NO782452L - PROCEDURE AND FACILITY FOR CARRYING OUT CHEMICAL AND MICRO-BIOLOGICAL ANALYSIS - Google Patents

Description

The present invention relates to devices for carrying out chemical and microbiological analyses, e.g. in urine or

other biological fluids, methods for producing such devices and analytical methods where these are used.

For rapid tests, it is known to use reagent-impregnated strips of paper or porous plastic or tablets. Only a limited number of chemical reactions occur correctly in such bands or tablets.

Said strips or tablets are dipped in the fluid to be analyzed after which they change color when a certain substance is present in the fluid. Such sampling methods generally provide data of a qualitative nature; in certain cases also semi-quantitative data. When using reagent strips in combination with a reflectometer, it can e.g. quantitative determinations are carried out with limited accuracy. For more accurate determinations, it is generally necessary to use more complicated procedures which are time-consuming and require more equipment. In principle, many tests can be automated, but in light of the expensive equipment required, this is only possible in larger laboratories where series of the same tests are carried out.

It is further known to carry out radioimmunoassays in plastic tubes (K Catt and G. W. Tregear in "Science", 158, 1570

(1967) or in capillaries with inner walls of high polymers

(including glass) (German patent no. 2530957). These tests are carried out with tubes or capillaries where the inner walls are coated with an antibody. According to the German patent, the diameter of these capillaries is preferably 1 mm or less.

It has now been found that a large number of analytical reactions are conveniently carried out in capillaries with diameters of over 1 mm. Such capillaries provide inexpensive, easy-to-handle containers for carrying out qualitative, semi-quantitative and quantitative determinations. The invention offers a device consisting of a capillary of an inert material with an internal diameter of more than 1 mm and with a reagent or several such reagents adhering to the internal surface. The amount of reagent can vary from a monomolecular layer to the maximum amount that can be taken up in the bore in the capillary tube without affecting the capillary effect.

In the present description, the term "reagent" denotes not only chemical substances, but also microorganisms that can be affected by the sample fluid.

The material in the capillary should be wettable by the fluid to be tested. For the examination of aqueous liquids, glass capillaries are most suitable. The inner diameter of the capillary should be such that the sample fluid rises in the capillary when it is dipped in the fluid. When the diameter is less than 1 mm, the reagent will only leave insufficient space for the fluid and when the capillary is too wide, the capillary action will be missing or there will be an insufficient one, resulting in only a very small volume of fluid being sucked up ,, which thus prevents

proper use. An inner diameter between 1 and 1.5 mm is preferred. The length of the capillary is preferably between 30 and 150 mm. Commercially available micropipettes of approx. 100 yl. can advantageously be used for the purposes of the invention.

When the cohesion of the reagent or its adhesion to the capillary material is insufficient to hold the material in the capillary, an adhesive can be used. Polymers of biological or synthetic origin such as proteins, cellulose derivatives or polyvinylpyrrolidone are examples of suitable adhesives. It is clear that an adhesive should be chosen that does not affect the test to be carried out.

According to a further feature of the invention, test capillaries are produced by immersing a capillary in a liquid with one or more reagents dissolved or suspended and, if necessary, an adhesive, so that the solution rises in the capillary under the influence of capillary forces, then removing the capillary from the solution and then removing the liquid in the capillary, e.g.

by lyophilization.

According to another feature of the invention, a chemical or microbiological determination is carried out by sucking up a liquid to which one or more reagents may be added in a capillary which has one or more such reagents for the substances to be determined adhering to its inner wall.

In both cases, the effect of the reagent in the capillary on the sample fluid is observed, e.g. color change or an inhibition of the growth of microorganisms.

The capillaries according to the invention can be used for qualitative, semi-quantitative and quantitative determinations. A qualitative determination can be carried out with a single capillary. When a reaction is observed between a sample fluid and the reagent in the capillary, this suggests that a specific substance is present above a certain minimum concentration. When a series of capillaries with varying and pre-selected amounts of reagents is used, a semi-quantitative determination can be carried out.

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

For those samples that involve normal chemical reactions, it is preferred to use reagents that are easily dissolved or suspended in the sample 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 in the fluid.

