A novel nitrite biosensor based on the direct electron transfer of hemoglobin immobilized on CdS hollow nanospheres

A novel nitrite biosensor based on the direct electron transfer of hemoglobin (Hb) immobilized on CdS hollow nanospheres (HS-CdS) modified glassy carbon electrode was constructed. The direct electron transfer of Hb showed a pair of redox peaks with a formal potential of -286 mV (vs. SCE) in 0.1M pH 7.0 phosphate buffer solution. It was a surface-controlled electrode process involving a single proton transfer coupled with a reversible one-electron transfer for each heme group of Hb. HS-CdS had a large specific surface area and good biocompatibility and had a better electrochemical response than that of solid spherical CdS. The immobilized Hb on HS-CdS displayed an excellent response to NO(2)(-) with one irreversible electrode process for NO reduction. Under optimal conditions, the biosensor could be used for the determination of NO(2)(-) with a linear range from 0.3 to 182 microM and a detection limit of 0.08 microM at 3 sigma based on the irreversible reduction of NO. HS-CdS provided a good matrix for protein immobilization and had a promising application in constructing sensors.     

Direct electron transfer and enzymatic activity of hemoglobin in a hexagonal mesoporous silica matrix

The direct electrochemistry of hemoglobin (Hb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Hb and the HMS was investigated using UV-Vis spectroscopy, FT-IR, and electrochemical methods. The direct electron transfer of the immobilized Hb exhibited two couples of redox peaks with the formal potentials of -0.037 and -0.232 V in 0.1 M (pH 7.0) PBS, respectively, which corresponded to its two immobilized states. The electrode reactions showed a surface-controlled process with a single proton transfer at the scan rate range from 20 to 200 mV/s. The immobilized Hb retained its biological activity well and displayed an excellent response to the reduction of both hydrogen peroxide (H2O2) and nitrate (NO2-). Its apparent Michaelis-Menten constants for H2O2 and NO2- were 12.3 and 49.3 microM, respectively, showing a good affinity. Based on the immobilization of Hb on the HMS and its direct electrochemistry, two novel biosensors for H2O2 and NO2- were presented. Under optimal conditions, the sensors could be used for the determination of H2O2 ranging from 0.4 to 6.0 microM and NO2- ranging from 0.2 to 3.8 microM. The detection limits were 1.86 x 10(-9) M and 6.11 x 10(-7) M at 3sigma, respectively. HMS provided a good matrix for protein immobilization and biosensor preparation.     

Direct electron transfer of hemoglobin in layered alpha-zirconium phosphate with a high thermal stability

A heme protein hemoglobin (Hb) was reacted with preexfoliated layered alpha-zirconium phosphate (alpha-ZrP) platelets. An X-ray diffraction (XRD) pattern of small range showed that the exfoliated alpha-ZrP platelets reassembled after the addition of Hb molecules, with the protein intercalated between the layers. UV-Vis and Fourier transform infrared (FTIR) spectra analysis displayed that no significant denaturation occurred to the intercalated protein. The bioactivity of Hb was also investigated by testing the electrochemical properties of the Hb/alpha-ZrP composite. Results showed that the intercalation of Hb into the layered material not only improved the thermal stability of Hb but also enhanced the direct electron transfer ability between protein molecules and electrode. The protein still showed bioactivity after treatment at a temperature as high as 85 degrees C. A pair of well-defined redox peaks at approximately -0.37 and -0.32V was observed on the cyclic voltammograms (CVs) of the Hb/alpha-ZrP composite modified electrode, and the electrode reactions showed a surface-controlled process with a single proton transfer. The resultant biosensor constructed by the Hb/alpha-ZrP composite displayed an excellent response to the reduction of hydrogen peroxide (H(2)O(2)) with good reproducibility.     

Botanical micelle and its application for direct electrochemical biosensor

This paper is concerned about the entrapment of horseradish peroxidase (HRP) within botanical inositol hexakisphosphoric (IP(6)) micelles for the preparation of enzyme biosensor. The good affinity of IP(6) micelles with the enzyme provides naturally biocompatible microenvironment for the enzyme immobilization, achieving the direct electron transfer between HRP and electrode surface. The resulting biosensor to H(2)O(2) detection exhibits a low detection limit of 0.1 μmol L(-1) (S/N = 3), a quick response time (3s), and a long-term stability. The apparent Michaelis-Menten constant is quite tiny about 0.0016 mmol L(-1).     

