Direct electrochemistry of hemoglobin on carbonized titania nanotubes and its application in a sensitive reagentless hydrogen peroxide biosensor

Carbonized TiO(2) nanotubes (TNT/C) prepared by carbonization with organic polymers possess advantages combined from high conductivity of carbon and nanostructure of TiO(2) nanotubes. The material was used as a supporting matrix to immobilize a redox protein, hemoglobin (Hb), to explore its direct electron transfer ability. The apparent heterogeneous electron transfer rate constant (k(ET)) of Hb on TNT/C is 108s(-1), which is much higher than that in the reported works, demonstrating excellent direct electrochemistry behavior. The TNT/C-Hb modified glassy carbon electrode (GCE) demonstrates significant electrocatalytic activity for reduction of hydrogen peroxide with a small apparent Michaelis-Menten constant (87.5 microM). The TNT/C-Hb based H(2)O(2) sensor has a low detection limit (0.92 microM), fast response time (3s) and high dynamic response range (10(-6) to 10(-4)M), a much better performance than the reported works. These results demonstrate that a direct electrochemistry behavior can be significantly enhanced through simple carbon coating on a nanostructured material for higher reaction surface area and better conductivity. This work suggests that Hb-immobilized TNT/C has potential applications in a sensitive H(2)O(2) sensor.     

Carbon nanotubes-polymer-redox mediator hybrid thin film for electrocatalytic sensing

Electrocatalytic sensing of NADH using a hybrid thin film derived from multi-wall carbon nanotubes (CNTs), Nafion (Nf) polymer and electrogenerated redox mediator is described. The redox mediator was electrochemically generated by the oxidation of serotonin on the hybrid thin film modified glassy carbon electrode (GC/Nf-CNT). Controlled potential electrolysis of serotonin at 0.1 V in neutral solution results in the generation of the redox mediator 5,5'-dihydroxy-4,4'-bitryptamine (DHB) on the hybrid thin film. The electrogenerated DHB has redox active quinone-imine structure and was electrochemically characterized by studying the pH dependent redox response. DHB on the hybrid thin film exhibits reversible redox peak at -0.05 V and the formal potential shifts by -55 mV while increasing the solution pH by 1 unit. The quinone-imine structure of DHB efficiently catalyzes the oxidation of NADH with a decrease in the overpotential of about 500 mV compared to the unmodified electrode. The CNTs of the hybrid thin film facilitates the mediated electrocatalytic oxidation of NADH. The hybrid thin film modified electrode exhibits stable amperometric response and it linearly responds to NADH (0.5-400 microM). This hybrid thin film modified electrode could detect NADH as low as 0.1 microM at -0.05 V with a sensitivity of 11.1 nA/microM in physiological pH.     

Overoxidized polypyrrole film directed single-walled carbon nanotubes immobilization on glassy carbon electrode and its sensing applications

In this paper, the films of overoxidized polypyrrole (PPyox) directed single-walled carbon nanotubes (SWNTs) have been electrochemically coated onto glassy carbon electrode (GCE). Electroactive monomer pyrrole was added into the solution containing sodium dodecyl sulfate (SDS) and SWNTs. Then, electropolymerization was proceeded at the surface of GCE, and a novel kind of conducting polymer/carbon nanotubes (CNTs) composite film with the orientation of CNTs were obtained correspondingly. Finally, this obtained polypyrrole (PPy)/SWNTs film modified GCE was oxidized at a potential of +1.8 V. It can be found that this proposed PPyox/SWNTs composite film modified GCE exhibited excellent electrocatalytic properties for some species such as nitrite, ascorbic acid (AA), dopamine (DA) and uric acid (UA), and could be used as a new sensor for practical applications. Compared with previous CNTs modified electrodes, SWNTs were oriented towards the outside of modified layer by PPyox and SDS, which made the film easily conductive. Moreover, this proposed film modified electrode was more stable, selective and applicable.     

A novel hydrogen peroxide sensor based on the direct electron transfer of horseradish peroxidase immobilized on silica-hydroxyapatite hybrid film

The direct electron transfer of immobilized horseradish peroxidase (HRP) on silica-hydroxyapatite (HAp) hybrid film-modified glassy carbon electrode (GCE) and its application as H(2)O(2) biosensors were investigated. On silica/HRP-HAp/GCE, HRP displayed a fast electron transfer process accompanied with one proton participate in. This sensor exhibited an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The proposed biosensor showed good reproducibility and high sensitivity to H(2)O(2) with the detection limit of 0.35 microM. In the range of 1.0-100 microM, the catalytic reduction current of H(2)O(2) was proportional to H(2)O(2) concentration. The apparent Michaelis-Menten constant (k(m)(app)) of the biosensor was calculated to be 21.8 microM, exhibiting a high enzymatic activity and affinity for H(2)O(2).     

Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay-chitosan-gold nanoparticle nanocomposite

Gold nanoparticles stabilized by chitosan (AuCS) were hybridized with exfoliated clay nanoplates through electrostatic interaction. The resulting clay-chitosan-gold nanoparticle nanocomposite (Clay/AuCS) was used to modify glassy carbon electrode (GCE). HRP, a model peroxidase, was entrapped between the Clay/AuCS film and another clay layer. UV-vis spectrum suggested HRP retained its native conformation in the modified film. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among HRP, AuCS and clay during the modified film drying. The immobilized HRP showed a pair of quasi-reversible redox peaks at -0.195 V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), and the biosensor displayed a fast amperometric response to H(2)O(2) with a wide linear range of 39 microM to 3.1 mM. The detection limit was 9.0 microM based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K(m) (Michaelis-Menten constant) were evaluated to be 0.53, 2.95+/-0.20s(-1) and 23.15 mM, respectively.     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

A sensitive determination of estrogens with a Pt nano-clusters/multi-walled carbon nanotubes modified glassy carbon electrode

On the top of a multi-walled carbon nanotubes (MWNTs) modified glassy carbon electrode (MWNTs/GCE), Pt nanoclusters were electrochemically deposited, fabricating a Pt/MWNTs composite modified electrode, Pt/MWNTs/GCE. X-ray photoelectron spectroscopy, powder X-ray diffraction and field emission scanning electron microscope were used for the surface characterization of the electrode, and demonstrated the formation and distribution of Pt clusters of Pt nanoparticles of 8.4 nm in averaged size in the MWNTs matrix. The preliminary study found that this composite modified electrode has strong electrocatalytic activity toward the oxidation of estrogens involving estradiol, estrone and estriol. The voltammetric behavior of estrogens on this electrode was investigated by cyclic voltammetry, linear sweep voltammetry and square-wave voltammetry. In comparison with the MWNTs/GCE or a Pt nanoparticles modified GCE prepared in the similar way, this composite modified electrode exhibited much higher current sensitivity and catalytic activity. This electrode is also stable. The linear range of square-wave voltammetric determination was 5.0 x 10(-7)-1.5 x 10(-5)mol/L for estradiol, 2.0 x 10(-6)-5.0 x 10(-5)mol/L for estrone, and 1.0 x 10(-6)-7.5 x 10(-5)mol/L for estriol. Under an assumption that the concentration ratio of estradiol:estrone:estriol is 2:2:1, the real sample of blood serums was tested for the determination using this electrode. Satisfactory result was obtained with averaged recovery of 105%.     

Application of a Cu–chitosan/multiwalled carbon nanotube film-modified electrode for the sensitive determination of rutin

A new sensitive electrochemical sensor, a glassy carbon electrode modified with chemically cross-linked copper-complexed chitosan/multiwalled carbon nanotubes (Cu–CS/MWCNT/GCE), for rutin analysis was constructed. Experimental investigations of the influence of several parameters showed that the rutin can effectively accumulate on the surface of the Cu–CS/MWCNT/GCE, which accumulation caused a pair of well-defined redox peaks in the electrochemical signal when measurements were carried out in Britton–Robinson buffer solution (pH 3, 0.04 M). The surface of the Cu–CS/MWCNT/GCE was characterized by field-emission scanning electron microscopy, transmission electron microscopy, and X-ray diffractometry analysis. In a rutin concentration range of 0.05–100 μM and under optimized conditions, a linear relationship between the oxidation peak current of rutin and its concentration was obtained with a detection limit of 0.01 μM. The Cu–CS/MWCNT/GCE showed good selectivity, stability, and reproducibility. Moreover, the sensor was used to determine the presence of rutin in fruits with satisfactory results.     

