Reagentless amperometric glucose biosensor based on the immobilization of glucose oxidase on a ferrocene@NaY zeolite composite

Ferrocene (Fc) was encapsulated in the cavities of a NaY zeolite by vapor diffusion via sublimation at below 100?°C. The resulting Fc@NaY zeolite composite was investigated by power X-ray diffraction, diffuse reflectance UV?Cvis and FT-IR spectroscopy, and by cyclic voltammetry. The results indicated that Fc was encapsulated into the zeolite whose microporous structure had remained intact. The Fc in the silica matrix had retained its electroactivity and did not leach out. A glucose biosensor was obtained by immobilization of the modified zeolite and glucose oxidase on a carbon paste electrode. It displays a linear response to glucose (from 0.8???M to 4.0?mM), a detection limit of 0.2???M, and a response time of 4?s. The good performance of the biosensor is ascribed to the biocompatibility of the zeolite and presence of Fc which facilitates the electron transfer from the enzyme to the surface of the electrode.

聚芳酰胺-多壁碳纳米管混合物固定漆酶电极的电化学行为

以聚芳酰胺-多壁碳纳米管混合物为载体,利用漆酶表面氨基与聚芳酰胺主链端羧基的共价偶联以及碳纳米管与漆酶间的疏水作用,构筑了具有较高稳定性和电催化活性的漆酶修饰电极.并对该固酶修饰电极的固酶量、酶活力、电化学行为及其电催化氧还原的性能进行了表征.对漆酶分子具有亲和力的聚芳酰胺芳环结构及聚芳酰胺端羧基与漆酶表面氨基的共价偶联避免了漆酶的脱落和变性.而碳纳米管与聚芳酰胺的混合使得该三维修饰电极具有良好的电子导电性,并成功地实现了漆酶的氧化还原活性位与电极之间的直接电荷转移,这一点可由在0.73和0.38V附近观察到漆酶的T1和T2(漆酶的T1,T2铜活性位的形式电位分别为0.78和0.39V(vsNHE))铜活性位的两对氧化还原峰确认.漆酶的担载量为56.0mg·g-1,具有电化学活性的漆酶占总担载漆酶量的68%.在pH=4.4磷酸盐缓冲溶液中,该修饰电极上氧气还原的起始电位为0.55V,其对氧气的米氏常数KM为55.8μmo·lL-1,对氧气的检测限为0.57μmo·lL-1.在4℃下保存两个月后能实现直接电荷转移的漆酶量仅下降了14%左右而氧还原超电势提高了约50mV.结果表明该修饰电极有望用作酶基生物燃料电池的阴极和电流型氧气传感器.     

Nitrogen‐Doped Carbon Hollow Spheres for Immobilization,Direct Electrochemistry,and Biosensing of Protein

Nitrogen‐doped carbon hollow spheres (NCHS) were designed for the immobilization and biosensing of proteins. Chitosan was first functionalized with glutaraldehyde to form cross‐linked chitosan with free ? CHO groups (GCS). The as‐prepared GCS was used for dispersion of nitrogen‐doped carbon hollow spheres. Using glucose oxidase (GOD) as a model, the NCHS was tested for immobilization of redox proteins and the design of electrochemical biosensors. GOD molecules immobilized in the nanocomposites showed direct electrochemistry with a formal potential of ?0.448 V and well electrochemical performance. The proposed biosensor exhibited a linear response to glucose concentrations ranging from 3.7 µM to 18.0 mM with a detection limit of 1.2 µM and a sensitivity of 11.85 µA mM^?1. This biosensor was also applied to detect glucose in human serum samples, accomplishing good recovery in the range of 92–105 %. The nanocomposites provided a good matrix for protein immobilization and biosensor fabrication.     

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers

This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV). Sensors and enzymatic biosensors for H₂O₂ and choline detection, respectively, have been developed, using choline oxidase (ChOD) as a model enzyme. In this case, hydrogel is used both as the enzymatic immobilization matrix and as a template for the electrosynthesis of polyluminol. The enzyme was immobilized by entrapment in the gel matrix during its formation before electropolymerization of the monomer. Several parameters have been optimized in terms of polymerization conditions, enzyme loading, and average pore size. Using calcium alginate or tetramethoxysilane (TMOS)-based silica as porous matrix, H₂O₂ and choline detection are reported down to micromolar concentrations with three orders of magnitude wide dynamic ranges starting from 4?×?10^?7 M. Polyluminol/hydrogel composites appear as suitable electrochemiluminescence (ECL)-active sensing layers for the design of new reagentless and disposable easy-to-use optical sensors and biosensors, using conventional TMOS-based silica gel or the more original and easier to handle calcium alginate, reported here for the first time in such a configuration, as the biocompatible hydrogel matrix. Figure