Highly sensitive and selective hydrogen peroxide biosensor based on hemoglobin immobilized at multiwalled carbon nanotubes-zinc oxide composite electrode

A highly sensitive and selective amperometric hydrogen peroxide (H(2)O(2)) biosensor based on immobilization of hemoglobin (Hb) at multiwalled carbon nanotubes-zinc oxide (MWCNT/ZnO) composite modified glassy carbon electrode (GCE) is reported. ZnO microsponges were electrochemically grown on MWCNT surface by the simple, cost-effective, green, electrochemical method at room temperature. The MWCNT/ZnO/Hb composite film showed a pair of well-defined, quasi-reversible redox peaks with a formal potential (E°') of -0.336V, characteristic features of heme redox couple of Hb. The electron transfer rate constant (k(s)) of immobilized Hb was 1.26s(-1). The developed biosensor showed a very fast response (>2s) toward H(2)O(2) with good sensitivity, wide linear range, and low detection limit of 0.02μM. The fabricated biosensor showed interesting features, including high selectivity, acceptable stability, good reproducibility, and repeatability along with excellent conductivity, facile electron mobility of MWCNT, and good biocompatibility of ZnO. The fabrication method of this biosensor is simple and effective for determination of H(2)O(2) in real samples with quick response, good sensitivity, high selectivity, and acceptable recovery.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

Direct electrochemistry of hemoglobin in dimethyldioctadecyl ammonium bromide film and its electrocatalysis to nitric oxide

Hemoglobin (Hb) was successfully immobilized in dimethyldioctadecyl ammonium bromide (DOAB) film at pyrolytic graphite (PG) electrode. Electrochemical experiments revealed that Hb in DOAB film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.160 V versus saturated calomel electrode (SCE) in pH 5.0 buffer, characteristic of the heme Fe(III)/Fe(II) redox couple of Hb. The electron transfer (eT) rate between Hb and the PG electrode was 0.10 s(-1). Positions of the Soret absorbance band indicated that the Hb retained its secondary structure and was similar to its native state. Furthermore, the Hb in DOAB film acted as a biological catalyst towards the reduction of nitric oxide (NO). The voltammetric response of NO at the Hb-DOAB modified electrode could be used to determine the concentration of NO in solution.     

One-step method embedding superoxide dismutase and gold nanoparticles in silica sol-gel network in the presence of cysteine for construction of third-generation biosensor

A new and one-step method has been developed for the fabrication of superoxide dismutase (SOD) based biosensor. This method was used to form a silica sol-gel (SG) thin film and to immobilize SOD and gold nanoparticles (GNPs) in silica SG network for the fabrication of biosensor. The immobilized superoxide dismutase realized direct electron transfer between the enzyme and electrode surface, and the rate constants of the electrochemical process (ks) of SOD was markedly enhanced by GNPs. The electrochemical performance and influencing factors of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response to superoxide anion. The calibration range of superoxide anion was from 0.05 to 0.4 micromol L(-1). The proposed method exhibited the benefits of the advantages of self-assembly, nanoparticles and SG techniques. The fabrication of the SOD-modified electrode was easy and simple. The biosensor exhibited high sensitivity and long-term stability.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

Direct electrochemistry of lactate dehydrogenase immobilized on silica sol-gel modified gold electrode and its application

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol-gel film on gold electrode was investigated, and an obvious cathodic peak at about -200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (alpha) of 0.79 and the apparent heterogeneous electron transfer rate constant (K(s)) of 3.2 s(-1). The activated voltammetric response and decreased charge-transfer resistance of Ru(NH(3))(6)(2+/3+) on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol-gel and the LDH embedded in silica sol-gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 x 10(-6) to 3.0 x 10(-5) mol L(-1) and the detection limit was 8.0 x 10(-7) mol L(-1) at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.     

