DD296762A5 - REAGENT CARRIER FOR THE PHOTOMETRIC QUICK DETERMINATION OF ANALYTES - Google Patents

Abstract

The invention relates to a reaction carrier for the photometric rapid determination of analytes in predominantly biological fluids. Gemaesz the invention, at least on a reaction and evaluation of the Reaktionsstraegers reagents are fixed in a dry form, which are stabilized by a non-affecting the detection reaction protective substance. The protective substance may either be added to a reagent layer as an admixture or applied as a final adherent layer. Aggressive or sensitive reagents are located separately from underlying layers in a separate layer. The coating according to the invention excludes transport losses of these layers and their temporal decomposition and ensures immediate release of the reagents for analysis purposes after addition of a defined amount of water in which the sample fluids to be analyzed are contained {photometry; Multiple measurement; Dry test; Microanalysis; microtiter plate; clinical Chemistry; Micro plate; Reader; Biochemistry; Rapid test}

Description

Field of application of the invention

The invention relates to prepared reagent carriers for the photometric determination of analytes in predominantly biological fluids, wherein after addition of defined volumes of water with or without further reaction components, the sample solutions to be analyzed are added immediately and a detail in the preparation of the prepared reagent carriers.

Characteristic of the known state of the art

For clinical-chemical determinations, a number of test kits are known, which can be used after dissolution of the reagent substance with defined predetermined amounts of liquid and following individual division for appropriate analyte determinations. These tests are particularly favorable if only one dissolution step has to be carried out. The so-called monotests allow after the addition of the predetermined amount of solvent, the implementation of several test provisions from the total amount to be divided into individual portions (test primer, Boehringer, Mannheim GmbH, 1987). Effective use therefore requires a minimum number of samples since the final amount of reagent can only be kept for a limited time. To avoid this drawback, another concept is to use ready-prepared single cuvettes per analysis, sample and reagent capillary being added to the reaction extra in the liquid cuvette filling (COMPUR Laboratory System M 2000, company publication Compur-Elektronic GmbH, Munich, 1985). However, these are individual tests that a single processing and evaluation z.T. even in special equipment, which also includes capillary enclosed reagent combinations in the dry state, which react after addition of diluted sample (DDR patent DD 225788). Both cuvettes and capillaries are specially sealed containers or reagents that require an increased production effort.

Other known analytical systems work with prefabricated individual test packs per determination, which are used only in special analysis machines primarily for emergency diagnostics. The test packs are tightly sealed containers whose manufacture and use is relatively expensive (e.g., ACA, Du Pont De Nemours GmbH, Bad Nauheim, 1978).

When using optical measuring method z. As for clinical laboratories, the measurement has been enforced in recent years with so-called microplate reader (Micro Plate Reader). Such photometric evaluation devices have been further developed for the implementation of quantitative determinations. Among other things, these photometers are also used for the serial determination of analytes in clinical chemistry. A combination of the analogue technology with dry reagents and the measurement with prepared microtitration plates (usually 96 measuring points) could reduce disadvantages in the measuring variant. Any excess of reagent would be dispensed with, and in addition the possibilities of error for the user are reduced by eliminating several dosing steps. Thus, the analytical method becomes more economical and more effective, yet with high analytical quality. However, when using conventional photometers, a single determination with a prepared single cuvette is possible. However, there is the difficulty to firmly connect the microtiter plate or cuvette with the reagent dry substance, otherwise by transport, etc., small losses of substance may occur and resulting in erroneous determinations. A covering with foil or placing a cover triggers

these difficulties not (smallest loss amounts per cavity cause errors), so that in particular for biochemical investigations such combinations do not exist, but only for immunological reactions or immunoassays (antigens or antibodies are bound to the walls of the cavities at the molecular level). However, the immune bodies remain coupled to the cavities during the detection reaction.

For clinical chemistry, however, the reagents must again easily and quickly dissolve in the added solution.

Also prepared microtitration plates for bacteriological investigations, for example for the determination of the minimum inhibitor concentration, are closed for the stabilization of the reagents (Sensititer Susceptibility System, company publication, Sensititer Ltd./Radiometer Copenhagen Com., 1988). In addition, the requirements for analytical quality are not as high as for quantitative, exact reaction sequences between several reagents and sample solution for a clinical chemical determination.

There are still no suitably prepared reagent carriers, such as microtitration plates or segments of these up to single cuvettes for reagent-free or poor further processing, which meet the above requirements for initially adherent reagent layer.

