Optimization of Nanohybrid Biosensors Based on Electro-Crosslinked Tannic Acid Capped Nanoparticles/Enzyme

Enzymes/Nanoparticles (NPs) bioconjugates are massively used nowadays to develop thin films for optical and electrochemical biosensors. Nevertheless, their full characterization as a thin coating onto electrodes remains little discussed, in particular the influence of NPs size and enzyme/NPs ratio used in the electrodeposition solution. In this study, GOx (160 kDa) and HRP (44 kDa) were used in association with tannic acid capped gold NPs (a series with sizes from 7 to 40 nm) to electrodeposit biosensor coatings, sensitive towards glucose and H₂O₂, respectively. The electrodeposition process was based on a mussel-inspired electro-crosslinking between gallol moieties of tannic acid (at the surface of NPs) and amine moieties of the enzymes. On one hand, the sensitivity of the GOx/NPs coatings depends strongly on the NP size and the enzyme/NPs molar ratio of the electrodeposition solution. An optimal sensitivity was obtained by electrodeposition of 11 nm NPs at a GOx/NPs molar ratio close to the theoretical value of the enzyme monolayer. On the other hand, a modest influence of the NPs size was found on the sensitivity in the case of the electrodeposited HRP/NPs coatings, reaching a plateau at the HRP/NPs molar ratio close to the value of the theoretical enzyme monolayer. In both cases, the enzyme/NPs molar ratio played a role in the sensitivity. To fully understand the parameters driving the biosensor sensitivity, a comprehensive evaluation of the colloidal state of the bioconjugates is proposed here.     

An XPS investigation on glucose oxidase and Ni/Al hydrotalcite interaction

In this work, a Ni/Al hydrotalcite (HT) was used as glucose oxidase (GOx) immobilizer. Small‐area and angle‐resolved X‐ray photoelectron spectra were recorded on HTs electrosynthesized on Pt in the absence and in the presence of GOx, and compared with those obtained for a Pt surface, modified with the electrosynthesized HT, on which a drop of GOx solution was deposited. The simultaneous electrodeposition of HT + GOx resulted in a compact deposit, thicker than the XPS sampling depth (>10 nm), that is not homogeneous in the lateral and in‐depth composition. The presence of GOx can be deduced comparing the N1s spectra of HT and HT + GOx: in the latter, the N1s component at 400 eV binding energy (BE) is predominant whilst, depending on the analyzed point, a small or no contribution from the component at 407.2 eV, due to nitrate, is revealed. Angle‐resolved XPS provides evidence on the in‐depth composition of anions, cations and GOx. The results highlight the crucial role played by nickel in GOx immobilization. On the basis of the results, it can be suggested that enzyme activity is unevenly distributed and is localized in small areas, where Ni concentration is higher. Copyright © 2010 John Wiley & Sons, Ltd.     

Minimally‐invasive Microneedle‐based Biosensor Array for Simultaneous Lactate and Glucose Monitoring in Artificial Interstitial Fluid

Here we report the first mediated pain free microneedle‐based biosensor array for the continuous and simultaneous monitoring of lactate and glucose in artificial interstitial fluid (ISF). The gold surface of the microneedles has been modified by electrodeposition of Au‐multiwalled carbon nanotubes (MWCNTs) and successively by electropolymerization of the redox mediator, methylene blue (MB). Functionalization of the Au‐MWCNTs/polyMB platform with the lactate oxidase (LOX) enzyme (working electrode 1) and with the FAD‐Glucose dehydrogenase (FADGDH) enzyme (working electrode 2) enabled the continuous monitoring of lactate and glucose in the artificial ISF. The lactate biosensor exhibited a high sensitivity (797.4±38.1 μA cm^?2 mM^?1), a good linear range (10–100 μM) with a detection limit of 3 μM. The performance of the glucose biosensor were also good with a sensitivity of 405.2±24.1 μA cm^?2 mM^?1, a linear range between 0.05 and 5 mM and a detection limit of 7 μM. The biosensor array was tested to detect the amount of lactate generated after 100 minutes of cycling exercise (12 mM) and of glucose after a normal meal for a healthy patient (10 mM). The results reveal that the new microneedles‐based biosensor array seems to be a promising tool for the development of real‐time wearable devices with a variety of sport medicine and clinical care applications.     

