Inclusion of metal micro-particles into poly(pyrrolylalkylammonium) films containing an enzyme for bioelectrocatalysis: a preliminary study

The electrocatalytic detection of products issued from enzymatic reactions occuring at biosensor interfaces can be improved owing to metal microparticle dispersions. This contribution describes the faesability of this concept for the modification poly(pyrrolylalkylammonium) films containing an enzyme through the inclusion of platinum. A model enzyme, glucose oxidase (GOx) was employed to demonstrate the reliability of this approach through the design of an amperometric glucose biosensors. The obtained composite assemblies were characterised regarding their sensing capabilities toward glucose. Platinum particles incorporation was operated within the biofilms to obtain a suitable sensitivity of the biosensor to glucose at a lower operational oxidation potential (eg 0.5 V/SCE) than the oxidation potential generally observed for hydrogen peroxide detection onto carbon electrodes. The incorporation procedure of platinum aggregates has been optimised to design biosensors exhibiting a sensitivity of about 8 mA. M(-1) cm(-2).     

Functionalization of poly-SNS-anchored carboxylic acid with Lys and PAMAM: surface modifications for biomolecule immobilization/stabilization and bio-sensing applications

Poly(2-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl) (SNS) acetic acid) was electrochemically deposited on graphite electrodes and functionalized with lysine (Lys) amino acid and poly(amidoamine) derivatives (PAMAM?G2 and PAMAM?G4) to investigate their matrix properties for biosensor applications. Glucose oxidase (GOx) was immobilized onto the modified surface as the model enzyme. X-Ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to report the surface properties of the matrices in each step of the biosensor construction. The biosensors were characterized in terms of their operational and storage stabilities and the kinetic parameters (K and I(max)). Three new glucose biosensors revealed good stability, featuring low detection limits (19.0 μM, 3.47 μM and 2.93 μM for lysine-, PAMAM?G2- and PAMAM?G4-functionalized electrodes, respectively) and prolonged the shelf lives (4, 5, and 6 weeks for Lys-, PAMAM?G2- and PAMAM?G4-modified electrodes, respectively). The proposed biosensors were tested for glucose detection on real human blood serum samples.     

基于Ru(bpy)■-三乙胺体系的电致化学发光生物传感器检测葡萄糖

联吡啶钌(Ru(bpy)■)拥有优良的电致化学发光(ECL)性能,但其较好的水溶性使其固载面临巨大问题。该文制备了Pt纳米粒子与Ru(bpy)■的复合物(Pt NPs-Ru),将其修饰于电极并进一步固载葡萄糖氧化酶(GOx)制得传感器。基于H2O2对Ru(bpy)■-三乙胺体系ECL信号的猝灭作用,随着葡萄糖浓度的增加,其在GOx的催化下原位产生的H2O2量增多,导致ECL信号逐渐减弱,从而实现葡萄糖的检测。ECL强度与葡萄糖浓度的对数在1.0×10-8~5.0×10-5 mol/L范围内呈良好的线性关系,检出限低至5.2×10-9 mol/L。传感器具有好的稳定性和高的选择性。Pt NPs-Ru复合物为ECL传感器的构建提供了良好平台,为葡萄糖检测提供了新方法。     

Amperometric glucose biosensor based on a gold nanorods/cellulose acetate composite film as immobilization matrix

We report on the utilization of gold nanorods to create a highly responsive glucose biosensor. The feasibility of an amperometric glucose biosensor based on immobilization of glucose oxidase (GOx) in gold nanorod is investigated. GOx is simply mixed with gold nanorods and cross-linked with a cellulose acetate (CA) medium by glutaraldehyde. The adsorption of GOx on the gold nanorods is confirmed by X-ray photoelectron spectroscopy (XPS) measurements. Circular dichroism (CD) and UV-spectrum results show that the activity of GOx was preserved after conjugating with gold nanorods. The current response of modified electrode is 10 times higher than that of without gold nanorods. Under optimal conditions, the biosensor shows high sensitivity (8.4 μA cm⁻² mM⁻¹), low detection limit (2 × 10⁻⁵ M), good storage stability and high affinity to glucose (). A linear calibration plot is obtained in the wide concentration range from 3 × 10⁻⁵ to 2.2 × 10⁻³ M.     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

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.     

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.     

