GB2185318A - Creatinine assay - Google Patents

Abstract

A sensor for use in the assay of creatinine in blood takes the form of a dry strip sensor comprising a substrate with a printed reference electrode and a printed working electrode, the working electrode comprising a conducting surface carrying an enzyme for converting creatine to creatine e.g. creatininase; an enzyme for converting creatinine to sarcosine and urea e.g. creatinase; an enzyme for converting sarcosine to glycine formaldehyde and hydrogen peroxide e.g. sarcosine oxidase; a peroxidase which can catalyse reduction of hydrogen peroxide to water; and a mediator which can transfer electrons between the conducting surface and the peroxidase when the peroxidase is catalytically active.

Description

SPECIFICATION Creatinine assay The present invention relates to an assayfor detection, measurement or monitoring of creatinine in blood, saliva, urine or othersample.

Background of the invention Creatinine, 1 -methyl-hydantoin-2-imide, is second in importance onlyto urea as a nitrogenous end product excreted by animals. Measurementofthe level of creatinine provides useful information on renal function. The normal range of serum creatinine is proportional to muscle mass and varies with age and sex. For men it lies between 80 and 133 mMol/l andforwomen between 62 and 115 mMol/l. Higher levels are an indication of impairment of kidney function.

Most modern methods for determination of creatinine are based on the Jaffe reaction, which was first described in 1886 and involves reaction between creatinine and alkaline picrate. A red-orange adduct develops, and the concentration of the adduct can be determined spectrophotometrically.

The Jaffe reaction is known to be non-specific when applied to the measurement of creatinine in plasma, due to interference by diverse, commonly-occuring compounds such as protein, glucose, ascorbic acid, guanidine, acetone, cephalosporins and ketoacids such as acetoacetate and pyruvate. Thus, for an assay based on the Jaffe reaction, careful preparation of a blood sample is needed.

Alternative assays for creatinine have been proposed, notably colourimetric methods based on enzymic procedures (see, for example, Clin Chem (1975)211422 orChem Pharm Bull (1980)28,3501).

The 1975 article describes the use of an enzymic cocktail of creatininase, creatine kinase, pyruvate kinase and lactate dehydrogenaseto change the NADH concentration which can be measured by spectrophotometry orfluorimetry. The 1980 article describes the use of creatininase, creatinase and sarcosineoxidaseto generate hydrogen peroxide which is then fluorophotometrically determined by coupling with a peroxidase and diacetyldichlorofluorescein.

Enzymatic colourimetric creatinine assays have recently become available, based on the generation of a quinone dye using the creatininase-creatinase-sarcosine oxidase-peroxidase system. It is recognized that such assays suffer from defects, notably the need to dilute blood samples and also the possibility of interference, for instance by bilirubin, as described in Clin Chem (1984)30,1389.

Objects ofthe invention The need still remains for novel assay procedures suitable for determination of creatinine. It is an object ofthis invention to provide an assay which can be performed rapidly, accurately and at low cost by persons such as general medical practitioners in their own surgery. It is a further, related objectto provide sensors for use in such an assay. Aspecific object ofthis invention is an assay which can be performedwithoutthe need for elaborate preliminary preparation of a blood sample or other form of sample.

Summary of the invention The present invention provides a sensor for use in the assay of creatinine. The sensor is a dry strip sensor, and comprises a substrate with a printed reference electrode and a printed working electrode.

The working electrode comprising a conducting surface, preferably of fineiy divided carbon optionally with carbon tracking, carrying a reagent mix. The reagent mix includes an enzyme for converting creatinineto creatine; an enzyme for converting creatineto sarcosine and urea; an enzyme for converting sarcosineto glycine, formaldehyde and hydrogen peroxide; a peroxidase which can catalyse reduction of hydrogen peroxide to water; and a mediator which can transfer electrons between the conducting surface and the peroxidase when the peroxidase is catalytically active.

In an assayforcreatinine using such a sensor, the conversion of creatinine through to glycine, formaldehyde and hydrogen peroxide results in catalytic activity of the peroxidase which is electrochemically detected by the sensor using the mediator to transfer electrons.

Thus,the present invention further provides an assay permitting detection, measurement or monitoring of creatinine content in a complex mixture, such as a blood sample which can be of undiluted whole blood. The assay is suited for other samples, such as a urine sample or a sample of saliva.

In the assay, creatinine is measured by setting up a sequence of enzyme-catalysed reactions in which the final step is a readily measurable oxidation of the mediator by hydrogen peroxide in the presence of peroxidase. The mediator is itself electrochemically reduced at the working electrode, producing a current related to the amount of creatinine present in the sample. To this end, the working electrode is poised at a small negative voltage relative to the reference electrode.

