IE47123B1

IE47123B1 - Analytical device

Info

Publication number: IE47123B1
Application number: IE1422/78A
Authority: IE
Original assignee: Gist Brocades Nv
Priority date: 1977-07-15
Filing date: 1978-07-14
Publication date: 1983-12-28
Also published as: IT7868681A0; NO782452L; DK317078A; FI782252A; AU3804178A; BE869022A; JPS5448592A; NZ187860A; IE781422L; FR2397636A1; DE2831083A1; SE7807844L; FR2397636B3; IT1108116B; LU79976A1; NL7807581A

Abstract

Analytical instrument comprises a capillary of inert matl. (esp. glass) with internal diam. (i.d.) >1mm and with >=1 reagent fixed to the inner surface. Pref. the i.d. is 1-1.5 mm and the vol. of the capillary bore is about 100 mu l. The wall thickness and i.d. of the tubes should not vary by >2% from the mean value. Kits contg. a number of these tubes are also claimed. Used for chemical and microbiological analysis partic. of body fluids; specifically for assaying lactate. Compared with very narrow capillaries previously used, these tubes are easier to handle, more reproducible and suitable for a wider range of qualitative and semi-quantitative assays.

Description

The invention relates to devices for carrying out chemical and micro-biological analyses, for instance in urine or other biological fluids, to methods for producing such devices and to analytic methods in which said devices are used.

It is known to use for rapid tests reagent-impregnated strips of paper or porous plastic or tablets. Only a limited number of chemical reactions proceed properly in such a test strip or tablet.

The strips or tablets are dipped into the fluid to be analysed and change colour when a certain substance is present in the fluid. Such test methods generally provide data of a qualitative character; in some instances semi-quantitative data are obtained. For instance, by using a reagent strip in combination with a reflectometer, quantitative determination of limited accuracy can be carried out. For more accurate determinations it is generally necessary to use more complicated procedures, which are time-consuming and require elaborate equipment In principle many tests can be automated but in view of the expensive apparatus required, this is only warranted in large laboratories in which large series of the same test have to be carried out.

It is. further known to carry out radio-immunoassay in plastic tubes (K. Catt and G.W. Tregear, Science, 158, 1570 (1967) or in capillaries with inner walls of high polymers (including glass) (German Patent No. 2 530 957). 47133 The assays are carried out with tubes or capillaries of which the inner wall is coated with antibody. According to German Patent specification the diameter of the capillaries should preferably be 1mm or less.

It has now been found that a great variety of analytical reactions are suitably performed in capillaries having a diameter of more than 1mm. Such capillaries provide cheap, easy to handle vessels for carrying out quantitative chemicals determinations.

The invention provides a method of preparing a device for the quantitative chemical analysis of liquids, the device comprising a capillary having a predetermined amount of reagent or reagents adhering to the inner surface thereof, which method comprises dipping a capillary of an inert material and having an internal diameter greater than 1mm into a liquid containing one or more reagents in suspension or IS solution and, if necessary, an adhesive, so that the liquid rises in the capillary action to a predetermined level, removing the capillary from the liquid and removing the liquid contained in the capillary by lyophilisation to leave the reagent or reagents adhering to the inner surface thereof.

The amount of reagent may vary from a monomolecular layer to the maximum amount that can be taken up in the bore of the capillary without interfering with the capillary action.

In this specification the term reagent refers not only to chemical substances but also to micro-organisms that may be effected by the test fluid.

The material of the capillary should be wettable by the fluid to be tested. For the examination of aqueous liquids glass capillaries are most suitable. The internal diameter of the capillary should be such that the test fluid will rise in the capillary when it is dipped in the fluid. When the diameter is less than 1 mm the reagent will leave insufficient room for the fluid and when the capillary is too wide, lack of capillary action or insufficient capillary action, resulting in a very small volume of liquid being sucked up, will preclude proper use. An inner diameter between 1 and 1.5 mm is preferred. The length of the capillary will preferably be between 30 and 150 mm. Commercially available micropipettes of approximately 100 μ 1 may be suitably used for the purpose of the invention.

When the cohesion of the reagent or its adhesion to the capillary material is insufficient to keep it in the capillary, an adhesive may be used. Polymers of biologic or synthetic origin, such as proteins, cellulose derivatives or polyvinylpyrroliodone are examples of suitable adhesives. It will be understood that an adhesive should be chosen that does not interfere with the test to be performed.

