Amperometric glucose biosensor based on polymerized ionic liquid microparticles

A glucose amperometric biosensor based on the immobilization of glucose oxidase (GOx) in microparticles prepared by polymerization of the ionic liquid 1-vinyl-3-ethyl-imidazolium bromide (ViEtIm⁺Br⁻) using the concentrated emulsion polymerization method has been developed. The polymerization of the emulsion dispersed phase, in which the enzyme was dissolved together with the ionic liquid monomer, provides poly(ViEtIm⁺Br⁻) microparticles with entrapped GOx. An anion-exchange reaction was carried out for synthesizing new microparticles of poly(ViEtIm⁺(CF₃SO₂)₂N⁻) and poly(ViEtIm⁺BF₄⁻). The enzyme immobilization method was optimized for biosensor applications and the following optimal values were determined: pH 4.0 for the synthesis medium, 1.23 M monomer concentration and 3.2% (w/w) cross-linking content. The performance of the biosensor as a function of some analytical parameters such as pH and temperature of the measuring medium, and enzymatic load of the microparticles was also investigated. The effect of the substances which are present in serum samples such as uric and ascorbic acid was eliminated by using a thin Nafion layer covering the electrode surface. The biosensor thus prepared can be employed in aqueous and in non-aqueous media with satisfactory results for glucose determination in human serum samples. The useful lifetime of this biosensor was 150 days.     

Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor

A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.     

An amperometric pyruvate biosensor based on pyruvate oxidase nanoparticles immobilized onto pencil graphite electrode

This present study was aimed to fabricate a sensitive and improved amperometric biosensor by the nanoparticles of pyruvate oxidase, which were prepared and immobilized covalently onto pencil graphite electrode. The biosensor showed ideal working within 5 s under defined conditions of pH 6.0 and incubation temperature of 30 °C at an applied voltage of -0.1 V. Under standard assay conditions, a linear response was obtained between pyruvate concentration ranging from 0.001 to 6000 μM and current (μA). A lower detection limit (0.58 μM) and an excellent correlation coefficient (R² = 0.999) with standard spectrophotometric assay was obtained for the present biosensor. Within and between batches of coefficients of variation were calculated and found to be 3.61 % and 3.33 %, respectively. The biosensor was put to continual use for over 210 days. The biosensor was employed for the measurement of pyruvate level in sera of normal healthy individuals and persons suffering from heart disease.     

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.     

Amperometric l-lysine determination biosensor amplified with l-lysine oxidase nanoparticles and graphene oxide nanoparticles

We describe the amplification of amperometric l-lysine biosensor using l-lysine oxidase nanoparticles (LOxNPs) and graphene oxide nanoparticles (GrONPs) immobilized onto pencil graphite electrode (PGE). LOxNPs and GrONPs were characterized by UV spectroscopy and transmission electron microscopy (TEM). The working electrode (LOxNPs/GrONPs/PGE) was studied by scanning electron microscopy (SEM) and cyclic voltammetry at different stages of its construction. The biosensor showed optimum current at 0.7 V, pH 6.5, 35 °C, a detection limit of 0.01 μM, response time as 3.95 s and a wider linear range 0.01–1000 μM. The analytical recovery of added lysine in sera was 97 %. The within assay and between batch coefficients of variation for the biosensor were 0.068 and 0.074 % respectively. The biosensor measured l-lysine levels in sera of healthy adults and human immunodeficiency virus (HIV) patients. The biosensor exhibited good correlation with standard spectrophotometric method (R² = 0.989). The biosensor lost 35 % of its original activity after its regular uses for a period of 180 days, while being stored dry at 4 °C.     

Amperometric bioelectrode for specific human immunoglobulin G determination: optimization of the method to diagnose American trypanosomiasis

Bioelectrodes to detect immunoglobulin G (IgG) antibodies occurring in sera of patients suffering from American trypanosomiasis were assembled. The device consisted of a gold electrode modified with a thiol sensitized with parasite proteins. The assemblage was accomplished by adsorbing IgG antibodies from confirmed infected patients followed by adsorption of anti-human IgG labeled with a redox enzyme. The appliance was used as a working electrode in a three-electrode cell containing a soluble charge-transfer mediator, also behaving as enzyme cosubstrate. The method is based on the measurement of the catalytic current after addition of the enzyme substrate, occurring when a positive serum is used to build up the biosensor. The discrimination efficiency between positive and negative sera was 100% for the samples studied. A 0.9525 correlation coefficient was obtained for results acquired by using this approach and one commercial diagnostic kit. The reproducibility, evaluated by the percentage coefficient of variation, varied between 7 and 20%. The sensitivity was 12.4 ng mL(-1) IgG, which is in the same order as that obtained with the commercial kit. Stability of the device was studied for a 7-day period and the results showed no significant change (p = 0.218). Leishmaniasic sera showed cross-reactivity when total parasite homogenate was used as antigen.     

