Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
  • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/58—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving urea or urease

Definitions

• the present invention relates to methods of determining the concentration of an analyte in each individual sample of a sequence of samples by means of an analyte sensor.
• the methods according to the invention has the features of claim 1 or claim 2 or claim 3.
• the preferred embodiments have the features of claims 4-9.
• the analyte sensor may be an electrochemical sensor, an optical sensor or any other suitable sensor means.
• the fluid element is stationary and is held by any suitable support means, such as a porous or fibrous member in the shape of a film, a sheet, a block, a cylinder or any other suitable shape, e.g. a ceramic pin or a thread, a hydrophilic or porous membrane, a gelled structure etc..
• a suitable support means such as a porous or fibrous member in the shape of a film, a sheet, a block, a cylinder or any other suitable shape, e.g. a ceramic pin or a thread, a hydrophilic or porous membrane, a gelled structure etc.
• the chemical composition of the fluid element may be maintained by conditioning the fluid element with the carrier fluid prior to the contact with each individual sample.
• the fluid element is preferably an aqueous composition.
• the sample may be fed to the analyzer in any conventional manner, e.g. by injection or aspiration.
• the sample is transported through the analyzer by $ means of a flowing carrier fluid.
• the carrier fluid may constitute the conditioning medium of controlled chemical composition.
• There may be direct contact between the sample and the carrier fluid or there may be a plug of an immiscible fluid such as a plug of oil or of gas separating sample and carrier fluid.
• the analyzer may be of the conventional flow injection analyzer type.
• the analyzer will be able to handle in series more than 30 samples per hour.
• Fig. 1(a) shows a FIA manifold for the enzymatic determination of urea
• Fig. 1(b) shows an exploded view of a gas diffusion/detection unit shown in Fig. 1(a).
• Fig. 2 shows a calibration graph for urea?
• Fig. 3 shows a FIA manifold for the detection of wea using an ion-selective electrode
• Fig. 4 shows a FIA manifold for the detection of glucose or lactate
• Fig. 5 shows calibration curves for glucose
• Fig. 6 shows a 25 mM glucose standard record
• Fig. 7 shows the glucose response for a glucose o standard with and without paracetamol.
• Fig. 1 (a) shows a FIA manifold for the enzymatic determination of urea, comprising an integrated gas diffusion/detection unit, an exploded view of which is shown in (b) .
• the sample (S) is injected into the donor stream (buffer) and propelled to part B of the flow cell, in which urea is degraded in the enzyme-containing membrane (M) .
• the generated ammonia diffuses via the gas permeable membrane (G) into the acceptor (indicator) stream, where the resulting pH change is monitored by reflectance measurement by an optical fibre (F), bifurcated at the remote end to communicate with a light source and a photo diode detector (D).
• I and O denote in- and outlets for the donor and acceptor streams, respectively; GF, gold foil; W, waste.
• the flow cell which is made part of a FIA microconduit, is assembled by means of 4 screws.
• Fig. 2 shows a calibration graph for urea in the range 0-35 mM as obtained with the FIA system depicted in Fig. 1.
• Fig. 3 shows a FIA manifold for -the detection of o urea using an ion-selective electrode with detection of enzymatically generated ammonium, comprising a syringe pump (PI); a peristaltic pump (P2); an injection valve; a pH meter; a urea electrode (E2) ; and a flow-through reference 5 electrode (El).
• PI syringe pump
• P2 peristaltic pump
• E2 a urea electrode
• El flow-through reference 5 electrode
• Fig. 4 shows a FIA manifold for the detection of glucose or lactate using an amperometric electrode with detection of enzymatically 0 generated hydrogen peroxide, comprising a syringe pump (PI); a peristaltic pump (P2); an injection valve; a BBC computer; a potentiostat; and a glucose electrode (GE) or a lactate electrode (LE) .
• PI syringe pump
• P2 peristaltic pump
• an injection valve a BBC computer
• GE glucose electrode
• LE lactate electrode
• Fig. 6 shows a 25 mM glucose standard recorded at io high paper speed to demonstrate the fast rate of reaction.
• Fig. 7 shows the glucose response for a 15 mM glucose standard with and without 360 uM paracetamol as a function of the applied is potential.
• a miniaturized flow injection system for the assay of urea in undiluted whole blood is described. Based on the optical determination of o ammonia, the system incorporates a flow cell combining gas diffusion and optosensing, the separating barrier betweeen the donor and accepting streams consisting of a sandwich of a hydrophilic membrane between which is contained a s gel of covalently immobilized urease.