Elaboration of electrochemiluminent polyluminol/hydrogel composite sensing layers     

A Nitrite Biosensor Based on the Direct Electron Transfer of Hemoglobin Immobilized on Carboxyl‐Functionalized Multiwalled Carbon Nanotubes/Polyimide Composite

The high electrically conductive carboxyl‐functionalized multiwalled carbon nanotubes (COOH‐MWCNTs) were used to combine with nonconducting polyimide (PI) to generate a PI/COOH‐MWCNTs membrane. PI served as a matrix to entrap COOH‐MWCNTs and hemoglobin (Hb). COOH‐MWCNTs can improve the conductivity of the composite. The direct electrochemistry measurement indicated that the PI/COOH‐MWCNTs composite enhanced the immobilization of Hb significantly. Besides, the Hb/PI/COOH‐MWCNTs/GCE biosensor possessed excellent electrocatalytic activity for the detection of nitrite. Therefore, PI is a good matrix for Hb immobilization and it has application in sensor construction. This work is promising in the development of sensitive biosensors based on PI/COOH‐MWCNTs composite film.     

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10^–6 M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

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.

Mathematical modeling of biosensor action in the region between diffusion and kinetic modes

We describe the action of electrochemical enzyme-based biosensor by applying mathematical modeling. We consider two types of biosensors: a biosensor containing a single heterogeneous enzyme layer and biosensor containing an additional protecting polymer-based layer. The initial parameters of the biosensor were selected on the basis of typical immobilized glucose oxidase-based electrochemical biosensor. A phenomenon of the accumulation of the electrochemically active product inside the biocatalytic layer was evaluated. It was shown that accumulation of the product can increase sensitivity of the biosensor no more than 2.6 times. Due to the asymmetric distribution of the electrochemically active product inside the enzyme-containing membrane and asymmetric diffusion of the substrate, it was shown that the thickness of the membrane possesses an optimal value. For the selected set of initial parameters, the optimal thickness of the enzyme-containing layer was 2.9–4.5  $\upmu $ m. Real profiles of the impact of the thickness of the membranes were evaluated. A method for the evaluation of acceptable fluctuations of the membrane diffusion parameters on biosensor response was created, and the profiles of the dependence were calculated. These dependencies can be used for development of the software for biosensor monitoring.     

A novel, disposable, screen-printed amperometric biosensor for ketone 3-β-hydroxybutyrate fabricated using a 3-β-hydroxybutyrate dehydrogenase–mesoporous silica conjugate

A disposable amperometric biosensor for ketone 3-β-hydroxybutyrate (3HB) has been developed successfully. The sensor is based on a screen-printed carbon electrode containing Meldola’s Blue (MB) and sensing components containing nicotinamide adenine dinucleotide (NAD⁺) and 3-β-hydroxybutyrate dehydrogenase (3HBDH) immobilized in mesoporous silica (FSM8.0) using an aqueous photo-cross-linkable polymer matrix of polyvinyl alcohol (O-391), and it requires only a small sample volume of 10 μL for the measurement. The behavior of a resulting biosensor, i.e., 3HBDH–FSM8.0/NAD⁺/MB-SPCE, was examined in terms of NAD⁺ concentration for construction, pH, applied potential, operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.6 and at an applied potential of ?50 mV. The determination range and the response time for 3HB were from 30 μM to 8 mM and approximately 30 s, respectively. In addition, the sensor was quite stable and maintained >90 % of its initial response after being stored for over 6 months. This result implies that our method provides a novel biosensor for ketone 3-β-hydroxybutyrate which is easy-to-use, cost-effective, and has good reproducibility, which are vital for commercial purposes.

Enzyme-based amperometric galactose biosensors: a review

This review (with 35 references) summarizes the various strategies used in biosensors for galactose, and their analytical performance. A brief comparison of the enzyme immobilization methods employed and the analytical performance characteristics of a range of galactose biosensors are first summarized in tabular form and then described in detail. Selected examples have been included to demonstrate the various applications of these biosensors to real samples. Following an introduction into the field that covers the significance of sensing galactose in various fields, the review covers biosensors based on the use of galactose oxidase, with a discussion of methods for their immobilization (via cross-linking, adsorption, covalent bonding and entrapment). This is followed by a short section on biosensors based on the use of galactose dehydrogenase. The conclusion section summarizes the state of the art and addresses current challenges.