Carbon nanotube/cobalt hexacyanoferrate nanoparticle-biopolymer system for the fabrication of biosensors

Cobalt hexacyanoferrate nanoparticles (CoNP) can be easily prepared by mixing hexacyanoferrate and cobalt chloride solution at room temperature. The nanoparticles were solubilized in aqueous solution of a biopolymer chitosan (CHIT). With the introduction of carbon nanotubes (CNT), the CoNP-CNT-CHIT system formed shows synergy between CNT and CoNP with the significant improvement of redox activity of CoNP due to the excellent electron-transfer ability of CNT. The CoNP-CNT-CHIT film modified glassy carbon electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. In particular, with the introduction of CNT, it amplified the H2O2 sensitivity by approximately 70 times compared to film of CoNP-CHIT. With the immobilization of glucose oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to glucose has been constructed. In pH 6.98 phosphate buffer, interference free determination of glucose has been realized at -0.2V versus saturated calomel electrode (SCE) with a linear range from 0.01 to 10 mM and response time<10s. The detection limit was 5 microM glucose (S/N=3).     

Digestion of restriction enzyme for the detection of single-base mismatch in DNA

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.     

Direct electrochemistry and electrocatalysis of myoglobin on redox-active self-assembling monolayers derived from nitroaniline modified electrode

The adsorption processes and electrochemical behavior of 4-nitroaniline (4-NA) and 2-nitroaniline (2-NA) adsorbed onto glassy carbon electrodes (GCE) have been investigated in aqueous 0.1M nitric acid (HNO(3)) electrolyte solutions using cyclic voltammetry (CV). Nitroaniline adsorbs onto GCE surfaces and upon potential cycling past -0.55 V is transformed into the arylhydroxylamine (ArHA), which exhibits a well-behaved pH dependent redox couple centered at 0.32 V (pH 1.5). This modified electrode can be readily used as an immobilization matrix to entrap proteins and enzymes. In our studies, myoglobin (Mb) was chosen as a model protein for investigation. A pair of well-defined reversible redox peaks for Mb(Fe(III)-Fe(II)) was obtained at the Mb/arylhydroxylamine modified glassy carbon electrode (Mb/HAGCE) by direct electron transfer between the protein and the GCE. The formal potential (E(0')), the surface coverage (Gamma) and the electron transfer rate constant (k(s)) were calculated as -0.317 V, 4.15+/-0.5 x 10(-11)mol/cm(2) and 51+/-5s(-1), respectively. Dramatically enhanced biocatalytic activity was exemplified at the Mb/HAGCE for the reduction of hydrogen peroxide (H(2)O(2)), trichloroacetic acid (TCA) and oxygen (O(2)). The Mb/ArHA film was also characterized by UV-vis spectra, scanning electron microscope (SEM) indicating excellent stability and good biocompatibility for protein in the film. The applicability of the method to the determination of H(2)O(2) ( approximately 3%) in a commercial antiseptic solution and soft-contact lenses cleaning solutions were demonstrated. This new Mb/HAGCE exhibited rapid electrochemical response (with in 2s) with good stability in physiological condition.     

Amperometric sensors based on Ni/Al and Co/Al layered double hydroxides modified electrode and their application for hydrogen peroxide detection

Two different hydrogen peroxide sensors were constructed with Ni/Al and Co/Al layered double hydroxides (LDHs) modified glassy carbon electrodes (GCE). Ni (Co)/Al-LDHs were synthesized by electrochemical method and were characterized by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The advantages and shortcoming of the two hydrogen peroxide sensors were described in detail. Compared to Co/Al-LDHs modified electrode, sensors fabricated by Ni/Al-LDHs showed quicker heterogeneous electron transfer rate constants (k(s)), lower detection and better reproducibility. But Co/Al-LDHs modified electrode held the advantages of wider linear range and higher sensitivity. Further more, the different catalytic redox mechanisms of hydrogen peroxide on the Ni/Al/GCE and Co/Al/GCE were firstly comparatively explored.     

Sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film

A novel strategy for the fabrication of sensitive immunosensor to detect alpha-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml(-1) with a detection limit of 0.6 ng ml(-1). The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP-anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.     

The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determinations

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2':5',2'-terthiophene-3'-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s(-1). The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H(2)O(2) by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 microM and 100 microM and the detection limit was determined to be about 0.3 microM. The response time of the biosensors was about 6s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

Determination of the absolute redox potential of Rutin: experimental and theoretical studies

The conditional formal potential, E degrees', of Rutin has been studied by cyclic voltammetry using a Rutin film deposited at the multi-wall carbon nanotubes modified glassy carbon electrode (GCE) as the working electrode in different pH phosphate buffered solutions. The experimental standard redox potential, E degrees, of Rutin is obtained to be 0.88 V versus SHE (Standard Hydrogen Electrode). High-level ab initio calculations have been also performed on a chemical model of Rutin and the absolute reduction potential has been calculated. The theoretical standard reduction potential relative to SHE (0.83 V) is in relatively good agreement with experiment.     