• the sample urea content is quantified by the colour change of an acid-base indicator contained within the acceptor stream. Within the physiological range the measurement is not affected by variations in 0 the pH value, the buffering capacity or the
• the system is 5 stable for at least one week of continuous use.
• Urea is the final product of human protein metabolism. Determination of blood urea makes it posible to trace and follow changes in protein ° and amino acid composition, and the concentration of urea in blood is an important index of renal, function. In clinical chemistry blood urea is traditionally asayed in the serum or plasma fraction, yet the ultimate goal is to perform the measurement in the original sample matrix since this requires a minimum of manipulations, and hence a shorter time to obtain the analytical result, such an apparoach furthermore facilitates the possibility of continuous monitoring. Besides, the importance to confine manual handling of blood samples has been emphasised in recent years due to increased occurrence of contagious diseases.
• urease is a rather inexpensive enzyme, procedures employing immobilized rather than soluble enzymes are preferred, because immobilization of the catalyst not only offers increased stability of the activity of the enzyme, but readily permits its integration into automated systems. Immobilization of urease can be -achieved in many ways (1-4), and the majority of these techniques provide excellent reactors and/or membranes which are stable for extended periods of time.
• Urea is frequently determined by the indophenol colorimetric method (5), yet most of the work reported in the literature has been devoted to development and use of enzyme electrodes.
• the nonactin/monactin electrode (2) provides poor o selectivity for ammonium ions over potassium, but the interference can be compensated for by introducing a second ammonium selective electrode for NH- and K background detection (4).
• Sensing of the pH changes generated in a 5 urease-gel, wrapped around a pH electrode, is another approach (6-8). This type of ammonia sensor yields excellent selectivity for ammonia over ions in the solution (9), yet besides being pH sensitive its response is rather slow.
• a o hybride urea sensor consisting of an immobilized layer of nitrifying bacteria with ultimate detection of oxygen with an oxygen electrode has been reported (10).
• An ammonia sensitive In/Pd MOS capacitor (11-13) has been suggested as an s alternative to the ammonia gas electrode with the added advantage of being inexpensive.
• a FIA microconduit system for the assay of urea in undiluted whole blood is described.
• the system incorporates a flow cell combining gas diffusion and optosensing, the separating barrier between the donor and accepting streams consisting of a sandwich of a hydrophobic gas permeable membrane and a hydrophilic membrane between which is contained a gel of covalently immobilized urease.
• the ammonia generated by the enzymatic degradation of the urea in the sample solution is determined via the colour change of an acid-base indicator contained within the acceptor stream, o the change in reflectance being a function of the ammonia diffused through the gas permeable membrane and thus the sample urea content.
• the measurement is not affected by variations in the pH value or the 5 buffering capacity of the sample solution, nor is it, due to the geometric construction of the flow cell, prone to interferences owing to the colour of the sample solution itself.
• FIA manifold depicted in Fig. la, comprising an integrated gas diffusion and detection unit (for details, see below) accommodating an 5 acceptor (indicator) stream and a donor (buffer) stream.
• the sample solution (20 ⁇ l) is injected into the latter, the distance between the injection port and the the gas diffusion cell being made as short as possible (2,5 cm, corresponding to 5 ⁇ l ) .
• the rotary injection valve (15) and the gas diffusion/detection unit were incorporated into a FIA microconduit (14, 15).
• the gas diffusion/detection unit (Fig. lb) was constructed of black PVC.
• the unit comprises two separate parts (A and B) between which, when assembled by four screws, the membrane sandwich is contained.
• part A Into part A is milled a 1 x 1 x 20 mm groove communicating with two perpendicularly drilled holes (0,5 mm ID) serving as in- (I) and outlets (0) for the acceptor stream.
• part B embodies a 1 x 1 x 10 mm groove served by in- and outlets for the donor stream.
• the membrane sandwich consists of a gas permeable membrane (G) ⁇ Celgard 2500, hydrophobic polypropylene polymer, thickness 25 ⁇ m, pore size 0.004 ⁇ i , Celanese, Belgium), and an enzyme membrane (M) (see below).
• G gas permeable membrane
• M enzyme membrane
• the optical measurement of the colour of the acid-base indicator is effected by reflectance, the nontransparent white gas permeable membrane serving as a reflecting opaque background, which, in order to further improve the reflected signal, is supplemented by the incorporaration of a thin foil of gold (GF) placed opposite the optical fibre.