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Graphical abstract Fabrication of a disposable screen-printed (a) electrochemical galactose biosensor (b) for real sample analysis and a dummy biosensor (c) for compensating the effect of interferences

     

Disposable screen-printed electrode coupled with recombinant Drosophila melanogaster acetylcholinesterase and multiwalled carbon nanotubes for rapid detection of pesticides

Recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE), multiwalled carbon nanotubes (MWCNTs), and Prussian blue have been combined for development of a three-electrode biosensor with more rapid responses and higher stability than in our previous study. A new disposable screen-printed electrode (SPE) was developed for rapid detection of organophosphate and carbamate pesticides. After optimization, 10 microg MWCNT and 5 microL enzyme immobilization solution consisting of 0.2% glutaraldehyde, 0.1% Nafion, 0.2% bovine serum albumin, 0.1 g/L MWCNT, and 1.5 mU R-DmAChE were fixed on each of the R-DmAChE/MWCNT SPEs. The LOD of this biosensor was 0.5 microg/L for pesticide standards of dichlorvos (DDV) and carbofuran. The performance of this biosensor was tested for vegetable and water samples at various spiked levels, and good stability and sensitivity were found. The obtained recoveries were from 82.6 to 110.5% for DDV at levels of 0.5-5 microg/L and 73.4 to 118.4% for carbofuran at 1-10 microg/L in lake and sea water samples, demonstrating that the proposed approach is an alternative means for rapid detection of pesticide residues and contaminants in food safety and environmental monitoring.     

Hydrothermal Growth of Ag‐Doped ZnO Nanoparticles on Electrospun Cellulose Nanofibrous Mats for Catechol Detection

We fabricated a novel hierarchical composite mat composed of electrospun cellulose nanofibers decorated with Ag‐doped ZnO (Ag‐ZnO) nanoparticles and further demonstrated its potential application as the efficient laccase (Lac) biosensor substrate material. The cyclic voltammograms revealed that the Ag‐ZnO/cellulose nanofibrous mat provided an excellent microenvironment for Lac immobilization and benefited direct electron transfer of Lac. The fabricated Lac/Ag‐ZnO/cellulose/GCE exhibited a highly sensitive detection of catechol with a wide linear range from 0.995 to 811 µM and a low detection limit of 0.205 µM. The results indicated that Ag‐ZnO/cellulose nanofibers were the promising nanostructured materials for the construction of different biosensors.     

Enzymatic biosensors based on the use of metal oxide nanoparticles

Over the past decades, various techniques have been developed to obtain materials at a nanoscale level to design biosensors with high sensitivity, selectivity and efficiency. Metal oxide nanoparticles (MONPs) are of particular interests and have received much attention because of their unique physical, chemical and catalytic properties. This review summarizes the progress made in enzymatic biosensors based on the use of MONPs. Synthetic methods, strategies for immobilization, and the functions of MONPs in enzymatic biosensing systems are reviewed and discussed. The article is subdivided into sections on enzymatic biosensors based on (a) zinc oxide nanoparticles, (b) titanium oxide nanoparticles, (c) iron oxide nanoparticles, and (d) other metal oxide nanoparticles. While substantial advances have been made in MONPs-based enzymatic biosensors, their applications to real samples still lie ahead because issues such as reproducibility and sensor stability have to be solved. The article contains 256 references.

Direct electron transfer of glucose oxidase promoted by carbon nanotubes is without value in certain mediator-free applications

We have investigated the direct electron transfer (DET) promoted by carbon nanotubes (CNTs) on an electrode containing immobilized glucose oxidase (GOx) with the aim to develop a third-generation glucose biosensor and a mediator-free glucose biofuel cell anode. GOx was immobilized via chitosan (CS) on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs). Cyclic voltammetric revealed that the GOx on the surface of such an electrode is unable to simultaneously demonstrate DET with the electrode and to retain its catalytic activity towards glucose, although the MWCNTs alone can promote electron transfer between GOx and electrode. This is interpreted in terms of two types of GOx on the surface, the distribution and properties of which are quite different. The first type exhibits DET capability that results from the collaboration of MWCNTs and metal impurities, but is unable to catalyze the oxidation of glucose. The second type maintains its glucose-specific catalytic capability in the presence of a mediator, which can be enhanced by MWCNTs, but cannot undergo DET with the electrode. As a result, the MWCNTs are capable of promoting the electron transfer, but this is without value in some mediator-free applications such as in third-generation glucose biosensors and in mediator-free anodes for glucose biofuel cells.

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.     

Urea biosensor based on amperometric pH-sensing with hematein as a pH-sensitive redox mediator

The natural dye hematein in water solution was used as a pH-sensitive redox-active mediator for amperometric pH-sensing. The electrochemical characteristics were studied using cyclic voltammetry and chronoamperometry. Several types of urea biosensors were constructed with urease on the surface of platinum and graphite composite electrodes or in the bulk of the graphite composite. They were used for the amperometric urea determination at a working potential of 0 mV (versus SCE) using 0.5 mM hematein. Detection limits and response linearity was in the micromolar range depending on the biosensor type, concentration and pH of buffers used. An interference study of various cations, anions, and substances, which may be present in real samples demonstrated good selectivity for the determination of urea. The biosensors showed good operational (>3 h) and storage (>3 months) stability. The results of urea determination in blood and urine obtained by biosensor correlated well with those obtained by a spectrophotometric reference method.     

