A novel amperometric biosensor based on gold nanoparticles-mesoporous silica composite for biosensing glucose

We report a novel bienzyme biosensor based on the assembly of the glucose oxidase (GOD) and horseradish peroxidase (HRP) onto the gold nanoparticles encapsulated mesoporous silica SBA-15 composite (AuNPs-SBA-15). Electrochemical behavior of the bienzyme bioconjugates biosensor is studied by cyclic voltammetry and electrochemical impedance spectroscopy. The results indicate that the presence of mesoporous AuNPs-SBA-15 greatly enhanced the protein loadings, accelerated interfacial electron transfer of HRP and the electroconducting surface, resulting in the realization of direct electrochemistry of HRP. Owing to the electrocatalytic effect of AuNPs-SBA-15 composite, the biosensor exhibits a sensitive response to H₂O₂ generated from enzymatic reactions. Thus the bienzyme biosensor could be used for the detection of glucose without the addition of any mediator. The detection limit of glucose was 0.5 μM with a linear range from 1 to 48 μM. Supported by the National Natural Science Foundation of China (Grant Nos. 20635020 & 90606016)     

Development of an amperometric glucose biosensor based on the immobilization of glucose oxidase in an ormosil-PVA matrix onto a Prussian Blue modified electrode

An amperometric glucose biosensor was developed based on the immobilization of glucose oxidase in the organically modified silicate (ormosil)-polyvinyl acetate (PVA) matrix onto a Prussian Blue (PB)-modified glassy carbon electrode. A higher stability PB-modified electrode was prepared by the electrochemical deposition of FeCl₃, K₃[Fe(CN)₆] and ethylenediamine tetraacetic acid (EDTA) under cyclic voltammetric (CV) conditions. The effects of the potential range of CV conditions, electrolyte cations, applied potential, pH, temperature and co-existing substances were investigated. The detection limit of the glucose biosensor was 8.1 μmol·L⁻¹ (S/N = 3) with a linear range from 20 μmol·L⁻¹ to 2 mmol·L⁻¹ (R = 0.9965). The biosensor presented a fast response and good selectivity. Additionally, excellent reproducibility and stability of the biosensor were observed. Supported by the National High Technical Development Project (863 project) Foundation (Grant No. 2006AA09Z160) and the National Natural Science Foundation of China (Grant No. 20775064)     

Sulfonate-based fluorescent probes for imaging hydrogen peroxide in living cells

Based on the mechanism of H₂O₂-mediated hydrolysis of sulfonates, two fluorescein disulfonates compounds (FS-1 and FS-2) were designed and synthesized as the highly selective and sensitive fluorescent probes for imaging H₂O₂ in living cells. The probes were detected with elemental analysis, IR, ¹H NMR and ¹³C NMR. Upon reaction with H₂O₂, the probes exhibit strong fluorescence responses and high selectivity for H₂O₂ over other reactive oxygen species and some biological compounds. Furthermore, the sulfonate-based probes, as novel fluorescent reagents, are cell-permeable and can detect micromolar changes in H₂O₂ concentrations in living cells by using confocal microscopy. Supported by the National Basic Research Program of China (Grant No. 2007CB936000), the National Natural Science Funds for Distinguished Young Scholar (Grant No. 20725518), Major Program of the National Natural Science Foundation of China (Grant No. 90713019), the National Natural Science Foundation of China (Grant No. 20875057), the Natural Science Foundation of Shandong Province, China (Grant No. Y2007B02), and the Science and Technology Development Programs of Shandong Province, China (Grant No. 2008GG30003012)     

Biocomposites of covalently linked glucose oxidase on carbon nanotubes for glucose biosensor

The formation of covalently linked composites of multi–walled carbon nanotubes (MWCNT) and glucose oxidase (GOD) with high-function density for use as a biosensing interface is described. The reaction intermediates and the final product were characterized by using FT–IR spectroscopy, and the MWCNT-coated GOD nanocomposites were examined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Interestingly, it was found that the GOD–MWCNT composites are highly water soluble. Electrochemical characterization of the GOD–MWCNT composites that were modified on a glassy carbon electrode shows that the covalently linked GOD retains its bioactivity and can specifically catalyze the oxidation of glucose. The oxidation current shows a linear dependence on the glucose concentration in the solution in the range of 0.5–40 mM with a detection limit of 30 μM and a detection sensitivity of 11.3 μA/mMcm². The present method may provide a way to synthesize MWCNT related composites with other biomolecules and for the construction of enzymatic reaction-based biofuel cells and biosensors. Supported by grants from the National Natural Science Foundation of China (NSFC, No. 20125515; 90206037; 20375016) and the Natural Science Foundation of Jiangsu Province (Grant No. BK 2004210)     