• GF thin foil of gold
• the donor and the acceptor streams were propelled by means of an Ismatec Mini-S-840 peristaltic pump at 0.1 ml/min. and 0.3 ml/min., respectively.
• the colour change in the acceptor stream was monitored by a home-made fibre optical diode photometer which transmits and receives through a single optical fibre.
• the photometer is constructed to emit chopped light at two different wavelengths, that is, at 820 nm, at which the absorbance of the acid-base indicator (bromothymol blue, BTB) is independent of pH (i.e., the ensuing reflected signal received at this wavelength serves as a reference level), and at 635 nm, where the absorbance of BTB is a function of pH.
• the signal from the optical fibre photometer was sent to a recorder (Radiometer Servograph REC 61, furnished with a REA-112 high-sensitivity interface), the peak height at peak maximum serving as the analytical readout.
• the carrier (donor) stream solution consisted of 0.1 M TRIS buffer adjusted to pH 9.2.
• the acceptor stream solution was 0.04% (w/v bromothymol blue (Merck) dissolved in 24% aqueous ethanol.
• pH of the solution Prior to use the pH of the solution was adjusted to 6.2-6.3 with 1.0 M NaOH, s corresponding to 5-10% of the indicator initially being on the alkaline form, the blue colour of which was monitored at 635 nm.
• aqueous urea standards in the range 1-35.0 mM used for calibrating the system were prepared by o appropriate dilutions with 0.01 M phosphate buffer of pH 7.2 of a 35.0 mM stock solution made by dissolving 2.102 g of urea (Riedel-de Haen) in 1 1 of 0.01 M phosphate buffer of pH 7.2.
• Immobilized Enzyme Membranes 40 mg of urease from "jack beans” (E.C. 3.5.1.5, Sigma, 70 U/mg) and 30 mg of bovine serum o albumine were completely dissolved in 0.3 ml of phosphate buffer (10 mM, pH 7.2) and 20 11 of glutaraldehyde (25% aqueous solution) was added.
• a controlled dispersion of the sample zone while it is passing through the system towards the s detector is essential in Flow Injection
• the sensing process in fact, occurs in two steps: In the first step, where the injected sample is in contact with the enzyme layer, the degradation of urea to ammonium takes place.
• the first step where the injected sample is in contact with the enzyme layer, the degradation of urea to ammonium takes place.
• the amount of ammonia ultimately diffusing into the acceptor stream of the system in Fig. 1 is of such magnitude that 5 the ensuing absorbance change registered by the optical system is sufficient to obtain a reliable signal.
• additional signal enhancement may be o obtained by operating the donor stream and especially the acceptor stream in a stopped-flow mode (14) .
• Assay of Standards, Plasma and Whole Blood Samples For actual urea assays the FIA manifold depicted in Fig. la was used. A typical calibration graph obtained with a series of aqueous urea standards is shown in Fig. 2.
• the dynamic measuring range is 0-35 mM urea with a linear range from 0 to 6 mM urea (the normal physiological range).
• the curvature of the calibration plot might be due to several factors. In order to obtain a linear relationship between the urea concentration and the recorded absorbance signal the following two conditions must be fulfilled:
• the activity of the membrane sandwich remained constant during at least one week of continuous daily operation, upon which some deterioration in the measured signal appeared.
• Urea in whole blood, enzymatically degraded by immobilized urease followed by colorimetric detection of the ammonia generated can be 5 determined in a FIA system provided that the dispersion coefficient of the injected sample at the point of quantification is as close to unity as possible. With this condition fulfilled, the analytical readout is independent of any ° variations in the pH, the buffering capacity and the hematocrit level of individual samples.
• ABSTRACT 5 A flow injection system for the assay of urea in undiluted whole blood is described. Quantification of urea is achieved by means of an ammonium ion-selective electrode furnished with a membrane incorporating immobilized urease o measuring the enzymatically generated ammonium which is directly related to the concentration of the urea. The interference from potassium is reduced by adjusting the potassium concentration in the carrier stream and in the aqueous s calibration solutions to 4.0 mM. However, a complete elimination of this interference is achieved by measuring the actual potassium concentration in the respective sample using an external instrument, thus mathematically 0 correcting for the additional contribution to the signal due to the presence of K .
• the linear measuring range is 1-40 mM urea with a sample frequency of 40 h ⁇ and a standard deviation of 1% for whole blood samples.
