Direct Electrochemistry Of Cytochrome Research Articles

Direct electrochemistry of cytochrome c immobilized on one dimensional Au nanoparticles functionalized magnetic N-doped carbon nanotubes and its application for the detection of H2O2

In recent years, the fabrication of noble metal functionalized magnetic carbon nanotubes for a variety of novel biosensors has aroused considerable interest owing to their synergistic improved sensitivity. In the paper, the Fe3O4 and Cu nanoparticles (NPs) were integrated into the NCNTs to obtain N-doped carbon@Fe3O4-Cu nanotubes (NCNTs@Fe3O4@Cu) through a one-pot high temperature decomposition procedure. Then, Au NPs were assembled on the magnetic NCNTs to obtain NCNTs@Fe3O4@Au composite by galvanic replacement with Cu NPs. The resultant composite had provided a friendly platform for enzyme immobilization to develop highly sensitive biosensors. After the cytochrome c(cyt c) was accumulated by NCNTs@Fe3O4@Au composite, the cyt c/NCNTs@Fe3O4@Au gathered to the surface of electrode with an external magnet. Thus, the sensitivity of designed biosensor could be further improved, and most of electrochemical interferences could be excluded. Benefiting from the unique hybrid structure of the composite, the direct electron transfer of cyt c was highly facilitated and the constructed biosensors exhibited good performance for the detection of H2O2 with an extraordinary low detection limit of 0.3 μM, which revealed potential application for fabricating novel electrochemical biosensors.

Enhancement of the direct electron transfer to encapsulated cytochrome c by electrochemical functionalization with a conducting polymer

The present work deals with the direct electrochemistry of cytochrome c (cyt c) encapsulated within a methyl-modified silica film prepared by the sol-gel method. It was observed that the voltammetric currents of the redox processes grow in proportion to the protein amount inserted within the gel up to a limiting value. From that point, recorded currents remain independent of the amount of protein loaded. Such a behavior indicates that a portion of cytochrome c molecules is located in non-accessible sites of the silica matrix. The electrochemical insertion of poly(3,4-ethylenedioxythiophene) (PEDOT) through silica pores was carried out to connect those electrically isolated protein molecules and in situ UV–vis spectroscopy was used to gain information on the redox process of encapsulated cyt c. It was observed that the presence of conducting polymer gives rise to a 3-fold enhancement in cyt c electrochemical reduction rate at the initial stages of the treatment, irrespective of the amount of inserted polymer. The faster electrochemical reduction rate was obtained after the insertion of 6.45μg PEDOT within a silica gel containing 0.5mg protein. The presence of low conductivity domains in the conducting polymer or the hindrance to the protein free movement could explain that higher amounts of inserted PEDOT cannot improve the electrochemical reduction rate.

Effects of Film Morphology and Surface Chemistry on the Direct Electrochemistry of Cytochrome c at Boron-Doped Diamond Electrodes

The effects of film morphology and surface termination on the direct electron transfer of horse heart cytochrome c on boron-doped ultrananocrystalline (B-UNCD) and microcrystalline (B-MCD) diamond thin-film electrodes were investigated. Quasi-reversible, diffusion-controlled cyclic voltammetric responses were observed on oxygen-terminated (atomic O/C ∼0.015), but not hydrogen-terminated (atomic O/C ∼0.02) diamond thin films. The effect of the surface termination was the same for both the nanostructured B-UNCD film with sp2-bonded carbon atoms in the grain boundaries and the well faceted B-MCD film with micron-sized grains and largely devoid of sp2 carbon. Stable cyclic voltammetric i-E curves were recorded with cycling for both oxygen-terminated films indicating the absence of protein denaturation and electrode fouling. The peak currents increased linearly with the square root of the scan rate and the protein concentration; both indicative of a reaction rate limited by semi-infinite linear diffusion of the protein. Similar heterogeneous electron-transfer rate constants were observed for oxygen-terminated B-UNCD (3.48 (±1.25)×10−3cm/s) and B-MCD films (2.38 (±0.72)×10−3cm/s). The results clearly reveal that the oxygen-terminated surface is more active for electron-transfer with this soluble redox protein than is the hydrogen-terminated surface. The film morphology does not influence the diffusion-controlled response of the redox protein.