The enzyme-amplified amperometric DNA sensor using an electrodeposited polymer redox mediator

A highly sensitive method for the detection of a breast cancer-associated BRCA-1 gene is reported. The detection is based on a classical sandwich-type assay using horseradish peroxidase (HRP) as a catalytic label and electrodeposited Os^2+/3+ conducting polymer (PAA-PVI-Os) as a redox mediator. Target DNA could be detected by the HRP-catalyzed reduction of H₂O₂, leading to a limit of detection as low as 10 fM. Supported by the National Natural Science Foundation of China (Grant Nos. 20725516, 20704043, 20873175 & 20805055), Shanghai Municipal Commission for Science and Technology (Grant Nos. 0752nm021 & 07ZR14136), Ministry of Science and Technology (Grant Nos. 2006CB933000, 2007CB936000 & 2007AA06A406), Ministry of Health (Grant No. 2009ZX10603), and China Postdoctoral Science Foundation and Shanghai Postdoctoral Scientific Program (Grant No. 07R214160).     

基于氧化铒-石墨烯氧化物复合纳米材料的葡萄糖氧化酶直接电化学性能及对葡萄糖的检测

将稀土纳米材料Er₂O₃用于构建葡萄糖生物传感器。Er₂O₃和氧化石墨烯形成复合基底,将葡萄糖氧化酶（GOD）固载在玻碳电极表面。首先利用SEM和XRD技术对所制备的Er₂O₃和氧化石墨烯纳米材料进行表征。利用EIS和CV对整个生物传感器制备过程进行表征。Er₂O₃的存在能有效的保持GOD的生物活性并加速其电子传递速率。由于Er₂O₃和氧化石墨烯之间的协同效应,使得制备的传感器具有一对良好的氧化还原峰,证实GOD和电极之间的直接传递性能。当用于对葡萄糖的电催化氧化时,传感器的CV响应随着葡萄糖浓度的增加而变弱。在葡萄糖浓度为1~10 mmol·L^-1范围内,CV响应值与葡萄糖浓度成线性关系。此外,传感器具有好的稳定性和重现性。     

基于氧化铒石墨烯氧化物复合纳米材料的葡萄糖氧化酶直接电化学性能及对葡萄糖的检测

将稀土纳米材料Er₂O₃用于构建葡萄糖生物传感器。Er₂O₃和氧化石墨烯形成复合基底,将葡萄糖氧化酶（GOD）固载在玻碳电极表面。首先利用SEM和XRD技术对所制备的Er₂O₃和氧化石墨烯纳米材料进行表征。利用EIS和CV对整个生物传感器制备过程进行表征。Er₂O₃的存在能有效地保持GOD的生物活性并加速其与电极之间的电子传递。由于Er₂O₃和氧化石墨烯之间的协同效应,使得制备的传感器在CV图中呈现一对明显的氧化还原峰,证实GOD和电极之间的直接电子传递性能。当用于对葡萄糖的电催化氧化时,传感器的CV响应随着葡萄糖浓度的增加而变弱。在葡萄糖浓度为1~10 mmol·L^-1范围内,CV响应值与葡萄糖浓度成线性关系。此外,传感器具有好的稳定性和重现性。     

Construction of bienzyme biosensors based on combination of the one-step electrodeposition and covalent-coupled sol-gel process

