Reagentless Glucose Biosensor Research Articles

The Development and Evaluation of Reagentless Glucose Biosensors Using Dendritic Gold Nanostructures as a Promising Sensing Platform.

Reagentless electrochemical glucose biosensors were developed and investigated. A graphite rod (GR) electrode modified with electrochemically synthesized dendritic gold nanostructures (DGNs) and redox mediators (Med) such as ferrocenecarboxylic acid (FCA), 1,10-phenathroline-5,6-dione (PD), N,N,N',N'-tetramethylbenzidine (TMB) or tetrathiafulvalene (TTF) in combination with glucose oxidase (GOx) (GR/DGNs/FCA/GOx, GR/DGNs/PD/GOx, GR/DGNs/TMB/GOx, or GR/DGNs/TTF/GOx) were developed and electrochemically investigated. A biosensor based on threefold-layer-by-layer-deposited PD and GOx (GR/DGNs/(PD/GOx)3) was found to be the most suitable for the determination of glucose. To improve the performance of the developed biosensor, the surface of the GR/DGNs/(PD/GOx)3 electrode was modified with polypyrrole (Ppy) for 5 h. A glucose biosensor based on a GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was characterized using a wide linear dynamic range of up to 39.0 mmol L-1 of glucose, sensitivity of 3.03 µA mM-1 cm-2, limit of detection of 0.683 mmol L-1, and repeatability of 9.03% for a 29.4 mmol L-1 glucose concentration. The Ppy-based glucose biosensor was characterized by a good storage stability (τ1/2 = 9.0 days). Additionally, the performance of the developed biosensor in blood serum was investigated.

Bifunctional (Zn,Fe)3O4 nanoparticles: Tuning their efficiency for potential application in reagentless glucose biosensors and magnetic hyperthermia

In a new approach based on a two-step procedure, co-precipitation method followed by hydrothermal treatment in a microwave field, Zn-substituted Fe3O4 nanoparticles (ZnxFe3-xO4) were synthesized. Results of XRD, FT–IR and TEM analysis clearly demonstrate that nanoparticles were single phase, crystallizing in the spinel structure type (S.G. Fd3¯m) with crystallite size in the range of 2–20 nm, which strongly depends on Zn concentration. The produced nanoparticles were used for fabrication of modified carbon paste electrodes as a novel system for electrochemical non-enzymatic glucose detection. It was found that the increase of zinc concentration up to the value of x = 0.56 (Zn0.56Fe2.44O4) of as-prepared nanoparticles was followed with an increase of a performance of the modified carbon paste electrode toward glucose detection. Linear working range from 0.1 to 2 mM was obtained with detection limit of 0.03 mM, and with fast response time (<3 s). Proposed sensor was successfully applied for the determination of glucose level in real samples with satisfactory recovery. The synthesized zinc-ferrite samples were also tested as potential heating agents in magnetic hyperthermia. The heating ability (SAR value) increases with x value, reaching maximum for x = 0.37. This is correlated with changes of particle size and magnetic characteristics which strongly depend on Zn concentration.

Self-assembly of glucose oxidase on reduced graphene oxide-magnetic nanoparticles nanocomposite-based direct electrochemistry for reagentless glucose biosensor

A novel approach of the immobilization of a highly selective and stable glucose biosensor based on direct electrochemistry was fabricated by a self-assembly of glucose oxidase (GOD) on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe3O4 NPs) modified on a magnetic screen-printed electrode (MSPE). The RGO-Fe3O4 nanocomposite has remarkable enhancement in large surface areas, is favorable environment for enzyme immobilization, facilitates electron transfer between enzymes and electrode surfaces and possesses superparamagnetism property. The morphology and electrochemical properties of RGO-Fe3O4/GOD were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, cyclic voltammetry (CV) and amperometry. The modified electrode was a fast, direct electron transfer with an apparent electron transfer rate constant (ks) of 13.78s−1. The proposed biosensor showed fast amperometric response (3s) to glucose with a wide linear range from 0.05 to 1mM, a low detection limit of 0.1μM at a signal to noise ratio of 3 (S/N=3) and good sensitivity (5.9μA/mM). The resulting biosensor has high stability, good reproducibility, excellent selectivity and successfully applied detection potential at −0.45V. This mediatorless glucose sensing used the advantages of covalent bonding and self-assembly as a new approach for immobilizing enzymes without any binder. It would be worth noting that it opens a new avenue for fabricating excellent electrochemical biosensors. Novelty statementThis is a new approach that reporting the immobilization of glucose oxidase on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe3O4 NPs) by electrostatic interaction and modified screen printed electrode. We propose the reagentless with fabrication method without binder and adhesive agents for immobilized enzyme. Fe3O4 NPs increasing surface area to enhance the immobilization and prevent the leaching of enzymes at electrode surfaces by magnetic stickers which is improve the stability of the biosensor. Based on this synthesis technique, it is a good new strategy and simple used to fabrication of third-generation glucose biosensor and this nanocomposite could be used as a platform for disposable biosensor and biofuel cell applications.