Direct electrochemistry of cytochrome c with three-dimensional nanoarchitectured multicomponent composite electrode and nitrite biosensing

A novel electrochemical nitrite ion (NO2−) biosensor was designed and fabricated by modifying a glassy carbon electrode (GCE) into three-dimensional nanoarchitectured electrode (3DNE) using a multicomponent nanocomposite (MCNC) film composed of graphene embedded titanium dioxide nanowires (TiO2(G) NWs), thiol-functionalized polyaniline (PANI(SH)), gold nanoparticles (Au), and immobilized cytochrome c (cyt c). The new NO2− biosensor was designated as cyt c/TiO2(G) NWs@PANI(SH)-Au MCNC/3DNE. The assembly of the 3DNE, involved sequential depositions of PANI(SH) and Au over previously synthesized TiO2(G) NWs by electrospinning-hydrothermal processes and immobilization of the cyt c onto the TiO2(G) NWs/PANI(SH)/Au MCNC film. The cyt c/TiO2(G) NWs@PANI(SH)-Au MCNC/3DNE exhibited direct electron transfer from cyt c to the electrode at a high rate constant (25.34s−1). The fabricated cyt c/TiO2(G) NWs@PANI(SH)-Au MCNC/3DNE showed high selectivity to NO2− ions with excellent sensitivity (9.2μA/mM), a wide linear concentration range (10μM–720mM), and a low detection limit (0.05μM). Additionally, the cyt c/TiO2(G) NWs@PANI(SH)-Au MCNC/3DNE exhibited high stability with good reproducibility and repeatability.

Direct electrochemistry of cytochrome c immobilized on gold electrode surface via Zr(IV) ion glue and its activity for ascorbic acid

Direct electrochemistry of cytochrome c (Cyt c) is achieved via Zr(IV) ion as an immobilization matrix to interface Cyt c on gold surface via thiol self-assembled monolayers. Steps of surface modification and electrocatalytic activity of the immobilized Cyt c are followed by voltammetry, impedance spectroscopy, chronoampetrometry, and attenuated total reflectance Fourier transform infrared (ATR–FTIR) spectroscopy. The results indicate that the native structure of Cyt c is conserved during the immobilization process. The immobilization method is rather simple, effective and free of immobilizing activators and reagents. Direct electron transfer rate constant and surface coverage of the immobilized Cyt c are found as 8.62(±1.98) s−1 and 1.15(±0.38)×10−11molcm−2, respectively. Bioactivity studies of the immobilized Cyt c toward oxidation of the ascorbic acid (AA) substrate show a linear response, from 10.0μM to 1.30mM AA, with a detection limit of 5.0(±1.8) μM AA and mean relative standard deviations varied from 13.7% to 3.7% for n=4 at each point. A value of 1.6(±0.8) mM AA is found for the Michaelis–Menten constant of Au-MPA-Zr(IV)-Cyt c toward AA for the first time. The tightly immobilized Cyt c maintains its bioactivity for more than 32days storage at 4°C.

Direct electrochemistry of cytochrome c immobilized on a graphene oxide–carbon nanotube composite for picomolar detection of hydrogen peroxide

A new bionanocomposite electrode based on cytochromecimmobilized graphene oxide-multiwalled carbon nanotube is used to fabricate a highly selective and sensitive amperometric biosensor for the picomolar level detection of hydrogen peroxide which may find application in bioimaging and healthcare

Oleylamine-Stabilized Gold Nanostructures for Bioelectronic Assembly. Direct Electrochemistry of Cytochrome c

