A biosensing platform based on horseradish peroxidase immobilized onto chitosan-wrapped single-walled carbon nanotubes

One-step, diameter-selective dispersion of single-walled carbon nanotubes (SWCNTs) has been accomplished through noncovalent complexation of the nanotubes with a water-soluble, biocompatible polymer chitosan at room temperature. Such chitosan-wrapped individual SWCNTs can be used for the immobilization of horseradish peroxidase (HRP) and be used to construct an electrode for direct bioelectrochemical sensing without an electron mediator. The direct electron transfer between HRP and the electrode surface was observed with a formal potential of approximately −0.35 V (vs. saturated calomel electrode) in phosphate buffer solution and the calculated heterogeneous electron transfer rate constant is approximately 23.5 s⁻¹. Experimental results indicate that the immobilized HRP retains its catalytic activity for the reduction of nitric oxide. Such an HRP–SWCNT–chitosan-based biosensor exhibited a rapid response time of less than 6 s and a good linear detection range for nitrite concentration, from 25 to 300 μM with a detection limit of 3 μM. The apparent Michaelis–Menten constant (K _m) and the maximum electrode sensitivity (i_max/K _m) are found to be 7.0 mM and 0.16 μA mM⁻¹, respectively. Both the unique electrical properties of SWCNTs and biocompatibility of chitosan enable the construction of an excellent biosensing platform for improved electrocatalysis of HRP, allowing, specifically, the detection of trace levels of nitric oxide.     

A new hydrogen peroxide biosensor based on gold nanoelectrode ensembles/multiwalled carbon nanotubes/chitosan film-modified electrode

Gold nanoelectrode ensembles were produced by electrodeposition using multiwalled carbon nanotubes (MWNTs) as template. A new third generation amperometric biosensor for hydrogen peroxide was developed based on adsorption of horseradish peroxidase (HRP) at the glassy carbon (GC) electrode modified with Au nanoelectrode ensembles/multiwalled carbon nanotubes/chitosan film. The resulting HRP biosensor offered an excellent detection for hydrogen peroxide at −0.11 V with a linear response range of 2.08 × 10⁻⁷ to 7.6 × 10⁻³ M with a correlation coefficient of 0.998, and response time <5 s. The detection limit was 1.02 × 10⁻⁷ M at 3σ. The biosensor displays rapid response, expanded linear response range, and excellent repeatability. The simple and fast fabrication of the sensor makes it superior to other techniques.     

Electrochemical sensing of DNA immobilization and hybridization based on carbon nanotubes/nano zinc oxide/chitosan composite film

A novel electrochemical DNA biosensor based on zinc oxide （ZnO） nanoparticles and multi-walled carbon nanotubes （MWNTs） for DNA immobilization and enhanced hybridization detection is presented. The MWNTs/nano ZnO/chitosan composite film modified glassy carbon electrode （MWNTs/ZnO/CHIT/GCE） was fabricated and DNA probes were immobilized on the electrode surface. The hybridization events were monitored by differential pulse voltammetry （DPV） using methylene blue （MB） as an indicator. The sensor can effectively discriminate different DNA sequences related to PAT gene in the transgenic corn, with a detection limit of 2.8× 10^-12 mol/L of target sequence.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hybrid organic-inorganic film of chitosan/sol-gel/carbon nanotubes

A hybrid organic-inorganic nanocomposite film of chitosan/sol-gel/multi-walled carbon nanotubes was constructed for the immobilization of horseradish peroxidase (HRP). This film was characterized by scanning electron microscopy. Direct electron transfer (DET) and bioelectrocatalysis of HRP incorporated into the composite film were investigated. The results indicate that the film can provide a favorable microenvironment for HRP to perform DET on the surface of glassy carbon electrodes with a pair of quasi-reversible redox waves and to retain its bioelectrocatalytic activity toward H₂O₂.     

Amperometric biosensor for hydrogen peroxide based on coimmobilized horseradish peroxidase and methylene green in ormosils matrix with multiwalled carbon nanotubes

A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV-vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H₂O₂ at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H₂O₂, less than 10 s, and excellent linear range of concentration from 5 × 10⁻⁷ to 2 × 10⁻⁵ M with the detection limit of 0.5 μM (S/N = 3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4 °C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability.     