Codeposited Poly(benzoxazine) and Os‐Complex Modified Polymethacrylate Layers as Immobilization Matrix for Glucose Biosensors

AbstractBenzoxazine oligomers synthesized by a Mannich type reaction of bisphenol A, tetraethylenepentamine and formaldehyde were electrochemically crosslinked in presence of both an Os‐complex modified poly(methacrylate) polymer and glucose oxidase. The crosslinking led to the formation of a biocatalytically active layer on an electrode surface exhibiting a swelling process after immersion in an electrolyte solution containing glucose most likely due to the local decrease of the pH value upon glucose oxidation. Optimization of the poly(benzoxazine) to Os‐complex modified poly(methacrylate) ratio was performed leading to a reagentless glucose biosensor with improved stability.

The use of different glucose oxidases for the development of an amperometric reagentless glucose biosensor based on gold nanoparticles covered by polypyrrole

ABSTRACT The amperometric glucose biosensors based on adsorbed electron transfer mediator (ETM) tetrathiafulvalene (TTF) or 1,10-phenanthroline-5,6-dione (PD) and glucose oxidase (GOx) from Aspergillus niger (GOx A.niger ), Penicillium adametzii (GOx P.adametzii ) or Penicillium funiculosum (GOx P.funiculosum ) cross-linked with glutaraldehyde were investigated. ETM and enzyme were immobilized layer by layer on bare graphite rod electrode (GR) premodified with gold nanoparticles (AuNP) of (i) 3.5 nm (GOx/ETM/AuNP 3.5 /GR), (ii) 6.0 nm (GOx/ETM/AuNP 6.0 /GR) and (iii) 13.0 nm (GOx/ETM/AuNP 13.0 /GR) size. The amperometric signals for all the developed biosensors were higher using PD in comparison with TTF. The biosensor based on GOx P.funiculosum showed higher analytical signal to glucose in a comparison to biosensors based on GOx A.niger and GOx P.adametzii . The registered current to glucose using GOx P.funiculosum /PD/AuNP 3.5 /GR electrode was linear in the glucose range from 0.1 to 10.0 mmol L −1 and the limit of detection was 0.024 mmol L −1 . Enzymatical synthesis of polypyrrole (Ppy) layer on the electrode was applied in order to expand the linear glucose detection range. After 22 h of polymerization the amperometric signal was linear in the glucose concentration range from 0.1 to 25.0 mmol L −1 , while after 69 h this rage was increased up to 50.0 mmol L −1 . Additionally Ppy layer on the electrode surface reduced the influence of interfering species on the amperometric signal. The performance of developed biosensor was investigated in human serum samples.

Efficient Immobilization of Glucose Oxidase on Nanocomposite via Layer-by-Layer Self-Assembly to Construct Bionanomultilayer Films

A reagentless glucose biosensor was constructed based on bionanomultilayer films composed of gold nanoparticles (GNps) wrapping multiwalled carbon nanotubes (MWNTs)-polyethylenimine (PEI) nanocomposite and glucose oxidase (GOD) through layer-by-layer (LBL) assembly. Firstly, a hybrid film of chitosan - Prussian blue (CS-PB) which acted as redox mediator had been prepared through directly electrodeposited on gold electrode. Then the biosensor was fabricated based on reliably electrostatic interaction of the positively charged GNps-MWNTs-PEI nanocomposite and the negatively charged GOD. The GNps-MWNTs-PEI nanocomposite was characterized by UV-vis spectra, transmission electron microscopy (TEM) and energy dispersive X-ray spectrometer (EDX). And the fabricated process and electrochemical behaviors of the biosensor was characterized by scanning electron microscopy (SEM) and cyclic voltammetry (CV). Under the optimal conditions, the biosensor exhibited excellent response performance to glucose in the linear range from 0.004 to 6.2 mM, with a low detection limit of 1.3 μM and a high sensitivity of 382.8 μA mM−1 cm−2. Moreover, the biosensor showed rapid response, good reproducibility and interference-free of other co-existing electroactive species.

