobilizing protein. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were adopted to characterize this composite film. Hemoglobin (Hb) was chosen as a model protein to investigate the composite system. UV‐vis absorbance spectra showed that Hb still maintained its heme crevice integrity in this composite film. By virtue of the Hb/DMPC/[bmim]PF₆ composite film‐modified glassy carbon electrode (GCE), a pair of well‐defined redox peaks of Hb was obtained through the direct electron transfer between protein and underlying GCE. Moreover, the reduction of O₂ and H₂O₂ at the Hb/DMPC/[bmim]PF₆ composite film‐modified GCE was dramatically enhanced.     

Optical and Bioelectrochemical Characterization of Water‐Miscible Ionic Liquids Based Composites of Multiwalled Carbon Nanotubes

Acidic treated multiwalled carbon nanotubes (AMWNTs) were ground with water‐miscible room temperature ionic liquids, 1‐butyl‐3‐methylimidazolium tetrafluoroborate ([bmim]BF₄), and resulted in AMWNTs‐[bmim]BF₄ composite. Its electrical‐ionic conductivity and optical properties were compared with the other two types of carbon materials‐[bmim]BF₄ composites: pyrolytic graphite powder (PGP), pristine multiwalled carbon nanotubes (PMWNTs), through the ac impedance technology and Raman spectroscopy. The impedance data show that AMWNTs‐[bmim]BF₄ composite exhibits the highest conductivity. Raman spectra study exhibits that the [bmim]BF₄ can form gel with PMWNTs and AMWNTs but only form a viscous liquid with PGP. AMWNTs‐[bmim]BF₄ gel modified GC electrode was applied in direct electrochemistry of heme proteins (Hb and HRP) and it catalysis to the reduction of H₂O₂ was investigated.     

Direct electron transfer of horseradish peroxidase at Co<Subscript>3</Subscript>O<Subscript>4</Subscript>–graphene nanocomposite modified electrode and electrocatalysis

In this paper, the mixture of Co₃O₄–graphene nanocomposite and horseradish peroxidase (HRP) was spread on the surface of carbon ionic liquid electrode (CILE). Then, Nafion film was used for the immobilization. The results of spectroscopy proved that HRP kept up its native structure in the complex material. Direct electrochemistry of HRP resulted in a couple of quasi-reversible redox waves on cyclic voltammograms, reflecting the realization of direct electron transfer of HRP with electrode. The improvement in electrochemical responses was due to the usage of highly conductive Co₃O₄–graphene nanocomposite with biocompatible interface. Electrochemical parameters such as the electron transfer coefficient (α) was estimated as 0.47, and the apparent heterogeneous electron transfer rate constant (k _s) was calculated as 2.90 s^?1. The HRP modified electrode exhibited good electrochemical catalytic ability toward the reduction of trichloroacetic acid and NaNO₂. As a consequence, an updated third-generation electrochemical HRP biosensor with Co₃O₄–GR/CILE was constructed successfully.     

Direct electrochemistry of hemoglobin in a nafion and CuS microsphere modified carbon ionic liquid electrode and its electrocatalytic behavior

Direct electrochemistry of hemoglobin (Hb) was realized on a Nafion and CuS microsphere composite film modified carbon ionic liquid electrode (CILE) with N-butylpyridinium hexafluorophosphate (BPPF6) as binder. Scanning electron microscopy (SEM), UV-Vis absorption spectroscopy and cyclic voltammetry were used to characterize the fabricated Nafion/CuS/Hb/CILE. Experimental results showed that a pair of well-defined quasi-reversible redox peaks appeared with the formal potential as ?0.386 V (vs. SCE) in pH 7.0 Britton-Robinson (B-R) buffer solution, which was attributed to the Hb heme Fe(III)/Fe(II) redox couples. The electrochemical parameters of Hb in the composite film were carefully investigated with the charge transfer coefficient (α), the electron transfer number (n) and the electron transfer rate constant (k _s) as 0.505, 1.196 and 0.610 s^?1, respectively. The composite film provided a favorable microenvironment for retaining the native structure of Hb. The presence of CuS microspheres showed great improvement on the electron transfer rate of Hb with the CILE, which maybe due to the contribution of specific characteristics of CuS microspheres and the inherent advantages of ionic liquid on the modified electrode. The fabricated Hb modified electrode showed good electrocatalytic ability in the reduction of H₂O₂. The proposed bioelectrode can be used as a new third generation H₂O₂ biosensor.     