Horseradish peroxidase (HRP) and glucose oxidase (GOD) bienzyme biosensor was constructed by in-situ formation of the organic-inorganic biocomposite film based on the one-step electrodeposition and covalent-coupled sol-gel process. The electrodeposition was performed in the solution containing functional inorganic precursor possessing the epoxy groups, γ-glycidoxypropyltrimethoxysiloxane (GPTMS), a biopolymer chitosan (CS), HRP and GOD. The covalent-coupled sol-gel process was formed by self-hydrolysis and self-condensation of GPTMS, followed by in-situ covalent cross-linking of CS, HRP and GOD through covalent reaction between amino groups and epoxy groups. The developed bienzyme biosensor presented high stability in acidic solution owing to the covalent-coupled organic-inorganic hybridization. Compared with the non-hybrid HRP-GOD/CS/Au electrode, the bienzyme biosensor of HRP-GOD/GPTMS/CS/Au showed improved sensitivity and a wider linear range for the determination of glucose. The linear response of the developed HRP-GOD/GPTMS/CS/Au biosensor for the determination of glucose ranged from 1 to 351 μmol/L with a detection limit of 0.3 μmol/L.     

Preparation of polymer-supported hydrated ferric oxide based on Donnan membrane effect and its application for arsenic removal

In the present study a novel technique was proposed to prepare a polymer-supported hydrated ferric oxide (D201-HFO) based on Donnan membrane effect by using a strongly basic anion exchanger D201 as the host material and FeCl3-HCl-NaCl solution as the reaction environment. D201-HFO was found to exhibit higher capacity for arsenic removal than a commercial sorbent Purolite ArsenX. Furthermore, it presents favorable adsorption selectivity for arsenic removal from aqueous solution, as well as satis- factory kinetics. Fixed-bed column experiments showed that arsenic sorption on D201-HFO could re- sult in concentration of this toxic metalloid element below 10 μg/L, which was the new maximum con- centration limit set recently by the European Commission and imposed by the US EPA and China. Also, the spent D201-HFO is amenable to efficient regeneration by NaOH-NaCl solution.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

一种基于壳聚糖固定葡萄糖氧化酶的葡萄糖生物传感器的研究

本文选用生物相容性好的壳聚糖作为基体材料，使其与戊二醛交联成网状结构包埋葡萄糖氧化酶制成电化学传感器。这种壳聚糖膜不仅可以减小葡萄糖氧化酶的流失，而且能为酶提供了适宜的微环境。用红外光谱、紫外光谱及透射电镜对膜的形态和性质进行了表征。实验结果表明该传感器具有很快的响应速度，很好的稳定性和重现性，能选择性地催化葡萄糖并测定其浓度。该传感器的制备方法简单，成本低，于冰箱中放置两周信号保持在90％以上，对葡萄糖测量的线性范围为1×10^-5 - 3.4×10^-3mol•L^-1，当信噪比为3:1时检测限为5×10^-6mol•L^-1。     

Glucose Chemiluminescence Biosensor Based on Covalent Immobilization of Enzyme in Glutaraldehyde-Functionalized Glass Cell and Direct Coupling of Chitosan-Induced Au/Ag alloy Nanoparticles

We report herein the development of a novel glucose chemiluminescence (CL) biosensor based on covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell and direct coupling of chitosan-induced Au/Ag alloy nanoparticles on it, and how it may be useful for determination of glucose due to CL detection of enzymatically generated H₂O₂. In addition, the nanoalloy offers excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose and increases stability of covalent-linked enzyme. Chitosan molecules act as both the reducing and stabilizing agents for the preparation of nanoparticles (NPs) and also as a coupling agent between GOD and Au/Ag alloy NPs, which made possible the fabrication of a sensitive, accurate and stable biosensor for glucose. Under the optimum conditions, the biosensor can be used for the determination of glucose in the range of 1.2 × 10^–6 to 6.25 × 10^–3 M with a detection limit of 5.0 × 10^–7 M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2 months storage at pH 7.0. The present CL biosensor has been used to determine glucose in real serum and urine samples and validated against colorimetric spectrophotometry method.     

