Highly selective amperometric glucose microdevice derived from diffusion layer gap electrode

Although most of enzyme catalytic reactions are specific, the amperometric detection of the enzymatic reaction products is largely nonselective. How to improve the detection selectivity of the enzyme-based electrochemical biosensors has to be considered in the sensor fabrication procedures. Herein, a highly selective amperometric glucose biosensor based on the concept of diffusion layer gap electrode pair which we previously proposed was designed. In this biosensor, a gold tube coated with a conductive layer of glucose oxidase/Nafion/graphite was used to create an interference-free region in its diffusion layer by electrochemically oxidizing the interfering electroactive species at proper potentials. A Pt probe electrode was located in this diffusion layer of the tube electrode to selectively detect hydrogen peroxide generated from the enzyme catalytic oxidation of glucose in the presence of oxygen in the solution. In practical performance of the microdevice, parameters influencing the interference-removing efficiency, including the tip-tube opening distance, the tube electrode potential and the electrolyzing time had been investigated systematically. Results showed that glucose detection free from interferents could be achieved at the electrolyzing time of 30s, the tip-tube opening distance of 3mm and the tube electrode potential of 0.4V. The electrochemical response showed linear dependence on the concentration of glucose in the range of 1 x 10(-5) to 4 x 10(-3) M (the correlation coefficient: 0.9936, without interferents; 0.9995, with interferents). In addition, the effectiveness of this device was confirmed by numerical simulation using a model system of a solution containing interferents. The simulated results showed good agreement with the experimental data.     

Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.     

A novel system combining biocatalytic dephosphorylation of L-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.     

Fabrication of a glucose sensor based on a novel nanocomposite electrode

The direct electrocatalytic oxidation of glucose in alkaline medium at nanoscale nickel hydroxide modified carbon ionic liquid electrode (CILE) has been investigated. Enzyme free electro-oxidation of glucose have greatly been enhanced at nanoscale Ni(OH)(2) as a result of electrocatalytic effect of Ni(+2)/Ni(+3) redox couple. The sensitivity to glucose was evaluated as 202 microA mM(-1)cm(-2). From 50 microM to 23 mM of glucose can be selectively measured using platelet-like Ni(OH)(2) nanoscale modified CILE with a detection limit of 6 microM (S/N=3). The nanoscale nickel hydroxide modified electrode is relatively insensitive to electroactive interfering species such as ascorbic acid (AA), and uric acid (UA) which are commonly found in blood samples. Long-term stability, high sensitivity and selectivity as well as good reproducibility and high resistivity towards electrode fouling resulted in an ideal inexpensive amperometric glucose biosensor applicable for complex matrices.     

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 K(M)(app) and maximum currents I(max)(app) 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 (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees 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 0.04 mM.     

Glucose-induced oscillatory changes in extracellular ionized potassium concentration in mouse islets of Langerhans.

Liquid membrane [K+]-sensitive microelectrodes (1-2 micron tip diameter) were used to measure the extracellular ionized potassium concentration in mouse pancreatic islets of Langerhans. With the tip of the microelectrode at the surface of the islet, the time course of the [K+]-sensitive electrode potential changes in response to the application of rapid changes in [K+]o (from 1.25 to 5 mM), could be reproduced by the equation for K+-diffusion through a 100-micron-thick unstirred layer around the islet (diffusion coefficient for K+ at 27 degrees C, DK,o, taken as 1.83 X 10(-5) cm2/s). The time to reach 63% of the steady-state electrode response with the tip in the chamber at the surface of the islet was from 5 to 6 s. When the tip of the [K+]-sensitive electrode was placed in the islet tissue, the time for the response to reach 63% of the steady-state level increased. The time course of the [K+]-sensitive electrode response could be reproduced using the same diffusion model assuming that K+ diffusion into the islet tissue takes place in a tortuous intercellular path with an apparent diffusion coefficient, DK,I, about half of DK,o, in series with the unstirred layer around the islet. In the absence of glucose the potassium concentration in the extracellular space, [K+]I, was found to be higher than the concentration in the external modified Krebs solution, [K+]o. The difference in concentration [K+]I - [K+]o was greater when [K+]o was smaller than 2 mM. In the presence of glucose (between 11 and 16 mM), under steady-state conditions, small oscillatory changes in the [K+], (1.48 +/- 0.94 mM) were detected. Simultaneous recording of membrane potential from one B-cell and [K+], in the same islet indicated that the potassium concentration increased during the active phase of the bursts of electrical activity. Maximum concentration in the intercellular was reached near the end of the active phase of the bursts. We propose that the space between islet cells constitutes a restricted diffusion system where potassium accumulates during the transient activation of potassium channels.     