Features of the invention It is a feature of this invention that the sensor is a dry strip sensor. A similar mix of reagents employed in a wet system would not give good results over a similar concentration range of creatinine.

It is a furtherfeature ofthis invention that the dry strip sensor is produced with the aid of printing, preferably screen printing.

Preferred embodiments For convenience, the sensorfunctions on a micro scale, with small adjacent printed electrodes over which single drop of blood can be located for direct readout of creatinine content.

Preferably, the enzymes inthereagentmixforthe working electrode comprise creatininase (creatinine amidohydrolase) (E.C. 3.5.2.10), creatinase (creatine amidinohydrolase) (E.C. 3.5.3.3), sarcosine oxidase (EC 1.5.3.1), and horse-radish peroxidase (EC 1.11.1.7) The amounts of such enzymes will normally be selected so that a linear response is obtained with variation in the amount of creatinine in the liquid sample. In general, itis preferredto employ approximately one part (expressed as enzyme units) of each of the creatininase, creatinase, and sarcosine oxidase, and from five to twenty parts ofthe peroxidase.

It is to be noted that sarcosine oxidase is itself a flavorprotein which couples to various mediator compounds. However, in the present invention, the peroxidase is also included in the reagent mix in order to achieve reproduceable results.

The mediator may be selected for instance from ferrocyanide, ruthenium compounds, carboranes, polyviologens, one-dimensional conductive salts of TCNQ, haloanils, alkylphenazines, and bis-cyclopentadienyl organometalliccomplexes such as ferrocene and its derivatives. The mediator is preferably ferrocene or a derivative thereof, especially a water-soluble ferrocene derivative.

In a particularly preferred embodiment, the dry strip sensor comprises an elongate, electrically-insulating substrate having a pair of longitudinal, substantially parallel, electrically-conducting tracks thereupon, each track being provided atthesameendwith meansfor electrical connection to a read-out means and provided at the other end with an electrode, one of the electrodes being the reference electrode and the other being the working electrode.

More specifically, such a sensor is suitably in the form of a supporting strip of electrically insulating material such as a synthetic polymer (for instance pvc) carrying at a location intermediate its ends the two printed electrodes supported on electrically conductive printed tracks. Forexample,the electrodes can taketheform oftwo rectangular areas side by side on the strip, one comprising the reagent mix and the other an Ag/AgCI composition as reference electrode, each contacting conductive silver-inktracking located on respective conductive carbon-inktrack. Such areas can be configured to be covered by a single drop of blood for testing for creatinine.If desired, non-rectangular electrode areas, for instance diamond-shaped, semicircular, or triangular areas, can be employed to provide a jointly occupied area for optimised contact by a liquid sample.

The present invention will be further illustrated by way of example and with reference to the accompanying drawings.

Sum mary ofthe drawings In the drawings, Figures 1(a) to 1(d)show diagrammatically successive stages in the screen printing of a sensor electrode ofthis invention capable of detecting creatinine; Figure 2 is a graph of creatinine concentration againstcurrentflow, as measured with an electrode combination manufactured in accordance with Figure 1; and Figure3is a graph showing a comparison between a creatinine assay of this invention and a commercially available creatinine assay kit.

Examples of the invention The manufacture of a sensor electrode of this invention is illustrated by the steps (a) to (d) of Figure 1. In practice, the electrodes will be mass-produced by large-scale screen printing on a substrate sheet, followed by cutting or punching to give individual sensors. For clarity and ease of description, the preparation of a single electrode is shown in the steps of Figure 1.

Figure 1 (b) shows an expanse 1 of PVC sheetfrom which a strip carrying the sensor electrode will eventually be cut, at dotted line 1 a. In a first screen printing, two conductive tracks 2a, 2b of a carbon ink are printed, each 45mm long and 2mm wide at 0.5mm separation.

Figure 1 (b) shows the second screen-printing of a conductive silver ink comprising silver particles in a carrier of resin and organic solvent. On existing carbon track 2a, the silver conductive ink is printed to give a rectangular outer end portion 3, say 2mm by 8mm, a thin connection track 4, and a rectangular inner end portion 5 of 2mm by 8mm. On track2bthe silver ink is printed to give a like rectangular outer end portion 6, a like thin connection track 7, and a minor inner end portion 8, in this case about 2mm square.

Figure 1 (c) shows a screen-printed rectangle 9 of silver/silver chloride reference electrode material of 2mm by 6mm printed over the outer part of end portion 5 of the silver ink on carbon track 1 a.