In immunoassay techniques, a reagent (e.g. antibody) is used which remains fixed to the wall so that the reaction takes place at the interface between the fluid and the reagent-covered capillary wall. In such cases, the capillaries have the advantage that the reagent layer is not easily damaged. The capillaries can be easily drained, e.g. by bringing them into contact with a piece of tracing paper and they can be refilled with a liquid to a certain level, which corresponds to a desired volume. Thus, the capillaries according to the invention can provide a means of easily carrying out multi-step analyzes without the use of additional equipment such as pipettes etc.

The occurrence of a reaction can be easily observed visually (color change, sediment formation, etc.) or by instruments (e.g., change in absorbance ion in the ultraviolet or infrared part of the spectrum).

It is obvious that the material in the capillary should be transparent to the wavelength(s) used when using optical methods.

Usual analytical techniques can be used for quantitative determination of the concentration of the reaction product in the capillary.

Optical instruments can. used to measure changes in color or absorption in the visible, ultraviolet or infrared part of the spectrum.

Radioactive measurements can be used in radioimmunoassays where either the antigen or the antibody is coated or covalently bound to the wall of the capillary or to an intermediate layer that adheres to the wall.

Where the reaction leads to changes in electrical properties (e.g. conductivity), such changes can be measured with suitable instruments.

In some cases, a single measurement after the reaction is complete is sufficient. In other cases, it may be necessary to follow the change of parameters during the test during a certain period of time, e.g. in enzyme immunoassays where the indicator enzyme reaction is monitored with an absorbiometer..

Clumping reactions can be followed by means of viscosity measurements, e.g. by noting the flow of fluid when the capillary is turned upside down or the time required to empty the capillary on a piece of tracing paper. The growth of microorganisms in the capillary can be measured by turbidimetry. In this way, the sensitivity to antibiotics can be measured or vice versa the concentration of antibiotics using a standard suspension of sensitive microorganisms.

This list of techniques in which the capillaries can be used is only intended as an example and does not limit the scope of the invention. Generally speaking, the capillaries can be used for the analysis of all biological fluids and secretions and for the determination of

all substances of diagnostic interest.

As a result of the relatively large diameter of the capillaries (compared to those used according to the above-mentioned German patent), minor variations in the dimension have no great influence on the reliability values for quantitative determinations. For certain purposes, especially in quantitative determinations using optical techniques, the variations should be. within certain limits. Preferably, the inner diameter and wall thickness do not deviate by more than 2% from the average value within the same capillary. When a determination involves the use of two or more capillaries in a comparative measurement (e.g. of light absorption), the difference between their dimensions should also preferably lie within these limits.

A further feature of the invention is therefore a "pack or set comprising two or more capillaries

of which the wall thickness and the inner diameter do not deviate by more than 2% from the average values for the capillaries in this package or this set.

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 acid oxaloacetic transaminase (SGOT), serum glutamic acid pyruvic transaminase (SGPT), creatine phosphokinase (CPK), 3-hydroxybutyrate, lactate dehydro- genase (LDH);

in amniotic fluids: enzymes or metabolites whose absence or presence suggests inherent metabolic defects, e.g. galactokinase, galacto-1-phosphateridyl, 1,4-glucosidase, aryl sulphatase A, α 1-iduronidase, pyruvic acid, lactic acid and 2-oxoglutaric acid;

in cerebro-spinal fluids: glucose, protein.

Generally known reactions can be used for these provisions. E.g. lactate can be determined with LDH and a dye, e.g. dichlorophenolindophenol (DCIP), LDH can be determined with lactate and a dye (eg DCIP); glucose can be determined with glucose oxidase, peroxidase and a dye (eg orthotoluidine); cholesterol can be determined with cholesterol oxidase, peroxidase and a dye; protein can be determined by a biuret-like reagent of pH-indicative dye, using the so-called protein effect; 3-hydroxybutyrate can be determined with 3-hydroxybutyrate dehydrogenase and a dye.

The production of a sample capillary according to the invention is described in the following example.

Example 1

2.4 millimoles of dichlorophenolindophenol (DCIP) were dissolved in one liter of distilled water and 10 µl of 1N NaOH was added to this solution. Pure oxygen was then bubbled through the solution to achieve total oxidation. This solution was diluted with 5 liters of a solution which per liter contained 0.18 moles of a potassium phosphate buffer and 1.2 millimoles of EDTA. Then yeast lactate dehydrogenase (Y-LDH) was dissolved in an amount containing 90,000 units and with a specific effect of 17.2 units per mg of protein. The final concentration of DCIP was "determined to be 0.4 m mol per liter. Glass capillaries with an inner diameter of 1.5 mm and a length of about 60 mm (capacity about 100 µl) were dipped into this solution and the solution was allowed to rise to a height of about 22 mm so that about 40 µl of the solution was taken up in each of the capillaries. Immediately thereafter these were frozen and lyophilized for about 17 hours. On

the dry capillaries were the limit to which they were filled clearly visible.