Highly sensitive and selective method to detect dopamine in the presence of ascorbic acid by a new polymeric composite film

A new approach was applied to modify gold electrode with a unique polymer composite for selectively detecting dopamine (DA), a neurotransmitter, in the presence of an electroactive species of ascorbic acid (AA). After self-assembly of 11-mercaptoundecanoic acid (MUA) monolayer on gold surface, polyethylene glycol (PEG) was used to perform electrochemical esterification with MUA. In general, AA is the main interference of DA detection in a biological system. The resulting composite layer showed high sensitivity to detect DA but selectively blocked the interference from AA. Furthermore, for the first time, an interesting mechanism was demonstrated from our experimental results, namely, that the catalytic effect of AA on DA is limited by DA concentration when AA/DA>1. The modified electrode showed good reproducibility (+/-2% relative standard deviation), a low detection limit (10 nM), a fast response time (<2s), high sensitivity (86 nA/microM), a wide dynamic range of detection (20 microM), and great selectivity (without AA interference). The discovery is very promising for applications of detection of DA in a physiological environment where a high concentration of AA always exists.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

A microvoltammetric study of permeation of ferrocene derivatives through a planer bilayer lipid membrane

Permeability of ferrocene derivatives through a planer bilayer lipid membrane (BLM) was examined by an electrochemical method using microelectrodes. Location of the microelectrode tip inside the unstirred layer enables the detection of electroactive substances permeating the membrane without unstirred layer perturbation.     

Cell-compatible array of three-dimensional tip electrodes for the detection of nitric oxide release

An array of electrodes on which cells could be grown directly was fabricated using silicon anisotropic etching and a thick-photoresist process and employed for the detection of nitric oxide (NO) released from a population of adherently growing human umbilical vein endothelial cells (HUVEC). The electrodes are tip-shaped and are 40 microm high of which only the top 15 microm are exposed Pt-tips. After electrochemical induced modification of the exposed Pt tips using Ni phthalocyanine the individual addressable electrode tips were sensitive and selective for the detection of NO at an applied constant potential of 750 mV. The silicon nitride insulation of the lower part of the tip electrodes prevented the death of the cells upon the application of the working potential at which NO was detected. It also helped to avoid the perturbation of the integrity of the sensing chemistry imparted on the electrode surface that could have resulted from the contact of the adherently growing cells with the active electrode surface. The release of nitric oxide from HUVEC was successfully monitored with different numbers of tip electrodes simultaneously connected as combined working electrode.     

An amperometric biosensor based on a composite of single-walled carbon nanotubes, plasma-polymerized thin film, and an enzyme

We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose oxidase (GOx) as an enzyme model. A mixture of the GOx and a CNT film is sandwiched with 10-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GOx, CNT was treated with nitrogen or oxygen plasma. The resulting device showed that the oxidizing current response due to enzymatic reaction was 4-16-fold larger than that with only CNT or PPF, showing that the PPF and/or plasma process is an enzyme-friendly platform for designing electrochemical communication from the reaction center of GOx to the electrode via CNTs. The optimized glucose biosensor showed high sensitivity (sensitivity of 42 microA mM(-1)cm(-2), correlation coefficient of 0.992, linear response range of 0.025-2.2 mM, and a detection limit of 6 microM at signal/noise ratio of 3, +0.8 V versus Ag/AgCl), high selectivity (almost no interference by 0.5 mM ascorbic acid) for glucose quantification, and rapid response (<4 s to reach 95% of maximum response). Additionally, the devices showed a small and stable background current (0.35+/-0.013 microA) compared with the glucose response (ca. 10 microA at 10mM glucose) and suitable reproducibility from sample-to-sample (<3%, n=4).     