• the result of the 5 measurement is obtained within 25 sec. from the time of injection. Variations in the hematocrit level of the sample has no effect on the measurement.
• the method is in excellent agreement with the one used routinely at a local 0 County Hospital.
• the sensor is stable for more than 30 days.
• Enzyme sensors or electrodes as they are conventionably called, consists of an electrode covered with a membrane layer containing one or possibly more enzymes. This type of electrodes have proved to be useful in biochemical analysis and have been developed for a variety of substrates. The enzyme catalyses the reaction of the substrate to be measured by generating or o consuming species for which the inner sensor is selective. In most cases, covalently immobilized rather than physically entrapped enzymes are preferred, because covalent immobilization techniques generally offers increased stability 5 of the enzyme activity. Enzyme electrodes have the advantage of being simple, reliable, essentially reagentless and sensitive, besides being highly selective due to the inherent selectivety of the enzymes.
• nonactin/monactin electrode used provides poor selectivity for.ammonium ions over potassium.
• An urea electrode of similar composition was used by Yasuda et.al. (2) in conjunction with a FIA system, where the interference from potassium was compensated for by using an additional ammonium ion-selective electrode (without enzyme) for K detection. This system proved to be s linear in the range 3.6-107 mM urea. However, attempts to determine urea in whole blood was impeded due to influence by the hematocrit level of the sample.
• the FIA manifold therefore should, for pratical assays, 5 be operated at a D-value as close as possible to 1, that is, the distance between the injection port and the detection position should be minimized (3, 4).
• E standard electrode potential 0 (temperature dependent)
• R is the gas constant
• T is the absolute temperature (K)
• F is the Faraday constant
• ax is the ion activity - ⁇ .
• y J x denotes the activity - ⁇ coefficient which is governed by the total ionic strength, I, of the sample.
• the ionic strength is defined as:
• c. concentration of each ionic species present in the sample and z. is the charge of each ionic species present in the sample.
• the activity coefficient can, up to an ionic
• a and B are general constants assuming a value of 0.5115 and 0.3291 respectively at 25 C and a is the specific ion size parameter.
• Kxi. values When.Kxi. values are known, it is p e ossible to compensate for interferences by making appropriate calculations. However, it must be remembered that the K value is not a constant. It varies to some degree with the concentration level of the primary ion (analyte) and of the interfering species, sample composition, time and temperature.
• K values can be determined in the following way: Firstly, the electrode potential is measured against a suitable reference electrode in a solution where the activity of the primary ion (analyte) is a (E. ) . Secondly, the electrode potential is measured in a solution where the activity of the primary ion (analyte) is identical to that of the first solution and the activity of the interfering ions is a.(E 2 ).
• E. activity of the primary ion
• E 2 activity of the interfering ions
• FIA manifold depicted in Fig. 3, comprising a flow through reference electrode (El -Radiometer A/S, K1702), a rotary injection valve, and an urea electrode (E2) .
• the sample solution (45 l) is injected into the carrier stream, the distance between the injection port and the 5 ⁇ l flowcell (FC2) being made as short as possible in order to ensure minimum dispersion (D value) of the sample zone.
• the urea electrode was made by placing an immobilized urease membrane (cf. below) on top of a small ammonium ion-selective electrode consisting of a actin/monactin PVC membrane. The diameter of the active surface was 1.5 mm.
• the enzyme electrode was connected to a Radiometer A/S PHM 64 pH meter and the potentials were recorded on a Servograph REC 61 recorder furnished with a REA 112 high-sensitivity interface (Radiometer A/S), the peak height at peak maximum serving as the analytical readout.
• the carrier stream was propelled by means of a Radiometer A/S ABU80 syringe pump (P2) at 0.125 ml/min..
• the concentration of potassium in actual whole blood samples was measured with a KNa instrument from Radiometer A/S. All connections were made with microline tubings (o,5 mm i.d. ) .
• the carrier stream solution consisted of an isotonic HEPES buffer comprisning 4 mM KCl, 86 mM NaCl, 60 mM Na-HEPES and 0.55 g NaEDTA/L. All chemicals were of analytical-reagent grade.
• aqueous urea standards in the. range 1-40 mM used for calibrating the system were prepared by dissolving appropriate amounts of urea in the isotonic HEPES buffer. Human blood were obtained from the clinical chemistry department of a local hospital (Copenhagen County Hospital in Herlev) .