An amperometric penicillin biosensor with enhanced sensitivity based on co-immobilization of carbon nanotubes, hematein, and β-lactamase on glassy carbon electrode

An amperometric penicillin biosensor with enhanced sensitivity was successfully developed by co-immobilization of multi-walled carbon nanotubes (MWCNTs), hematein, and β-lactamase on glassy carbon electrode using a layer-by-layer assembly technique. Under catalysis of the immobilized enzyme, penicillin was hydrolyzed, decreasing the local pH. The pH change was monitored amperometrically with hematein as a pH-sensitive redox probe. MWCNTs were used as an electron transfer enhancer as well as an efficient immobilization matrix for the sensitivity enhancement. The effects of immobilization procedure, working potential, enzyme quantity, buffer concentration, and sample matrix were investigated. The biosensor offered a minimum detection limit of 50 nM (19 μg L⁻¹) for penicillin V, lower than those of the conventional pH change-based biosensors by more than two orders of magnitude. The electrode-to-electrode variation of the response sensitivity was 7.0% RSD.     

An optical catechol biosensor based on a desert truffle tyrosinase extract immobilized into a sol–gel silica layered matrix

An optical biosensor for the determination of catechol, a widely used yet toxic and carcinogenic molecule, is proposed using a crude extract of desert truffle (Terfezia leonis Tul.) as an enzymatic source of tyrosinase. The biosensor is constructed by the immobilization of tyrosinase crude extract in a bi-layered silica gel film prepared by dip-coating of an alkoxide/colloidal silica solution containing the enzyme on glass slide. Encapsulation has a moderate effect of the enzyme optimal pH stability but largely increases its thermal stability. Immobilized enzymes have a higher substrate affinity towards catechol but smaller maximum conversion velocity. The optical biosensor provides a linear response for catechol in the concentration range of 50–400?µM and a limit of detection was 52?µM. AFM studies show that the enzymes impact on the silica gel structure, preventing further deposition of additional layers. Comparison with similar dopamine biosensors points out that the impact of encapsulation on enzymatic activity may depend on the considered substrate.

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Biosensors designed for environmental and food quality control based on screen-printed graphite electrodes with different configurations

Graphite electrodes fabricated by screen-printing have been used as amperometric detectors in biosensors based on NAD(+)-dependent dehydrogenases, tyrosinase, or genetically modified acetylcholinesterases. The mono-enzyme sensors have been optimized as disposable or reusable devices for detection of a variety of substrates important in the food industry ( D-lactic acid, L-lactic acid, acetaldehyde) or in environmental pollution control (phenols and dithiocarbamate, carbamate and organophosphorus pesticides). The sensors were prepared in four configurations differing in enzyme confinement, enzyme immobilization and location of the immobilization agent in the biosensor assembly. Tests on real samples have been performed with the biosensors; D-lactic acid and acetaldehyde have been detected in wine and phenols in air.     

Three-dimensional network polyamidoamine dendrimer-Au nanocomposite for the construction of a mediator-free horseradish peroxidase biosensor

A three-dimensional network PAMAM-Au nanocomposite (3D-PAMAM-Au NC) was prepared by using the first generation polyamidoamine dendrimer (G1 PAMAM) as the dispersant agent. The resultant 3D-PAMAM-Au NC was successfully used as an immobilization matrix for the construction of a reagentless mediator-free horseradish peroxidase (HRP)-based H(2)O(2) biosensor on a multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode. With the advantages of the three-dimensional network, the organic-inorganic hybrid materials dramatically facilitate the direct electron transfer of HRP, and good bioelectrocatalytic activity towards H(2)O(2) was demonstrated. Under optimum conditions, the current response of the enzyme modified electrode at -0.30 V was detected. The current response is linearly correlated to H(2)O(2) concentration within the range of 18.00 μM to 20.80 mM with a correlation coefficient of 0.9992 and a sensitivity of 377.78 μA mM(-1) cm(-2). The detection limit was down to 6.72 μM (S/N = 3). Furthermore, the biosensor exhibits some other excellent characteristics, such as high selectivity, short response time, and long-term stability. The 3D-PAMAM-Au NC has proved to be a promising biosensing platform for the construction of mediator-free biosensors, and may find wide potential applications in biosensors, biocatalysis, bioelectronics and biofuel cells.