Direct electrochemistry and electrocatalysis of hemoglobin on chitosan-room temperature ionic liquid-TiO(2)-graphene nanocomposite film modified electrode

TiO₂-graphene nanocomposite was prepared by hydrolysis of titanium isopropoxide in colloidal suspension of graphene oxide and in situ hydrothermal treatment. The direct electrochemistry and electrocatalysis of hemoglobin in room temperature ionic liquid 1-Butyl-3-methylimidazolium hexafluorophosphate, chitosan and TiO₂-graphene nanocomposite modified glassy carbon electrode were investigated. The biosensor was examined by using UV-vis spectroscopy, scanning electron microscopy and electrochemical methods. The results indicated that hemoglobin remained its bioactivity on the modified electrode, showing a couple of well-defined and quasi-reversible redox peaks, corresponding to hemoglobin Fe^III/Fe^II couple. The kinetic parameters for the electrode reaction, such as the formal potential (E^o'), the electron transfer rate constant (k_s), the apparent coverage (Γ), and Michaelis–Menten constant (K_m) were evaluated. The biosensor showed good electrochemical responses to the reduction of H₂O₂ in the ranges of 1–1170 μM. The detection limit was 0.3 μM (S/N = 3). The properties of this composite film, together with the bioelectrochemical catalytic activity, could make them useful in the development of bioelectronic devices, and investigation of electrochemistry of other heme proteins at functional interface.     

An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres

A new kind of magnetic dextran microsphere (MDMS) with uniform shape and narrow diameter distribution has been prepared from magnetic iron nanoparticles and dextran. Horseradish peroxidase (HRP) was successfully immobilized on the surface of an MDMS-modified glassy-carbon electrode (GCE), and the immobilized HRP displayed excellent electrocatalytic activity in the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ). The effects of experimental variables such as the concentration of HQ, solution pH, and the working potential were investigated for optimum analytical performance. This biosensor had a fast response to H₂O₂ of less than 10 s and an excellent linear relationship was obtained in the concentration range 0.20 μmol L⁻¹–0.68 mmol L⁻¹, with a detection limit of 0.078 μmol L⁻¹ (S/N = 3) under the optimum conditions. The response showed Michaelis–Menten behavior at larger H₂O₂ concentrations, and the apparent Michaelis–Menten constant was estimated to be 1.38 mmol L⁻¹. Moreover, the selectivity, stability, and reproducibility of the biosensor were evaluated, with satisfactory results. Figure Amperometric response of the biosensor to successive additions of H₂O₂ and the plot of amperometric response vs. H₂O₂ concentration     

Electron‐Transfer Mediator Microbiosensor Fabrication Based on Immobilizing HRP‐Labeled Au Colloids on Gold Electrode Surface by 11‐Mercaptoundecanoic Acid Monolayer

In this paper, a new strategy for constructing a mediator‐type amperometric hydrogen peroxide (H₂O₂) microbiosensor was described. An electropolymerized thionine film (PTH) was deposited directly onto a gold electrode surface. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly cross‐linked network structure. Consequently, horseradish peroxidase (HRP) was successfully immobilized on nanometer‐sized Au colloids, which were supported by thiol‐tailed groups of 11‐mercaptoundecanoic acid (11‐MUA) monolayer covalently bound onto PTH film. With the aid of the PTH mediator, HRP‐labeled Au colloids microbiosensor displayed excellent electrocatalytical response to the reduction of H₂O₂. This matrix showed a biocompatible microenvironment for retaining the native activity of the covalent HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H₂O₂. The proposed H₂O₂ microbiosensor exhibited linear range of 3.5 μM–0.7 mM with a detection limit of 0.05 μM (S/N=3). The response showed a Michaelis‐Menten behavior at larger H₂O₂ concentrations. The K_M^app value for the biosensors based on 24 nm Au colloids was found to be 47 μM, which demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H₂O₂ with no loss of enzymatic activity. This microbiosensor possessed good analytical performance and storage stability.     