Voltammetric discrimination of mandelic acid enantiomers

We report a novel electrochemical biosensor for direct discrimination of d- and l-mandelic acid (d- and l-MA) in aqueous medium. The glassy carbon electrode (GCE) surface was modified with reduced graphene oxide (rGO) and γ-globulin (GLOB). Electrochemical characterization of the modified electrodes was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The modified electrode surfaces were also characterized by scanning electron microscopy. Electrochemical response of the prepared electrode (GCE/rGO/GLOB) for discrimination of d- and l-MA enantiomers was investigated by cyclic voltammetry and was compared with bare GCE in the concentration range of 2 to 10 mM. Whereas the bare GCE showed no electrochemical response for the MA enantiomers, the GCE/rGO/GLOB electrode exhibited direct and selective discrimination with different oxidation potential values of 1.47 and 1.71 V and weak reduction peaks at potential values of −1.37 and −1.48 V, respectively. In addition, electrochemical performance of the modified electrode was investigated in mixed solution of d- and l-MA. The results show that the produced electrode can be used as electrochemical chiral biosensor for MA.     

Voltammetric detection of anti-HIV replication drug based on novel nanocomposite gold-nanoparticle–CaCO<Subscript>3</Subscript> hybrid material

A novel bionanocomposite, horse radish peroxidase- gold-nanoparticle–Calcium carbonate (HRP-AuNPs-CaCO₃), hybrid material was encapsulated by silica sol on a glassy carbon electrode (GCE). The fabricated modified electrode was used as a novel voltammetric sensor for electrochemical sensing of anti-HIV replication drug i.e. deferiprone. The surface morphology of the modified electrode was characterized by scanning electron microscopy (SEM). Results obtained from the voltammetric measurements show that HRP-AuNPs-CaCO₃ modified GCE offers a selective and sensitive electrochemical sensor for the determination of deferiprone. Under experimental conditions, the proposed voltammetric sensor has a linear response range from 0.01 to 10,000 μM with a detection limit of 0.01 μM. Furthermore, the fabricated sensor was successfully applied to determine deferiprone level in spiked urine and serum samples.     

Electrochemical detection of the MT-ND6 gene and its enzymatic digestion: Application in human genomic sample

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome.     

Bimetallic PtM (M=Pd, Ir) nanoparticle decorated multi-walled carbon nanotube enzyme-free, mediator-less amperometric sensor for H₂O₂

A new highly catalytic and intensely sensitive amperometric sensor based on PtM (where M=Pd, Ir) bimetallic nanoparticles (NPs) for the rapid and accurate estimation of hydrogen peroxide (H(2)O(2)) by electrooxidation in physiological conditions is reported. PtPd and PtIr NPs-decorated multiwalled carbon nanotube nanocatalysts (PtM/MWCNTs) were prepared by a modified Watanabe method, and were characterized by XRD, TEM, ICP, and XAS. The sensors were constructed by immobilizing PtM/MWCNTs nanocatalysts in a Nafion film on a glassy carbon electrode. Both PtPd/MWCNTs and PtIr/MWCNTs assemblies catalyzed the electrochemical oxidation of H(2)O(2). Cyclic voltammetry characterization measurements revealed that both the PtM (M=Pd, Ir)/MWCNTs/GCE possessed similar electrochemical surface areas (～0.55 cm(2)), and electron transfer rate constants (～1.23 × 10(-3)cms(-1)); however, the PtPd sensor showed a better performance in H(2)O(2) sensing than did the PtIr counterpart. Explanations were sought from XAS measurements to explain the reasons for differences in sensor activity. When applied to the electrochemical detection of H(2)O(2), the PtPd/MWCNTs/GC electrode exhibited a low detection limit of 1.2 μM with a wide linear range of 2.5-125 μM (R(2)=0.9996). A low working potential (0V (SCE)), fast amperometric response (<5s), and high sensitivity (414.8 μA mM(-1)cm(-2)) were achieved at the PtPd/MWCNTs/GC electrode. In addition, the PtPd/MWCNTs nanocatalyst sensor electrode also exhibited excellent reproducibility and stability. Along with these attractive features, the sensor electrode also displayed very high specificity to H(2)O(2) with complete elimination of interference from UA, AA, AAP and glucose.