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     

碳纳米管/TiO2薄膜固定酶的生物传感器及电催化氧化H2O2的研究

采用酸性溶胶法在碳纳米管上负载纳米TiO2颗粒,并制备了CNT-TiO2薄膜固定辣根过氧化物酶的生物传感器。研究了该生物传感器在磷酸盐缓冲溶液中对H2O2的电催化氧化作用及实验条件的影响。结果表明该生物传感器对H2O2表现出良好的电催化性能,在最佳实验条件下,对H2O2检测的线性范围为:4.2×10-7mol/L~3.2×10-3mol/L。检出限为:7.22×10-8mol/L。     

Hydrogen peroxide sensor based on horseradish peroxidase immobilized on a silver nanoparticles/cysteamine/gold electrode

A third-generation hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) on a gold electrode modified with silver nanoparticles. A freshly-cleaned gold electrode was first immersed in a cysteamine–ethanol solution, and then silver nanoparticles were immobilized on the cysteamine monolayer, and finally HRP was adsorbed onto the surfaces of the silver nanoparticles. This self-assemble process was examined via atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited an excellent electrocatalytic response toward the reduction of hydrogen peroxide. The linear range of the biosensor was 3.3 M to 9.4 mM, and the detection limit was estimated to be 0.78 M. Moreover, the biosensor exhibited a fast response, high sensitivity, good reproducibility, and long-term stability.     

Amperometric glutamate biosensor based on self-assembling glutamate dehydrogenase and dendrimer-encapsulated platinum nanoparticles onto carbon nanotubes

A novel amperometric biosensor based on self-assembling glutamate dehydrogenase (GLDH) and poly(amidoamine) dendrimer-encapsulated platinum nanoparticles (Pt-PAMAM) onto multiwall carbon nanotubes (CNTs) has been developed for the determination of glutamate. The formation of the self-assembled (GLDH/Pt-PAMAM)_n/CNTs construction was investigated by ζ-potential and high resolution transmission electron microscopy (HRTEM). The results indicated the uniform growth of the layer-by-layer nanostructures onto carboxyl-functionalized CNTs. The electrocatalytic property of the (GLDH/Pt-PAMAM)_n/CNTs modified electrode to glutamate in presence of NAD⁺ (β-nicotinamide adenine dinucleotide, 0.1 mM) was investigated at a low overpotential 0.2 V by electrochemical measurements. The results showed it had series of attractive characteristics, such as a large determination range (0.2-250 μM), a short response time (within 3 s), a high sensitivity (433 μA/mM⁻¹ cm²) and good stability (85% remains after 4 weeks).     

Enhancement of a conducting polymer-based biosensor using carbon nanotube-doped polyaniline

A biosensor with improved performance was developed through the immobilization of horseradish peroxidase (HRP) onto electropolymerized polyaniline (PANI) films doped with carbon nanotubes (CNTs). The effects of electropolymerization cycle and CNT concentration on the response of the biosensor toward H₂O₂ were investigated. It was found that the application of CNTs in the biosensor system could increase the amount and stability of the immobilized enzyme, and greatly enhanced the biosensor response. Compared with the biosensor without CNTs, the proposed biosensor exhibited enhanced stability and approximately eight-fold sensitivity. A linear range from 0.2 to 19 μM for the detection of H₂O₂ was observed for the proposed biosensor, with a detection limit of 68 nM at a signal-to-noise ratio of 3 and a response time of less than 5 s.     

Third generation biosensor based on myoglobin-TiO2/MWCNTs modified glassy carbon electrode

TiO2 nanoparticles were homogeneously coated on multi-walled carbon nanotubes by hydrothermal deposition, this nanocomposite may be a promising material for myoglobin immobilization in view of its high biocompatibility and large surface. The glassy carbon electrode modified with Mb-TiO2/MWCNTs films exhibited a pair of weU defined, stable and nearly reversible cycle voltammetric peaks. The electron transfer between Mb and electrode surface, Ks of 3.08 s^-1, was greatly facilitated in the TiO2/ MWCNTs film. The electrocatalytic reductions of hydrogen peroxide were studied, the apparent Michaelis-Menten constant is calculated to be 83.10 μmol/L, which shows a large catalytic activity of Mb in the TiO2/MWCNTs film to H2O2.     

基于二氧化锆和碳纳米管协同作用的过氧化氢生物传感器

过氧化氢作为一种重要的化工产品在纺织行业、化工行业、造纸工业、环保行业、电子行业、食品卫生行业及其他领域得到广泛的应用~([1]).     