Methods to prepare ultrathin gold nanowires and monodisperse nanoparticles based on the intrinsic property of gold(I) ions to form aurophilic interactions stabilized by oleylamine and long-chain alkylamine have been widely explored. Due to the low thermodynamic stability of the high aspect ratio nanostructures, their conjugation and assembly into functional nanosystems have not been explored so far. One of the reasons for this is that the surface of the nanostructures is insulated by stabilization compounds, which preserve the integrity of the nanostructures but at the same time form an insulating barrier in electronic and electrochemical systems in contact areas and for the charge transfer reactions. Conjugation of a metalloprotein cytochrome c (Cyt c) with oleylamine-stabilized gold ultrathin nanowires and nanoparticles into a bioelectrochemically active nanoarchitecture is presented here for the first time. Methods of preparing and assembling the ultrathin nanowires and nanoparticles on the thin-film g...

Direct Electrochemistry of Cytochrome c Based on Poly(diallyldimethylammonium Chloride)‐ Graphene Nanosheets/Gold Nanoparticles Hybrid Nanocomposites and Its Biosensing

AbstractA novel biosensor was developed by entrapping cytochrome c (Cyt c) in thin films of the room temperature ionic liquid (RTIL) containing nanocomposites of poly(diallyldimethylammonium chloride)‐graphene nanosheets‐gold nanoparticles (PDDA‐Gp‐AuNPs) at a 11‐mercaptoundecanoic acid‐6‐mercapto‐1‐hexanol modified gold electrode. The synthesized PDDA‐Gp‐AuNPs hybrid nanocomposites were characterized by UV‐vis spectroscopy, Raman spectroscopy, scanning electron microscopy and atomic force microscopy. The PDDA‐Gp‐AuNPs nanocomposites could increase the effective surface of the electrode, enhance the fixed amount of Cyt c on the electrode surface, promote the electron transfer and facilitate the catalytic activity of Cyt c. The RTIL could provide a biocompatible microenvironment to keep Cyt c biological activities, act as an effective mediator to immobilize a large number of Cyt c on the electrode and have good conductivity to improve electron transfer. Therefore, the resultant electrode exhibited good electrochemical performance and electrocatalytic activity. It could be used for electrochemical detection of H2O2 with rapid response, high sensitivity, wide linear range and low detection limit, as well as good stability, repeatability and selectivity. The sensor might be promising for practical application.

Bienzymatic glucose biosensor based on direct electrochemistry of cytochrome c on gold nanoparticles/polyaniline nanospheres composite

Gold nanoparticles (GNPs) assembled polyaniline nanospheres (PANS) composite were prepared by noncovalent strategy. Cytochrome c (Cyt c) was electrostatically adsorbed on the GNPs/PANS modified electrode. The direct electron transfer (DET) between Cyt c and GNPs/PANS modified electrode was investigated. Cyt c shows a couple of quasi-reversible and well-defined cyclic voltammetry (CV) peaks with a formal potential (E0′) of −0.27V (versus Ag/AgCl) in pH 7.0 phosphate buffer solution (PBS). The Cyt c/GNPs/PANS modified electrode gives an improved electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2). The catalysis currents increase linearly to the H2O2 concentration in a wide range of 0.01–2.4mM with a correlation coefficient 0.998. After immobilized glucose oxidase (GOD) on the Cyt c/GNPs/PANS modified electrode by Nafion polymer, a bienzymatic glucose biosensor was further prepared for sensitive detection of glucose. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at −0.2V versus Ag/AgCl to reduce the enzymatically produced H2O2 with minimal interference from the coexisting electroactive compounds. The proposed glucose biosensor exhibits good response performance to glucose with a wide linear range from 0.01 to 3.2mM with a correlation coefficient of 0.997. The electrode has high sensitivity of 63.1μAmM−1cm−2 and a detection limit 0.01mM at the signal-to-noise ratio of 3. Moreover, the glucose biosensor possesses good stability and reproducibility.