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.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 &#119 l each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 μl each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

Amperometric ethanol biosensor based on poly(vinyl alcohol)-multiwalled carbon nanotube-alcohol dehydrogenase biocomposite

A novel amperometric ethanol biosensor was constructed using alcohol dehydrogenase (ADH) physically immobilized within poly(vinyl alcohol)–multiwalled carbon nanotube (PVA–MWCNT) composite obtained by a freezing–thawing process. It comprises a MWCNT conduit, a PVA binder, and an ADH function. The measurement of ethanol is based on the signal produced by β-nicotinamide adenine dinucleotide (NADH), the product of the enzymatic reaction. The homogeneity of the resulting biocomposite film was characterized by atomic force microscopy (AFM). The performance of the PVA–MWCNT–ADH biocomposite modified glassy carbon electrode was evaluated using cyclic voltammetry and amperometry in the presence of NADH and in the presence of ethanol. The ethanol content in standard solutions was determined and a sensitivity of 196 nA mM⁻¹, a linear range up to 1.5 mM, and a response time of about 8 s were obtained. These characteristics allowed its application for direct detection of ethanol in alcoholic beverages: beer, red wine, and spirit.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Construction of a triglyceride amperometric biosensor based on chitosan-ZnO nanocomposite film

A method is described for construction of a novel amperometric triglyceride (TG) biosensor based on covalent co-immobilization of lipase, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) onto chitosan (CHIT) and zinc oxide nanoparticles (ZnONPs) composite film deposited on the surface of Pt electrode. The enzymes-ZnONPs-CHIT composite was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The sensor showed optimum response within 6 s at pH 7.5 and temperature of 35 °C. The sensor measures current due to electrons generated at 0.4 V against Ag/AgCl from H₂O₂, which is produced from triolein by co-immobilized enzymes. A linear relationship was obtained between a wide triolein concentration range (50-650 mg/dl) and current (mA) under optimum conditions. The biosensor showed high sensitivity, low detection limit (20 mg/dl) and good storage stability (half-life of 7 months at 4 °C). The biosensor was unaffected modified by a number of serum substances at their physiological concentrations. The biosensor was evaluated and employed for determination of TG in sera in apparently healthy subjects and persons suffering from hypertriglyceridemia.     

Electrochemical DNA biosensor for the study of ciprofloxacin-DNA interaction

The interaction of ciprofloxacin with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of ciprofloxacin was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current at +0.9 V was used as an indicator for the interaction mechanism in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 1.33+/-0.02 x 10(4) and 1.32+/-0.08 x 10(4) M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak currents was observed in the range of 40-80 microM ciprofloxacin, with a detection limit of 24 microM with r=0.995 and 9 microM with r=0.999 by using constant current potentiometry and differential pulse voltammetry, respectively. Moreover, the influence of sodium and calcium ions was also studied to elucidate the mechanism of ciprofloxacin-DNA interaction at different solution conditions, and this proved to be helpful in understanding the ciprofloxacin-DNA interaction.     

Development of an oral biosensor for salivary amylase using a monodispersed silver for signal amplification

An amperometric biosensor for monitoring the level of protein amylase in human saliva is described. A novel design and the preparation of amylase antibodies and antigens, essential for the development of the biosensor, are reported. The biosensor sensing elements comprise a layer of salivary antibody (or antigen) self-assembled onto Au-electrode via covalent attachment. Molecular recognition between the immobilized antibody and the salivary amylase proteins was monitored via an electroactive indicator (e.g., K(3)Fe(CN)(6)) or a monodispersed silver layer present in solution or electrochemically deposited onto the solid electrode. This electroactive indicator was oxidized or reduced and the resulting current change provided the analytical information about the concentration of the salivary proteins. The limit of detection of 1.57 pg/ml was obtained, in comparison to detection limits of 4.95 pg/ml obtained using potassium ferrocyanide as the redox probe and 10 ng/ml obtained using enzyme-linked immunosorbent assay. Cross-reactivity was tested against cystatin antibodies and was found to be less than 2.26%.     

An amperometric biosensor for determining amino acids using a bienzymatic system containing amino acid oxidase and protease

An amperometric biosensor for rapid determination of the concentration of l-amino acids has been developed using l-amino acid oxidase (l-AAO) immobilized by gel entrapment with poly(carbamoyl) sulfonate hydrogel. The broad substrate range of l-AAO allows this biosensor to be flexible in application. The artificial sweetener, aspartame, was determined by coupling l-AAO with pronase.     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

Comparative study of hydrogel-immobilized l-glutamate oxidases for a novel thick-film biosensor and its application in food samples

Novel, thick-film biosensors have been developed for the determination of l-glutamate in foodstuffs. The sensors were prepared by immobilization of l-glutamate oxidase by using polycarbamylsulfonate-hydrogel on a thick-film sensor. l-Glutamate oxidases obtained from Streptomyces sp. with different degree of purification were compared with their characteristic response to l-glutamate at different conditions and for their specificity, inhibition, and storage properties. These sensors were applied to determine monosodium glutamate in soy sauce samples and show good correlation with colorimetric method.     

Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybrids platform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a model protein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels and secondary antibodies (Ab₂) linked to GNPs at high HRP/Ab₂ molar ratio. The approach provided a linear response range between 0.125 and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.