According to another feature of the invention a chemical or microbiological determination is made by sucking up a liquid to which one or more reagents, diluents or other auxiliary substances may be added, into a capillary, having adhered to its inner wall a reagent or reagents for the subtance to be determined.

In both cases the effect of the reagent in the capillary on the test fluid is observed, e.g. change of colour or inhibition of micro-organism growth.

Examples of substances that are suitably added to the test liquid are unlyophilisable reagents such as alcohols. Reagents which on lyophilisation interact with one another can be kept apart by having the bulk of the reagent compounds lyophilised in the capillary and any other reagents necessary for the reaction added to the liquid to be tested.

The capillaries according to the invention may be used for semiquantitative and quantitative determinations. It will be appreciated that a qualitative determination can be carried out with a single capillary.

When a reaction is observed between the test fluid and the reagent in the It capillary, this indicates that a specific substance is present above a certain minimum concentration. Accordingly, when a series of capillaries is used with varying, preselected amounts of reagents, a semi-quantitative determination can be made.

Capillaries of an inert material having preselected amounts of one or more reagents adhering to their inner surface are therefore another feature of the invention. The amount of reagent in the capillary can be controlled by sucking up a liquid with a known concentration of the reagent to a chosen level in the capillary. The determination may be based on common chemical reactions, but the reagent may also be a component of an antigen-body reaction so that the capillaries may be used in immunoassay techniques.

In tests involving common chemical reactions, it is preferred to use reagents that are easily dissolved or suspended by the test fluid. In such cases the concentration of the reagent in the fluid will be determined by the dimensions of the capillary and the amount of reagent contained by it. 47133 In immuno-assay techniques, a reagent (e.g. antibody) is used that remains fixed to the wall, so that a reaction takes place at the interface of fluid and reagent-covered capillary wall. In such cases the capillaries have the advantage that the reagent layer will not easily be damaged. The capillaries may be easily drained, e.g. by contacting them with a piece of blotting paper, and refilled with a liquid to a desired level, corresponding to a desired volume. Thus the capillaries of the invention provide a means to carry out easily multi-step analyses without using dispensing equipment, such as pipettes etc.

The occurrence of a reaction can be observed visually (change of colour, forming a precipitate etc.), or by instruments (e.g. change of absorbance in the U.V or I.R. portion of the spectrum). It will be understood that the material of the capillary should be transparent for the wave length(s) involved when optical methods are used.

Common analytical techniques may be used for quantitative determination of the concentration of the reaction product in the capillary.

Optical instruments can be used to measure changes in colour or absorbance in the visible, U.V. or I.R. portion of the spectrum.

Radioactivity measurements can be used in radio-immuno-assay determinations where either the antigen or the antibody is coated or covalently bound to the wall of the capillary or to.an intermediary layer adhesing to the wall.

Where the reaction involves changes of electrical properties (e.g. conductivity), such changes can be measured by the appropriate instruments.

In some cases a single measurement after the reaction is completed will suffice. In other instances it will be necessary to follow the change of the parameteruider test during a certain period of time, e.g. in enzyme-immuno-assays where the indicator-enzyme reaction is followed with an absorptiometer.

Clotting reactions may be followed by means of viscosity measurement, e.g. by observing the flow of the fluid when the capillary is turned upsidedown or the time needed to empty the capillary on a piece of blotting paper. The growth of micro-organisms in the capillary can be measured by turbidimetry. In this way sensitivity to antibiotics can be measured or vice-versa the concentration of antibiotics can be determined with the help of a standard suspension of sensitive micro-organisms.

This enumeration of techniques in which the capillaries can be applied is by way of example only and does not limit the scope of the invention. Generally speaking the capillaries can be used in the analysis of all biological fluids and secretions and in the determination of all substances of diagnostic interest.

As a consequence of the relative large diameter of the capillaries (as compared to those used according to German Patent No. 2 530 957) small variations in dimensions have no large influence on the confidence limits of quantitative determinations. For certain purposes, however, particularly in quantitative determinations using optical techniques, the variations should remain within certain limits. Preferably the inner diameter and the wall thickness should not deviate more than 2% from the mean value within the same capillary. When a determination 47133 involves the use of two or more capillaries in a comparative measurement (e.g. of light absorbance), the differences between their dimensions should preferably also be within the same limits.

A further feature of the invention is therefore a package or kit comprising two or more capillaries of which the wall thickness and inner diameters do not deviate more than 2% from the mean values of the capillaries in the package or kit.

