Interference elimination of an amperometric glucose biosensor using poly(hydroxyethyl methacrylate) membrane

A permselective membrane fabricated from photo‐cross‐linked poly(hydroxyethyl methacrylate) (pHEMA) was studied as a potential selective membrane that can eliminate electrochemical interferences commonly faced by a hydrogen peroxide‐based biosensor. The quantitative selection of the permselective membrane was based on the permeabilities of hydrogen peroxide and acetaminophen (AC). AC was used as a model of the interfering substance due to its neutral nature. pHEMA membrane with the cross‐linking ratio of 0.043 was found to achieve a selectivity of hydrogen peroxide over AC of 10, while maintaining an acceptable degree of hydrogen peroxide response. A two‐layer glucose biosensor model consisting of glucose oxidase entrapped within a freeze‐thawed poly(vinyl alcohol) matrix and the cross‐linked pHEMA membrane was challenged with AC, ascorbic acid and uric acid. 0.2 mM AC and 0.2 mM ascorbic acid were completely eliminated. However, 0.2 mM uric acid could not be completely eliminated and still gave a bias of approximately 6.6% relative to 5 mM glucose. The results showed that cross‐linked pHEMA was quite promising as an interference eliminating inner membrane.     

Design of an amperometric biosensor using polypyrrole-microgel composites containing glucose oxidase

A new material consisting of a water-dispersed complex of polypyrrole-polystyrensulfonate (PPy) embedded in polyacrylamide (PA) has been prepared and tested as enzyme immobilizing system for its use in amperometric biosensors. Glucose oxidase (GOx) and the water-dispersed polypyrrole complex were entrapped within polyacrylamide microgels by polymerization of acrylamide in the dispersed phase of concentrated emulsions containing GOx and PPy. Polymerization of the dispersed phase provides microparticles whose size lies between 3.5 and 7 microm. The aim of incorporating polypyrrole into the polyacrylamide microparticles was to facilitate the direct transfer of the electrons released in the enzymatic reaction from the catalytic site to the platinum electrode surface. The conductivity of the microparticles was measured by a four-point probe method and confirmed by the successful anaerobic detection of glucose by the biosensor. Thus, the polyacrylamide-polypyrrole (PAPPy) microparticles combine the conductivity of polypyrrole and the pore size control of polyacrylamide. The effects of the polyacrylamide-polypyrrole ratio and cross-linking on the biosensor response have been investigated, as well as the influence of analytical parameters such as pH and enzymatic loading. The PAPPy biosensor is free of interferences arising from ascorbic and uric acids, which allows its use for quantitative analysis in human blood serum.     

Monitoring glutamine in mammalian cell cultures using an amperometric biosensor.

An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

BioMEMS device with integrated microdialysis probe and biosensor array

The fabrication of a microdevice for continuous sampling and on-line monitoring of glucose is described. The device comprised a microdialysis sampling system integrated on the flow through channel of a microfabricated enzyme sensor. The sensor was produced by thin film technology and was assembled to a printed circuit board (PCB) that provided the means for both electrical and fluidic connections. A polyacrilonitrile fibre, with a cut-off of 50 kDa, was used in the fabrication of the sampling probe. The performance of the device was evaluated in-vitro. High sampling efficiency of the microdialysis probe was achieved by appropriate selection of the perfusion fluid flow rate. Response times varying from 1.5 to 3.0 min were determined for flow rates ranging between 1 and 0.2 micro l/min. The linear response range was up to 30 mM glucose and interference from other electroactive substances was almost negligible. The device showed excellent stability under continuous operation for at least 5 days and sensitivity variation less than 3% over a period of 15 days.     

Fast and easy preparation of an amperometric glucose biosensor

Summary Electrochemical polymerization of pyrrole in aqueous KCl solution containing glucose oxidase produces adherent films at platinum electrode surface. Such coated electrodes are prepared in 20 min and can determine glucose in the range 0 to 100mm. The useful lifetime of the electrode is 85 assays. Its stability is at least 75 days under storage in PBS at 4°C.     

Polypyrrole-based bilayer nitrate amperometric biosensor with an integrated permselective poly-ortho-phenylenediamine layer for exclusion of inorganic interferences

A bilayer amperometric nitrate biosensor with an integrated permselective layer has been developed for exclusion of inorganic anion and cation interferences. The inner PPy(polypyrrole)-NaR-NADH layer of the biosensor is formed by galvanostatic polymerization of pyrrole (Py) in presence of nitrate reductase (NaR) and nicotinamide adenine dinucleotide (NADH), followed by formation of the outer permselective poly-ortho-phenylenediamine (P-o-PDA) layer by potentiodynamic polymerization of ortho-phenylenediamine (o-PDA). The exclusion efficiency (E(eff)) of the outer layer in rejecting inorganic cation and anion interferences is evaluated by a new proposed relationship. 73-87% and 47-84% of anion and cation interferences, respectively, were efficiently rejected with the permselective layer. Further improvement in the exclusion efficiency for cations was accomplished by combining the use of the outer layer with the addition of 1mM EDTA into the measurement solution. The addition of EDTA improved the E(eff) achieved for cation rejection by 10-40% to give net E(eff) of 89-94%. The inclusion of the outer layer also aided the retention of NaR and NADH in the inner PPy-NaR-NADH layer and, hence, enabled improved amperometric detection of nitrate, achieving a detection limit of 0.20 μM and a linear concentration range of 10-500 μM with a 3.4%rsd (n=10).     