Improvement of carbon paste-based enzyme electrode using a new ionic liquid [Pmim][PF6] as the binder

A new carbon ionic liquid electrode (CILE) has been constructed using a low melting point (39?°C) hydrophobic ionic liquid (IL) 1-propyl-3-methylimidazolium hexafluorophosphate ([Pmim][PF₆]) as the binder. Both cyclic voltammetry and electrochemical impedance spectroscopy demonstrate that, in addition to the composition optimization of the IL/graphite composite, heating the composite at a temperature a little higher than the melting point of [Pmim][PF₆] can also lower the background current and enhance the mechanical strength of the CILE. The heated CILE is more sensitive than the traditional carbon paste electrode for the detection of H₂O₂. Glucose oxidase (GOx) can be easily entrapped in the bulk IL/graphite composite. Heating the GOx-modified CILE (GOx-CILE) at the melting point of [Pmim][PF₆] does not lower the catalytic activity of GOx. As compared with n-octylpyridinium hexafluorophosphate (melting point 65?°C) as the binder, [Pmim][PF₆]-based CILE is much better in signal-to-noise ratio. Under the optimum conditions, the [Pmim][PF₆]-based GOx-CILE has a linear amperometric response to glucose over a concentration range of 2.0–26?mM with the detection limit as low as 0.39?mM. It follows that choosing an IL with a melting point of ca. 40?°C as a binder to fabricate enzyme-entrapped CILEs is a good strategy for the enhancement of the performance of the electrode.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol–gel-derived tin oxide/gelatin composite films

Horseradish peroxidase (HRP) was immobilized into a new type of sol–gel-derived nano-sized tin oxide/gelatin composite film (SnO₂ composite film) using a sol–gel film/enzyme/sol–gel film “sandwich” configuration. Direct electrochemistry and electrocatalysis of HRP incorporated into the composite films were investigated. HRP/SnO₂ composite film exhibited a pair of stable and quasi-reversible cyclic voltammetric peaks for the HRP Fe(III)/HRP Fe(II) redox couple with a formal potential of about −0.25 V (vs. SCE) in a pH 6.0 phosphate buffer solution. The electron transfer between the enzyme and the underlying electrode was greatly enhanced in the microenvironment with nano-SnO₂ particles and nanoporous structures. Morphologies and microstructures of the composite films and HRP/composite films were characterized with TEM, AFM. Electrochemical impedance spectroscopy (EIS) was also used to feature the HRP incorporated into composite films. FTIR and UV–Vis spectroscopy demonstrated that HRP in the composite film could retain its native secondary structure. With the advantages of organic–inorganic hybrid materials, the HRP/SnO₂ composite film modified electrode displayed good stability and electrocatalytic activity to the reduction of H₂O₂, The apparent Michaelis-Menten constant was estimated to be 0.345 mM, indicating a high affinity of HRP entrapped into the composite film toward H₂O₂.     

Electrochemical Biosensor Based on Carbon Ionic Liquid Electrode Modified with Nafion/Hemoglobin/DNA Composite Film

A new electrochemical biosensor was constructed by immobilization of hemoglobin (Hb) on a DNA modified carbon ionic liquid electrode (CILE), which was prepared by using 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (EMIMBF₄) as the modifier. UV‐vis absorption spectroscopic result indicated that Hb remained its native conformation in the composite film. The fabricated Nafion/Hb/DNA/CILE was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A pair of well‐defined redox peaks was obtained on the modified electrode, indicated that the Nafion and DNA composite film provided an excellent biocompatible microenvironment for keeping the native structure of Hb and promoting the direct electron transfer rate of Hb with the basal electrode. The electrochemical parameters of Hb in the composite film were further calculated with the results of the charge transfer coefficient (α) and the apparent heterogeneous electron transfer rate constant (k_s) as 0.41 and 0.31 s^?1. The proposed electrochemical biosensor showed good electrocatalytic response to the reduction of trichloroacetic acid (TCA), H₂O₂, NO and the apparent Michaelis–Menten constant (K_M^app) for the electrocatalytic reaction was calculated, respectively.     

