Mesoporous Materials Promoting Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase

The direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) immobilized on hexagonal mesoporous silicas (HMS) matrix was studied. The interaction between HRP and HMS was examined by using Fourier transform infrared spectroscopy, nitrogen adsorption isotherms and electrochemical methods. The immobilized HRP at a modified glassy carbon electrode showed a good direct electrochemical behavior, which depended on the specific properties of the HMS. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the HRP intercalated in the mesopores and adsorbed on the external surface of the HMS were observed with the formal potentials of ?0.315 and ?0.161 V in 0.1 M pH 7.0 PBS, respectively. The amount of HRP intercalated in the mesopores of HMS proved to be related to the pore size. The HRP intercalated in the mesopores showed a surface controlled electrode process with a single proton transfer. The immobilized HRP displayed an excellent electrocatalytic response to the reduction of hydrogen peroxide (H₂O₂) without the aid of an electron mediator. The HMS provided a novel matrix for protein immobilization and direct electron transfer study of the immobilized protein.     

A whisker-like carbon composite for the immobilization of laccase and its bioelectrochemistry

A novel mesoporous carbon/whisker-like carbon (MCWC) composite was used for the immobilization of laccase (Lac) and its bioelectrochemical behaviors were studied. It was confirmed by XPS that Lac was strongly adsorbed on the surface of the MCWC composite. The cyclic voltammetric results showed that the immobilized Lac underwent a direct quasi-reversible electrochemical reaction. The value of the electron transfer rate constant k _s was estimated to be 0.770 s^?1, indicating a reasonably fast electron transfer between the immobilized Lac and the underlying electrode. The surface concentration (Γ) of Lac was estimated to be 2.730 × 10^?12 mol/cm². Further experimental results showed that the immobilized Lac displayed an appreciable electrocatalytic activity to the electrochemical reduction of O₂. These properties could be attributed to the particular structure of loosely packed nanometer-scale carbon whiskers and the existence of a large amount of oxygen-containing groups. The immobilization method and the novel carrier (MCWC) may find new applications in fabricating the biocatalysts for biofuel cells.     

An amperometric biosensor using toluidine blue as an electron transfer mediator intercalated in α-zirconium phosphate-modified horseradish peroxidase immobilization matrix

A novel H₂O₂ biosensor was constructed employing α-zirconium phosphate as a new support substrate to hold an electron shuttle toluidine blue between a glassy carbon electrode and horseradish peroxidase. Toluidine blue was intercalated into α-zirconium phosphate-modified horseradish peroxidase immobilization matrix cross-linked on a glassy carbon electrode surface via bovine serum albumin-glutaraldehyde. This co-immobilization matrix of the mediator and the enzyme was formed from the α-zirconium phosphate (α-ZrP)-toluidine blue (TB) inclusion colloid in which horseradish peroxidase (HRP) was dissolved. Intercalation of TB in layered α-ZrP was investigated by scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and electrochemical measurements. TB immobilized in this way underwent a quasi-reversible electrochemical redox reaction at the electrode. Cyclic voltammetry and amperometric measurements demonstrated good stability and efficiently-shuttled electrons between HRP and the electrode. The sensor responded rapidly to H₂O₂ with a detection limit of 3.0 × 10^–7 mol/L.     

Direct electron transfer and biocatalytic activity of iron storage protein molecules immobilized on electrodeposited cobalt oxide nanoparticles

Ferritin was immobilized on a glassy carbon electrode with electrodeposited cobalt oxide nanoparticles, and its direct electron transfer behavior was studied. It exhibits a pair of redox peaks due to direct electron transfer between ferritin and the nanoparticles. Electrochemical parameters including the formal potential (E^0??), the charge transfer coefficient (??), and the apparent heterogeneous electron transfer rate constant (k_s) were determined. The sensor displays excellent biocatalytic activity in terms of reduction of hydrogen peroxide, and this was applied to electrochemical sensing of hydrogen peroxide.