Direct electrochemistry of cytochrome c on a graphene/poly (3,4-ethylenedioxythiophene) nanocomposite modified electrode

The direct electrochemistry of cytochrome c (Cyt c) embedded in a novel support matrix of graphene/poly (3,4-ethylenedioxythiophene) (G-PEDOT) nanocomposite on a glassy carbon electrode was studied. The prepared G-PEDOT nanocomposite is of good biocompatibility, enhanced electric conductivity and large specific surface area. The immobilized Cyt c in G-PEDOT matrix (graphene weight percentage<2%) displays excellent direct electrochemistry and retains its biocatalytic activity toward the reduction of hydrogen peroxide. The electrochemical signal shows a linear response to H2O2 in the concentration range from 5.0×10−7 to 4.0×10−4M with a detection limit of 2.49×10−7M. The results demonstrate that the G-PEDOT nanocomposite offers a biocompatible material for the construction of biofuel cells, bioelectronics and biosensors.

Direct Electron Transfer of Cytochrome c on ZnO Nanoparticles Modified Carbon Paste Electrode

The direct electrochemistry of cytochrome c (cyt c) immobilized on a modified carbon paste electrode (CPE) was described. The electrode was modified with ZnO nanoparticles. Direct electrochemistry of cytochrome c in this paste electrode was easily achieved, and a pair of well-defined quasireversible redox peaks of a heme Fe (III)/Fe(II) couple appeared with a formal potential (E0) of −0.303 V (versus SCE) in pH 7.0 phosphate buffer solution (PBS). The fabricated modified bioelectrode showed good electrocatalytic ability for reduction of H2O2. The preparation process of the proposed biosensor was convenient, and the resulting biosensor showed high sensitivity, low detection limit, and good stability.

Direct Electrochemistry of Cytochrome c Adlayer on the ITO Electrode

The electrochemical redox reaction of a cytochrome c (Cyt c) adlayer on an indium tin oxide (ITO) electrode was directly monitored and the surface concentrations of Cyt c versus solution concentrations were obtained from cyclic voltammograms. The results indicate that the surface concentration increases from 0.35 × 10 - 12 to 1.53 × 10 - 12 mol·cm - 2 when the solution concentration is increased from 2 to 10 μmol·L - 1 . A quasi-linear relationship between the reciprocals of surface concentration and solution concentration was observed, indicating that Cyt c adsorption on the ITO electrode closely obeys the Langmuir isothermal adsorption model. The cyclic voltammograms of the Cyt c solutions with the ITO electrode reveal that both adsorbed and dissociated Cyt c molecules are involved in the electrode reaction and that the contribution of dissociated molecules is much larger than that of adsorbed ones. The electrode reaction is basically diffusion controlled and quasi-reversible. Based on the Nicholson method, the average standard heterogeneous rate constant was determined to be 1.65×10 - 3 cm·s - 1 . The electrochemical

Direct electrochemistry of cytochrome c entrapped in agarose hydrogel in room temperature ionic liquids

Direct electrochemistry of cytochrome c (cyt- c) entrapped in agarose hydrogel on gold electrode (Au), edge plane pyrolytic graphite electrode (EPPGE) and glassy carbon electrode (GC) in two room temperature ionic liquids was investigated. The effects of the addition of N, N-dimethylformamide (DMF) in the agarose-cyt- c film, water concentration in ionic liquids and exterior metal ions on the electrochemical behavior of cyt- c were monitored, and electrocatalytic properties of cyt- c were also done. Results showed that a good quasi-reversible redox behavior of cyt- c could be found after adding DMF in agarose-cyt- c film, and peak shape would not change after continuously scanning for 50 cycles. In addition, a certain amount of water in hydrophilic ionic liquids is necessary to maintain electrochemical activities of cyt- c, electrochemical performance of cyt- c is the best when the water content is 5.2% and 5.8% for 1-butyl-3-methylimidazolium bromide ([Bmim][Br]) and 1-butyl-3-methylimidazolium tetrafluoroborate([Bmim][BF 4]) respectively. However, electrochemical activities of cyt- c are inhibited by exterior metal ions. Interestingly, cyt- c entrapped in agarose hydrogel on EPPGE and GC could catalyze the electroreduction of trichloroacetic acid (TCA) and tert-butyl hydroperoxide ( t-BuOOH) in [Bmim][BF 4], but could not in [Bmim][Br]. Reasons for above-mentioned differences of electrochemical properties of cyt- c in different ionic liquids were preliminarily discussed.