Composite Multienzyme Amperometric Biosensors for an Improved Detection of Phenolic Compounds

A biosensor design, in which glucose oxidase and peroxidase are coimmobilized by simple physical inclusion into the bulk of graphite‐Teflon pellets, is reported for the detection of phenolic compounds. This design allows the “in situ” generation of the H₂O₂ needed for the enzyme reaction with the phenolic compounds, which avoids several problems detected in the performance of single peroxidase biosensors as a consequence of the presence of a high H₂O₂ concentration. So, a much lower surface fouling was found at the GOD‐HRP biosensor in comparison with a graphite‐Teflon‐HRP electrode, suggesting that the controlled generation of H₂O₂ makes more difficult the formation of polymers from the enzyme reaction products. The construction of trienzyme biosensors, in which GOD, HRP and tyrosinase were coimmobilized into the graphite‐Teflon matrix is also reported, and their performance was compared with that of GOD‐HRP bienzyme electrodes. The practical applicability of the composite multienzyme amperometric biosensors was evaluated by the estimation of the phenolic compounds content in waste waters from a refinery, and the results were compared with those obtained by using a colorimetric official method based on the reaction with 4‐aminoantipyrine.     

一步电沉积制备交联型有机-无机生物杂化膜构筑双酶传感器的研究

利用一步电沉积法,以含有环氧基团的γ-环氧丙氧丙基三甲氧基硅烷（GPTMS）为无机杂化试剂和功能性交联试剂,通过壳聚糖（Chitosan,CS）、辣根过氧化物酶（HRP）和葡萄糖氧化酶（GOD）分子中-NH2与环氧基团的反应,在金电极表面原位制备交联型有机-无机生物杂化膜,得到共固定HRP和GOD的新型双酶生物传感器．实验证实了这种有机-无机生物杂化膜在不同酸、碱条件下都具有高的稳定性和耐用性,克服了CS酸溶的不足,从而扩大了其使用范围．在葡萄糖检测中,交联型双酶传感器HRP-GOD／GPTMS／CS,Au比无交联的双酶传感器HRP-GOD／CS／Au具有更高的灵敏度、更宽的线性范围,其线性范围为1μmol／L-351μmol／L,检期4限为0．3μmol／L．     

A fluorescent sensing membrane for iodine based on intramolecular excitation energy transfer of anthryl appended porphyrin

A single anthryl appended meso-tetraphenylporphyrin (TPP) dyad has been synthesized and applied in fluorescence sensing of iodine based on the intramolecular excitation energy transfer. The molecular recognition of the sensor is based on the interaction of iodine with inner anthracene moiety of the dyad, while the signal reporter for the recognition process is the TPP fluorescence quenching. Because the emission spectrum of anthracene is largely overlapped with the Soret band absorption of TPP, intramolecular excitation energy transfer interaction occurs between the donor, anthracene and acceptor, TPP. This energy transfer leads to TPP fluorescence emission by excitation of anthracene. The sensor was constructed by immobilizing the dyad in a plasticized poly(vinyl chloride) (PVC) membrane. The sensing membrane shows higher sensitivity compared to the sensors by using anthracene, TPP, or a mixture of anthracene and TPP as sensing materials. Under the optimum conditions, iodine in a sample solution can be determined from 2.04 to 23.6 mmol·L⁻¹ with a detection limit of 33 nmol·L⁻¹. The sensing membrane shows satisfactory response characteristics including good reproducibility, reversibility and stability, as well as the short response time of less than 60 s. Except for Cr₂O₇²⁻ and MnO₄⁻, other common metal ions and anions in foodstuff do not interfere with iodine determination. The proposed method was applied in the determination of iodine in table salt samples. The results agree well with those obtained by other methods. Supported by the National Outstanding Youth Science Foundation of China (Grant No. 20525518), the National Natural Science Foundation of China (Grant No. 20775005), and the National Natural Science Foundation of Hunan province (Grant No. JJ076021)     

Biosensing of Aromatic Amines in Reversed Micelles with Self‐Generation of Hydrogen Peroxide at Glucose Oxidase‐Peroxidase Bienzyme Electrodes