Direct electrochemistry-based hydrogen peroxide biosensor formed from single-layer graphene nanoplatelet-enzyme composite film

A novel electrochemical sensing system for direct electrochemistry-based hydrogen peroxide biosensor was developed that relied on the virtues of excellent biocompatibility, conductivity and high sensitivity to the local perturbations of single-layer graphene nanoplatelet (SLGnP). To demonstrate the concept, the horseradish peroxidase (HRP) enzyme was selected as a model to form the SLGnP-TPA (tetrasodium 1,3,6,8-pyrenetetrasulfonic acid)-HRP composite film. The single-layer graphene composite film displayed a pair of well-defined and good reversible cyclic voltammetric peak for Fe(III)/Fe(II) redox couple of HRP, reflecting the enhancement for the direct electron transfer between the enzyme and the electrode surface. Analysis using electrochemical impedance spectroscopy (EIS) revealed that electrostatic attractions existed between graphene monolayers and enzyme molecules. The intimate graphene and enzyme interaction was also observed using scanning electron microscopy (SEM), which resulted in the special properties of the composite film. Ultraviolet visible spectroscopy (UV-vis) indicated the enzyme in the composite film retained its secondary structure similar to the native state. The composite film demonstrated excellent electrochemical responses for the electrocatalytic reduction of hydrogen peroxide (H₂O₂), thus suggesting its great potential applications in direct electrochemistry-based biosensors.     

Direct electron transfer of horseradish peroxidase and its electrocatalysis based on carbon nanotube/thionine/gold composites

Horseradish peroxidase (HRP) was incorporated into multiwalled carbon nanotube/thionine/Au (MTAu) composite film by electrostatic interactions between positively charged HRP and negatively charged MTAu composite. The results of electrochemical impedance spectroscopy (EIS) confirmed adsorption of HRP on the surface of MTAu modified GC electrode. Moreover, the electrochemical results showed that HRP retained its bioactivity and bioelectrocatalytical activity, and also showed good direct electron transfer behavior on such a composite film.     

Solid-state electrochemiluminescence sensor based on the Nafion/poly(sodium 4-styrene sulfonate) composite film

An effective electrochemiluminescence (ECL) sensor based on Nafion/poly(sodium 4-styrene sulfonate) (PSS) composite film-modified ITO electrode was developed. The Nafion/PSS/Ru composite film was characterized by atomic force microscopy, UV-vis absorbance spectroscopy and electrochemical experiments. The Nafion/PSS composite film could effectively immobilize tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) via ion-exchange and electrostatic interaction. The ECL behavior of Ru(bpy)₃²⁺ immobilized in Nafion/PSS composite film was investigated using tripropylamine (TPA) as an analyte. The detection limit (S/N = 3) for TPA at the Nafion/PSS/Ru composite-modified electrode was estimated to be 3.0 nM, which is 3 orders of magnitude lower than that obtained at the Nafion/Ru modified electrode. The Nafion/PSS/Ru composite film-modified indium tin oxide (ITO) electrode also exhibited good ECL stability. In addition, this kind of immobilization approach was simple, effective, and timesaving.     

Direct Electrochemistry of Horseradish Peroxidase Immobilized in a Low Molecular Weight Gel

The ternary system of dodecylpyridinium bromide (DDPB)/acetone/H₂O with appropriate composition can form a gel spontaneously and the gel is stable in hydrophobic ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([Bmim]PF₆). Based on the gelation phenomenon we observed, the low molecular weight gelator (LMWG) was first tried to immobilize horseradish peroxidase (HRP) on glassy carbon electrode (GCE). The scanning electron microscope (SEM) images, the UV‐Vis spectra and the bioactivity measurement indicate that the gel is suitable for the immobilization of HRP. The direct electrochemistry of the HRP‐gel modified GCE (HRP‐gel/GCE) in [Bmim]PF₆ shows a pair of well‐defined and quasi‐reversible redox peaks with the heterogeneous electron transfer rate constant (k_s) being 14.4 s^?1, indicating that the direct electron transfer between HRP and GCE is fast. The HRP‐gel/GCE is stable and reproducible. Also the electrode exhibits good electrocatalytic effect on the reduction of trichloroacetic acid (TCA), showing good promise in bioelectrocatalysis.     

Hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on ��-Al2O3 nanoparticles/chitosan film-modified electrode

An amperometric biosensor based on horseradish peroxidase (HRP) and ??-Al₂O₃/chitosan composite film at a glassy carbon electrode has been developed. Hydrogen peroxide (H₂O₂) was detected with the aid of ferrocene monocarboxylic acid mediator to transfer electrons between the electrode and HRP. The morphology and composition of the modified electrode were characterized by scanning electron microscopy and electrochemical impedance spectroscopy. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and amperometry. The effects of HRP concentration, the applied potential, and the pH values of the buffer solution on the response of the sensor were investigated for optimum analytical performance. The proposed biosensor showed high sensitivity (0.249?A M^?1?cm^?2) and a fast response (<5?s) to H₂O₂ with the detection limit of 0.07???M. The linear response range of the enzyme electrode to H₂O₂ concentration was from 0.5 to 700???M with a correlation coefficient of 0.9998. The apparent Michaelis-Menten constant of the biosensor was calculated to be 0.818?mM, exhibiting a high enzymatic activity and affinity for H₂O₂.     