Direct electrochemistry of cytochrome c entrapped in agarose hydrogel by protein film voltammetry

Cytochrome c (cyt-c) entrapped in agarose hydrogel was electrostatically bound to the edge plane pyrolytic graphite electrode (EPPGE), and then stable agarose-cyt-c/EPPGE was prepared. Direct electrochemistry between cyt-c and the EPPGE was monitored by protein film voltammetry. The effects of ionic strength, pH and exterior substances on the direct electrochemistry, and electrocatalytic properties of cyt-c were also studied. Results show that the electrochemical behavior of heme Fe(III)/Fe(II) in cyt-c is quasireversible. Its electrochemical performance is the best when the concentration of supporting electrolyte phosphate buffer solution is 0.2 mol/l and pH is 7.0, and greatly inhibited by exterior metal ions. Catalytic reduction of oxygen could also be achieved at the agarose-cyt-c/EPPGE.

Direct electrochemistry of cytochrome c immobilized on a novel macroporous gold film coated with a self-assembled 11-mercaptoundecanoic acid monolayer

We have successfully constructed a novel gold film with open interconnected macroporous walls of nanoparticles by combining the hydrogen bubble dynamic template synthesis with galvanic replacement reaction. After modified by a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA), the three-dimensionally (3D) interconnected macroporous Au film has been used as a biocompatible substrate for the immobilization of cytochrome c. The morphology, structure and electrochemical features of the modified and unmodified macroporous Au films were characterized by field-emission scanning electron microscopy (FESEM), energy-dispersive X-ray (EDX), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results reveal that the resultant films had a large electroactive surface area for high protein loading, enhanced electron transfer of cytochrome c, retained electrochemical activity, good stability and repeatability. And the excellent electrochemical behaviors could be attributed to the hierarchical structure of the macroporous Au film constructed by nanoparticles.

Direct electrochemistry of cytochrome c on nanotextured diamond surface

Nanotextured diamond surfaces with geometrical properties close to protein dimensions were used for the realization of direct electron transfer of cytochrome c (cyt c) without any covalent bonding. The peroxidase activity of native and denatured cyt c was also investigated. Cyclic voltammograms of native cyt c show quasi-reversible electron transfer reactions, while no heme redox activity is detected for denatured cyt c. Unfolding (denaturation) of cyt c can be achieved in the presence of hydrogen peroxide. Partially or fully denatured cyt c showed higher peroxidase activity than native cyt c. This is because denatured cyt c loses its tertiary structure and hydrogen peroxide is easier to access the heme redox center. The apparent Michaelis–Menten constant K m for native and denatured cyt c has been determined to be 0.23 mM and 0.08 mM.

Direct Electrochemistry of Cytochrome c at Modified Si(100) Electrodes

This paper demonstrates the direct electron transfer between the heme moiety of horse hearth cytochrome c and a pyridinyl group on self-assembled-monolayer-modified Si(100) electrodes. Self-assembled monolayers (SAMs) containing the putative receptor ligand were prepared by a step-wise procedure using "click" reactions of acetylene-terminated alkyl monolayers and isonicotinic acid azide derivatives. Unoxidized Si(100) electrodes, possessing either isonicotinate or isonicotinamide receptor ligands, were characterized using X-ray photoelectron spectroscopy, contact-angle goniometry, cyclic voltammetry, and electrochemical impedance spectroscopy. The ability of isonicotinic acid terminated layers to coordinatively bind the redox center of cytochrome c was found to be restricted to pyridinyl assemblies with a para-ester linkage present. The protocol detailed here offers an experimentally simple modular approach to producing chemically well-defined SAMs on silicon surfaces for direct electrochemistry of a well-studied model redox protein.