Novel reagentless glucose biosensor based on ferrocene cored asymmetric PAMAM dendrimers

A novel amperometric glucose biosensor is fabricated with immobilization of glucose oxidase (GOx) onto PAMAM-Fc dendrimers decorated gold electrode. Series of asymmetric PAMAM dendrimers containing a single ferrocene unit located in the focal point of these macromolecules have been synthesized and characterized. Surface of the gold electrode was covalently modified with 3-mercaptopropionic acid, PAMAM-Fc dendrimers and GOx enzyme by using coupling agents, respectively. The PAMAM-Fc/GOx biosensor shows an excellent performance for glucose at +0.25 V with a high sensitivity (6.54 μA/mM) and lower response time (∼3 s) in a wide concentration range of 1–22 mM (correlation coefficient of 0.9988). In addition, PAMAM-Fc/GOx biosensor possesses better reproducibility, storage stability and there is negligible interference from other electroactive components.

An reagentless glucose biosensor based on direct electrochemistry of glucose oxidase immobilized on poly(methylene blue) doped silica nanocomposites

Conducting polymer nanocomposites have attracted a wide attention in material chemistry and analytical chemistry. In this work, poly(methylene blue) doped silica nanocomposites (PMB@SiO2(nano)) on glassy carbon electrode (GCE) were synthesized based on a two-step electropolymerization. Compared with poly(methylene blue) film, PMB@SiO2(nano) had more advantages in facilitating electron transfer between glucose oxidase (GOx) and the electrode surface. GOx immobilized on PMB@SiO2(nano) underwent a direct and reversible electrochemical reaction involving two-electron and two-proton exchange. A reagentless glucose biosensor using the nanocomposite modified electrode was developed. The calibration curve was linear over a glucose concentration range of 0.01–1.11mM with a correlation coefficient of 0.998 and a detection limit (3σ) of 3μM. The apparent Michalis–Menten constant, Km, for the enzymatic reaction was 0.50mM. The developed biosensor presented good accuracy, repeatability, reproducibility and stability as well as an excellent ability to resist interference from ascorbic acid and uric acid species. The investigation indicates that PMB@SiO2(nano) has a good potential for application in construction of the third-generation enzyme biosensor as a conducting matrix material.

Construction of reagentless glucose biosensor based on ferrocene conjugated polypyrrole

Glucose oxidase (GOx) was covalently immobilized onto an electrochemically prepared novel copolymer of ferrocene branched polypyrrole for the construction of an amperometric glucose biosensor. Ferrocene-pyrrole monomers were prepared by condensation reaction between ferrocene ethanol and 3-(1H-pyrrol-1-yl)propanoic acid and ferrocene-pyrrole, 3-(1H-pyrrol-1-yl)propanoic acid, and pyrrole monomers were electrochemically polymerized on the surface of the electrode. GOx was covalently immobilized onto the co-polymer coated electrode surface by using coupling agents to create amine bonds. The prepared copolymers were utilized as conducting films for amperometric glucose sensing after immobilization of GOx. Amperometric response was measured as a function of concentration of glucose at a fixed potential of +0.38V vs. Ag/AgCl in a phosphate-buffered saline solution (pH 7.0). Results clearly showed that the ferrocene groups on the co-polymer play the role of electron transfer mediator between the redox center of glucose oxidase (GOx) and the surface of the electrode. Also, the effects of pH and temperature, storage, reusability and interference of the amperometric glucose biosensor were investigated.

Fabrication of reagentless glucose biosensors: A comparison of mono-enzyme GOD and bienzyme GOD–HRP systems

Glucose oxidase (GOD) and horseradish peroxidase (HRP), entrapped alone and simultaneously in silica sol–gel (SG) network on gold nanoparticles (GNPs) modified indium tin oxide (ITO) electrode, were used to fabricate mono-enzyme GOD and bienzyme GOD–HRP glucose biosensors. The optimal conditions for the construction and the analytical performances of the biosensors were studied and compared. The optimal GOD concentration in mixed solution for the mono-enzyme and bienzyme glucose biosensors was about 250 U/mL, and the optimal GOD/HRP ratio was approximately 2 for the bienzyme glucose biosensors. Amperometric response of glucose was evaluated by holding the biosensors at 0.1 V (versus SCE). The linear ranges of detection for glucose were between 0.05–4.0 mmol/L and 0.02–3.2 mmol/L, respectively. The sensitivity of the bienzyme glucose biosensor was approximately 2.4-fold higher than that of mono-enzyme glucose biosensor. Both glucose biosensors showed rapid response, high selectivity and long-term stability.