A simple method to fabricate a chitosan-gold nanoparticles film and its application in glucose biosensor

A novel film of chitosan-gold nanoparticles is fabricated by a direct and facile electrochemical deposition method and its application in glucose biosensor is investigated. HAuCl(4) solution is mixed with chitosan and electrochemically reduced to gold nanoparticles, which can be stabilized by chitosan and electrodeposited onto glassy carbon electrode surfaces along with the electrodeposition of chitosan. Then a model enzyme, glucose oxidase (GOD) is immobilized onto the resulting film to construct a glucose biosensor through self-assembly. The resulting modified electrode surfaces are characterized with both AFM and cyclic voltammetry. Effects of chitosan and HAuCl(4) concentration in the mixture together with the deposition time and the applied voltage on the amperometric response of the biosensor are also investigated. The linear range of the glucose biosensor is from 5.0 x 10(-5) approximately 1.30 x 10(-3) M with a Michaelis-Menten constant of 3.5 mM and a detection limit of about 13 microM.     

A biosensor prepared by co-entrapment of a glucose oxidase and a carbon nanotube within an electrochemically deposited redox polymer multilayer

A glucose biosensor based on a nanocomposite made by layer-by-layer electrodeposition of the redox polymer into a multilayer containing glucose oxidase (GOx) and single-walled carbon nanotubes (SWCNT) on a screen-printed carbon electrode (SPCE) surface was developed. The objectives of the electrodeposition of redox polymer are to stabilize further the multilayer using a coordinative cross-linked redox polymer and to wire the GOx. The electrochemistry of the layer-by-layer assembly of the GOx/SWCNT/redox polymer nanocomposite was followed by cyclic voltammetry. The resultant biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of layers. The biosensor displayed a linear range from 0.5 to 6.0mM, a sensitivity of 16.4μA/(mMcm(2)), and a response time of about 5s. It shows no response to 0.05mM of ascorbic acid, 0.32mM of uric acid and 0.20mM of acetaminophen using a Nafion membrane covering the nanocomposite-modified electrode surface.     

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.     

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．     

Anti‐Interferent Properties of Oxidized Nickel Based on Layered Double Hydroxide in Glucose Amperometric Biosensors

An amperometric biosensor based on Pt electrodes modified with a thin film of a Ni, Al layered double hydroxide (LDH), submitted to a preliminary oxidative treatment in order to have the nickel centers at the oxidation state +4, and glucose oxidase (GOx) is presented. The oxidized LDH acts both as a system to support the enzyme and as a barrier to anions since it acquires an overall negative charge, as demonstrated by electrochemical impedance spectroscopy. Even if the biosensor response is due to the detection of H₂O₂ at anodic potentials, glucose can be accurately determined in the presence of ascorbic acid or other interferences, commonly present in real matrices in anionic form, since they can not reach the electrode surface. The effectiveness of the developed biosensor has been demonstrated by measuring glucose in samples of fruit juices containing ascorbic acid at high levels.     

纳米金-还原氧化石墨烯修饰葡萄糖氧化酶传感器的制备及其电流法检测饮料中的葡萄糖

利用纳米金(Au NPs)与还原氧化石墨烯(rGO)复合纳米材料制备了葡萄糖氧化酶生物传感器并用于饮料中葡萄糖含量的检测。将壳聚糖作为还原剂及稳定剂,通过一步法合成了Au NPs-rGO复合材料,并通过物理吸附固定葡萄糖氧化酶(GOx)来制作GOx生物传感器。该传感器在磷酸盐缓冲溶液(0.1 mol/L,p H6.0)中,-0.45 V(vs.Ag/Ag Cl)电位下电流法检测葡萄糖含量,线性检测范围为0.01~0.88 mmol/L,灵敏度为22.54μA·mmol-1·L·cm-2,检出限为1.01μmol/L,且表观米氏常数为0.497 mmol/L。该传感器用于多种饮料中葡萄糖含量的直接检测,结果满意。     

In situ chemo-synthesized multi-wall carbon nanotube-conductive polyaniline nanocomposites: characterization and application for a glucose amperometric biosensor