• an isotonic buffer (1-0.15 M) solution should be employed as carrier stream to prevent the whole blood from hemolysing.
• a phosphate buffer is often used in conjunction with enzyme analysis because it is compatible with many enzymes.
• phosphate has the ability to form ammonium complexes, thus reducing the analytical signal due to masking effect.
• a biological HEPES buffer was chosen as carrier stream.
• the "naked" ammonium ion-selective electrode exhibits a linear calibration curve in the range 10 -3- 100 M ammonium. Since the physiological range of urea is 1-40 mM it is obvious that the enzymatic conversion of urea to ammonium has to be greater than 50% (1/2 mol of urea is converted into 1 mol of ammonium) to obtain an ammonium concentration within the linear range of the ASE.
• a membrane containing covalently immobilized urease (cf. above) was made and tested in the FIA system.
• the linear range of the sensor was 1-40 mM urea with a sample frequency ' of 40 h ⁇ and a standard deviation of 1%.
• the enzyme membrane was found to be stable for more than 1 month due to the increased stability of the covalently immobilized enzymes.
• K 1. from electrode to electrode was evaluated with and without the incorporation of an enzyme membrane using 5 different electrodes (ASE.-ASAg).
• the K. value was only determined in the range 1-10 M urea and 1-10 mM ammonium. At higher concentration levels the interference from potasiu is of less importance. As expected, the "naked" ASE can be made very reproducible (cf. Table 2) owing to the uniformity of the PVC ion-selective membranes. TABLE 2 Determination of K. as a function of the
• K indicates the K value at an ammonium x concentration of x mM
• Kx indicates the K value at an urea concentration of x mM
• a typical calibration graph is obtained with a series of aqueous urea standards. At urea 5 concentrations higher than 40 mM the calibration graph is no longer lineari The curvature of the calibration plot at concentrations above 40 M urea might be due to several factors.
• the following three conditions must be fulfilled: (a) The kinetics of the urease enzyme has to follow that of pseudo-first order reaction conditions, i.e. the amount of urease must be in excess and CureaD « K-, s (Michaelis-Menten constant).
• the coefficient of variation for 10 measurements l o of a blood sample was 1.06%.
• the concentration of potassium in the actual whole blood samples were 1 measured using an external static apparatus and mathematical
• the correlation coefficient is 0.999. up to - 15% can occur. Even though the actual determinations of the potassium have been made in a static system, there is no reason, whatsoever, that a FIA system similar to that used for the derermination of urea, should not be used in conjunction with a potassium ion-selective electrode, thus making a parallel determination of these two analytes.
• a computer controlled flow ' injection system for the assay of D-glucose and L-lactic acid in undiluted plasma is described. Quantification of glucose/lactate is achieved by coupling of an immobilized glucose oxidase/lactate oxidase enzyme membrane with an amperometric electrode, thus measuring the enzymatically generated hydrogen peroxide, which is directly related to the concentration of glucose/lactate.
• the amperometric electrodee consists of a platinum anode, to which a potential of +600 mV is applied, and a Ag/AgCl cathode. The diameter of the exposed electrode surface is 2.5 mm. The linear range is 0-40 mM and 0-10 mM for glucose and lactic acid respectively.
• the sample ffrreeqquueennccyy i iss 66C0 h with a standard deviation of less than 1.5%.
• Analysis of blood glucose are used to detect hyper- and hypo-glycaemia, both of which can result from a variety of different disorders, many of them of endocrine origin.
• hyperglycaemia is diabetes mellitus, and elderly patients may present in hyperosmolar non-ketotic coma with blood glucose levels as high as 60 mmol/L.
• serial blood glucose levels are required to monitor diabetes on treatment, and to detect and ultimately to prevent gestational hyperglycaemia which although not necessarily harmful to the mother, may be damaging to the foetus (1) .
• the arterial lactate concentration is now recognized as a very good indicator of severity and prognosis (2, 3).
• the increases in blood lactate are due to a critical reduction in oxygen delivery such that aerobic metabolism through the Krebs cycle cannot be sustained.
• the severity of the defect in oxygen delivery is proportional to the increase in lactate generated through the emergency pathway of glucose metabolism from pyruvate.
• Samples (25 ⁇ l ) were injected into the single line FIA system depicted in Fig. 4 by means of a 5 rotary valve operated by a step motor.
• the distance between,the injection port and the 10 JJ.1 flow cell (FC) was made as short as possible in order to ensure minimum dispersion of the sample zone.