A novel approach to construct a horseradish peroxidase|hydrophilic ionic liquids|Au nanoparticles dotted titanate nanotubes biosensor for amperometric sensing of hydrogen peroxide

The direct electrochemistry of horseradish peroxidase (HRP) on a novel sensing platform modified glassy carbon electrode (GCE) has been achieved. This sensing platform consists of Nafion, hydrophilic room-temperature ionic liquid (RTIL) and Au nanoparticles dotted titanate nanotubes (GNPs-TNTs). The composite of RTIL and GNPs-TNTs was immobilized on the electrode surface through the gelation of a small amount of HRP aqueous solution. The composite was characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and infrared spectroscopy (IR). UV-Vis and IR spectroscopy demonstrated that HRP in the composite could retain its native secondary structure and biochemical activity. The HRP-immobilized electrode was investigated by cyclic voltammetry and chronoamperometry. The results from both techniques showed that the direct electron transfer between the nanocomposite modified electrodes and heme in HRP could be realized. The biosensor responded to H(2)O(2) in the linear range from 5×10(-6) to 1×10(-3) mol L(-1) with a detection limit of 2.1×10(-6) mol L(-1) (based on the S/N=3).     

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl₂ produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential (E^0′) on the solution pH (56 mV pH^− 1) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10^− 11 mol cm^− 2_, indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant (k_s) was 1.43 s^− 1, indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K_m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.     

A nitric oxide biosensor based on the multi-assembly of hemoglobin/montmorillonite/polyvinyl alcohol at a pyrolytic graphite electrode

Direct electron transfer of hemoglobin (Hb) can be achieved in a Hb/montmorillonite (MMT)/polyvinyl alcohol multi-assembly at a pyrolytic graphite (PG) electrode. Accordingly, a novel nitric oxide (NO) biosensor is proposed. The reduction of NO is observed at a potential of approximately -783 mV (vs. SCE) at pH 5.5. At optimum pH, this biosensor shows a wide linear range of 1.0x10(-6)-2.5x10(-4) mol/l with a detection limit of 5.0x10(-7) mol/l. The sensor-to-sensor reproducibility is good consideringmacr; a relative standard deviation of 3.5% in five independent determinations at 5.0x10(-5) mol/l NO. The modified electrode is conveniently constructed and durable in long-term operations.     

Direct electron transfer and electrocatalysis of hemoglobin adsorbed on mesoporous carbon through layer-by-layer assembly

Using chitosan as an effective linker between CMK-3 and glassy carbon electrode surface, {Hb/CMK-3}n multilayer film-modified electrodes were constructed through layer-by-layer assembly. The morphology of thus-formed {Hb/CMK-3}n film was characterized by scanning electron micrographs, and the interaction of hemoglobin (Hb) with CMK-3 was studied by UV-vis spectroscopy and electrochemical methods. Under optimal conditions, {Hb/CMK-3}6 film showed a couple of stable and well-defined redox peaks at about -377 and -296 mV in pH 7.0 buffers. Furthermore, the {Hb/CMK-3}6 film displayed excellent electrocatalysis to the reduction of both H2O2 and O2. Based on thus-formed film and its direct electron transfer behavior, a novel biosensor was presented for the determination of H2O2 ranging from 1.2 to 57 muM with the detection limit of 0.6microM at S/N=3. CMK-3 provided a desirable matrix for protein immobilization and biosensor preparation.     

Electrochemical properties of heme-protein in lauric acid films and its application as a biosensor

In this research, the enhancement of electron-transfer activity of hemoglobin (Hb) in lauric acid film was investigated for the first time. This type of composite film was made on a glassy carbon electrode by a casting method. Cyclic voltammetric result of the modified electrode displays a well-defined redox peak, which was attributed to the direct electrochemical response of Hb. Our results illustrate that Hb exchange electrons directly with electrode and exhibits the characteristics of peroxidase. When we use this modified electrode as a biosensor, it gives excellent performance in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). The parameters such as pH and applied potential of the biosensor influencing in H₂O₂ detection were optimized carefully. Through the optimal conditions, the proposed biosensor shows the linear range for H₂O₂ determination was from 1×10⁻⁵ to 1.25×10⁻⁴ mol L⁻¹ with a detection limit of 1×10⁻⁷ mol L⁻¹. The biosensor retained more than 90% of the initial response after 14 d.     