Examples of tests that can be carried out are: in urine: lactate, glucose; in serum or plasma: glucose, cholesterol, blood urea nitrogen (B.U.N)., uric acid, triglycerides, total proteins, alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), creatine phosphokinase (CPK), β-hydroxybutyrate, lactate dehydrogenase (LDH); in amniotic fluid: enzyme or metabolites the presence or absence of which is indicative of inborn errors of metabolism, e.g. galactokinase, glactose-l-phosphate eridyl, 1.4-glucosidase, aryl suphatase A,a 1-iduronidase, pyruvic, lactic and 2-oxoglutaric acids: in cerebro-spinal fluid: glucose, protein.

Generally known reactions can be used in these determinations. For instance, lactate can be determined with LDH and a dye, e.g. dichlorophenolindophenol (DC1P); LDH can be determined with lactate and a dye (e.g. DC1P) - glucose can be determined with glucose-oxidase, peroxidase and a dye (e.g. o-toluidine); cholesterol can be determined with cholesterol-oxidase, peroxidase and a dye; protein can be determined with a biuret-like reagent or a pH-indicating dye, using the so-called protein effect; β-hydroxybutyrate can be determined with β-hydroxybutyrate dehydrogenase and a dye.

The capillaries of the invention are suitably used in determinations involving the enzymatic reduction of the coenzyme NAD or NADP (nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phospate) to NADH or NADPH (the reduced forms) coupled with the reduction of a redox indicator dye, dichlorophenol underphenol(DClP), in the presence of an electron carrier, phenazine methosulphate, (PMS).

Any substance or enzyme which takes part in reactions which result in the reduction of NAD or NADP can be used by this method of detection, so that the system provides a method for measuring a number of diagnostically important metabolites or enzymes present in biological fluids.

The preparation-of a test-capillary according to the invention is described in the following Example. 47133 EXAMPLE 1 In one litre of distilled water 2.4 millimoles of dichlorophenol underphenol(DC1P) were dissolved and to this solution 10 microlitres of 1 N NaOH were added. Pure oxygen was then bubbled through the solution in order to obtain complete oxidation. This solution was diluted with 5 litres of a solution which contained per litre 0.18 moles of potassium phosphate buffer and 1.2 millimoles of EDTA. Then yeast-lactate dehydrogenase (Y-LDH) (Y-LDH = L-lactate ferricytochrome c oxydoreductase, E.C. nr. 1.1.2.3.) was dissolved in an amount containing 90,000 units and having a specific activity of 17.2 units per mg of protein. The final concentration of DC1P was determined to be 0.4 mm mmol per litre. Glass capillaries having an inner diameter of 1.5 mm and a length of about 60 mm (capacity about 100 microlitres) were dipped into this solution and the solution was allowed to rise to a height of about 22 mm so that about 40 microlitres of the solution were taken up in each of the capillaries. Immediately thereafter the capillaries were frozen and lyophilised for about 17 hours. On the dry capillaries the limit to which they had been filled was clearly visible.

The Y-LDH used in the above procedure was prepared in the following way. 800 g fresh, compressed baker's yeast was lyophilised and finely powdered. It was then extracted and fractionated with acetone as was described in detail by A. Spyridakis and L.F. Naslin in 47133 Biochemie 53, 195-205 (1971) except that the autolysis was performed for 20 hours at 4°C. The pink precipitate was extracted for 1 hour at -2°C to 0°C with an aqueous solution which contained per litre 0.15 mole of sodium lactate, 0.1 millimole of ethylenediamine tetraacetic acid (EDTA), 1 millimole of MgSO^ and 150 g acetone. The pH of the solution was 6.8. This avoids the dialysis of the pink, sticky precipitate, which would result in severe loss of activity.

The extract was then treated as described by R.K. Morton and K. Shepley, Biochem J. 89, pages 257-262 (1963) until the phyrophosphate extract was obtained and this was saturated with ammoniumsulphate to 70% and this solution was stored overnight at -20°C. After centrifugation for 50 minutes at 11,000 g and 0°C the crude Y-LDH was dissolved in 10 ml of a 0.1 molar buffer (tri(hydroxymethyl) aminomethane-HCL at pH 8.0 and chromatographed at 4°C on a column of diethylaminoethylcellose (*Sephadex A 25 supplied by AB, Uppsala, Sweden), having a length of 30 cm and a diameter of 2.5 cm. The Y-LDH was eluted with a 0.1 molar solution of a buffer at pH 8.0 (tri(hydromethyl) aminomethane-HCL). The enzyme solution, saturated with ammonium sulphate to 70% can be stored at -20°C and it can be concentrated by centrifugation. The specific activity of this product was found to be up to 17 units of enzyme per mg of total protein.