Properties of Poly(sodium 4‐styrenesulfonate)‐Ionic Liquid Composite Film and Its Application in the Determination of Trace Metals Combined with Bismuth Film Electrode

A new kind of bismuth film modified electrode to sensitively detect trace metal ions based on incorporating highly conductive ionic liquids 1‐butyl‐3‐methyl‐imidazolium hexafluorophosphate (BMIMPF₆) in solid matrices at glassy carbon (GC) was investigated. Poly(sodium 4‐styrenesulfonate) (PSS), silica, and Nafion were selected as the solid matrices. The electrochemical properties of the mixed films modified GC were evaluated. The electron transfer rate of Fe(CN)₆^4?/Fe(CN)₆^3? can be effectively improved at the PSS‐BMIMPF₆ modified GC. The bismuth modified PSS‐BMIMPF₆ composite film electrodes (GC/PSS‐BMIMPF₆/BiFEs) displayed high mechanical stability and sensitive stripping voltammetric performances for the determination of trace metal cations. The GC/PSS‐BMIMPF₆/BiFE exhibited well linear response to both Cd(II) and Pb(II) over a concentration range from 1.0 to 50 μg L^?1. And the detection limits were 0.07 μg L^?1 for Cd(II) and 0.09 μg L^?1 for Pb(II) based on three times the standard deviation of the baseline with a preconcentration time of 120 s, respectively. Finally, the GC/PSS‐BMIMPF₆/BiFEs were successfully applied to the determination of Cd(II) and Pb(II) in real sample, and the results of present method agreed well with those of atomic absorption spectroscopy.     

Electrochemical behavior of horseradish peroxidase on WS2 nanosheet‐modified electrode and electrocatalytic investigation

In this paper, a WS₂ nanosheet was modified on the surface of a carbon ionic liquid electrode (CILE), and horseradish peroxidase (HRP) was further fixed on the electrode with a Nafion film. Direct electrochemistry and bioelectrocatalysis of HRP incorporated on the modified electrode were investigated in detail. On Nafion/HRP/WS₂/CILE, a pair of well‐defined quasi‐reversible redox peaks appeared on the cyclic voltammogram, indicating that the presence of the WS₂ nanosheet on the electrode surface could provide a specific interface with large surface area for HRP and its direct electron transfer rate was greatly enhanced. The formal potential (E⁰) obtained was –0.179 V, which was the typical feature of heme Fe(III)/Fe(II) in HRP. The electron transfer coefficient (α) and the heterogeneous electron transfer rate constant (k_s) of HRP were calculated as 0.44 and 1.01 s^–1, respectively. This HRP‐modified electrode showed excellent electrocatalytic activity for the reduction of trichloroacetic acid and NaNO₂ with a wide linear range and low detection limit. Real samples were detected by this proposed method, indicating the successful fabrication of a new third‐generation electrochemical enzyme sensor utilizing the WS₂ nanosheet.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

Direct electrochemistry and electrocatalysis of myoglobin based on silica-coated gold nanorods/room temperature ionic liquid/silica sol-gel composite film

A novel biosensor based on the silica-coated gold nanorods (GNRs@SiO₂) and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium tetrafluroborate ([bmim][BF₄]) was fabricated for the determination of hydrogen peroxide (H₂O₂) and nitrite. GNRs@SiO₂ can not only act as a binder to hinder [bmim][BF₄] (RTIL) leaking from the electrode surface, but also provide a favorable microenvironment for direct electrochemistry of myoglobin (Mb). A pair of well-defined and quasi-reversible redox peaks of Mb was obtained at the GNRs@SiO₂-Mb/RTIL-sol-gel composite film modified GCE (GNRs@SiO₂-Mb/RTIL-sol-gel/GCE) through direct electron transfer between Mb and the underlying electrode. This biosensor showed an excellent electrocatalytic activity towards hydrogen peroxide and nitrite. The linear range for the determination of H₂O₂ was from 0.2 to 180 μM with a detection limit of 0.12 μM based on the signal-to-noise ratio of 3. In addition, the biosensor also exhibited high selectivity, good reproducibility, and long-term stability. Therefore, this kind of composite film can provide an ideal matrix for protein immobilization and biosensor fabrication.     