The amperometric biosensing of aromatic amines using a composite glucose oxidase (GOD)‐peroxidase (HRP) biosensor in reversed micelles is reported. Rigid composite pellets of graphite and Teflon, in which GOD and HRP were coimmobilized by simple physical inclusion, were employed for the biosensor design. This design allows the in situ generation of the H₂O₂ needed for the enzyme reaction with the aromatic amines, thus preventing the negative effect that the presence of a high H₂O₂ concentration in solution has on HRP activity. The H₂O₂ in situ generation is performed by oxidation of glucose catalyzed by GOD. The effect of the composition of the reversed micelles, i.e., the nature of the organic solvent used as the continuous phase, the nature and concentration of the surfactant used as emulsifying agent, the aqueous 0.05 mol L^?1 phosphate buffer percentage used as the dispersed phase, and the glucose concentration in the aqueous phase, on the biosensor response was evaluated. Reversed micelles formed with ethyl acetate, a 5% of phosphate buffer (pH 7.0) containing 3.0×10^?3 mol L^?1 glucose, and 0.1 mol L^?1 AOT (sodium dioctylsulfosuccinate), were selected as working medium. Well‐defined and reproducible amperometric signals at 0.00 V were obtained for p‐phenylenediamine, 2‐aminophenol, o‐phenylenediamine, m‐phenylenediamine, 1‐naphthylamine, o‐toluidine and aniline. The useful lifetime of one single biosensor was of 60 days. The trend in sensitivity observed for the aromatic amines is discussed considering the effect of their structure on the stabilization of the radicals formed in the enzyme reaction which are electrochemically reduced. The behavior of the composite bienzyme electrode was also evaluated in a FI (flow injection) system using reversed micelles as the carrier. The suitability of the composite bienzyme electrode for the analysis of real samples was demonstrated by determining aniline in spiked carrots.     

Enzyme-catalyzed reaction of voltammetric enzyme-linked immunoassay system based on OAP as substrate

The o-aminophenol (OAP)-H_2O_2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay new system has extremely high sensitivity. HRP can be measured with a detection limit of 6.0×10~-(10) g/L and a linear range of 1.0×10~(-9)—4.0×10~(-6) g/L. The pure product of H_2O_2 oxidizing OAP catalyzed by HRP was prepared with chemical method. The enzyme-catalyzed reaction has been investigated with electroanalytical chemistry, UV/Vis spectrum, IR spectrum, ~(13)C NMR, ~1H NMR, mass spectrum, elemental analysis, etc. Under the selected enzyme-catalyzed reaction conditions, the oxidation product of OAP with H_2_O2 catalyzed by HRP is 2-aminophe-noxazine-3-one. The processes of the enzyme-catalyzed reaction and the electroreduction of the product of the enzymecatalyzed reaction have been described.     

Carbon-decorated ZnO nanowire array: A novel platform for direct electrochemistry of enzymes and biosensing applications

We report here the direct electron transfer of GOD and a novel glucose biosensor based on carbon-decorated ZnO(C–ZnO) nanowire array electrode. The C–ZnO nanowire array provides a novel platform for fast direct electrochemistry of GOD, and its based biosensor shows very high sensitivity and low detection limit. Based on the direct electrochemistry of horseradish peroxidase (HRP), the H₂O₂ biosensing application is further demonstrated using this new C–ZnO array architecture. The high conductivity of carbon and good electron transfer capability of ZnO nanowires, along with their low cost and biocompatibility make the C–ZnO nanowire array a promising platform for direct electrochemistry of enzymes and mediator-free enzymatic biosensors.     

Nano Au/TiO2 hollow microsphere membranes for the improved sensitivity of detecting specific DNA sequences related to transgenes in transgenic plants

Gold nanoparticles (nano Au)/titanium dioxide (TiO₂) hollow microsphere membranes were prepared on the carbon paste electrode (CPE) for enhancing the sensitivity of DNA hybridization detection. The immobilization of nano Au and TiO₂ microsphere was investigated with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored with EIS using [Fe(CN)₆]^3−/4− as indicator. The sequence-specific DNA of the 35S promoter from cauliflower mosaic virus (CaMV35S) gene was detected with this DNA electrochemical sensor. The dynamic detection range was from 1.0×10⁻¹² to 1.0×10⁻⁸ mol/L DNA and a detection limit of 2.3×10⁻¹³ mol/L could be obtained. The polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase (NOS) gene from the real sample of a kind of transgenic soybean was also satisfactorily detected. Supported by the National Natural Science Foundation of China (Grant Nos. 20635020 and 20375020), Doctoral Foundation of the Ministry of Education of China (Grant No. 20060426001) and Natural Science Foundation of Qingdao City (Grant No. 04-2-JZP-8)