Direct electrochemistry of cytochrome c and biosensing for hydrogen peroxide on polyaniline grafted multi-walled carbon nanotube electrode

Cytochrome c ( cyt c) was immobilized into a matrix consisting of polyaniline (PANI) and multi-walled carbon nanotubes (MWNT) by a new strategy. First, PANI chains were grafted onto MWNT through electropolymerization. Second, the amine groups in PANI chains were oxidized at an applied potential of +0.80 V to acquire positive charges that would effectively immobilize negatively charged cyt C. The ITO/MWNT-g-PANI(O)/ cyt c electrode exhibited a pair of redox peaks with a peak potential separation (anodic to cathodic) of 0.25 V (vs Ag/AgCl) in 0.1 M phosphate buffer (pH 7.0). The results demonstrated that ITO/MWNT-g-PANI(O)/ cyt c promoted direct electron transfer between cyt c and electrode with a high electron transfer rate constant (17 s −1). The ITO/MWNT-g-PANI(O)/ cyt c electrode catalyzes the reduction of H 2O 2. The ITO/MWNT-g-PANI(O)/ cyt c biosensor displays an amperometric response to H 2O 2 with a linear concentration range from 0.5 μM to 1.5 mM ( r = 0.99, n = 12), a high sensitivity (32.2 μAm M −1) and fast response (9 s) and detection limit of 0.3 μM (S/N = 3).

Direct electrochemistry of cytochrome c at a novel gold nanoparticles-attached [formula omitted] ions implantation-modified indium tin oxide electrode

A NH 2 + ions implantation-modified indium tin oxide (NH 2/ITO) electrode was prepared and the existence of amino group on the electrode was verified by FT-IR spectra and X-ray photoelectron spectroscopy analysis (XPS). The gold nanoparticles (AuNPs) have been fabricated on the surface of NH 2/ITO electrode, resulting novel gold nanoparticles-attached NH 2 + ions implantation-modified ITO (AuNPs/NH 2/ITO) electrode, which could provide a biocompatible surface for the adsorption of cytochrome c (Cyt c). The Ultraviolet–visible (UV–vis) spectra indicated that Cyt c adsorbed on the AuNPs interface retained the native structure. Electrochemical impedance spectra (EIS) and cyclic voltammetric techniques were employed to evaluate the electrochemical behaviors of Cyt c. The surface of the AuNPs/NH 2/ITO electrode was exhibited using the scanning electron microscopy (SEM) comparing with the surface of the gold nanoparticles-attached ITO electrode (AuNPs/ITO). We found that the functional surface of the NH 2/ITO electrode resulted in a high AuNPs loading. The obtained Cyt c/AuNPs/NH 2/ITO electrode gave an excellent electrocatalytic activity towards the reduction of H 2O 2, and the catalysis currents were proportional to the H 2O 2 concentration in the range of 2.0–300.0 μM with a detection limit of 0.5 μM ( S/ N = 3).

Direct electrochemistry of cytochrome c on a phosphonic acid terminated self-assembled monolayers

The direct electrochemistry of cytochrome c (cyt c) on a gold electrode modified with 3-mercaptopropylphosphonic acid [HS–(CH 2) 3–PO 3H 2, MPPA] self-assembled monolayers (SAMs) was for the first time investigated. Electrochemical measurements and surface-enhanced infrared absorption spectroscopic reveal that the adsorption kinetics of cyt c on the MPPA-SAMs is very fast (saturation adsorption is completed within 5 s) and the immobilized cyt c molecules retain their native secondary protein structure. The nature of interaction between cyt c and –PO 3H 2 groups is mainly the electrostatic interaction. The direct electrochemistry of the immobilized cyt c on the –PO 3H 2 terminated SAMs with short chain is nearly reversible. Its formal potential ( E 0′ = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution ( E 0′ = 18–22 mV vs. SCE). In addition, the electron transfer rate of cyt c immobilized on –PO 3H 2 terminated SAMs is relatively slow as compared to –SO 3H and –COOH terminated SAMs, indicating excess negative charge density on the SAMs surface will decrease the electron transfer rate of cyt c.