Nonenzymatic electrochemical detection of glucose using well-distributed nickel nanoparticles on straight multi-walled carbon nanotubes

A nonenzymatic electrochemical sensor device was fabricated for glucose detection based on nickel nanoparticles (NiNPs)/straight multi-walled carbon nanotubes (SMWNTs) nanohybrids, which were synthesized through in situ precipitation procedure. SMWNTs can be easily dispersed in solution after mild sonication pretreatment, which facilitates the precursor of NiNPs binding to their surface and results in the homogeneous distribution of NiNPs on the surface of SMWNTs. The morphology and component of the nanohybrids were characterized by scanning electron microscopy (SEM) and X-ray powder diffraction (XRD), respectively. Cyclic voltammetry (CV) and amperometry were used to evaluate the catalytic activity of the NiNPs/SMWNTs nanohybrids modified electrode towards glucose. It was found that the nanohybrids modified electrode showed remarkably enhanced electrocatalytic activity towards the oxidation of glucose in alkaline solution compared to that of the bare glass carbon electrode (GCE), the NiNPs and the SMWNTs modified electrode, attributing to the synergistic effect of SMWNTs and Ni²⁺/Ni³⁺ redox couple. Under the optimal detection conditions, the as-prepared sensors exhibited linear behavior in the concentration range from 1 μM to 1 mM for the quantification of glucose with a limit of detection of 500 nM (3σ). Moreover, the NiNPs/SMWNTs modified electrode was also relatively insensitive to commonly interfering species such as ascorbic acid (AA), uric acid (UA), dopamine (DA), galactose (GA), and xylose (XY). The robust selectivities, sensitivities, and stabilities determined experimentally indicated the great potential of NiNPs/SMWNTs nanohybrids for construction of a variety of electrochemical sensors.     

Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel

A novel amperometric biosensor, based on electrodeposition of platinum nanoparticles onto multi-walled carbon nanotube (MWNTs) and immobilizing enzyme with chitosan-SiO(2) sol-gel, is presented in this article. MWNTs were cast on the glass carbon (GC) substrate directly. An extra Nafion coating was used to eliminate common interferents such as acetaminophen and ascorbic acids. The morphologies and electrochemical performance of the modified electrodes have been investigated by scanning electron microscopy (SEM) and amperometric methods, respectively. The synergistic action of Pt and MWNTs and the biocompatibility of chitosan-SiO(2) sol-gel made the biosensor have excellent electrocatalytic activity and high stability. The resulting biosensor exhibits good response performance to glucose with a wide linear range from 1 microM to 23 mM and a low detection limit 1 microM. The biosensor also shows a short response time (within 5s), and a high sensitivity (58.9 microAm M(-1)cm(-2)). In addition, effects of pH value, applied potential, rotating rate, electrode construction and electroactive interferents on the amperometric response of the sensor were investigated and discussed in detail.     

Development and study of an amperometric biosensor for the in vitro measurement of low concentration of putrescine in blood

An amperometric biosensor was developed for the in vitro determination of putrescine in blood samples because elevated level of putrescine in blood can be a diagnostic indicator of certain kinds of cancer. The electrochemical transducer consisted of a flat form, three electrode amperometric micro-cell fabricated with thin film photolithography on flexible Kapton substrate. An immobilized putrescine oxidase (PUO) layer provided the biocatalytic oxidation of the putrescine, while the generated hydrogen peroxide was detected on the platinum-working electrode. An electropolymerized poly(m-phenylenediamine) (pPDA) size-exclusion layer was used to protect the working electrode from fouling and to prevent signal generation by common electroactive interferents present in blood. The preparation of the biocatalytic enzyme- and outer protective layers was optimized for improved sensitivity and response time. A detection limit of 50 nM was achieved in pH-adjusted whole blood samples, which is below pathological levels.     