Figure 1 (d) shows a screen-printed rectangle 10 of working electrode material (the reagent mix as a printable paste) of 2mm by 6mm printed directly on to the conductive carbon of track 2b, at a small spacing 11 from portion 8 of the silver ink electrode.

Bythis expedient of providing the spacing 11, the enzyme content of the working electrode material is not deactivated by contact with silver, but conduction of current is still predominantly by the silver ink ratherthan the carbon ink of lesserconductivity.

If desired, a mesh can be applied over the printed electrodes to give protection. The mesh can be coated with one or more of the electrode reagents, which then need not be included in the screen printed ink.

In use, the sensor is pressed into a socket of a suitable supply and readout equipment (not shown) so that electrical contact is achieved at end portions 5 of the conductive silver ink printing. A drop of blood is placed to cover all of the active electrode 10 and reference electrode 9. The joint areas 10 and 9 occupy a relatively compact region 4.5mm wide and 6mm long, and accordingly a small drop of blood can be used. If desired, a mask region exposing the electrodes 9 and 10 in an uncovered portion thereof, for easy assessment of blood drop coverage, can be provided (not shown) or the electrodes can be, for instance, semicircularwith straight edges spaced apart (also not shown).

The steady state currentfrom the electrochemical reduction is then monitored, for example at - 100 mV. The total charge passed is dependent upon the amount of hydrogen peroxide available to the peroxidase and hence to the concentration of creatinine in the sample.

Such an electrode was calibrated for test purposes against hydrogen peroxide, sarcosine, creatine and creatinine. Standard solutions were prepared by appropriate dilution of a stock 50 mM solution with buffer pH 8.0 containing 0.9% (w/v) KCI as supporting electrolyte.

A sample of buffer containing hydrogen peroside, sarcosine, creatine or creatinine was applied to a target area covering both the working electrode and the reference/counter electrode. The potential was poised at - 100 mV (v SCE) and the current/time transient was recorded.

Calibration curveswereconstructedforthe analytes.

A linear current response with respect to analyte concentration was obtained over respective concentration ranges of O to 3 mM for creatinine, creatine, and for hydrogen peroxide. Such a linear response could not be obtained over the range Oto 3mM for sarcosine, as sarcosine oxidase does not coupleto the mediator at high sarcosine concentrations. The resultsforcreatinineasanalyte are shown in Figure 2.

In further experiments, the response of a sensor electrode of this invention was compared with a commercially available assay kit based on a spectrophotometric enzyme procedure. Figure 3 shows this comparison. There is a good correlation between the two methods.

Thus, a dry chemistry based novel electrochemical kinetic assay which responds in a linear mannerto changing concentrations of hydrogen peroxide, creatine and creatinine has been obtained, which could be used for creatine detection if desired, as well asforthe intended purpose of creatinine detection.

Comparative Example An electrochemical glass ceil was set up using a glassy carbon working electrode, a standard calomel reference electrode, and a platinum gauze counter electrode. Mediator, buffer, peroxidase, and sarcosine oxidase were added in a total volume of 0.9 ml. Additions of sarcosine were made and the potential poised at O mV after a 2 minute incubation period.

There was an increase in current with increasing sarcosine concentration until towards 0.3m M sarcosine. Around this point, a decrease in current was observed. It was concluded that at concentrations of sarcosine above 0.25mM,the sarcosine oxidase was inhibited or was mediated with the oxidized form ofthe mediator.

This problem was not encountered on the dry strip electrode, which istherefore crucial to the electrochemical detection of creatinine.

Claims (6)

1. A sensorfor use in the assay of creatinine in a sample, the sensor being a dry strip sensor comprising a substrate with a printed reference electrode and a printed working electrode, the working electrode comprising a conducting surface carrying an enzyme for converting creatinineto creatine; an enzyme for converting creatine to sarcosine and urea; an enzyme for converting sarcosine to glycine, formaldehyde and hydrogen peroxide; a peroxidase which can catalyse reduction of hydrogen peroxide to water; and a mediator which can transfer electrons between the conducting surface and the peroxidase when the peroxidase is catalytically active.

2. Asensorofclaim 1, which comprises an elongate, electrically-insulating substrate having a pair of longitudinal, substantially parallel, electrically-conducting tracks thereupon, each track being provided atthesameendwith means for electrical connection to a read-out means and provided at the other end with an electrode, one of the electrodes being the reference electrode and the other being the working electrode.

3. A sensor of claim 1, wherein the electrodes are configured to be covered by a single drop of sample.

4. An assay for creatinine in a sample, wherein the creatinine is amperometrically quantitated using a sensoras defined in claim 1.

5. An assay of claim 4, wherein the sample is undiluted whole blood.

6. An assay of claim 5, wherein the sample is a single drop of undiluted whole blood.