Hemoglobin co-immobilized with silver–silver oxide nanoparticles on a bare silver electrode for hydrogen peroxide electroanalysis

Hemoglobin (Hb) and silver–silver oxide (Ag–Ag₂O) nanoparticles were co-immobilized on a bare silver electrode surface by cyclic voltammetry, and were characterized by UV–vis reflection spectroscopy, scanning electron microscopy, and electrochemical impedance spectroscopy. The immobilized Hb was shown to maintain its biological activity well. Direct electron transfer between Hb and the resulting electrode was achieved without the aid of any electron mediator. The reduction currents to hydrogen peroxide (H₂O₂) at co-immobilized electrodes showed a linear relationship with H₂O₂ concentration over a concentration range from 6.0?×?10^?6 to 5.0?×?10^?2 mol L^?1, and a detection limit of 2.0?×?10^?6 mol L^?1 (S/N?=?3).     

Cytochrome c′ from Chromatium vinosum on gold electrodes

Cytochrome c′ from Chromatium vinosum (CVCP) was immobilized at a surface-modified gold electrode. Characterization of the CVCP electrode showed a quasi-reversible, diffusionless electrochemical redox behavior of the surface adsorbed protein with a formal potential of −128±5 mV vs. Ag/AgCl. The heterogeneous electron transfer rate constant of adsorbed CVCP was determined to be about 50 s⁻¹. Different immobilization strategies were compared. The interaction of the immobilized CVCP with nitric oxide (NO) was investigated and applied for a primary amperometric detection of NO in solution.     

Development of a peroxide biosensor made of a thiolated-viologen and hemoglobin-modified gold electrode

A gold electrode modified with thiolated-viologen is used to design a biosensor in corporate with hemoglobin (Hb). A highly stable self-assembled monolayer (SAM) of thiol-based viologen is immobilized onto the gold electrode. Hb is then immobilized onto the viologen-modified electrode. The modified electrode is very stable. By incorporating with SAMs of viologen and Hb, viologen can act as an electron transfer mediator for Hb to the gold electrode. The potential of Hb was found to be about − 135 mV versus Ag/AgCl for ferro and ferri active centers. The electrochemistry of Hb provides an opportunity to manufacture a third generation of biosensors. Experimental conditions influencing the biosensor performances such as pH, and potential are optimized and assessed. This sensor offered an excellent electrochemical response for H₂O₂ concentration below the μmol level with high sensitivity and selectivity with a short time response.     

Modelling of the impedimetric responses of an aflatoxin B1 immunosensor prepared on an electrosynthetic polyaniline platform

Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. An electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) was developed with AFB₁antibody (AFB₁-Ab) immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of the Pt/PANi–PSSA electrode, the Pt/PANi–PSSA/AFB₁-Ab immunosensor and Pt/PANi–PSSA/AFB₁-Ab incubated in bovine serum albumin (BSA) were 0.458, 720 and 1,066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for the Pt/PANi–PSSA electrode to 1,066 kΩ for the Pt/PANi–PSSA/AFB₁-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB₁ immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩ L/mg.     

The effects of carbon nanotubes on the electrocatalysis of hydrogen peroxide by metallo-phthalocyanines