Hydrogen peroxide biosensor with a supramolecular layer-by-layer design

A new sensor design is reported for the construction of an amperometric enzyme biosensor toward H (2)O(2). It was based in the supramolecular immobilization of alternating layers of horseradish peroxidase (either modified with 1-adamantane or beta-cyclodextrin-branched carboxymethylcellulose residues) on Au electrodes coated with polythiolated beta-cyclodextrin polymer. The analytical response of the electrodes, using 1 mM hydroquinone as an electrochemical mediator, increases when the number of enzyme layers increases. The biosensor having three enzyme layers showed a sensitivity of 720 microA/M cm (2) and a detection limit of 2 microM and retained 96% of its initial activity after 30 days of storage. The host-guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.     

亲水金和憎水二氧化硅纳米颗粒对葡萄糖生物传感器响应灵敏度的增强作用

用亲水金、憎水二氧化硅纳米颗粒固定葡萄糖氧化酶(GOD),采用聚乙烯醇缩丁醛(PVB)为辅助固酶膜基质来制备葡萄糖生物传感器,并考察了亲水金、憎水二氧化硅纳米颗粒对酶电极电流响应的影响.实验表明,引入纳米粒子可显著增强电极响应灵敏度.并对两种不同性质纳米颗粒所起作用的可能机理进行讨论,从理论和实验上证明了纳米颗粒对固定酶的作用.为制备有实用价值的葡萄糖生物传感器提供了可供参考的实验和理论依据.     

New CNT/poly(brilliant green) and CNT/poly(3,4-ethylenedioxythiophene) based electrochemical enzyme biosensors

A combination of the electroactive polymer poly(brilliant green) (PBG) or conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) with carbon nanotubes to obtain CNT/PBG and CNT/PEDOT modified carbon film electrodes (CFE) has been investigated as a new biosensor platform, incorporating the enzymes glucose oxidase (GOx) as test enzyme, alcohol oxidase (AlcOx) or alcohol dehydrogenase (AlcDH). The sensing parameters were optimized for all biosensors based on CNT/PBG/CFE, CNT/PEDOT/CFE platforms. Under optimized conditions, both GOx biosensors exhibited very similar sensitivities, while in the case of AlcOx and AlcDH biosensors, AlcOx/CNT/PBG/CFE was found to give a higher sensitivity and lower detection limit. The influence of dissolved O₂ on oxidase-biosensor performance was investigated and was shown to be different for each enzyme. Comparisons were made with similar reported biosensors, showing the advantages of the new biosensors, and excellent selectivity against potential interferents was successfully demonstrated. Finally, alcohol biosensors were successfully used for the determination of ethanol in alcoholic beverages.     

Direct electrochemical sensing of glucose using glucose oxidase immobilized on functionalized carbon nanotubes via a novel metal chelate-based affinity method

We report on a novel amperometric glassy carbon biosensing electrode for glucose. It is based on the immobilization of a highly sensitive glucose oxidase (GOx) by affinity interaction on carbon nanotubes (CNTs) functionalized with iminodiacetic acid and metal chelates. The new technique for immobilization is exploiting the affinity of Co(II) ions to the histidine and cysteine moieties on the surface of GOx. The direct electrochemistry of immobilized GOx revealed that the functionalized CNTs greatly improve the direct electron transfer between GOx and the surface of the electrode to give a pair of well-defined and almost reversible redox peaks and undergoes fast heterogeneous electron transfer with a rate constant (k _s) of 0.59?s^?1. The GOx immobilized in this way fully retained its activity for the oxidation of glucose. The resulting biosensor is capable of detecting glucose at levels as low as 0.01?mM, and has excellent operational stability (with no decrease in the activity of enzyme over a 10?days period). The method of immobilizing GOx is easy and also provides a model technique for potential use with other redox enzymes and proteins.

Glucose microfluidic biosensors based on immobilizing glucose oxidase in poly(dimethylsiloxane) electrophoretic microchips

Here we reported a novel microfluidic biosensor with an on-column immobilized enzyme microreactor. The fabrication approach of this biosensor is simple and the enzyme microreactors with controlled sizes can be placed at any desired position on the microchip. Taking glucose oxidase (GOx) as an example, electroosmotic flow (EOF) as a driving force and amperometry as a detection method, the performance of biosensors were modulated by changing the length of enzyme reactor from 0.5 cm to 3 cm, and the linear ranges were changed from 0-8.0 mM to 0-30.0 mM with the detection limits from 42 microM to 6.5 microM. The enzyme reactor remained its 65% activity after 23 days storage. It also showed good anti-interference ability and was used to quantify glucose in human serum samples.     

Reduced Graphene Oxide/Pt Nanoparticles/Zn‐MOF‐74 Nanomaterial for a Glucose Biosensor Construction

In this study, a new glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on platinum nanoparticles (Pt NPs) decorated reduced graphene oxide (rGO)/Zn‐MOF‐74 hybrid nanomaterial. Herein, the biosensor fused the advantages of rGO with those of porous Zn‐MOF and conductive Pt NPs. This has not only enlarged the surface area and porosity for the efficient GOx immobilization and faster mass transport, but also provided favorable electrochemical features such as high current density, remarkable electron mobility through metal nanoparticles, and improved electron transfer between the components. The GOx‐rGO/Pt NPs@Zn‐MOF‐74 coated electrode displayed a linear measurement range for glucose from 0.006 to 6 mM, with a detection limit of 1.8 μM (S/N: 3) and sensitivity of 64.51 μA mM^?1 cm^?2. The amperometric response of the enzyme biosensor demonstrated the typical behavior of Michaelis‐Menten kinetics. The obtained satisfying sensitivity and measurement range enabled fast and accurate glucose measurement in cherry juice using the fabricated biosensor. The water‐stable Zn‐MOF‐74 demonstrated higher enzyme loading capacity and can be potent supporting material for biosensor construction.     

Integrated Microcentrifuge Carbon Entrapped Glucose Oxidase Poly (N-Isopropylacrylamide) (pNIPAm) Microgels for Glucose Amperometric Detection

This study demonstrates a miniaturized integrated glucose biosensor based on a carbon microbeads entrapped by glucose oxidase (GOx) immobilized on poly (N-isopropylacrylamide) (pNIPAm) microgels. Determined by the Lowry protein assay, the pNIPAm microgel possesses a high enzyme loading capacity of 31?mg/g. The pNIPAm GOx loaded on the microgel was found to maintain a high activity of approximately 0.140?U determined using the 4-aminoantipyrine colorimetric method. The integrated microelectrochemical cell was constructed using a microcentrifuge vial housing packed with (1:1, w/w) carbon entrapped by pNIPAm GOx microgels, which played the dual role of the microbioreactor and the working electrode. The microcentrifuge vial cover was used as a miniaturized reference electrode and an auxiliary electrode holder. The device can work as biosensor, effectively converting glucose to H₂O₂, with subsequent amperometric detection at an applied potential of ?0.4?V. The microelectrochemical biosensor was used to detect glucose in wide linear range from 30?µM to 8.0?mM, a low detection limit of 10?µM, a good linear regression coefficient (R²) of 0.994, and a calibration sensitivity of 0.0388?µA/mM. The surface coverage of active GOx, electron transfer rate constant (ks), and Michaelis–Menten constant (K_M^app) of the immobilized GOx were 4.0?×?10^?11?mol/cm², 5.4?s^?1, and 0.086?mM, respectively. To demonstrate the applicability and robustness of the biosensor for analysis of high sample matrix environment, glucose was analyzed in root beer. The microelectrochemical device was demonstrated for analysis of small sample (<50?µL), while affording high precision and fast signal measurement (≤5?s).     

蜘蛛丝素和聚乙烯醇固定葡萄糖氧化酶 制备葡萄糖传感器

用蜘蛛丝素和聚乙烯醇的混合材料把葡萄糖氧化酶固定在氧电极表面,制成葡萄糖氧化酶电极。传感器对葡萄糖有灵敏的响应,平均响应时间为20s,电位变化值与葡萄糖浓度在3.0×10     

A Novel Enzymatic Glucose Biosensor and Nonenzymatic Hydrogen Peroxide Sensor Based on (3‐Aminopropyl) Triethoxysilane Functionalized Reduced Graphene Oxide

In the present study, a novel enzymatic glucose biosensor using glucose oxidase (GOx) immobilized into (3‐aminopropyl) triethoxysilane (APTES) functionalized reduced graphene oxide (rGO‐APTES) and hydrogen peroxide sensor based on rGO‐APTES modified glassy carbon (GC) electrode were fabricated. Nafion (Nf) was used as a protective membrane. For the characterization of the composites, Fourier transform infrared spectroscopy (FTIR), X‐ray powder diffractometer (XRD), and transmission electron microscopy (TEM) were used. The electrochemical properties of the modified electrodes were investigated using electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry. The resulting Nf/rGO‐APTES/GOx/GC and Nf/rGO‐APTES/GC composites showed good electrocatalytical activity toward glucose and H₂O₂, respectively. The Nf/rGO‐APTES/GC electrode exhibited a linear range of H₂O₂ concentration from 0.05 to 15.25 mM with a detection limit (LOD) of 0.017 mM and sensitivity of 124.87 μA mM⁻¹ cm⁻². The Nf/rGO‐APTES/GOx/GC electrode showed a linear range of glucose from 0.02 to 4.340 mM with a LOD of 9 μM and sensitivity of 75.26 μA mM⁻¹ cm⁻². Also, the sensor and biosensor had notable selectivity, repeatability, reproducibility, and storage stability.     