Amperometric hydrogen peroxide biosensor based on the immobilization of heme proteins on gold nanoparticles-bacteria cellulose nanofibers nanocomposite

A novel matrix, gold nanoparticles-bacterial cellulose nanofibers (Au-BC) nanocomposite was developed for enzyme immobilization and biosensor fabrication due to its unique properties such as satisfying biocompatibility, good conductivity and extensive surface area, which were inherited from both gold nanoparticles (AuNPs) and bacterial cellulose nanofibers (BC). Heme proteins such as horseradish peroxidase (HRP), hemoglobin (Hb) and myoglobin (Mb) were successfully immobilized on the surface of Au-BC nanocomposite modified glassy carbon electrode (GCE). The immobilized heme proteins showed electrocatalytic activities to the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ), which might be due to the fact that heme proteins retained the near-native secondary structures in the Au-BC nanocomposite which was proved by UV-vis and IR spectra. The response of the developed biosensor to H₂O₂ was related to the amount of AuNPs in Au-BC nanocomposite, indicating that the AuNPs in BC network played an important role in the biosensor performance. Under the optimum conditions, the biosensor based on HRP exhibited a fast amperometric response (within 1 s) to H₂O₂, a good linear response over a wide range of concentration from 0.3 μM to 1.00 mM, and a low detection limit of 0.1 μM based on S/N = 3. The high performance of the biosensor made Au-BC nanocomposite superior to other materials as immobilization matrix.     

Colorimetric detection of hydrogen peroxide and lactate based on the etching of the carbon based Au-Ag bimetallic nanocomposite synthesized by carbon dots as the reductant and stabilizer

In this report, carbon-based gold core silver shell Au-Ag bimetallic nanocomposite (Au-Ag/C NC) was synthesized using carbon dots (C-dots) as the reductant and stabilizer by a facile green sequential reduction approach. The structure and morphology of the nanocomposite are characterized by ultraviolet–visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The as synthesized Au-Ag/C NC exhibits good optic response toward hydrogen peroxide (H₂O₂) without adding any other chromogenic agents. The characteristic surface plasmon resonance (SPR) absorbance peak of Au-Ag/C NC declined and red-shifted with the solution color changing from reddish orange to light pink when adding H₂O₂ owing to the etching effect of H₂O₂ towards Ag. Thus, a simple colorimetric and UV strategy for sensitive detection of H₂O₂ is proposed. It provides the wide linear range for detection of H₂O₂ from 0.8–90 μM and 90–500 μM, and the detection limit was as low as 0.3 μM (S/N = 3). In addition, this colorimetric strategy can also be applied to directly distinguish and detect of lactate by naked eye and UV–Vis. The linear range of colorimetric sensing towards lactate was 0.1–22 μM and 22–220 μM, which was successfully applied in the analysis of lactate in human serum.     

Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator

An amperometric biosensor has been developed for the determination of H₂O₂ in plant samples. Horseradish peroxidase (HRP) is immobilized on a sandwiched nano-Au particle / m-phenylenediamine polymer film by glutaraldehyde cross-linking. The film is formulated on the carbon paste electrode (CPE) blended with ferrocene as an electron transfer mediator. On the low concentration range, the current response is related to the H₂O₂ concentration linearly from 0 to 8×10^-6 M with a detection limit of 1.3×10^-7 M. On a wider concentration range of 8×10^-6 to 1.4×10^-4 M, the reciprocal of current response is linearly related to the reciprocal of H₂O₂ concentration. The apparent Michaelis-Menten constant (K_m^app) was calculated to be 0.0334 mM. The sensor has been tested by determining H₂O₂ concentration in plant leaf samples.     

Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Prussian Blue electrodeposited on MWNTs-PANI hybrid composites for H2O2 detection

A Prussian Blue (PB)/polyaniline (PANI)/multi-walled carbon nanotubes (MWNTs) composite film was fabricated by step-by-step electrodeposition on glassy carbon electrode (GCE). The electrode prepared exhibits enhanced electrocatalytic behavior and good stability for detection of H₂O₂ at an applied potential of 0.0 V. The effects of MWNTs thickness, electrodeposition time of PANI and rotating rate on the current response of the composite modified electrode toward H₂O₂ were optimized to obtain the maximal sensitivity. A linear range from 8 × 10⁻⁹ to 5 × 10⁻⁶ M for H₂O₂ detection has been observed at the PB/PANI/MWNTs modified GCE with a correlation coefficient of 0.997. The detection limit is 5 × 10⁻⁹ M on signal-to-noise ratio of 3. To the best of our knowledge, this is the lowest detection limit for H₂O₂ detection. The electrode also shows high sensitivity (526.43 μA μM⁻¹ cm⁻²) for H₂O₂ detection which is more than three orders of magnitude higher than the reported.     