Anodic stripping voltammetric determination of mercury using multi-walled carbon nanotubes film coated glassy carbon electrode

An electrochemical method for the determination of trace levels of mercury based on a multi-walled carbon nanotubes (MWNT) film coated glassy carbon electrode (GCE) is described. In 0.1 mol L^–1 HCl solution containing 0.02 mol L^–1 KI, Hg²⁺ was firstly preconcentrated at the MWNT film and then reduced at –0.60 V. During the anodic potential sweep, reduced mercury was oxidized, and then a sensitive and well-defined stripping peak at about –0.20 V appeared. Under identical conditions, a MWNT film coated GCE greatly enhances the stripping peak current of mercury in contrast to a bare GCE. Low concentrations of I^– remarkably improve the determining sensitivity, since this increases the accumulation efficiency of Hg²⁺ at the MWNT film coated GCE. The stripping peak current is proportional to the concentration of Hg²⁺ over the range 8×10^–10–5×10^–7 mol L^–1. The lowest detectable concentration of Hg²⁺ is 2×10^–10 mol L^–1 at 5 min accumulation. The relative standard deviation (RSD) at 1×10^–8 mol L^–1 Hg²⁺ was about 6% (n=10). By using this proposed method, Hg²⁺ in some water samples was determined, and the results were compared with those obtained by atomic absorption spectrometry (AAS). The two results are similar, suggesting that the MWNT-film coated GCE has great potential in practical analysis.     

An amperometric biosensor based on acetylcholinesterase immobilized onto iron oxide nanoparticles/multi-walled carbon nanotubes modified gold electrode for measurement of organophosphorus insecticides

An acetylcholinesterase (AChE) purified from maize seedlings was immobilized covalently onto iron oxide nanoparticles (Fe₃O₄NP) and carboxylated multi walled carbon nanotubes (c-MWCNT) modified Au electrode. An organophosphorus (OP) biosensor was fabricated using this AChE/Fe₃O₄/c-MWCNT/Au electrode as a working electrode, Ag/AgCl as standard and Pt wire as an auxiliary electrode connected through a potentiostat. The biosensor was based on inhibition of AChE by OP compounds/insecticides. The properties of nanoparticles modified electrodes were studied by scanning electron microscopy (SEM), Fourier transform infrared (FTIR), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Fe₃O₄NP and c-MWCNT showed excellent electrocatalytic activity at low potential (+0.4 V). The optimum working conditions for the sensor were pH 7.5, 35 °C, 600 μM substrate concentration and 10 min for inhibition by pesticide. Under optimum conditions, the inhibition rates of OP pesticides were proportional to their concentrations in the range of 0.1–40 nM, 0.1–50 nM, 1–50 nM and 10–100 nM for malathion, chlorpyrifos, monocrotophos and endosulfan respectively. The detection limits were 0.1 nM for malathion and chlorpyrifos, 1 nM for monocrotophos and 10 nM for endosulfan. The biosensor exhibited good sensitivity (0.475 mA μM⁻¹), reusability (more than 50 times) and stability (2 months). The sensor was suitable for trace detection of OP pesticide residues in milk and water.     

基于石墨烯-壳聚糖-辣根过氧化物酶的H2O2生物传感器的研制

制备了石墨烯-壳聚糖(GR-CS)纳米复合材料,并将之与辣根过氧化物酶(HRP)混合,构建了基于石墨烯-壳聚糖-辣根过氧化物酶的生物传感器(GR-CS-HRP/GC)。探针及循环伏安研究表明,该界面具有优异的电子传导能力、较大的比表面积和良好的生物相容性,对H2O2的还原显示出较好的电催化活性,在工作电位为-0.2 V,0.05 mol/L的磷酸盐缓冲盐溶液(PBS,pH 6.8)中,该酶传感器对过氧化氢响应灵敏度高,检测范围宽,测定H2O2的线性范围为5.0×10-7～2×10-3mol/L(相关系数为0.998)。检出限为2.0×10-7mol/L(S/N=3)。并且表现出良好的稳定性和高选择性。该电极用于实际样品中H2O2的测定,结果令人满意。     

Voltammetric determination of quercetin at a multi-walled carbon nanotubes paste electrode