• a special designed electrode comprising a central 0.4 mm diameter platinum working electrode with an outer 2.5 mm-diameter silver ring as the reference electrode was polarized at + 600 mV and used for hydrogen peroxide detection.
• the electrode system was connected to a home-built potentiostat and the current intensity as a function of time was recorded by a BBC computer from Acorn DFS utilizing the built-in analogue to digital facility.
• the carrier stream 0 was propelled by means of a Radiometer A/S ABU80 syringe pump (PI) at 1.0 ml/min.
• PI Radiometer A/S ABU80 syringe pump
• the BBC computer facilitated the data aquisition and the operation of the sampling pump, the syringe pump and the injection valve.
• the isotonic phosphate buffer used as carrier stream was prepared by dissolving 1.12 g of NaEDTA, 1.84 g NaBenzoate, 3.04 g NaH 2 P0 4 , 13.80 g Na 2 HP0 4 and 5.44 g NaCl in 2 1 of distilled water.
• high rejective glucose or high rejective lactate membranes (3.0 mm diameter) purchased from Yellow Springs Instrument Co. was placed on the electrode tip and fixed with an outer polycarbonate membrane (Nucleopore, 5 ⁇ m thick, 0.08 urn pore size) held with an o-ring.
• control of the sampling and syringe pump 5) presentation of data i.e. the concentration in mM, the time of peak maximum and the sample no.
• Eqn. (11) is not valid for enzyme systems which require additional cosubstrate(s) or coenzyme(s). Nevertheless eqn. (11) has proved 5 to be a very useful approximation as long as the cosubstrate(s) or coenzyme(s) are in excess. In order to obtain a linear relationship between the glucose or the lactate concentration and the recorded signal the two following conditions must be fulfilled:
• the physiological range of glucose and lactate is larger than K M for each of the two enzymes.
• first order kinetics can only be achieved by including an outer membrane with low permeability to restrict diffusion of the analyte to the underlying enzyme layer.
• restricted diffusion of analyte to the enzyme can be controlled by the time of exposure of the sample to the electrode, the dependent variable being the flow rate.
• Fig. 5 This is illustrated in Fig. 5, where the linear range of the glucose electrode is expanded from 20 to 40 mM glucose by increasing the flow rate from 0.5 to 1.0 ml/min.
• the influence of the oxygen level on the measured glucose value was investigated.
• a series of aqueous glucose standards containing 10, 20 and 30 mM glucose was prepared and the pOfact level was adjusted in the range 1-140 mm Hg by means of a tonometer.
• the samples were injected into the FIA system and the glucose concentration as a function of p0 2 was monitored (cf. Table 6).
• glucose and hydrogen peroxide are not present in the enzyme membrane which is equilibrated with oxygen from the carrier solution.
• glucose diffuses into- the enzyme membrane and the degradation of glucose takes place, while the undiluted sample zone is swept away by the carrier stream.
• oxygen diffuses between three and four times faster than glucose (23,24). Since the time of exposure of the sample to the electrode is very short the oxygen from the carrier stream is transferred to the enzyme membrane so fast, that the oxygen concentration in the membrane is always close to the oxygen concentration of the carrier solution. Thus, it is possible to disregard the p0 2 level of the sample as a limiting factor with respect to the enzymatic reaction.
• a typical calibration run is obtained with a series of aqueous glucose standards. It is worth while to notice that the result of the analysis is completed within 5 sec. from the time of injection.
• the linear range is 0-40 mM glucose with a sample frequency of 60 h " and a standard deviation of less than 1.5%.
• a potential of 600-650 mV is chosen because the current is not affected by small changes of the potential in this range.
• a potential of +300 V can be used in conjunction with a three electrode system.
• the high rejective glucose oxidase membrane was then replaced with a high rejection lactate oxidase membrane, and a series of 11 blood plasma samples, supplied by the Copenhagen County 5 Hospital in Herlev (CCHH), were assayed in order to compare the FIA method with the YSI lactate analyzer.
• a FIA system has been applied for detection of glucose and lactate in plasma using an amperometric electrode with detection of enzymatically generated hydrogen peroxide.
• the analysis is completed within 5 sec. and there is no interference from other species.
• the linear range is 0-40 mM glucose and 0-10 mM lactate with aa ssaammppllee ffrreeqquueennccyy ooff 6600 hh ⁇ and a standard deviation of less than 1.5%.

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