1-丁基-3-甲基咪唑六氟磷酸盐+水+醇体系的相行为

在298.15 K, 常压下研究了1-丁基-3-甲基咪唑六氟磷酸盐([bmim][PF₆])+水+甲醇、[bmim][PF₆]+水+乙醇、[bmim][PF₆]+水+2-丙醇、[bmim][PF₆]+水+1-丙醇三元体系的相行为. 结果表明, 对于含甲醇、乙醇和2-丙醇的体系, 醇在水+醇溶液中摩尔分数分别为0.55-1.00、0.40-0.75 和0.35-0.50 时, 醇的水溶液与[bmim][PF₆]可以互溶. 而水+1-丙醇体系没有此类现象. 这说明, 这类三元系的相行为不但取决于醇分子的大小, 而且取决于其结构.     

Direct electrochemistry and electrocatalysis of myoglobin immobilized in hyaluronic acid and room temperature ionic liquids composite film

A novel biocomposite film based on hyaluronic acid (HA) and hydrophilic room temperature ionic liquid 1-ethyl-3-methyl-imidazolium tetrafluoroborate ([EMIM][BF₄]) was explored. Here, HA was used as a binder to form [EMIM][BF₄]-HA composite film and help [EMIM][BF₄] to attaching on glass carbon electrode (GCE) surface, while doping [EMIM][BF₄] in HA can effectively reduce the electron transfer resistance of HA. The composite film can be readily used as an immobilization matrix to entrap myoglobin (Mb). A pair of well-defined and quasi-reversible redox peaks of Mb was obtained at the Mb-[EMIM][BF₄]-HA composite film modified GCE (Mb-[EMIM][BF₄]-HA/GCE) through direct electron transfer between Mb and the underlying electrode. The Mb-[EMIM][BF₄]-HA/GCE showed an excellent electrocatalytic activity toward the reduction of H₂O₂. Based on the [EMIM][BF₄]-HA biocomposite film, a third-generation reagentless biosensor could be constructed for the determination of H₂O₂.     

Sensitive Voltammetric Detection of Trace Heavy Metals in Real Water Using Multi‐Wall Carbon Nanotubes/Nafion Composite Film Electrode

Multi‐wall carbon nanotubes (MWCNTs) and Nafion composite film (MWCNTs/Nafion) were used for fabricating electrochemical sensors for the voltammetric detection of trace lead(II) and cadmium(II) in several water samples. The morphology and structure of MWCNTs/Nafion film were characterized by scanning electron microscopy (SEM) and infrared spectrum (IR). The electron transfer of MWCNTs/Nafion composite film was examined by cyclic voltammetry (CV) and electrochemical impedance spectrum (EIS). Various experimental parameters, which influenced the response of MWCNTs/Nafion/GC to target metals, were optimized. The results showed that the synergistic effect was obtained on the MWCNTs/Nafion/GC whose sensitivity and stability were better than those of Nafion‐coated electrode (Nafion/GC) or CNTs/GC. Stability of the Pb(II) and Cd(II) stripping signals was excellent with relative standard deviations (RSD) within 5% (n=10) from one electrode preparation to another, and RSD of 30 µg·L^?1 Pb(II) and Cd(II) were 2.8% and 3.2% for 20 repeated analysis on one single CNTs/Nafion/GC. Over 50 runs, the stability of Pb and Cd detection at the MWCNTs/Nafion conposites electrode was still satisfactory with RSD lower than 6.0%. The determination limits (S/N=3) of the proposed method were determined to be 100 ng·L^?1 for Pb and 150 ng·L^?1 for Cd. Finally, the MWCNTs/Nafion/GC was successfully applied to determine Pb(II) and Cd(II) in different water samples with recoveries of 97%–103% for Pb and 96%–104% for Cd.     

Cationic ring-opening polymerization of 3,3-bis（chloromethyl）oxacyclobutane in ionic liquids

Cationic ring-opening polymerization of 3,3-bis(chloromethyl)oxacyclobutane catalyzed by BF_3·OEt_2 was carried out in ionic liquids [bmim]BF_4 and [bmim]PF_6.The influences of BCMO concentration and molar ratio of BCMO/BF_3·OEt_2 on the molecular weights and yield of PBCMO were investigated.The polymerization in ionic liquids proceed to high conversions,although molecular weights are limited,similar to polymerization in organic solvent such as CH_2Cl_2.Follow a viewpoint of green chemistry, we feel ionic liquid [bmim]BF_4 is superior to [bmim]PF_6.Extracting [bmim]PF_6 from the product using organic solvent as extractant limits its advantage as a green reaction media.