A new glucose amperometric biosensor, based on electrodeposition of platinum nanoparticles onto the surface of multi-wall carbon nanotube (MWNT)-polyaniline (PANI) nanocomposites, and then immobilizing glucose oxidase (GOD) with covalent interaction and adsorption effect, was constructed in this paper. Firstly, the MWNT-PANI nanocomposites had been synthesized by in situ polymerization and were characterized through transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and ultraviolet and visible (UV-vis) absorption spectra. The assembled process of the modified electrode was probed by scanning electron microscopy (SEM) and cyclic voltammetry (CV). Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. The glucose biosensor exhibited a linear calibration curve over the range from 3.0 μM to 8.2 mM, with a detection limit of 1.0 μM and a high sensitivity of 16.1 μA mM⁻¹. The biosensor also showed a short response time (within 5 s). Furthermore, the reproducibility, stability and interferences of the biosensor were also investigated.     

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 × 10^–4 to 1.5 × 10^–2 mol L^–1 with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.     

Design variations of a polymer–enzyme composite biosensor for glucose: Enhanced analyte sensitivity without increased oxygen dependence

The glucose sensitivity and oxygen dependence of a variety of implantable biosensors based on glucose oxidase (GOx), incorporating an electrosynthesized poly-o-phenylenediamine (PPD) permselective barrier on 125-μm diameter Pt disks (Pt_D) and cylinders (Pt_C, 1-mm length), were measured and compared. Full glucose calibrations and experimental monitoring of solution oxygen concentration allowed us to determine apparent Michaelis–Menten parameters for glucose and oxygen. In the linear region of glucose response, the most sensitive biosensor design studied was Pt_D/PPD/GOx (enzyme deposited over polymer) that was 20 times more sensitive than the more widely used Pt_C/GOx/PPD (enzyme immobilized before polymer deposition) configuration. The oxygen dependence, quantified as K_M(O₂), of both active and less active designs was surprisingly similar, a finding that could be rationalized in terms of an increase in K_M(G) with increased enzyme loading. The Pt_D/PPD/GOx design will now enable us to explore glucose concentration dynamics in smaller and layered brain regions with good sensitivity and minimal interference from fluctuations in tissue pO₂.     

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 x 10(-4) to 1.5 x 10(-2) mol L(-1) with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.     

Encapsulation of glucose oxidase within poly(ethylene glycol) methyl ether methacrylate microparticles for developing an amperometric glucose biosensor

Poly(ethylene glycol) methyl ether methacrylate (PEGMEM) microparticles were synthesized and glucose oxidase (GOx) was immobilized within the microparticles. An amperometric biosensor was fabricated using the microparticles with GOx as biological component. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content used in the preparation of the microparticles in the response of the biosensor. The best analytical results were obtained with the microparticles prepared with 0.21 M PEGMEM and 0.74% cross-linking. Furthermore, we have investigated the influence on the biosensor behaviour of parameters such as working potential, pH, temperature and enzymatic load. In addition, analytical properties such as sensitivity, linear range, response time and detection limit were determined. The biosensor was used to determine glucose in human serum samples and to avoid common interferents present in human serum such as uric and ascorbic acids. A Nafion layer was deposited on the electrode surface with satisfactory results. The useful lifetime of the biosensor was at least 520 days.     

A Novel Glucose Biosensor Based on Glucose Oxidase Immobilized on AuPt Nanoparticle – Carbon Nanotube – Ionic Liquid Hybrid Coated Electrode

A novel amperometric glucose biosensor is presented in this article, which is based on the adsorption of glucose oxidase on gold‐platinum nanoparticle (AuPt NP)‐multiwalled carbon nanotube (MWNT) – ionic liquid (i.e., 1‐octyl‐3‐methylimidazolium hexafluorophosphate, [OMIM]PF₆) composite. The gold‐platinum nanoparticles is prepared through direct electrodeposition. Owing to the synergistic action of AuPt nanoparticle, MWNT and [OMIM]PF₆, the biosensor shows good response to glucose, with wide linear range (0.01 to 9.49 mM), short response time (3 s), and high sensitivity (3.47 μA mM⁻¹). With the biosensor the determination of glucose in human serum is performed.     

