Sequential measurement of aminotransferase activities by amperometric biosensors

An l-glutamate biosensor modified by cation exchanger membrane on a palladium (Pd) electrode was designed for the purpose of preventing interferences and electrode fouling during the measurement of serum AST and ALT activities. The rate of signal increase obtained by our sensor for the determination of AST and ALT activity was 0.259 and 0.596 nA/min U(-1)l and the response of the sensor to AST and ALT activity were linear over the range of 8-200 and 8-250 Ul(-1), respectively. Both AST and ALT activities could be measured sequentially by injecting the serum into a solution containing l-aspartate and alpha-ketoglutarate. The rate of current increase was relative to AST activity. The activity of ALT was sequentially determined after addition of l-alanine into the solution. The change in the current increase rate after the addition of l-alanine was proportional to the ALT activity. By using the proposed biosensor, the interference of 1mM ascorbic acid was negligible on a dynamical aminotransferase determination when the dynamic data are taken after the steady state of an elevated baseline has been reached. The proposed l-glutamate biosensor provides adequate sensitivity for the measurement of AST and ALT and is expectable to be applied for rapid blood screening of AST and ALT activity in clinical sample.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

NO(2) Sensor which uses immobilized nitrite oxidizing bacteria

A biosensor consisting of immobilized nitrite oxidizing bacteria and an oxygen electrode has been developed for the amperometric determination of NO(2) (nitrogen dioxide) gas. The response time for the determination of NO(2) was within 3 min. A linear relationship was observed between the current decrease and the NO(2) concentration below 255 ppm. The minimum concentration for the determination of NO(2) was 0.51 ppm. The current decrease was reproducible within +/-4% of the relative error. The selectivity of the microbial sensor for NO(2) was satisfactory. The current output of the sensor was almost constant for more than 24 days and 400 assays.     

Amperometric sulfide detection using Coprinus cinereus peroxidase immobilized on screen printed electrode in an enzyme inhibition based biosensor

In the present work, an amperometric inhibition biosensor for the determination of sulfide has been fabricated by immobilizing Coprinus cinereus peroxidase (CIP) on the surface of screen printed electrode (SPE). Chitosan/acrylamide was applied for immobilization of peroxidase on the working electrode. The amperometric measurement was performed at an applied potential of -150 mV versus Ag/AgCl with a scan rate of 100 mV in the presence of hydroquinone as electron mediator and 0.1M phosphate buffer solution of pH 6.5. The variables influencing the performance of sensor including the amount of substrate, mediator concentration and electrolyte pH were optimized. The determination of sulfide can be achieved in a linear range of 1.09-16.3 μM with a detection limit of 0.3 μM. Developed sensor showed quicker response to sulfide compared to the previous developed sulfide biosensors. Common anions and cations in environmental water did not interfere with sulfide detection by the developed biosensor. Cyanide interference on the enzyme inhibition caused 43.25% error in the calibration assay which is less than the amounts reported by previous studies. Because of high sensitivity and the low-cost of SPE, this inhibition biosensor can be successfully used for analysis of environmental water samples.     

Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin

A simple tissue biosensor for measuring Na⁺ channel blockers such as tetrodotoxin (TTX) and saxitoxin (STX) has been developed. The membrane of frog bladder has Na⁺ channels which control the passage of Na⁺. It is well known that TTX blocks Na⁺ channels. The tissue biosensor consists of a Na⁺ electrode integrated within a flow cell. The tip of the electrode was covered with frog bladder membrane sandwiched between two sheets of cellulose acetate membrane, and the electrode was set in a flow cell.

A solution of 8% NaCl was carried in the cell and the output of the electrode allowed to stabilize. TTX was injected into the sensor system and measured from the inhibition ratio of the sensor peak output. One assay took approximately 5 min. The lower limit of detection was 86 fg. The continuous determination of TTX was feasible for 250 h in the presence of 0·003% NaN₃. A Linear correlation was obtained between TTX activities of F-niphobles and F-parudale determined by the methods of TTX sensor and mouse assay.     

Hydrogen peroxide sensitive amperometric biosensor based on horseradish peroxidase entrapped in a polypyrrole electrode

The enzyme horseradish peroxidase (HRP) has been entrapped in situ by electropolymerization of pyrrole onto a platinum electrode. The latter was previously coated by a polypyrrole layer for better adhesion of the biocatalyst film and in order to avoid the enzyme folding onto the Pt electrode. The biosensor allowed the determination of hydrogen peroxide in the concentration range comprised between 4.9 x 10(-7) and 6.3 x 10(-4) M. The biosensor retained more than 90% of its original activity after 35 days of use.     

Amperometric biosensor for determining human salivary phosphate

An amperometric biosensor was constructed for analysis of human salivary phosphate without sample pretreatment. The biosensor was constructed by immobilizing pyruvate oxidase (PyOD) on a screen-printed electrode. The presence of phosphate in the sample causes the enzymatic generation of hydrogen peroxide (H(2)O(2)), which was monitored by a potentiostat and was in proportion to the concentration of human salivary phosphate. The sensor shows response within 2s after the addition of standard solution or sample and has a short recovery time (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 to 625 microM phosphate with a detection limit of 3.6 microM. A total of 50 salivary samples were collected for the determination of phosphate. A good level of agreement (R(2)=0.9646) was found between a commercial phosphate testing kit and the phosphate sensor. This sensor maintained a high working stability (>85%) after 12h operation and required only a simple operation procedure. The amperometric biosensor using PyOD is a simple and accurate tool for rapid determinations of human salivary phosphate, and it explores the application of biosensors in oral and dental research and diagnosis.     

Three-dimensional interdigitated electrode array as a transducer for label-free biosensors

A new transducer for biosensor applications has been developed based on a three-dimensional interdigitated electrode array (IDEA) with electrode digits separated by an insulating barrier. Binding of molecules to a chemically modified surface of the transducer induces important changes in conductivity between the electrodes. Three-dimensional sensor shows considerable improvement compared with a standard planar IDEA design. The potential of the developed device as a sensor transducer to detect immunochemical and enzymatic reactions, as well as DNA hybridization events is demonstrated. The immunosensor allows direct detection of the antibiotic sulfapyridine and shows the IC(50) parameter value of 5.6 microgL(-1) in a buffer. Immunochemical determination occurs under competitive configurations and without the use of any label. Each modified sensor is of a single use. Nevertheless, biochemical reagents can be easily cleaned off the sensor surface for its reuse. Layer-by-layer method of used to deposit polyethyleneimine and glucose oxidase showed that the sensor is also highly effective for detecting single and multilayered molecular assemblies.     

Electrosynthesized poly(pyrrole)/poly(2-naphthol) bilayer membrane as an effective anti-interference layer for simultaneous determination of acethylcholine and choline by a dual electrode amperometric biosensor

Several neural diseases appear related to the neurotransmitter acethylcholine (ACh) and its metabolite choline (Ch) brain levels so that their simultaneous determination is essential. A cross-talk and interference free dual electrode amperometric biosensor for the simultaneous determination of both analytes has been developed. Acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized by glutaraldehyde co-crosslinking with bovine serum albumin. A very efficient rejection of electroactive interferents has been achieved by a novel electrosynthesized polymeric bilayer membrane composed by overoxidised poly(pyrrole) and poly(2-naphthol) films. Sensitivities towards several electroactive interferents ranged from ca. 0.04% (e.g. ascorbate) to ca. 0.3% (e.g. dopamine) of those relevant to ACh and Ch (11 and 15 microA/microM, respectively). Detection limits (at S/N=3) in flow injection analysis were ca. 100 nM for both ACh and Ch at the ChO-AChE electrode and ca. 40 nM for Ch at the ChO sensor. Biosensor performances appear more than adequate for brain tissue homogenates and cerebrospinal fluids analysis where average levels in the low micromolar range are typically found.     

A novel L-lactate-selective biosensor based on flavocytochrome b2 from methylotrophic yeast Hansenula polymorpha

A novel amperometric biosensor highly selective to L-lactate has been developed using L-lactate-cytochrome c oxidoreductase (flavocytochrome b2) isolated for the first time from thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. Different immobilization methods and low-molecular free-diffusing redox mediators have been tested for optimising the electrochemical communication between the immobilized enzyme and the electrode surface. Moreover, the possibility of direct electron transfer from the reduced form of FCb2 to carbon electrodes has been evaluated. The bioanalytical properties of FCb2-based biosensors, such as signal rise time, dynamic range, dependence of the sensor output on the pH value, the temperature and the storage stability were investigated, and the proposed biosensor demonstrated a very fast response and a high sensitivity and selectivity for L-lactate determination.     

Studies on the electrochemical performance of glucose biosensor based on ferrocene encapsulated ORMOSIL and glucose oxidase modified graphite paste electrode

The electrochemical performance of a new glucose biosensor is reported. The glucose biosensor is developed using glucose oxidase (GOD) and ferrocene encapsulated palladium (Pd)-linked organically modified sol-gel glass (ORMOSIL) material incorporated within graphite paste electrode. The ORMOSIL material incorporated within graphite paste electrode behaves as an excellent electrocatalyst for the oxidation of enzymatically reduced GOD. The electrochemical behavior of new glucose biosensor has been examined by cyclic volammetry and amperometric measurements. The bioelectrocatalysis of ORMOSIL embedded within graphite paste as a function of storage time and varying concentration of ORMOSIL is reported. The initial amperometric response on glucose sensing is recorded to be 145 microA at 15% (w/w) concentration of the ORMOSIL which is decreased to 20 microA at 5% of the same keeping GOD concentration constant. The variation of electrochemical behavior of the ORMOSIL embedded within graphite paste as a function of time has also been studied based on cyclic voltammetry. The voltammograms showing the reversible electrochemistry of ORMOSIL encapsulated ferrocene is changed into a plateau shape as a function of time, however, the electrocatalytic behavior is still retained. The practical usability of new glucose sensor has been compared with earlier developed glucose sensor. The sensitivity, response time and linearity of the new glucose biosensor are found to be excellent over earlier reported glucose biosensor. The amperometric response, calibration curve and practical applications of new glucose sensor are reported.     

A novel biosensor based on l-homocysteine desulfhydrase enzyme immobilized in eggshell membrane

A novel biosensor for homocysteine determination has been developed. The biosensor was fabricated with l-homocysteine desulfhydrase immobilized on the ammonium selective electrode by means of eggshell membrane. The measurement principle is based on determination of ammonia due to the enzymatic reaction in the medium by ammonium selective electrode. The effects of enzyme loading, glutaraldehyde concentration, pH, buffer concentration, temperature, dithiotreitol (DTT) concentration and ionic strength adjustment buffer (ISA) on the biosensor response were investigated in detail. The linear detection range and limit of detection (LOD) for homocysteine were found to be 0.15–1.8 mM and 55 μM, respectively. Finally, the homocysteine biosensor has been applied to plasma samples for determination of total homocysteine contents.     

Development of a micro-planar amperometric bile acid biosensor for urinalysis

The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.     

Palladium hexacyanoferrate hydrogel as a novel and simple enzyme immobilization matrix for amperometric biosensors

An amperometric glucose biosensor with glucose oxidase (GOx) immobilized into palladium hexacyanoferrate (PdHCF) hydrogel has been prepared and evaluated. The sensor was based on a two-layer configuration with biocatalytic and electrocatalytic layers separately deposited onto the electrode. To reduce the overpotential for reduction of hydrogen peroxide liberated in the enzyme catalyzed oxidation of glucose, an inner thin layer of nickel hexacyanoferrate (NiHCF) electrodeposited onto the surface of graphite electrode was used as an electrocatalyst. As an outer layer, the hydrogel of palladium hexacyanoferrate with entrapped glucose oxidase was used. Under optimal operating conditions (pH 5.0 and E = -0.075 V versus calomel (3.0 M KCl) reference electrode), sensor showed high sensitivity to glucose (0.3-1.0 microA/mM) and a response time of less than 30s. The linear response to glucose was obtained in the concentration range between 0.05 and 1.0 mM in batch analysis mode and 0-7.0 mM in FIA. During the 32 days testing period, no significant decrease in the sensor sensitivity was observed. The sensor was applied for the determination of glucose concentration in fruit juice and yoghurt drink, and the results obtained showed good correlation with results obtained by reference spectrophotometric enzyme method.     

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.     

A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxidase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 micromoll(-1), and the linear range was from 10.0 micromoll(-1) to 7.0 mmoll(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the studied biosensor exhibited good current repeatability and good fabrication reproducibility.     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

Electrochemical analysis of copper ion using a Gly–Gly–His tripeptide modified poly(3-thiopheneacetic acid) biosensor

A novel biosensor harnessing a conducting polymer functionalized with a copper ion specific peptide proved to be highly effective for electrochemical analysis of copper ions. The developed sensor comprised a transducer based on a conducting polymer (poly(3-thiopheneacetic acid)) electrode and a probe (tripeptide, Gly–Gly–His) selectively cognitive of copper ions. For functionalization of the electrode, the carboxylic group of the polymer was covalently coupled with the amine group of the tripeptide, and its structural features were confirmed by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection infrared (ATR-IR) spectroscopy. The peptide modified polythiophene biosensor was used for the electrochemical analysis of various trace metal ions by square wave voltammetry. The electrode was found to be highly sensitive and selective to Cu²⁺ in the range of 0.02–20 μM with almost no cross binding to other metal ions such as Ni²⁺ and Pb²⁺. Furthermore, the developed sensor exhibited a high stability and reproducibility despite the repeated use of the sensor electrode and probe. With the advent of more diverse affinity bioprobes specific towards a broad range of analytes, the demonstrated strategy harnessing peptide modified polythiophene biosensor is likely to provide an excellent platform for the selective determination of trace amount of analytes whose detection is otherwise cumbersome.     

Biosensor for continuos glucose monitoring

A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and glucose oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Recharging of the biosensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. Diffusion membranes made of silastic latex-rubber coatings over a microporous polycarbonate membrane are used. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations-up to 500 mg/dL (28 mM), covering hypoglycemic, normoglycemic, and hyperglycemic conditions. Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months. (c) 1994 John Wiley & Sons, Inc.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.