Quercetin can effectively accumulate at multi-walled carbon nanotubes-paraffin oil paste electrodes (CNTPE) and cause a sensitive anodic peak at around 0.32 V (vs. SCE) in a 0.10 M phosphate buffer solution (pH = 4.0). Under optimized conditions, the anodic peak current is linear to quercetin concentration in the ranges of 2.0 × 10^− 9−1.0 × 10^− 7 M and 1.0 × 10^− 7−2.0 × 10^− 5 M, and the regression equations are i_p (μA) = 0.0017 + 0.928c (μM, r = 0.999) and i_p (μA) = 0.183 + 0.0731c (μM, r = 0.995), respectively. This paste electrode can be regenerated by repetitively cycling in a blank solution for about 2 min. A 1.0 × 10^− 6 M quercetin solution is measured for 10 times using the same electrode regenerated after every determination, and the relative standard deviation of the peak current is 1.7%. The method has been applied to the determination of quercetin in hydrolysate product of rutin and the recovery is 99.2–102.6%. In comparison with graphite paste electrode, carbon nanotubes-nujol paste electrode and carbon nanotubes casting film modified glassy carbon electrode, the CNTPE gives higher ratio of signal to background current and better defined voltammetric peak.     

A glucose biosensor based on chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains formed by one-step electrodeposition

In this paper, a simple one-step electrodeposition method is described to fabricate chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains (CS-PB-MWNTs-H-PtCo) film onto the gold electrode surface, then glucose oxidase (GOD) and Nafion were modified onto the film subsequently to fabricate a glucose biosensor. The morphologies and electrochemistry of the composite were investigated by using Fourier transform infrared (FTIR) spectrometry, scanning electron microscopy (SEM) and electrochemical techniques including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. The performances of the biosensor have been investigated by chronoamperometry method under the optimized conditions. This biosensor showed a linear response to glucose range from 1.5 μM to 1.12 mM with a detection limit of 0.47 μM (S/N=3), a high sensitivity of 23.4 μA mM(-1) cm(-2), and a fast response time. The apparent Michaelis-Menten constant (K(M)(app)) was 1.89 mM. In addition, the biosensor also exhibited strong anti-interference ability, excellent stability and good reproducibility.     

An enhanced biosensor for glutamate based on self-assembled carbon nanotubes and dendrimer-encapsulated platinum nanobiocomposites-doped polypyrrole film

An enhanced amperometric biosensor based on incorporating one kind of unique nanobiocomposite as dopant within an electropolymerized polypyrrole film has been investigated. The nanobiocomposite was synthesized by self-assembling glutamate dehydrogenase (GLDH) and poly(amidoamine) dendrimer-encapsulated platinum nanoparticles (Pt-DENs) onto multiwall carbon nanotubes (CNTs). ζ-Potentials and high-resolution transmission electron microscopy (HRTEM) confirmed the uniform growth of the layer-by-layer nanostructures onto the carboxyl-functionalized CNTs. The size of Pt nanoparticles is approximately 3 nm. The (GLDH/Pt-DENs)_n/CNTs/Ppy hybrid film was obtained by electropolymerization of pyrrole onto glassy carbon electrodes and characterized with scanning electron microscopy (SEM), cyclic voltammetry (CV) and other electrochemical measurements. All methods indicated that the (GLDH/Pt-DENs)_n/CNTs nanobiocomposites were entrapped within the porous polypyrrole film and resulted in a hybrid film that showed a high electrocatalytic ability toward the oxidation of glutamate at a potential 0.2 V versus Ag/AgCl. The biosensor shows performance characteristics with high sensitivity (51.48 μA mM⁻¹), rapid response (within 3 s), low detection limit (about 10 nM), low level of interference and excellent reproducibility and stability.     

Amperometric glucose biosensor based on boron-doped carbon nanotubes modified electrode

Doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications due to their unique physicochemical properties. In this paper, the boron-doped carbon nanotubes (BCNTs) were used in amperometric biosensors. It has been found that the electrocatalytic activity of the BCNTs modified glassy carbon (GC) electrode toward the oxidation of hydrogen peroxide is much higher than that of the un-doped CNTs modified electrode due to the large amount of edge sites and oxygen-rich groups located at the defective sites induced by boron doping. Glucose oxidase (GOD) was selected as the model enzyme and immobilized on the BCNTs modified glassy carbon electrode by entrapping GOD into poly(o-aminophenol) film. The performance of the sensor was investigated by electrochemical methods. At an optimum potential of +0.60 V and pH 7.0, the biosensor exhibits good characteristics, such as high sensitivity (171.2 nA mM(-1)), low detection limit (3.6 microM), short response time (within 6s), satisfactory anti-interference ability and good stability. The apparent Michaelis-Menten constant (K(m)(app)) is 15.19 mM. The applicability to the whole blood analysis of the enzyme electrode was also evaluated.     

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.