Synthesis and Characterization of Poly(toluidine blue) Nanowires and Their Application in Amperometric Biosensors

Poly(toluidine blue) nanowires (PTBNWs) with an average diameter of ca. 200 nm and length of ca. 5 μm were synthesized for the first time using a template‐directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template. Their morphological characterization was carried out by scanning electron microscopy (SEM) and transmission electron microscope (TEM). By electrochemical polymerization, horseradish peroxidase (HRP) was encapsulated in situ in PTBNWs (denoted as PTBNWs‐HRP) for potential biosensor applications. PTBNWs‐HRP was then modified on a glassy carbon (GC) electrode. In the system obtained, the PTBNWs served as an excellent redox mediator and exhibited high efficiency of electron transfer between the HRP and the GC electrode for the reduction of H₂O₂. The proposed electrode can be used as an excellent amperometric sensor for H₂O₂ at ?0.1 V with a linear response range covering from 1 μM to 28 mM, a detection limit of 1 μM (based on S/N=3) and a fast response time of less than 8 s.     

Dendritic Silver/Silicon Dioxide Nanocomposite Modified Electrodes for Electrochemical Sensing of Hydrogen Peroxide

A novel biosensor for hydrogen peroxide was prepared by immobilizing horseradish peroxidase (HPR) on newly synthesized dendritic silver/silicon dioxide nanocomposites, which were coated on a glassy carbon electrode. The modified electrode was characterized with XPS, SEM, and electrochemical methods. This biosensor showed a very fast amperometric response to hydrogen peroxide with a linear range from 0.7 to 120 μM, a limit of detection of 0.05 μM and a sensitivity of 1.02 mA mM^?1 cm^?2. The Michaelis‐Menten constant of the immobilized HRP was estimated to be 0.21 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity. The preparation of the proposed biosensor was convenient, and it showed high sensitivity and good stability.     

Layer by layer assembly of catalase and amine-terminated ionic liquid onto titanium nitride nanoparticles modified glassy carbon electrode: Study of direct voltammetry and bioelectrocatalytic activity

A novel, simple and facile layer by layer (LBL) approach is used for modification of glassy carbon (GC) electrode with multilayer of catalase and nanocomposite containing 1-(3-Aminopropyl)-3-methylimidazolium bromide (amine terminated ionic liquid (NH₂-IL)) and titanium nitride nanoparticles (TiNnp). First a thin layer of NH₂-IL is covalently attached to GC/TiNnp electrode using electro-oxidation method. Then, with alternative self assemble positively charged NH₂-IL and negatively charged catalase a sensitive H₂O₂ biosensor is constructed, whose response is directly correlated to the number of bilayers. The surface coverage of active catalase per bilayer, heterogeneous electron transfer rate constant (k_s) and Michaelis–Menten constant (K_M) of immobilized catalase were 3.32 × 10⁻¹² mol cm⁻², 5.28 s⁻¹ and 1.1 mM, respectively. The biosensor shows good stability, high reproducibility, long life-time, and fast amperometric response with the high sensitivity of 380 μA mM⁻¹ cm⁻² and low detection limit of 100 nM at concentration range up to 2.1 mM.     

Direct Electrochemistry of Horseradish Peroxidase Embedded in Nano－Fe304 Matrix on Paraffin Impregnated Graphite Electrode and Its Electrochemical Catalysis for H2O2