Nanomechanics of opposing glycosaminoglycan macromolecules

In this study, the net intermolecular interaction force between a chondroitin sulfate glycosaminoglycan (GAG)-functionalized probe tip and an opposing GAG-functionalized planar substrate was measured as a function of probe tip-substrate separation distance in aqueous electrolyte solutions using the technique of high resolution force spectroscopy. A range of GAG grafting densities as near as possible to native cartilage was used. A long-range repulsive force between GAGs on the probe tip and substrate was observed, which increased nonlinearly with decreasing separation distance between probe tip and substrate. Data obtained in 0.1 M NaCl was well predicted by a recently developed Poisson-Boltzmann-based theoretical model that describes normal electrostatic double layer interaction forces between two opposing surfaces of end-grafted, cylindrical rods of constant volume charge density and finite length, which interdigitate upon compression. Based on these results, the nanomechanical data and interdigitated rod model were used together to estimate the electrostatic component of the equilibrium modulus of cartilage tissue, which was then compared to that of normal adult human ankle cartilage measured in uniaxial confined compression.     

Amperometric ATP biosensor based on polymer entrapped enzymes

A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1).     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

Nonenzymatic amperometric response of glucose on a nanoporous gold film electrode fabricated by a rapid and simple electrochemical method

An enzyme-free amperometric method was established for glucose detection using a nanoporous gold film (NPGF) electrode prepared by a rapid one-step anodic potential step method within 5 min. The prepared NPGF had an extremely high roughness and was characterized by scanning electron microscopy (SEM) and cyclic voltammetry. Electrochemical responses of the as-prepared NPGF to glucose in 0.1M phosphate buffer solution (PBS, pH 7.4) with or without Cl(-) were discussed. In amperometric studies carried out at -0.15 V in the absence of Cl(-), the NPGF electrode exhibited a high sensitivity of 232 μA mM(-1)cm(-2) and gave a linear range from 1mM up to 14 mM with a detection limit of 53.2 μM (with a signal-to-noise ratio of 3). In addition, the oxidation of ascorbic acid (AA) and uric acid (UA) can be completely eliminated at such a low applied potential. On the other hand, the quantification of glucose in 0.1M PBS (pH 7.4) containing 0.1M NaCl offered an extended linear range from 10 μM to 11 mM with a sensitivity of 66.0 μA mM(-1)cm(-2) and a low detection limit of 8.7 μM (signal-to-noise ratio of 3) at a detection potential of 0.2V.     

In situ synthesis of palladium nanoparticle-graphene nanohybrids and their application in nonenzymatic glucose biosensors

A nonenzymatic electrochemical biosensor was developed for the detection of glucose based on an electrode modified with palladium nanoparticles (PdNPs)-functioned graphene (nafion-graphene). The palladium nanoparticle-graphene nanohybrids were synthesized using an in situ reduction process. Nafion-graphene was first assembled onto an electrode to chemically adsorb Pd(2+). And Pd(2+) was subsequently reduced by hydrazine hydrate to form PdNPs in situ. Such a PdNPs-graphene nanohybrids-based electrode shows a very high electrochemical activity for electrocatalytic oxidation of glucose in alkaline medium. The proposed biosensor can be applied to the quantification of glucose with a wide linear range covering from 10 μM to 5mM (R=0.998) with a low detection limit of 1 μM. The experiment results also showed that the sensor exhibits good reproducibility and long-term stability, as well as high selectivity with no interference from other potential competing species.     

Rapid Prototyping of a High Sensitivity Graphene Based Glucose Sensor Strip

A rapid prototyping of an inexpensive, disposable graphene and copper nanocomposite sensor strip using polymeric flexible substrate for highly sensitive and selective nonenzymatic glucose detection has been developed and tested for direct oxidization of glucose. The CuNPs were electrochemically deposited on to the graphene sheets to improve electron transfer rates and to enhance electrocatalytic activity toward glucose. The graphene based electrode with CuNPs demonstrated a high degree of sensitivity (1101.3±56 μA/mM.cm²), excellent selectivity (without an interference with Ascorbic Acid, Uric Acid, Dopamine, and Acetaminophen), good stability with a linear response to glucose ranging from 0.1 mM to 0.6 mM concentration, and detection limits of 0.025 mM to 0.9 mM. Characterization of the electrodes was performed by scanning electron microscopy (FESEM and SEM). The electrochemical properties of the modified graphene electrodes were inspected by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry.