Flow Injection Analysis Biosensor Research Articles

Flow injection analysis biosensor for urea analysis in adulterated milk using enzyme thermistor

Urea in adulterated milk is one of the major health concern, it is especially harmful to pregnant women, children, and the sick. A sophisticated and reliable detection system is needed to replace current diagnostic tools for the urea in the milk. In this work, we report a flow injection analysis-enzyme thermistor (FIA-ET) bio-sensing system for monitoring of urea in adulterated milk. This biosensor was made of the covalently immobilized enzyme urease (Jack bean) on controlled pore glass (CPG) and packed into a column inside thermistor, which selectively hydrolysed the urea present in the sample. The specific heat registered from the hydrolysis of urea was found proportional to the concentration of urea present in the milk sample. The biosensor showed a linear range 1–200 mM, with % R.S.D. 0.96 for urea in 100 mM phosphate buffer, pH 7.2. Good recoveries were obtained (97.56–108.7%) for urea up to 200 mM in the spiked milk samples with % R.S.D. 0.95. In the adulterated milk, a simple filtration strategy and matrix matching technique was used to analyse urea. The response time of the sensor was evaluated for urea, which was 2 min, and it gives satisfactory output. A good comparison was observed between the urea concentrations measured through FIA-ET and the colorimetric method. These results indicate that utilizing this system could be very effective to detect low and high level of urea in adulterated milk. The immobilized urease column exhibited a good operational stability up to 180 days when used continuously at room temperature.

Analysis of fluid flow and reaction kinetics in a flow injection analysis biosensor

This paper presents the development of a convection–diffusion-reaction model to simulate the behavior of a flow injection analysis (FIA) biosensor with respect to its design and operational parameters, such as flow rate, fluidic channel dimensions, flow cell geometry and volume of substrate and enzyme injected. The problem is described by the steady-state incompressible Navier–Stokes equations for fluid flow, and transient convection–diffusion-reaction equations for the different dissolved species. The enzyme–substrate interaction is described by Michaelis–Menten kinetics. The model was successfully validated on a glucose FIA-biosensor by comparing measured and simulated H 2O 2 concentration versus time profiles. Both peaks matched well, however, a slightly longer tailing was observed in the measurements due to simplifications in the geometrical model of the flow cell. The simulated and measured calibration curves for glucose closely matched in the range between 0 and 2.5 mM. The model was also applied to simulate the substrate, enzyme and product concentration profiles in the fluidic channels and the flow cell as a function of time. A parabolic velocity profile obtained from a pressure driven flow resulted in more peak broadening in the flow cell than a uniform velocity profile that occurs in packed column flow. Given the specified set of parameters, the concentration profiles of substrate, enzyme and product illustrated that not all substrate was converted into product when the flow reached the inlet of the flow cell, indicating a suboptimal sensitivity of the designed biosensor. This modeling approach has a high potential in the design and development of high accuracy FIA-biosensors, regardless of the chosen enzyme substrate system.

An electrochemiluminescence-based fibre optic biosensor for choline flow injection analysis.

A fibre optic biosensor based on luminol electrochemiluminescence (ECL) integrated in a flow injection analysis (FIA) system was developed for the detection of choline. The electrochemiluminescence of luminol was generated by a glassy carbon electrode polarised at +425 mV vs. a platinum pseudo-reference electrode. Choline oxidase (Chx) was immobilised either covalently on polyamide (ABC type) or on UltraBind preactivated membranes, or by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) alone or after absorption on a weak anion exchanger, DEAE (diethylaminoethyl) Sepharose. The optimisation of the reaction conditions and physicochemical parameters influencing the FIA biosensor response demonstrated that the choline biosensor exhibited the best performances in a 30 mM veronal buffer containing 30 mM KCl and 1.5 mM MgCl2, at pH 9. The use of a 0.5 ml min-1 flow rate enabled the measurement of choline by the membrane-based ECL biosensors in 8 or 5 min, with ABC or UltraBind membranes, respectively, whereas the measurement required only 3 min with the DEAE-PVA system. For comparison, the detection of choline was performed with Chx immobilised using the four different supports. The best performances were obtained with the DEAE-PVA-Chx sensing layer, which allowed a detection limit of 10 pmol, whereas with the ABC, the UltraBind and the PVA systems, the detection limits were 300 pmol, 75 pmol and 220 pmol, respectively. The DEAE-based system also exhibited a good operational stability since 160 repeated measurements of 3 nmol of choline could be performed with an RSD of 4.5% whereas the stability under the best conditions was 45 assays with the other supports.

Luminol Electrochemiluminescence-Based Biosensor for Total Cholesterol Determination in Natural Samples

ABSTRACT A new cholesterol flow injection analysis biosensor is described. It is based on a luminol/hydrogen peroxide electrochemiluminescence (ECL) reaction induced by a glassy carbon electrode polarised at + 425 mV vs a Pt pseudo reference. The cholesterol b8543158 sensing layer is based on cholesterol oxidase (COD) immobilised on either UltraBindTM membrane or Immunodyne membrane. The physicochemical properties of the UltraBindTM type membranes enable a high-performance biosensor to be obtained. A high salt concentration, 3 M NaCl, is shown to induce a 400% increase of the immobilised COD activity, and intermediate salt concentrations, from 1 to 1.5 M NaCl, induce a 230% increase of the H2O2 sensor performances. Both phenomena enable the achievement of a sensitive ECL cholesterol biosensor which exhibited a 800% increase of its performance in the presence of 2 M NaCl. In those optimised conditions, the determination of free cholesterol could be performed with a detection limit of 0.6 nmol and a detection ranging over at least two decades. When used in conjunction with a cholesterol esterase in solution, the biosensor enables the detection of free and total cholesterol in human sera, in 15 minutes with a good accuracy. The biosensor operational stability was satisfactory either with buffered standard solutions or with complex matrices since a total of 55 and 43 assays, respectively, could be performed with good reproducibility (CV = 4.8% and 8.3%) and without detectable loss of sensitivity.

Development of a highly sensitive chemiluminescence flow-injection analysis sensor for phosphate-ion detection using maltose phosphorylase

A chemiluminescence flow-injection analysis biosensor has been constructed for phosphate-ion detection. This system is coenzymeless and employs a maltose phosphorylase, mutarotase, and glucose-oxidase (MP–MUT–GOD) reaction system combined with an Arthromyces ramosus peroxidase–luminol reaction system. The system consists of a column packed with MP–MUT–GOD immobilized on N-hydroxysuccinimide beads, a mixing joint for the chemiluminescence reaction, and a photomultiplier. The response provided by this system was linear, with a wide range between 10 nM and 30 μM phosphate ion, and a measuring time of 3 min per sample. Under the optimal condition, the sensor was able to detect 1.0 μM phosphate ion for at least 2 weeks. For a practical application, the determination of phosphate ion in river water was examined using ethylenediaminetetraacetic acid, and the results were estimated by comparing with the molybdenum-blue method.

Determination of Damaged Starch and Diastatic Activity in Wheat Flour Using a Flow‐Injection Analysis Biosensor Method

ABSTRACTThe determination of damaged starch and diastatic activity in flour was studied using a flow‐injection analysis (FIA) biosensor system. The system consisted of an oxygen electrode and an immobilized enzyme column containing purified glucoamylase and glucose oxidase immobilized on activated aminopropyl glass beads. The biosensor system has an optimum pH between 6.5 and 7.5 and an optimum temperature of 35°C for glucose measurement. The response of the FIA biosensor was linear up to 1.000 g/L of glucose with a lower detection limit of 0.025 g/L. Each assay took about 20 min, and the system showed good reproducibility (r = 0.998, n = 8). When applied to the measurement of damaged starch and diastatic activity in wheat flour, the results obtained agreed with those obtained using the conventional methods of measurement. This biosensor system is a rapid practical alternative for the measurement of damaged starch and diastatic activity in wheat flour.

A Microdialysis Probe Coupled with A Miniaturized Thermal Glucose Sensor for In Vivo Monitoring

A miniaturized thermal flow injection analysis biosensor has been coupled with a microdialysis probe for continuous subcutaneous glucose monitoring. Thermal biosensors are based on the principle of measuring the heat evolved during enzyme catalysed reactions. The system presented here consists of a miniaturized thermal biosensor with a small column containing coimmibolized glucose oxidase and catalase. The analysis buffer passes through the column at a flow rate of 60μL/min via an 1μL sample loop which is connected to a microdialysis probe. Invitro results showed constant permeability of the probe and stability of the biosensor response during 24 hours. The response time was 85 sec giving a sample rate of 42 samples/hour. During a load experiment, the glucose profile in a healthy volunteer was followed both in the subcutaneous tissue and blood using the microdialysis set-up proposed and comparing to blood glucose analyser.

Monitoring of beef aging using a two-line flow injection analysis biosensor consisting of putrescine and xanthine electrodes

A two-line flow injection analysis (FIA) biosensor was developed which can simultaneously detect bacterial spoilage and the progress of aging. This FIA biosensor was composed of a putrescine oxidase immobilized electrode and a xanthine oxidase immobilized electrode as detectors. The putrescine electrode measures putrescine and cadaverine which are produced by bacteria, and the xanthine electrode measures hypoxanthine and xanthine which accumulate in meat with aging. The analytical conditions for this system were set as follows; flow rate, 1 ml/ min; water bath temperature, 30 °C; flow buffer, 0.1 M phosphate buffer (pH 7.0); injection volume for putrescine electrode, 200 μl; injection volume for xanthine electrode, 40 μl; and measurement cycle, 2 min. The linear relationship for standard solution was between 20 and 800 nmol/ml in the putrescine electrode and between 0.1 and 3.0 μmol/ml in the xanthine electrode. The coefficients of variation in standard solutions were 2.14% with the putrescine electrode and 2.83% with the xanthine electrode. The coefficients of variation values in the specimen solution were 3.22% and 3.76%, respectively. This two-line FIA biosensor was applied to the vacuum-packed beef stored at 0, 5 and 10 °C. The progress of aging could be monitored at all temperatures, and the bacterial spoilage could be detected before the appearance of putrid odor at 5 and 10 °C. However, at 0 °C the putrid odor did not appear during storage, and neither putrescine nor cadaverine was detected. Thus, this FIA biosensor was confirmed to be useful for the quality control of beef aging at 5 and 10 °C, but not at 0 °C.

Determination of Glucose in Diluted Blood with a Thermal Flow Injection Analysis Biosensor

Abstract The glucose concentration in diluted whole blood has been measured, using a miniaturized thermal biosensor based on the enzyme thermistor principle. The biosensor is a small flow injection system. A sample volume of 20μl is injected into a flow of 50μl/min. The heat produced when the sample passes the enzyme column is measured with a thermistor connected to a Wheatstone bridge. The enzyme column contains glucose oxidase and catalase co-immobilized on a solid support material. Samples of whole blood usually cause problems in flow-systems. The blood cells tend to block the enzyme column and the back pressure increases. We have tested a superporous agarose material as enzyme support material using tenfold diluted samples of whole human blood. The blood was collected from the finger-tip and diluted with buffer containing an anticoagulant and sodium fluoride. The number of samples possible to inject and the accuracy compared to the Boehringer Mannheim Reflolux have been determined. At least 100 ten-fold diluted blood samples could be injected on a micro-column of superporous agarose. The obtained glucose concentration correlated well with the one obtained with the reference instrument.

An Amperometric Flow-Injection Analysis Biosensor for Glucose Based on Graphite Paste Modified with Tetracyanoquinodimethane

A biosensor system using flow injection analysis (FIA) has been developed for the analysis of glucose in human serum. The system consists of the enzyme glucose oxidase incorporated into graphite paste modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for oxidation of the reduced enzyme at 200 mV vs Ag/AgCl. The flow injection assay described has detection limits of 2 mM glucose using a 100-μl sample injection through a 250-μl sample loop. Data are presented to show the effect of sample injection volume and flow rate on the response of the FIA sensor. The biosensor exhibited excellent reproducibility for 800 injections. The loss of response after 800 injections was due to leaching of TCNQ from the graphite paste. Each assay takes 3 min giving a sample throughput of 20 per hour at a flow rate of 30 ml/h. The sensor was applied to the determination of glucose in human serum. The glucose measurements are in good agreement with those of a commercially available spectrophotometric method. Data showing the effect of interfering substances, ascorbic acid and acetaminophen, on the response of the sensor are also reported.

An improved FIA biosensor for the determination of aspartame in dietary food products.

A flow injection analysis (FIA) biosensor system was developed for the determination of the artificial sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester). The system consisted of an enzyme column of pronase immobilized on activated arylamine glass beads and a L-amino acid oxidase electrode connected in series. The dipeptide bond of aspartame was cleaved by immobilized pronase to release phenylalanine, which was in turn monitored by the enzyme electrode that used L-amino acid oxidase immobilized on a preactivated nylon membrane in combination with an amperometric electrode (platinum vs silver/silver chloride, 700 mV). The response of the FIA biosensor was linear up to 1 mM aspartame with a lower detection limit of 25 microM and had good reproducibility (rsd 0.3%). The FIA biosensor was stable for at least 30 h of continuous use at Tr. Each assay takes 4 min giving a sample throughput of 15 h-1. When applied to aspartame in dietary food products the results obtained agreed well with those reported by the product manufacturers.

Novel FIA amperometric biosensor system for the determination of glutamine in cell culture systems

A flow injection analysis (FIA) biosensor system has been developed for determining glutamine in insect cell and in murine hybridoma cell cultures. Glutamate oxidase and glutaminase, respectively, were covalently immobilized onto porous aminopropyl glass beads to form an immobilized enzyme column. The hydrogen peroxide produced by the enzyme reactions was detected by an amperometric electrode (platinum vs. silver/silver chloride) posied at +0.7 V. Among several anion exchange resins tested, endogenous glutamate was completely adsorbed onto acetate anion exchange resins. Such an ion exchanger also effectively adsorbed aspartic acid, uric acid, ascorbic acid, and acetaminophen. The FIA biosensor was linear up to 1 m m glutamine, with a lower detection limit of 10 μ m, and possessed good reproducibility (relative error of ± 1.2%). Each analysis could be performed in 3.5 min including sampling and washing with a corresponding throughput of 17 h −1. The anion exchange column could be operated continuously for 12 h for 200 analyses with diluted cell culture. The immobilized enzyme column was stable for at least 500 repeated analyses without significant loss of activity. When the biosensor system was applied to glutamine measurement in insect cell and mammalian cell cultures, the results obtained compared well with those of HPLC.

Determination of urinary glucose by a flow injection analysis amperometric biosensor and ion-exchange chromatography.

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 +/- 2.4 RU microM-1 (RU or relative unit is defined as 2.86 microV at the detection output) for glucose with a minimum detection level of 10 microM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25-30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.

Amperometric Assay of Creatinine in Urine by Flow Injection Analysis Based on Conjugated Reactions of Immobilized Enzymes

A flow-injection analysis biosensor system was developed for the amperometric assay of creatinine based on coupled reactions of three immobilized enzymes, using an oxygen electrode as the detection device. The ammonia produced by creatinine deiminase-catalyzed hydrolysis of creatinine was further converted into L-glutamate with two sequentially aligned enzyme reactors: glutamate dehydrogenase and glutamate oxidase. Endogenous ammonia was simultaneously compensated with a double peak recording system, where the flow was split after sample injection and rejoined before the glutamate dehydrogenase reactor. The system gave linear calibration in a range of 0.1-2.0 mM for creatinine and the first peak of ammonia, and 0.1-3.0 mM for the second peak of ammonia. One run was completed within two minutes. The system can be readily applied to the assay of creatinine in urine and showed good correlation with that from the currently used Jaffe method.

Flow injection analysis with immobilized reagents.

Immobilized reagent phase flow injection analysis can be configured as discrete reagent cells upstream of the sensor element or as an integral reagent/transduction system (flow injection analysis-biosensor). The former approach has attracted greater attention because several assays can be assembled with greater versatility in reagent column units employing a single sensor, than can be co-immobilized on the surface of a transducer.

Determination of aspartame in dietary food products by a FIA biosensor

A flow injection analysis (FIA) biosensor system was developed for the determination of the artificial sweetener aspartame (N- l-α-aspartyl- l-phenylalanine methyl ester). The system consisted of two enzyme columns, containing purified peptidase and aspartate aminotransferase respectively, immobilized on activated aminopropyl glass beads and an enzyme electrode connected in series. The dipeptide bond of aspartame was cleaved by immobilized peptidase to release aspartic acid which was in turn transaminated by aspartate aminotransferase. The glutamate produced by transamination was monitored by the enzyme electrode which used l-glutamate oxidase immobilized on a nylon membrane in combination with a hydrogen peroxide electrode. The response of the FIA biosensor was linear up to 1 m m aspartame with a lower detection limit of 20 μ m and had good reproducibility (r.s.d.2·2%). The FIA biosensor was stable for at least 30 h of continuous use at room temperature and the immobilized enzymes were stable for at least 1 month when stored in buffer at 4°C. Each assay takes ca. 7–8 min giving a sample throughput of 8 h −1. When applied to aspartame measurements in dietary food products, the results obtained agreed well with those reported by the product manufacturer.

An FIA biosensor system for the determination of phosphate

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and zanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0·7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 μM phosphate, with a minimum detectable concentration of 1·25 μM. phosphate. Each assay could be performed in 5–6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.