One unit of Y-LDH is defined in this specification to be the amount which will reduce one micromole of ferricyanide per minute at 25°C in a solution which contains per litre 0.1 mole of a phosphate buffer (pH 7.2), 5.0 millimole of ferricyanide ion, 20.0 millimole L (+) lithiumlactate as measured by following the extinction decrease at 420 nm (absorption maximum of ferricyanide).

♦Sephadex is a trade mark 11 712 3 The Y-LDH obtained by this procedure has a sufficiently high specific acitivity for a lactate determination as described in Example II It has been found that a specific activity between 10 and 20 units per mg of total protein is suitable for said determination. A lower specific activity would require rather large quantities of total protein in order to have sufficient enzyme and this tends to clog the capillaries.

A higher specific activity would imply insufficient protein to cement the solid reagent to the walls of the capillaries and in that case an external cementing material would be needed.

The determination of lactate with a capillary prepared according to Example I is described in the following Example.

EXAMPLE II Some of the dry capillaries were tested by filling them with 40 microlitres of a solution containing known amounts of sodium lactate per litre, shaking to obtain thorough mixing and observing any colour change. The solutions containing up to 0.4 millimoles per litre did not decolourise the capillaries within 2 minutes, but the solutions containing more than 0.4 millimoles of sodium lactate caused decolourisation within 2 minutes. The time needed for decolourisation was noted and the data were used to construct a standardisation curve. This curve is shown in the Figure. From this curve the excess of lactate ion can be estimated. A lactate concentration above 0.4 millimoles per litre is considered an excess as concentrations up to this value are normal in human urine and higher concentrations are an indication of a pathological condition, such as diabetes, glycogen storage disease, congenital lactic acidosis, neoplastic proliferative disorders or impaired renal function.

Urine samples containing varying known amounts of sodium lactate and ascorbic acid were tested in the following way: First to each sairole a small amount of powdered active carbon was added and the mixture was shaken to obtain a homogenous dispersion. Immediately thereafter the carbon was filtered off the filtrate was tested using ε capillary as described in Example I. It was found that the adsorption of the ascorbic acid on the active carbon was practically complete and the adsorption of the lactate on the carbon was negligible. Removal of ascorbic acid is necessary as this compound may also be oxidised in this test. 1-hydroxy butyrate has also been found in urine but this compound does not influence the lactate test.

A number of the capillaries prepared according to Example 1 were stored for periods up to 6 months at -20°C in darkness. When the stored capillaries were tested using the procedure of Example 11, and the results were compared with fresh capillaries from the same lot, no significant differences were found.

In the test as described in Example 11 LDH obtained from muscles of mammals may be used instead of Y-LDH. The former LDH has an optimum activity at pH = 10 and it works satisfactorily at pH between 9.6 and 10.3.

Any other comparable enzyme may also be used. Whichever enzyme is used, it will be always necessary to adjust the pH to a suitable value near the optimum pH of the enzyme by adding a buffer mixture to the reagent, since the pH of urine can vary widely.

In addition to DC1P there exist various coloured oxidants which have a suitable oxidation potential which will not oxidize most of the compounds which occur regularly or sometimes in urine in the absence of a catalysing enzyme and which in the presence of an LDH will selectively oxidize lactate ion.

Examples of other oxidants suitable for use with Y-LDH are ferricyanide ion, methylene blue and o-phenanthroline. 56444- ion.

However when using muscle LDH a mixture of nicotinamide-adenine dinucleotide, phenazine methosulphate and nitroblue tetrazoliumchloride or, preferably, DC1P, may be used. In the oxidized form each of these compounds is coloured and in the reduced form each is either colourless or it has a different colour. Preferably the oxidant used should undergo a rapid and strong change of colour at the moment when all the oxidant has been reduced and the reduced form of the oxidant should not be subject to easy reoxidation by air as this may obscure the results. The oxidant should not be hydroscopic and it should preferably be rapidly soluble in water.

A very good combination of such properties is found in DC1P, which has a strong blue colour in the form of its oxidized alkali salts (pH over about 6) and which is colourless in either its oxidized acid form or in its reduced form.