Immobilizing Pt nanoparticles and chitosan hybrid film on polyaniline naofibers membrane for an amperometric hydrogen peroxide biosensor

A convenient and effective way for fabricating amperometric hydrogen peroxide (H₂O₂) biosensor was designed in this paper. First, the polyaniline (PANI) nanofibers membrane with good conductance and high surface area was electropolymerized on a gold electrode surface. Then, Pt nanoparticle (PtNP) was electrochemically deposited on the PANI nanofibers membrane. Finally, the hybrid film of gold nanoparticle, chitosan, and horseradish peroxidase (HRP) was cast onto the modified electrode to form a stable biofunctional film, which was also employed as a protective layer to PtNP. The proposed biosensor exhibited a rapid response to H₂O₂ with the linear range from 7.0 × 10⁻⁶ to 1.4 × 10⁻² M and a detection limit of 2.8 × 10⁻⁶ M (S/N = 3). The sensitivity of 558 μA mM⁻¹ cm⁻² was obtained. The Michaelis–Menten constant, K_\textM^\textapp K_{\text{M}}^{\text{app}} value was 1.90 mM suggesting a high affinity. Moreover, it displayed a good reproducibility and long-term stability.     

Kinetic analysis of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase using an amperometric biosensor

An amperometric biosensor for the detection of cellobiose has been introduced to study the kinetics of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase. By use of a sensor in which pyrroloquinoline quinone-dependent glucose dehydrogenase was immobilized on the surface of electrode, direct and continuous observation of the hydrolysis can be achieved even in a thick cellulose suspension. The steady-state rate of the hydrolysis increased with increasing concentrations of the enzyme to approach a saturation value and was proportional to the amount of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism consisting of the adsorption of the free enzyme onto the surface of the substrate, the reaction of the adsorbed enzyme with the substrate, and the liberation of the product. The catalytic constant of the adsorbed enzyme was determined to be 0.044+/-0.011s(-1).     

Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

A high sensitivity amperometric biosensor using laccase as biorecognition element

An amperometric flow biosensor, using laccase from Rigidoporus lignosus as bioelement was developed. The laccase was kinetically characterized towards various phenolics both in solution and immobilized to a hydrophilic matrix by carbodiimide chemistry. A bioreactor connected to an amperometric flow cell by a FIA system was filled with the immobilized enzyme and the operational conditions of this biosensor were optimized as regards pH. Under the adopted experimental conditions, the immobilized enzyme oxidizes all the substrate molecules avoiding the need of cumbersome calibration procedures. The biosensor sensitivity, which was found to be 100 nA/microM for some of the tested substrates, resulted to be constant for more than 100 working days. This biosensor permits the detection of phenolics in aqueous solutions at concentrations in the nanomolar range and was successfully used to detect phenolics in wastewaters from olive oil mill without sample preparation.     

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H(2)O(2). The pH effect on amperometric response to H(2)O(2) was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.     

The development of an amperometric microbial biosensor using Acetobacter pasteurianus for lactic acid

A microbial biosensor, using Acetobacter pasteurianus cells and an oxygen electrode, was developed for the determination of lactic acid. The bacterial cells were retained on a nylon membrane and attached to the surface of the oxygen electrode. In view of response time, stability and sensitivity, the biosensor performed best at 26°C and in pH 6 phthalate buffer containing magnesium sulfate. The activity of the retained cells was stable for approximately 170 h and was regenerable. The biosensor exhibited a hyperbolic response to both D- and L-lactic acid in the range of 10⁻⁴ M to 25 × 10⁻³ M. However, in the range 10⁻⁴ M to 15 × 10⁻⁴ M the response was linear. The microbial biosensor was applicable for detecting lactate concentration in yogurt and milk, since it was not sensitive to lactose, sucrose and glucose — three major components of such dairy products.     