The pre-grafted screen-printed gold electrode modified with phenyl-amino monolayer was investigated for covalent immobilization of phenyl-amine functionalized single-walled carbon nanotubes (PA-SWCNT) and metal tetra-amino phthalocyanine (MTAPc) using Schiff-base reactions with benzene-1,4-dicarbaldehyde (BDCA) as cross-linker. The PA-SWCNT and MTAPc modified electrodes were applied as hybrids for electrochemical sensing of H₂O₂. The step-by-step fabrication of the electrode was followed using electrochemistry, impedance spectroscopy, scanning electron microscopy and Raman spectroscopy and all these techniques confirmed the fabrication and the immobilization of PA-SWCNT, MnTAPc and CoTAPc onto gold surfaces. The apparent electron transfer constant (k_app) showed that the carbon nanotubes and metallo-phthalocyanines hybrids possess good electron transfer properties compared to the bare, pre-grafted and the MTAPc modified gold electrode surfaces without PA-SWCNT. The electrochemical sensing of hydrogen peroxide was successful with PA-SWCNT-MTAPc hybrid systems showing higher electrocatalytic currents compared to the other electrodes. The analytical parameters obtained using chronoamperometry gave good linearity at H₂O₂ concentrations ranging from 1.0 to 30.0 μmol L⁻¹. The values for the limit of detection (LoD) were found to be of the orders of 10⁻⁷ M using the 3δ for all the electrodes. The PA-SWCNT-MTAPc modified SPAuEs were much more sensitive compared to PA-MTAPc modified SPAuEs.     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current-voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3−/4− as the redox indicator. The (CA-G_NP/SPAN)_n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G_NP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)₆]^3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G_NP/SPAN)_n films, the target DNA with 20-base could be detected up to 2.13 × 10⁻¹³ mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.     

An amperometric biosensor using toluidine blue as an electron transfer mediator intercalated in α-zirconium phosphate-modified horseradish peroxidase immobilization matrix

A novel H₂O₂ biosensor was constructed employing α-zirconium phosphate as a new support substrate to hold an electron shuttle toluidine blue between a glassy carbon electrode and horseradish peroxidase. Toluidine blue was intercalated into α-zirconium phosphate-modified horseradish peroxidase immobilization matrix cross-linked on a glassy carbon electrode surface via bovine serum albumin-glutaraldehyde. This co-immobilization matrix of the mediator and the enzyme was formed from the α-zirconium phosphate (α-ZrP)-toluidine blue (TB) inclusion colloid in which horseradish peroxidase (HRP) was dissolved. Intercalation of TB in layered α-ZrP was investigated by scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and electrochemical measurements. TB immobilized in this way underwent a quasi-reversible electrochemical redox reaction at the electrode. Cyclic voltammetry and amperometric measurements demonstrated good stability and efficiently-shuttled electrons between HRP and the electrode. The sensor responded rapidly to H₂O₂ with a detection limit of 3.0 × 10^–7 mol/L. Received: 1 July 1997 / Revised: 13 October 1997 / Accepted: 21 October 1997     

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.     

Direct electrochemistry of hemoglobin on an ionic liquid carbon electrode modified with zinc tungstate nanorods

We have constructed a new electrochemical biosensor by immobilization of hemoglobin (Hb) and ZnWO₄ nanorods in a thin film of chitosan (CTS) on the surface of carbon ionic liquid electrode. UV–vis and FT-IR spectra reveal that Hb remains in its native conformation in the film. The modified electrode was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. A pair of well-defined redox peaks appears which indicates direct electron transfer from the electrode. The presence of CTS also warrants biocompatibility. The electron transfer coefficient and the apparent heterogeneous electron transfer rate constant were calculated to be 0.35 and 0.757 s^?1, respectively. The modified electrode displays good electrocatalytic activity for the reduction of trichloroacetic acid with the detection limit of 0.613 mmol L^?1 (3σ). The results extend the protein electrochemistry based on the use of ZnWO₄ nanorods.

Immobilization and electrochemical behavior of gold nanoparticles modified leukemia K562 cells and application in drug sensitivity test

A new strategy for immobilization of tumor cells on electrode surface and accelerating electron transfer between electrode and the immobilized cells was proposed to study the electrochemical behavior of cells and the effect of antitumor drug on cell viability. The leukemia K562 cells immobilized in a microporous cellulose membrane were firstly modified with colloidal gold nanoparticles to retain efficiently the activity of immobilized living tumor cells and promote electron transfer between electroactive centers of the cells and the electrode, exhibiting a well-defined anodic peak of guanine at +0.830 V at 50 mV s⁻¹. The electrochemical response could be used to describe cell growth and evaluate the effectiveness of antitumor drug methotrexate on tumor cells. The proposed method offered potential advantages for drug sensitivity test with little usage of cells. It could be developed as a convenient means for the study of the tumor cells growth and the cytotoxicity of antitumor drugs.     