Fe3O4 particles coated with acrylic copolymer (ACP) of about 5--8 nm in diameter were synthesized and used for immobilization of horseradish peroxidase (HRP). Direct electrochemistry of HRP embedded in the nanosized Fe304 solid matrix modified paraffin impregnated graphite electrode (PIGE) was achieved,which is related to the heine Fe(Ⅲ)/Fe(Ⅱ) conversion of HRP. Cyclic voltammetry gave a pair of reproducible and welldefined redox peaks at about Ea of -0.295 V vs. SCE. The standard rate constant k, was determined as 2.7 s^-1. It demonstrated that the nano-Fe3O4 solid matrix offers a friendly platform to assemble the HRP protein molecules and enhance the electron transfer rate between the HRP and the electrode. UV-Vis absorption spectra and WrIR spectra studies revealed that the embedded HRP retained its native-like structure. The HRP/Fe3O4/PIGE showed a strong catalytic activity toward H2O2. The voltammetric response was a linear function of H2O2 concentration in the range of 10-140μmol/L with detection limit of 7.3 μmol/L (s/n = 3 ). The apparent Michaelis-Menten constant is calculated to be 0.42 mmol/L.     

Amplification of bioelectrocatalytic signalling based on silver nanoparticles and DNA-derived horseradish peroxidase biosensors

Horseradish peroxidase (HRP) was immobilized onto a polyion complex membrane containing positively charged silver nanoparticles (nanosilver), double stranded DNA and poly(thionine) to fabricate highly sensitive and selective electrochemical hydrogen peroxide (H₂O₂) biosensor on a glassy carbon electrode. The presence of nanosilver provided a biocompatible microenvironment for enzyme molecules, greatly amplified the surface coverage of HRP on the electrode surface, and most importantly could act as a charge carrier. The process of the biosensor construction was characterized by scanning electron microscopy. Voltammetric and time-based amperometric techniques were employed to characterize the properties of the derived biosensor. Under optimal conditions, the biosensor has an electrocatalytic behavior towards the H₂O₂ reduction, and exhibits a linear range from 1.1 μM to 5.2 mM, with a lower detection limit of 0.2 μM. The apparent Michaelis–Menten constant of the biosensor to H₂O₂ was estimated to be 1.02 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and acceptable stability. Importantly, the properties of composite film, together with the bioelectrochemical catalytic activity, could make them useful in the development of bioelectronic devices and investigation of protein electrochemistry at functional interface. Correspondence: Yan Liu, College of Chemistry, Chongqing Normal University, Chongqing 400047, P.R. China     

Hydrogen Peroxide Biosensor Based on the Electrochemistry of the Myoglobin‐TATP Composite Film

Abstract

Direct electrochemistry of the myoglobin‐triacetone triperoxide (Mb‐TATP) composite on carbon paste (CP) electrode is reported. This electrode gives a well‐defined and quasi‐reversible cyclic voltammogram for the Mb Fe^III/Fe^II redox coupled with the formal potential (E?′) of ?0.302 V (vs. Ag/AgCl) in pH 6.92 phosphate buffer. Electronic and vibrational spectroscopies show that the Mb in the composite retains a structure similar to its native form. The enzymatic reactivity to the reduction of H₂O₂ has been studied for the Mb‐TATP film. The analytical performances have been obtained with the linear range of 78.32–1135.64 µM, the detection limit of 55 µM (S/N=3), and the apparent Michaelis‐Menten constant (K _m) of 662.8 µM. This H₂O₂ biosensor based on the electrocatalysis of the immobilized Mb presents a higher stability within two weeks.     

Novel Protocol for Covalent Immobilization of Horseradish Peroxidase on Gold Electrode Surface

A novel protocol for immobilization of horseradish peroxidase (HRP) onto diazonium functionalized screen‐printed gold electrode (SPGE) has been successfully developed. This protocol involved 1) electrochemical reduction of p‐nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution to graft a layer of p‐nitrophenyl on SPGE, 2) electrochemical reduction of the nitro groups to convert to amines, 3) chemical reaction with nitrous acid to transform the amine to diazonium derivative and 4) chemical coupling of the enzyme with the diazonium group to form a covalent diazo bond. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediator. The biosensor exhibited fast amperometric response to H₂O₂. The catalytic current increased with increasing H₂O₂ concentration from 5 μM to 30 μM and the detection limit of the biosensor was 2 μM. The biosensor exhibited acceptable sensitivity, good reproducibility and long‐term stability.     