Urchinlike MnO2 nanoparticles for the direct electrochemistry of hemoglobin with carbon ionic liquid electrode

In this paper an urchinlike MnO(2) nanoparticle was synthesized by hydrothermal method and applied to the protein electrochemistry for the first time. By using a carbon ionic liquid electrode (CILE) as the basal electrode, hemoglobin (Hb) was immobilized on the surface of CILE with chitosan (CTS) and MnO(2) nanoparticle composite materials. Spectroscopic results indicated that Hb molecules retained its native structure in the composite film. A pair of well-defined redox peaks appeared on the cyclic voltammogram with the formal peak potential as -0.180 V (vs. SCE), which indicated that direct electron transfer of Hb was realized on the modified electrode. The result can be attributed to the specific characteristic of MnO(2) nanoparticle and the advantages of CILE, which facilitated the electron transfer rate. The fabricated CTS-MnO(2)-Hb/CILE showed good electrocatalytic ability to the reduction of trichloroacetic acid (TCA). Under the optimal conditions the catalytic current was in linear to TCA concentration in the range from 0.5 to 16.0 mmol L(-1) with the detection limit calculated as 0.167 mmol L(-1) (3σ). The result indicated that urchinlike MnO(2) nanoparticle had the potential application in the third generation electrochemical biosensors.     

A bioelectrochemical polypyrrole-containing Fe(CN)6(3-) interface for the design of a NAD-dependent reagentless biosensor

Ferricyanide ions were immobilized on a platinum electrode surface by means of an electrochemically grown polypyrrole film. The entrapped Fe(CN)6(3-)/Fe(CN)6(4-) redox system displayed a high heterogeneous electron transfer rate. The resulting modified electrode was efficient for the ferricyanide-mediated NADH oxidation catalyzed by a diaphorase. The bioelectrochemical interface was applied to the design of a reagentless amperometric D-lactate biosensor. A weakly polarized two polypyrrole-containing Fe(CN)6(3-) modified electrode system was involved without any reference. An enzymatic solution containing D-lactate dehydrogenase, diaphorase and NAD-dextran was further confined on the sensing electrode using a semi-permeable membrane. The sensitivity and the response time of the reagentless biosensor were similar to those of the analogous sensor working with soluble mediator and cofactor, i.e. 25 microA mM(-1) cm(-2) and 120 s, respectively. The other analytical performances were less satisfactorily: the detection limit was 5 x 10 mmol L(-1) and the linearity range was comprised between 0.1 and 0.5 mmol L(-1).     

Novel voltammetric biosensor for determining acrylamide in food samples

Recent findings showing that acrylamide is formed in heat-treated foods rich in asparagine and reducing sugars such as glucose, have accelerated the needs for the development of new analytical methods to determine this potential human carcinogen. Acrylamide forms adduct with hemoglobin (Hb) as a result of the reaction with the alpha-NH2 group of N-terminal valine of Hb. This interaction is the basis of a new voltammetric biosensor to detect acrylamide. The biosensor was constructed using a carbon-paste electrode modified with hemoglobin (Hb), which contains four prosthetic groups of heme--Fe(III). Such an electrode displays a reversible reduction/oxidation process of Hb-Fe(III)/Hb-Fe(II). Interaction between Hb and acrylamide was observed through decreasing of the peak current of Hb-Fe(III) reduction. The electrodes presented a very low detection limit (1.2 x 10(-10)M). The validation made in the matrix obtained by water extraction of potato chips showed that the electrodes presented are suitable for the direct determination of acrylamide in food samples.     

Immobilization of creatininase, creatinase and sarcosine oxidase on iron oxide nanoparticles/chitosan-g-polyaniline modified Pt electrode for detection of creatinine

Commercial enzymes, creatininase (CA) from Pseudomonas sp, creatinase (CI) from Pseudomonas sp, sarcosine oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosan-graft-polyaniline (Fe(3)O(4)-NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe(3)O(4)-NPs. A creatinine biosensor was fabricated using Enzymes/Fe(3)O(4)-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2s at pH 7.5 and 30 °C, when polarized at 0.4V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from 1 to 800 μM. The sensitivity of the biosensor was 3.9 μA μM(-1) cm(-2), with a detection limit of 1 μM (S/N=3). Apparent Michaelis-Menton (K(m)) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 °C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method (r=0.99).     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.