The Y-LDH used in the above-mentioned method was prepared as follows: 800 g of fresh, pressed baker's yeast was lyophilized and pulverized. It was then extracted and fractionated with acetone as described in detail by A. Spyridakis and L.F. Naslin in "Biochemie", 53, 195-205 (1971) except that the autolysis was carried out 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 per liter contained 0.15 mol sodium lactate, 0.1 mmol ethylenediaminetetraacetic acid (EDTA), 1 mmol MgSO^ and 150 g acetone. The pH value of the solution was 6.8. This prevented dialysis of the pink sticky precipitate, which would result in severe loss of activity. The extract was then processed as described by R.K. Morton and K. Shepley in "Biochem", J. 89, pp. 257-262

(1963) until the pyrophosphate extract was obtained and this was saturated with ammonium sulphate to 70% and this solution was stored overnight at -20°C. After centrifugation for 50 minutes at 11,000 g and 0°C, the impure Y-LDH was dissolved in 10 ml of a 0.1 mol buffer (trihydroxymethylaminomethane-HCl) at pH 8.0 and chromatographed at 4°C on a column of diethylaminoethylcellulose ("Sephadex A 25") with a length of 30 cm and a diameter of 2.5 cm. The obtained Y-LDH was eluted with a 0.1 mol solution of a buffer with pH 8.0 (trihydroxymethylaminomethane-HCl). The enzyme solution, saturated with ammonium sulfate to 70%, can be stored at -20°C and it can be concentrated by centrifugation. The specific effect for this product was found to be up to 17 units of enzyme per mg total protein.

A unit of Y-LDH is defined in the present description as the amount that reduces one micromole of iron(III) cyanide per minute at 25°C in a solution containing per liter 0.1 mol• of a phosphate buffer with pH 7.2, 5.0 millimoles iron(III) cyanide ion, 20.0 millimoles L (+) lithium lactate as measured by following the extinction reduction at 420 nm (iron(III) cyanide absorption maximum).

The Y-LDH obtained by this procedure had a sufficiently high specific activity for lactate determination as described in Example 2. It was found that a specific activity between 10 and 20 units per mg total protein is appropriate for the determination. A lower specific activity will require rather large amounts of total protein to have sufficient enzyme and this tends to happen. to stuff the capillaries.

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

The determination of lactate with a capillary prepared according to example 1 is described in the following example 2.

Example 2

Some of the dry capillaries were tested by filling them with 40 µl of a solution containing known amounts of sodium lactate per litres, shaken to achieve thorough mixing and observation of color change. The solutions that contained up to 0.4 millimol per liter did not decolorize the capillaries within 2 minutes, but solutions containing more than 0.4 millimole of sodium lactate caused decolorization within 2 minutes. The time required for decolorization was noted and the data entered were used to construct a standardization curve. This curve is shown in fig. 1. From this curve, the excess of lactation ions can be estimated. A lactate concentration above 0.4 millimol per liters is considered excess, as concentrations up to this value are common in human urine and higher concentrations are an indication of a pathological condition such as diabetes, glycogen storage diseases, congenital lactic acidosis, neoplastic proliferative defects or impaired renal functions.

Urine samples containing varying known amounts of

sodium lactate and ascorbic acid were tested as follows: First, a small amount of powdered activated carbon was added to each sample and the mixture was shaken to obtain a homogeneous dispersion. Immediately thereafter carbon was filtered off and the filtrate was sampled using a capillary as described in Example 1. It was found that the adsorption of ascorbic acid on the activated carbon was practically complete and the adsorption of lactate on the carbon was negligible. Removal of ascorbic acid is necessary, as this compound can also be oxidized in this sample.

1-Hydroxybutyrates have also been found in urine, but this

compound does not affect the lactate test.

A number of the capillaries produced according to Example 1 were stored for periods of up to 6 months at -20°C in the dark. When testing the stored capillaries using the procedure in example 2, no significant differences were found when the results were compared with new capillaries from the same lot.