Improved Performance of an Amperometric Biosensor with Polydiaminonaphthalene on Electrochemically Deposited Au Nanoparticles

The performance of an enzyme sensor fabricated through covalent bond formation on the HRP‐bonded poly(1,8‐diaminonaphthalene) (polyDAN) layer with gold nanoparticles (AuNPs) was applied to catalyze the electrochemical reduction of H₂O₂. The surface characteristics of the sensor probe were studied using cyclic voltammetry, SEM, XPS, QCM, and impedance spectroscopy. The AuNP‐deposited surface resulted in higher conductivity and sensitivity for H₂O₂ detection in phosphate buffer solution. A linear calibration plot was obtained in the H₂O₂ concentration range between 10.0 μM and 25.0 mM with detection limit 5.0±1.25 μM. The lifetime of HRP/polyDAN/AuNP/GC probe was over 70 days without response loss.     

Bilayer lipid membrane biosensor with enhanced stability for amperometric determination of hydrogen peroxide

In this paper, a polydopamine (PDA) film is electropolymerized on the surface of bilayer lipid membrane (BLM) which is immobilized with horseradish peroxidase (HRP). The coverage of the PDA film on HRP/BLM electrode is monitored by electrochemical impedance spectroscopy (EIS). The electrocatalytic reduction of H₂O₂ at the PDA/HRP/BLM electrode is studied by means of cyclic voltammetry (CV). The biosensor has a fast response to H₂O₂ of less than 5 s and an excellent linear relationship is obtained in the concentration range from 2.5 × 10⁻⁷ to 3.1 × 10⁻³ mol L⁻¹, with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The response current of BLM/HRP/PDA biosensor retains 84% of its original response after being stored in 0.1 mol L⁻¹ pH 7.0 PBS at 4 °C for 3 weeks. The selectivity, repeatability, and storage stability of PDA/HRP/BLM biosensor are greatly enhanced by the coverage of polydopamine film on BLM.     

The Modification of Screen‐Printed Carbon Electrodes with Amino Group and Its Application to Construct a H2O2 Biosensor

Electrooxidation of thionine on screen‐printed carbon electrode gives rise to the modification of the surface with amino groups for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The biosensor was constructed using multilayer enzymes which covalently immobilized onto the surface of amino groups modified screen‐printed carbon electrode using glutaraldehyde as a bifunctional reagent. The multilayer assemble of HRP has been characterized with the cyclic voltammetry and the faradaic impedance spectroscopy. The H₂O₂ biosensor exhibited a fast response (2 s) and low detection limit (0.5 μM).     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Covalently Immobilized on Electrografted Organic Film on Screen‐Printed Carbon Electrode

A new convenient strategy to fabricate a third‐generation hydrogen peroxide biosensor was described. The screen‐printed carbon electrode (SPCE) was first modified with a layer of 4‐nitrophenyl assembled from the 4‐nitroaniline diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were converted to amines followed by crosslinking to the horseradish peroxidase (HRP) by glutaraldehyde. The redox chemistry of the active center of the HRP was observed and the HRP‐modified electrode displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. H₂O₂ was determined in a linear range from 5.0 μM to 50.0 μM, with a detection limit of 1.0 μM. Furthermore, the biosensor exhibited fast amperometric response, good reproducibility and long‐term stability.     

基于一维DNA组装磁性纳米探针的夹心型安培免疫传感器研究

采用Fe3O4(核)/ZrO2(壳)纳米磁珠(ZMPs)标记待测物识别抗体,并用HRP酶封闭和DNA链接,建立了一类新型的"珠链状"一维磁性纳米探针制备方法。将甲胎蛋白(AFP)一抗固定于纳米金修饰的玻碳电极表面,构建了免疫电极(GCE?AFP Ab1)。基于该电极和上述合成探针,通过双抗体夹心法测定免疫产物上HRP酶对过氧化脲(CP)氧化对苯二酚反应的催化电流,研制了一类基于一维纳米结构组装的夹心型安培免疫传感器。研究表明:此一维纳米结构探针不仅大大增加了酶在电极表面的富集量,成倍扩增了催化电流,显著提高了传感器的灵敏度,而且易于通过外磁场与背景液可控分离,简化了分析步骤,并提高了结果的重复性。此传感器对AFP检测的线性范围为0.01~25 mg/L;检出限达4 ng/L(3σ),并被用于人血清中痕量AFP的测定,结果满意。