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.

Enzyme Deposition by Polydimethylsiloxane Stamping for Biosensor Fabrication

High‐performance biosensors were fabricated by efficiently transferring enzyme onto Pt electrode surfaces using a polydimethylsiloxane (PDMS) stamp. Polypyrrole and Nafion were coated first on the electrode surface to act as permselective films for exclusion of both anionic and cationic electrooxidizable interfering compounds. A chitosan film then was electrochemically deposited to serve as an adhesive layer for enzyme immobilization. Glucose oxidase (GOx) was selected as a model enzyme for construction of a glucose biosensor, and a mixture of GOx and bovine serum albumin was stamped onto the chitosan‐coated surface and subsequently crosslinked using glutaraldehyde vapor. For the optimized fabrication process, the biosensor exhibited excellent performance characteristics including a linear range up to 2 mM with sensitivity of 29.4±1.3 μA mM⁻¹ cm⁻² and detection limit of 4.3±1.7 μM (S/N=3) as well as a rapid response time of ∼2 s. In comparison to those previously described, this glucose biosensor exhibits an excellent combination of high sensitivity, low detection limit, rapid response time, and good selectivity. Thus, these results support the use of PDMS stamping as an effective enzyme deposition method for electroenzymatic biosensor fabrication, which may prove especially useful for the deposition of enzyme at selected sites on microelectrode array microprobes of the kind used for neuroscience research in vivo .     

New Urea Biosensor Based on Urease Enzyme Obtained from Helycobacter pylori

The urease enzyme of Helicobacter pylori was isolated from biopsy sample obtained from antrum big curvature cell extracts. A new urea biosensor was prepared by immobilizing urease enzyme isolated from Helicobacter pylori on poly(vinylchloride) (PVC) ammonium membrane electrode by using nonactine as an ammonium ionophore. The effect of pH, buffer concentration, and temperature for the biosensor prepared with urease from H. pylori were obtained as 6.0, 5 mM, and 25 °C, respectively. We also investigated urease concentration, stirring rate, and enzyme immobilization procedures in response to urea of the enzyme electrode. The linear working range of the biosensor extends from 1 × 10(-5) to 1 × 10(-2) M and they showed an apparent Nernstian response within this range. Urea enzyme electrodes prepared with urease enzymes obtained from H. pylori and Jack bean based on PVC membrane ammonium-selective electrode showed very good analytical parameters: high sensitivity, dynamic stability over 2 months with less decrease of sensitivity, response time 1-2 min. The analytical characteristics were investigated and were compared those of the urea biosensor prepared with urease enzyme isolated from Jack bean prepared at the same conditions. It was observed that rapid determinations of human serum urea amounts were also made possible with both biosensors.     

Physicochemical Characteristics of Polypyrrole/(Glucose oxidase)/(Prussian Blue)‐based Biosensor Modified with Ni‐ and Co‐Hexacyanoferrates

In this work, three types of electrodes suitable for amperometric glucose biosensors were designed. One type of electrode was based on bio‐selective layer of polypyrrole/(glucose oxidase)/(Prussian Blue) (Ppy/GOx/PB) and it was used as a control electrode regarding to which electrochemical properties of two other types of electrodes were compared. During the formation of Prussian blue layers graphite electrodes were additionally modified by Ni‐hexacyanoferrate (NiHCF) and by Co‐hexacyanoferrate (CoHCF) in order to design Ppy/GOx/PB‐NiHCF and Ppy/GOx/PB‐CoHCF electrodes, respectively. Some physicochemical characteristics of all three types of electrodes were evaluated and compared. The Ppy/GOx/PB‐NiHCF electrode showed wider linear range of the calibration curve than Ppy/GOx/PB and Ppy/GOx/PB‐CoHCF electrodes. The effect of temperature on analytical performance of the Ppy/GOx/PB‐NiHCF based biosensor has been evaluated and activation energy of enzyme catalysed reaction has been calculated within the temperature range of 15 °C to 30 °C.