The blue form of DC1P has an absorption maximum at 600 nanometres 3 2 with an extinction coefficient of 20.1 x 10 cm /millimole. At this wave length LDH isolated from muscles has a quite noticeable light absorption, at least in the preferably used form where it still contains some of the original accompanying protein material and for this reason it is preferred to use this oxidant in combination with LDH obtained from yeast, which does not show such absorption. Y-LDH has the further advantage that is optimum pH is nearer the pH values which are normally met with in urine and which may be as low as 4.4.

Use of an enzyme with lower optimum pH results in smaller amounts of buffer material needed to establish the optimum pH independently of the pH of the urine sample.

Another difference between the two types of LDH is that with LDH obtained from muscles the reoxidation of the oxidant by air is faster and this of course is undesirable.

The following Examples illustrate determinations involving the enzymatic reduction of NAD or NADP, coupled with the reduction of DC1P in the presence of PMS.

EXAMPLE III Lactate in urine or blood The following reaction sequence is made use of: Lactate X /NAD Pyruvate PMS oxidised DC1P oxidised (blue) >DC1P reduced (colourless) A reagent mixture is made up as follows: 4-712 3 ml 0.4% DC1P 1 ml 10% NAD ml muscle-lactate dehydrogenase suspension (400-500 units/mg protein) 7 ml 2.5M Tris-HCL pH 9.0 (muscle - LDH = L-lactate: NAD oxydoreductase; E.C. nr. 1.1.1.27.) pi reagent solution are taken up into a 100 μΐ capillary (internal diameter 1.2 mm), frozen and lyophilised. 1 ml urine is placed in a tube containing 1 mg phenazine methosulphate. The tube is mixed and the capillary containing the lyophilised reagents is dipped into the tube containing the urine until 50 μΐ are taken up. The change in colour observed after completion of the reaction (approx. minute) is related to the lactate concentration present in the urine.

A gradation of colour from dark blue in the absence of lactate to green, yellow and finally colourless at very high lactate concentrations is observed.

The same principle is used for plasma as for urine determination, except that in this case the plasma is diluted approximately 1:5 in diluent present in the tube. 50 μΐ diluted plasma are then taken up in the capillary, as before, and the colour change after one minute is observed.

Using this method there is a clear difference between the colour observed at lactate concentrations of 0.1 and 0.2 mM, with further distinct changes in colour with increasing lactate concentration up to approximately 15 mM/1.

The normal ranges for lactate in man are: 47133 Urine Ο - 0.4 mM/Ι Blood (fasting) 0.5 - 1.0 mM/1 Blood (Diabetic coma) 1.5 - 10 mM/1 By adjusting the amounts of the dyes used in the reaction, it is possible to alter the range over which a distinct colour change takes place.

EXAMPLE IV 3-OH-Butyrate The following reactions are used: NAD· NADH PMS reduced PMS oxidised DC1P oxidised DC1P reduced The reagent mixture is prepared as follows: ml 0.4% DC1P ml 10% NAD ml 0H-Butyrate dehydrogenase suspension (20-30 units/mg 15 protein) ml 2.5 M Tris-HCl pH 8.2 μΐ of above mixture are taken up into a capillary of 100 μΐ (internal diameter 1.2 mm), frozen and lyophilised. mg PMS is contained in a separate tube.

The procedure is exactly the same as for lactate determination described in Example III and can be used both with urine and blood, as described.

The detectable range is 0.02 - 5.0 mM.