Simple construction of an enzymatic glucose biosensor based on a nanocomposite film prepared in one step from iron oxide, gold nanoparticles, and chitosan

The one-step synthesis is reported of a nanofilm composed of iron oxide and gold nanoparticles in a chitosan matrix that can act as a novel matrix for the immobilization of glucose oxidase (GOx) to fabricate a glucose biosensor. The use for the composite film strongly increased the effective electrode surface for loading of GOx. The size and shape of the iron oxide nanoparticles were examined by transmission electron micrograph. Direct electron transfer and electrocatalysis by GOx was investigated via cyclic voltammetry and chronoamperometry. Under optimized conditions, the biosensor has a response time of 6?s and a linear response in the range between 3???M and 0.57?mM of glucose, with a detection limit of 1.2???M at a signal-to-noise ratio of 3. This novel and disposable mediatorless glucose biosensor may form the basis for a future mass-produced glucose biosensor.

Direct electrodeposition of a biocomposite consisting of reduced graphene oxide, chitosan and glucose oxidase on a glassy carbon electrode for direct sensing of glucose

We have electrodeposited a composite film consisting of graphene oxide, chitosan and glucose oxidase directly on a glassy carbon electrode (GCE) through electrochemical reduction of a solution of the 3 components under controlled direct electrical potential. The procedure takes only several minutes, and the thickness of the resulting film is uniform and controllable. The GOx has uncompromised bioactivity and exhibits reversible 2-proton and 2-electron transfer in presence of glucose. It therefore can be used amperometric sensing of glucose. The biosensor has a fast response (<3 s), a detection limit of 0.4 μM (which is 50-fold lower compared to the biosensor prepared by drop-casting solutions of the same materials onto an GCE), and a linear response in the 0.4 μM to 2 mM concentration range (which again is much better than that of the biosensor prepared by the drop-casting method). Other features include high reproducibility, long-time storage stability, and satisfactory selectivity. We presume that the direct single-step electrodeposition of this nanocomposite offers a promising approach towards novel types of highly sensitive and stable electrochemical biosensors.

鱼鳔膜为基质的生物传感器测定葡萄糖的研究

以鱼鳔膜为基质同定葡萄糖氧化酶,偶联氧电极,构建了葡萄糖生物传感器,通过测定溶解氧浓度的变化定量测定葡萄糖.考察了酶浓度、pH值、缓冲液浓度对传感器的影响,优化了实验条件:即酶浓度为1 mg,pH 7.0,缓冲液浓度为100 mmol/L.此传感器具有较宽的线性范围(0.016～1.2 mmol/L),较短的响应时间(...     

Amperometric Glucose Sensor Fabricated by Combining Glucose Oxidase Micelle Membrane and Aminated Glassy Carbon Electrode

Abstract

An amperometric glucose biosensor based on the detection of the reduction of oxygen has been developed by combining an aminated glassy carbon electrode with a polystyrene (PS) membrane containing glucose oxidase (GOD) micelles. The structure of GOD micelles contained in PS membrane was observed by scanning electron microscope. The micelle has a roughly spherical shape, and the enzyme colony is contained inside the micelle. This glucose sensor exhibited good sensitivity with short response time (within 2 min). A good linear relationship was observed in the concentration range of 0.2 mM to 2.6 mM when the applied potential was ? 0.45 V vs. Ag/AgCl.     

Non-enzymatic Glucose Biosensor Based on Cu/SWNTs Composite Film Fabricated by One-step Electrodeposition

Based on the adsorption of copper ions on single-walled carbon nanotubes(SWNTs) in electrolyte, Cu/SWNTs nanocomposite film was initially prepared on indium-doped tin oxide(ITO) substrate by one-step electrodeposition. This method may provide a versatile and facile pathway to fabricate other SWNTs-supported metal composite films. Electrochemical experiments revealed that the obtained Cu/SWNTs/ITO electrode offered an excellent electrocatalytic activity towards the oxidation of glucose and could be applied to the construction of non-enzymatic glucose biosensor. The linear range of the sensor was 1.0×10–6 to 6.0×10–4 mol/L and the response time was within 2 s. Particularly, its sensitivity reached as high as 1434.67 μA·L·mmol–1·cm–2, which was superior to any other non-enzymatic glucose biosensor based on copper-carbon nanotubes electrode reported previously.