1,4-二氧六环介质中辣根过氧化物酶电极的性能研究

用交联法制备辣根过氧化物酶(HRP)电极, 在1,4-二氧六环介质中研究其电化学行为。实验表明, 固定化的HRP在有机相中仍保持活性并可与电极进行直接电子传递, 因而能在没有其它电子传递体存在的条件下催化H~2O~2的电化学还原反应。当亚铁氰化物与酶共修饰至电极上之后, 它起着电子传递体的作用, 使HRP电极的性能大为改善。根据不同条件下得到的动力学参数, 讨论了影响酶电极性能的因素。     

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.     

Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins

Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b₅ (Cyt b₅) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand.     

Immobilization of functional light antenna structures derived from the filamentous green bacterium Chloroflexus aurantiacus

The integration of highly efficient, natural photosynthetic light antenna structures into engineered systems while their biophotonic capabilities are maintained has been an elusive goal in the design of biohybrid photonic devices. In this study, we report a novel technique to covalently immobilize nanoscaled bacterial light antenna structures known as chlorosomes from Chloroflexus aurantiacus on both conductive and nonconductive glass while their energy transducing functionality was maintained. Chlorosomes without their reaction centers (RCs) were covalently immobilized on 3-aminoproyltriethoxysilane (APTES) treated surfaces using a glutaraldehyde linker. AFM techniques verified that the chlorosomes maintained their native ellipsoidal ultrastructure upon immobilization. Results from absorbance and fluorescence spectral analysis (where the Stokes shift to 808/810 nm was observed upon 470 nm blue light excitation) in conjunction with confocal microscopy confirm that the functional integrity of immobilized chlorosomes was also preserved. In addition, experiments with electrochemical impedance spectroscopy (EIS) suggested that the presence of chlorosomes in the electrical double layer of the electrode enhanced the electron transfer capacity of the electrochemical cell. Further, chronoamperometric studies suggested that the reduced form of the Bchl- c pigments found within the chlorosome modulate the conduction properties of the electrochemical cell, where the oxidized form of Bchl- c pigments impeded any current transduction at a bias of 0.4 V within the electrochemical cell. The results therefore demonstrate that the intact chlorosomes can be successfully immobilized while their biophotonic transduction capabilities are preserved through the immobilization process. These findings indicate that it is feasible to design biophotonic devices incorporating fully functional light antenna structures, which may offer significant performance enhancements to current silicon-based photonic devices for diverse technological applications ranging from CCD devices used in retinal implants to terrestrial and space fuel cell applications.     

Direct electrochemistry of horseradish peroxidase based on hierarchical porous calcium phosphate microspheres

Biomorphic calcium phosphate (CaP) microspheres with hierarchical porous structure were synthesized using natural cole pollen grains as templates and were further employed for the immobilization of horseradish peroxidase (HRP). Scanning electron microscopy and Fourier transform infrared spectroscopy revealed (a) the porous structure of the CaP microspheres, (b) the effective immobilization, and (c) the retention of the conformation of HRP on CaP. The immobilized HRP was placed on a glassy carbon electrode where it underwent a direct, fully reversible, and surface-controlled redox reaction with an electron transfer rate constant of 1.96 s^?1. It also exhibits high sensitivity to the reduction of H₂O_2. The response to H₂O₂ is linear in the 5.00 nM to 1.27 μM concentration range, and the sensitivity is 30357 μA?mM^?1?cm^?2. The detection limit (at an SNR of 3) is as low as 1.30 nM. The apparent Michaelis–Menten constant (K _M ^app ) of the immobilized enzyme is 0.92 μM. This new CaP with hierarchical porous structure therefore represents a material that can significantly promote the direct electron transfer between HRP and an electrode, and is quite attractive with respect to the construction of biosensors.