EXAMPLE V GLUCOSE The following reaction sequence is used; Glucose + ATP hexoklnase Glucose-6-phosphate + ADP G-6-P Glucose-6-phosphate + NADP ____ 6-phosphogluconate dehydrogenase + NADPH + H >DC1P reduced DC1P oxidised The reaction mixture contains the following: ml 0.4% DC1P 1 ml 10% NADP i ml 0.1 M ATP 0.5 ml glucose-6-phosphate dehydrogenase suspension (300-400 units/mg protein) 0.5 ml hexokinase suspension (300 units/mg protein) ml IM Mg Cl2 ml 0.5 M Phosphate buffer pH 7.3 μΐ of above mixture are taken up into a 100 pi capillary (internal diameter 1.2 mm), frozen and lyophilised. mg PMS is contained in a separate tube The same procedure as in Example III is followed The detectable range is 0.02 - 10 mM/1 Normal fasting levels in blood are 3-5 mM/1 EXAMPLE VI Lactate in Urine or Blood Preparation of Test Capillaries The reagent mixture is prepared as follows: 0.5 ml 0.4% DC1P 0.5 ml 1% PMS 1.0 ml muscle-lactate dehydrogenase suspension (400-500 units/mg protein) 1.0 ml 10% NAD 7.0 ml 0.075M Phyrophosphate-HCL Buffer pH 9.0 μΐ reagent solution are taken up into a 100 μΐ capillary (internal diameter 1.2 mm), frozen and lyophilised. In this form, the contents of the capillary are stable for several months when the capillary is stored in a dark container, dessicated (e.g. on silica gel), and refrigerated. 47133 Test Procedure yl urine (or 50 yl plasma diluted approximately 1:5 in the case of blood) are taken up by capillary action. A mark on the capillary tube indicates the level when 50 yl are present. The capillary is then gently tilted back and forth a few times to secure uniform mixing of the test samples with the lyophilised reagents. Two minutes after the introduction of the sample into the capillary the colour is observed and compared with a colour chart. This will indicate a range of lactate concentrations within which the test sample falls.

Claims

1. A method of preparing a device for the quantitative chemical analysis of liquids, the device comprising a capillary having a predetermined amount of reagent or reagents adhering to the inner surface thereof, which method comprises dipping a capillary of an inert material and having an internal diameter greater than 1 mm into a liquid containing one or more reagents in suspension or solution and, if necessary, an adhesive, so that the liquid rises in the capillary by capillary action to a predetermined level, removing the capillary from the liquid and removing the liquid contained in the capillary by lyophilisation to leave the reagent or reagents adhering to the inner surface thereof.

2. A method according to Claim 1 wherein the liquid contains dichlorophenolindophenol and yeast-lactate dehydrogenase.

3. A method according to Claim 1 wherein the liquid contains dichlorophenolindophenol, NAD and Muscle-LDH.

4. A method according to Claim 3, wherein the liquid also contains phenazine methosulphate.

5. A method according to Claim 1, wherein the liquid contains dichlorophenolindophenol, NAD and OH-butyrate-dehydrogenase.

6. A method according to Claim 1, wherein the liquid contains dichlorophenolindophenol, NADP, ATP, glucose-6-phosphate dehydrogenase and hexokinase.

7. A method of preparing a device for the chemical analysis of liquids, substantially as described in any of the examples.

8. A device for the quantitative chemical analysis of liquids, when prepared by a method according to any preceding claim.

9. - A device according to Claim 8 carrying a volume mark on its surface.

10. A device according to Claim 8 or 9, wherein the capillary is made of glass.

11. A device according to Claim 8, 9 or 10, wherein the internal diameter of the capillary is between 1 mm and 1.5 mm.

12. A device according to Claim 8, 9, 10 or 11, wherein the internal volume of the capillary is approximately 100 yl.

13. A device according to any of Claims 8 to 12, wherein the deviations of the wall-thickness and the inner diameter are not more than 2% of their means values.

14. A device according to any of Claims 8 to 13, wherein the or each reagent remains fixed to the wall through an intermediary layer.

15. A set of two or more devices according to Claim 13, wherein the inner diameter and wall-thickness of each capillary does not deviate more than 2% from the mean value for the set.

16. A package or kit containing a set of devices according to Claim 15.

17. A method of quantitative chemical or microbiological determination, wherein a test liquid to which one or more reagents have been added is sucked up to a predetermined volume into the capillary of the device as claimed in any of Claims 8 to 14 containing a reagent or reagents for the substance to be determined and the quantitative effect of the reagent or reagents in the capillary on the test liquid is measured.

18. A method according to Claim 17, wherein the quantitative measurement is carried out directly in the capillary.

19. A method according to Claim 17, characterised in that a device prepared by a method as claimed in Claim 2 is used for the determination of lactate.

20. A method according to Claim 17, characterised in that a device prepared by a method as claimed in Claim 3 is used for the determination of lactate and that phenazine methosulphate is added to the liquid to be tested.

21. A method according to Claim 17, characterised in that a device prepared by a method as claimed in Claim 5 is used for the 10 determination of 3-0H-butyrate and that phenazine methosulphate is added to the liquid to be tested.

22. A method according to Claim 17, characterised in that a device prepared by a method as claimed in Claim 6 is used for the determination of glucose and that phenazine methosulphate is added 15 to the liquid to be tested.

23. A method of chemical or microbiological determination, substantially as described in any of the examples.