Microdevice with integrated dialysis probe and biosensor array for continuous multi-analyte monitoring

The simultaneous on-line determination of glucose and lactate using a microdevice that consisted of a dialysis sampling system incorporated to the flow-through cell of a microfabricated biosensor array is presented. The fluidic connections between the different device's components were realized by subsequent processing of stacked dry resist layers on a plastic support that provided also the means for electric connections. The performance of the device was evaluated in vitro. The cross-talk effect on the downstream sensor was investigated and found to be negligible. Recoveries of over 95% for both analytes were achieved when flow rates of the perfusion fluid 相似文献   

Development and analytical application of an uric acid biosensor using an uricase-immobilized eggshell membrane

An uric acid biosensor fabricated from a uricase-immobilized eggshell membrane and an oxygen electrode was presented. The detection schemes involve the enzymatic reactions of the uricase leading to the depletion of dissolved oxygen level upon exposure to uric acid solution. The decrease in oxygen level was monitored and related to the uric acid concentration. The scanning electron micrographs show the microstructure of the eggshell membrane within which the uricase is successfully immobilized. The effects of enzyme loading, pH, temperature, and phosphate buffer concentration on the response of the biosensor were investigated in detail. The uric acid biosensor has a linear response range of 4.0-640 microM with a detection limit of 2.0 microM (S/N=3). The response time was less than 100 s. The biosensor exhibited good repeatable response to a 0.10mM uric acid solution with a relative standard deviation of 3.1% (n=7). The reproducibility of fabrication of the biosensors using four different membranes was good with a R.S.D. of 3.2%. The biosensor showed extremely good stability with a shelf-life of at least 3 months. Some common potential interferents in samples such as glucose, urea, ascorbic acid, lactic acid, glycine, DL-alpha-alanine, DL-cysteine, KCl, NaCl, CaCl2, MgSO4, and NH4Cl showed no interferences on the response of the uric acid biosensor. The biosensor was successfully applied to determine the uric acid level in some human serum and urine samples, and the results agreed well with those obtained by a commercial colorimetric assay kit.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Simultaneous monitoring of glucose and lactate by an interference and cross-talk free dual electrode amperometric biosensor based on electropolymerized thin films.

An interference and cross-talk free dual electrode amperometric biosensor integrated with a microdialysis sampling system is described, for simultaneous monitoring of glucose and lactate by flow injection analysis. The biosensor is based on a conventional thin layer flow-through cell equipped with a Pt dual electrode (parallel configuration). Each Pt disk was modified by a composite bilayer consisting of an electrosynthesised overoxidized polypyrrole (PPYox) anti-interference membrane covered by an enzyme entrapping gel, obtained by glutaraldehyde co-crosslinking of glucose oxidase or lactate oxidase with bovine serum albumin. The advantages of covalent immobilization techniques were coupled with the excellent interference-rejection capabilities of PPYox. Ascorbate, cysteine, urate and paracetamol produced lactate or glucose bias in the low micromolar range; their responses were, however, completely suppressed when the sample was injected through the microdialysis unit. Under these operational conditions the flow injection responses for glucose and lactate were linear up to 100 and 20 mM with typical sensitivities of 9.9 (+/- 0.1) and 7.2 (+/- 0.1) nA/mM. respectively. The shelf-lifetime of the biosensor was at least 2 months. The potential of the described biosensor was demonstrated by the simultaneous determination of lactate and glucose in untreated tomato juice samples; results were in good agreement with those of a reference method.     

Detection of β-glucans using an amperometric biosensor based on high-affinity interaction between Dectin-1 and β-glucans

Early diagnosis of fungal infection plays an important role in increasing antifungal therapeutic response, but meaningful tests such as microbiological cultures and histopathological diagnosis are usually insensitive and time-consuming. A sensitive amperometric biosensor for β-glucans was fabricated by immobilizing Dectin-1 onto Nafion-thionine-gold nanoparticle-chitosan multilayer films to trap its corresponding ligand from sample solution. On formation of ligand-receptor complex, detection of β-glucans was accomplished by monitoring the decrease of the electrochemical signal of the modified electrode due to the inhibition of the transmission of electrons. Dectin-1 was constructed by cloning the extracellular carbohydrate recognition domain of the mouse Dectin gene into the pET28a(+) prokaryotic expression vector. Optimal conditions and analytical performances of the described biosensor were investigated. Under the optimal conditions, the biosensor response for β-glucans presented good accuracy, stability, and reproducibility. The proposed biosensor not only could be used for rapid analysis of serum β-glucans but also provided a screening procedure for the determination of fungal infections.     

AFM membrane roughness as a probe to identify oxidative stress-induced cellular apoptosis

The morphological change of cellular apoptosis initiates from the change of membrane roughness. In order to identify cellular apoptosis in its early stage, atomic force microscope was adapted to reveal the change of membrane roughness in unprecedented details, providing an image in nanometer-scaled resolution. The mouse monocyte/macrophage cell line RAW 264.7 was the subject studied and subjected to apoptotic induction by hydrogen peroxide. A finding of the qualitative correlation between cell membrane roughness and oxidative stress level is disclosed stating that roughness is increasing with the increasing level of oxidative stress.     

Detection of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system

A new highly sensitive amperometric method for the detection of organophosphorus compounds has been developed. The method is based on a ferophthalocyanine chemically modified carbon paste electrode coupled with acetylcholinesterase and choline oxidase co-immobilized onto the surface of a dialysis membrane. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetylcholine as substrate; choline produced by hydrolysis in the enzymatic layer was oxidized by choline-oxidase and subsequently H(2)O(2) produced was electrochemically detected at +0.35 V vs. Ag/AgCl. The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, up to 10(-10) M of paraoxon and carbofuran can be detected.