Combinatorial Polymer Synthesis as a Tool in Biosensor and Biofuel Cell Development and Optimization

A strategy was established for the synthesis of redox electrodepo¬sition polymers (redox EDP) consisting of the synthesis of monomers with attached imidazolyl anchor groups, followed by radical copolymerization with a variety of monomers bearing side chains with different steric, hydrophobic, and electronic properties. The synthesized EDP were coordinatively modified by reaction with Os-bis-N,N'-(2,2'-bipyridyl)-dichloro complexes. The obtained redox-EDPs were precipitated on Pt electrodes by means of an electrochemically induced pH modulation in the presence of a pyrrolo¬quinoline quinone-dependent glucose dehydrogenase (PQQ-sGDH). The impact of the redox-EDPs composition on the electron transfer properties of the resulting reagentless glucose biosensors was investigated.

Study of Ferrocene-modified G4 PAMAM Dendrimer for Reagentless Biosensor Develoment

A fourth generation amine-terminated dendrimer (G4-PAMAM) was chemically functionalized through ferrocene (Fc) tethered groups (length of tether = C11). The electrochemical properties of the new dendrimer molecule immobilized by using polyazetidine prepolymer (PAP) onto multi-walled carbon nanotubes (MWCNTs) screen printed electrode (SPE) surface, have been evaluated; then the Fc modified SPE was coupled to glucose oxidase (GOx) for the development of a reagentless glucose biosensor. The analytical performances of this device has been studied providing a linear response for one decade in the range 0.7 - 7.0 mM glucose with an RSD always below 4 %.

Synthesis of Ferrocene‐Branched Chitosan Derivatives: Redox Polysaccharides and their Application to Reagentless Enzyme‐Based Biosensors

AbstractFerrocene‐branched chitosan derivatives (CHIT‐Fc) are synthesized by reductive N‐alkylation of chitosan with ferrocenecarboxaldehyde. The structures of the products are determined by 1H NMR and FT‐IR spectra. CHIT‐Fc is used as a functionalized matrix to immobilize GOD on glassy carbon electrodes. Ferrocenyls in CHIT‐Fc exhibit an excellent redox activity and establish efficient electrical communication between GOD and the electrodes for the oxidation of glucose. The development of a reagentless glucose biosensor is described.magnified image

Layer-by-layer fabrication and direct electrochemistry of glucose oxidase on single wall carbon nanotubes

Layer-by-layer assembly of glucose oxidase (GOx) with single-wall carbon nanotubes (SWCNTs) is achieved on the electrode surface based on the electrostatic attraction between positively charged GOx in pH 3.8 buffer and negatively charged carboxylic groups of CNTs. The cyclic voltammetry and electrochemical impedance spectroscopy are used to characterize the formation of multilayer films. In deaerated buffer solutions, the cyclic voltammetry of the multilayer films of {GOx/CNT}n shows two pairs of well-behaved redox peaks that are assigned to the redox reactions of CNTs and GOx, respectively, confirming the effective immobilization of GOx on CNTs using the layer-by-layer technique. The redox peak currents of GOx increase linearly with the increased number of layers indicating the uniform growth of GOx in multilayer films. The dependence of the cyclic voltammetric response of GOx in multilayer films on the scan rate and pH is also studied. A linear decrease of the reduction current of oxygen at the {GOx/CNT}-modified electrodes with the addition of glucose suggests that such multilayer films of GOx retain the bioactivity and can be used as reagentless glucose biosensors.

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants KMapp and maximum currents Imaxapp were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020mM for glucose, and 0.023mM for maltose at 48°C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0–0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04mM.

Supramolecular multilayer structures of wired redox enzyme electrodes

Supramolecular multilayer structures comprised of glucose oxidase (GOx), and Os complex derivatised poly(allylamine) (PAH-Os) have been built by alternate layer-by-layer (LBL) electrostatic adsorption in a self-assembly process. The resulting modified electrodes with integrated mediator were tested as reagentless glucose biosensors. The enzyme kinetic parameters and the surface concentration of "wired" enzyme GammaE have been obtained by analysis of the catalytic current dependence on glucose concentrations for the ping-pong mechanism of glucose oxidation. An average osmium volume concentration was estimated by integration of the redox charge in the absence of glucose and the ellipsometric thickness. The total enzyme surface concentration was measured with a quartz crystal microbalance (QCM) during each adsoption step and the fraction of "wired" enzyme and the bimolecular rate constant for FADH2 oxidation by the redox polymer for the different multilayers. The catalytic current increases with the number of LBL layers because the increase in the enzyme loading while the efficiency of enzyme FADH2 oxidation by the Os redox polymer, except for the first dipping cycle remains almost constant at about 2 x 10(4) M(-1) S(-1).

チオニン修飾金ナノ粒子を用いた電極表面の機能化と電気化学的グルコースセンサへの応用

The electrochemically effective oxidation of NADH has drawn striking attention because it is widely applicable for fabricating biosensors. The mediators are normally used as electron shuttle molecules between the electrode and enzymes. Phenothiazine compounds deserve as suitable mediators due to theirs negative formal potential and large rate constant. For the practical use, both the enzyme and mediator are required to be co-immobilized on the electrode surface. In this study, gold nano-particles modified with thionine (Th+) were immobilized on the surface of the gold electrode by physical adsorption. Th+ was covalently bound to the surface of the gold nano-particles using 3,3'-dithiobis (succinimidyl propionate) (DSP) as a cross linker. The electrochemistry of Th+ modified on the gold nano-particles was characterized by voltammetry. Th+ molecules on the gold nano-particle modified electrodes showed two redox couples at −30mV and 240mV vs Ag/AgCl at pH7. A well-defined peak for the electrocatalytic oxidation of NADH was observed at 240mV. The nano-particle electrodes modified with glucose dehydrogenase (GDH) and Th+ mediators were fabricated and characterized aiming at fabricating a reagentless glucose biosensor. Upon the addition of glucose to the electrochemical cell, the oxidation current dramatically increased due to an electrocatalytic reaction.

Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors

Glucose oxidase was embedded in organic films through a layer-by-layer approach, where the enzyme demonstrated significantly enhanced electron-transfer reactivity and finely tuned enzymatic activity. An unmediated, reagentless glucose biosensor was accordingly prepared with two polyethylenimine/glucose oxidase bilayers-modified pyrolytic graphite electrode. A calibration linear range of glucose was 0.5–8.9 mM with a detection limit of 50 μM and sensitivity of 0.76 μA mM −1.

Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode

The direct electrochemistry of glucose oxidase (GOD) adsorbed on a colloidal gold modified carbon paste electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of −(449±1) mV in 0.1 M pH 5.0 phosphate buffer solution. The response showed a surface-controlled electrode process with an electron transfer rate constant of (38.9±5.3)/s determined in the scan rate range from 10 to 100 mV/s. GOD adsorbed on gold colloid nanoparticles maintained its bioactivity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a high sensitivity (8.4 μA/mM), a linear range from 0.04 to 0.28 mM and a detection limit of 0.01 mM at a signal-to-noise ratio of 3 σ. The sensor could exclude the interference of commonly coexisted uric and ascorbic acid.

A reagentless glucose biosensor based on glucose oxidase entrapped into osmium-complex modified polypyrrole films

A reagentless glucose biosensor based on glucose oxidase entrapped within an osmium-complex (Os-complex) modified conducting polymer has been developed. Optimisation of the electrochemical-induced deposition of the conducting-polymer film has been achieved by variation of key parameters, such as the concentration of glucose oxidase and the concentration of pyrrole. In order to unequivocally reveal the electron-transfer from the enzyme via the polymer-bound Os-complexes to the electrode surface, glassy-carbon electrodes have been used to prevent co-oxidation of enzymatically generated H 2O 2 in the presence of molecular oxygen. In addition, a significant enhancement of the glucose-dependent current could be obtained by increasing the hydrophilicity of the polymer film which was attained by changing the counter anion of the Os-complexes and introduction of carboxylic acid side chains at the polypyrrole backbone. The improved swelling of the polymer in aqueous solution leads to an improved substrate transport within the polymer film and faster electron-transfer kinetics due to a higher flexibility of the polymer backbone.