In the samples described in example 2, LDH obtained from mammalian muscle can be used instead of Y-LDH. the first-mentioned LDH has an optimal activity at a pH value equal to 10 and it works satisfactorily at pH values between 9.6 and 10.3.

Any other comparable enzyme can also be used. Regardless of which enzyme is used, it will always be necessary to adjust the pH to a suitable value close to the optimum pH for the enzyme by adding a buffer mixture to the reagent because the pH in urine can vary widely.

In addition to DCIP, there are various colored oxidants that have a suitable oxidation potential that do not oxidize most of the compounds that occur regularly or occasionally in urine in the absence of a catalyzing enzyme and that in the presence of an LDH selectively oxidize the lactate ions.

Examples of other oxidants which are suitable for use with Y-LDH are iron (III) cyanide ion, methylene blue and o-phenanthroline ■ iron (III) ion. However, when using muscle LDH, a mixture of nicotinamide adenine dinucleotide, phenazine methosulfate and nitro blue tetrazolium chloride can be used. In oxidized form each of these compounds is colored and in reduced form they are either colorless or have a different color. Preferably, the oxidant used should undergo a rapid and strong color change at the moment when all the oxidant has been reduced and the reduced form of the oxidant should not too easily be subject to reoxidation by air as this can confuse 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 DCIP, which has a strong blue color in the form of the oxidized alkali salts (pH above approx. 6) and which is colorless both in the oxidized acid form and in the reduced form.

The blue form of DCIP has an absorption maximum at 600 nanometers with an extinction coefficient of o 20.1 x 10<3>cm 2 /millimol. At this wavelength, LDH isolated from muscle has a very remarkable light absorption, at least in the form preferably used where it still contains some of the originally accompanying protein substances 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 additional advantage that the optimal pH value is closer to the pH value normally present in urine, which can be as low as 4.4. The use of an enzyme with a lower optimum pH results in the need for smaller amounts of buffer material to create the optimum pH regardless of the pH of the urine sample.

Another difference between the two types of LDH lies in the fact that LDH obtained from muscles is subjected to a faster reoxidation by the oxidant and this is of course undesirable.

Claims (16)

1. Analysis device, characterized in that it consists of a capillary of an inert material with an inner diameter of over 1 mm and with one or more reagents adhering to the inner surface. 2. Analysis device according to claim 1, characterized in that it is made of glass. 3. Analysis device according to claim 1 or 2, characterized in that the inner diameter is between 1 and 1.5 mm. 4. Analysis device according to claim 1, 2 or 3, characterized in that the volume of the borehole is approx. 100 yl. 5. Analysis device according to claims 1 to 4, characterized in that the deviation for the wall thickness and the inner diameter is not more than 2% from the mean value. 6. Set of two or more devices according to claim 5, characterized in that the inner diameter and wall thickness of each capillary does not deviate by more than 2% from the average value for the set. 7. Packing or set, characterized in that it contains a number of capillaries according to claim 6. 8. Method for manufacturing an analysis device according to claims 1-5, characterized in that a suitable capillary is dipped in a solvent or a suspension containing one or more reagents and, if necessary, an adhesive, so that the liquid rises in the capillary under the influence of the capillary forces, after which the capillary is removed from the liquid and the solvent or suspending agent contained in the capillary is removed. 9. Method according to claim 8, characterized in that the solvent or suspending agent is removed by lyophilization. 10. Method for carrying out a chemical or microbiological determination, characterized in that a liquid to which one or more reagents have been added is sucked up into a capillary according to any one of claims 1-5 containing one or more such reagents for the substance which is to be determined, whereby the action of the reagent or rea the genes in the capillary and the sample fluid are observed. 11. Device according to any one of claims 1-5, characterized in that it contains one or more preselected amounts of one or more reagents. 12. Device according to any one of claims 1-5 or 11, characterized in that the reagent or reagents are easily dissolved or suspended by the sample fluid. 13. Capillary according to any one of claims 1-5 or 11, characterized in that the reagent or reagents remain attached to the wall, directly or by means of an intermediate layer. 14. Device according to any one of claims 1-5, 11 or 12, characterized in that it contains the reagents dichlorophenolindophenol and yeast lactate dehydrogenase. 15. Method according to claim 8 or 9, characterized in that the liquid which is sucked up into the capillary contains dichlorophenolindophenol and yeast lactate dehydrogenase. 16. Method according to claim 10, characterized in that the device required in claim 14 is used for the determination of lactate.