Object of the invention

It is the object of the invention to increase the transport safety of easily handled, analyte-specific reagent carriers and their storage stability and to reduce both pipetting and thus measuring errors as well as time and reagent losses in the preparation of the analysis by reducing pipetting steps.

Explanation of the essence of the invention

The object of the invention is to form a firmly adhering to a reagent carrier applied reagent layer, in particular to exclude transport losses of the substances of this layer and their temporal decomposition and to ensure immediate release of the reagents for analysis after addition of a defined amount of water in which to be analyzed, predominantly biological sample liquids are contained or in which such sample liquids are subsequently dosed defined.

According to the invention, the object is achieved by a sample carrier for the photometric rapid determination of analytes in predominantly biological liquids, in which Reaklions- and evaluation are identical by at least on the reaction and evaluation reagents are fixed in dry form, by a detection reaction not influencing protective substance are stabilized. The reagents are applied in at least one layer, of which at least one contains the protective substance as an admixture. Another solution is to apply the protective substance as a final adherent layer.

When using analyte-specific reagents with aggressive or sensitive components that are not stable together in a dry layer, the proportion of aggressive or sensitive components of the reagents is localized in a separate layer, which is preferably located above underlying layers. As a result of such a coating, dry, stable analyte-specific reagent carriers result after rapid evaporation of the solvents.

As a protective substance, which is mainly suitable as an admixture, a sodium salt of polyacrylic acid is used with a concentration between 5% and 25%. In addition, phenol and phenol derivatives may also be included at a concentration of up to 15%. Preferred as the adherent layer is a polyvinyl pyrolidone (MW 20000-350000) dissolved in ethanol or water at a concentration of between 5% and 25%. It is possible to introduce into this solution phenol or phenol derivatives with a concentration of up to 15%. It is advantageous to add wetting agents in amounts of 0.05 to 1% to the solutions of the protective substances. In addition, it is advantageous if the solutions of the protective substances ethylene glycol between 50ml / l and 200ml / l solution.

embodiments example 1

Determination of total protein with biuret reagent

Solution 1: K-Na tartrate p.a

CuSO ₄ - 2H ₂ O

KJ

NaOH solution 2: KJ

NaOH

Solution3: 5ml solution1 and 20ml solution2 Solution 4: Scopacryl®LW300

ethylene glycol

Wetting agent NM 1783

H ₂ O

In NaOH to adjust to pH 11

4.5 g

1.5g

0.5 g

0.6 g in 25 ml of 0.2N NaOH

0,6gin25ml0,2nNaOH

1.5g (Na salt of polyacrylic acid, product of VEB Chemical plants Buna) 1.25ml

50 μΙ (product of the VEB Chemical Works Buna) 25ml

Solution 5: NaOH 0.2 η

Tween 0.2% based on O, 2N NaOH solution 6; test serum

40 μΙ of the solution 3 and 50 μΙ of the solution 4 are successively pipetted onto the reagent carrier. The solutions are completely dried (eg air drying with heating, freeze-drying).

Thereafter, the reaction carrier is sealed with a self-adhesive cover and can be stored and transported in this form.

To determine the total protein, 160 μl of the solution 5 are added to the reagent carrier to dissolve the dried reagents. In the solution 8 μ | the solution 6 added to the determination. The analyte determination is carried out in a known manner.

Example 2

Determination of glucose in serum (modified PAP method)

Solution 1: Tris-phosphate buffer 5,25ml

(consisting of:

300mmol / l of sodium hydrogen phosphate-12-hydrate

150mmol /! TRIS 0.1 η HCl to adjust pH8) GOD (11040nkat / l) POD (9600 nkat / l)

4-Aminoantipyrine (30mmol / L) Solution 2: Phenol Conz.

Ethanol 96% polyvinyl pyrolidone (MW ca. 25000) NM1783

0.5ml 0.5m, l 0.5ml

1.0ml 13.5ml

1.5 g (products of the company Serva, Heidelberg) ΙΟ, ΟμΙ

On the reagent carrier 50 μΙ of the solution 1 are added and completely dried. Thereafter, 40 .mu.l of the solution are added and again dried completely. Completed with a self-adhesive cover film, the reagent carriers are storable and transportable. For the determination of glucose, the contents of the reagent carrier in 150μΙ Aq. least. dissolved and 20μΙ of the test sample (preparation of the sample according to AB) entered. The analyte determination is carried out in a known manner. The protective substance polyvinylpyrolidone is also suitable as an admixture.

Example 3

Determination of uric acid

(Modification of the enzyrnatic color test according to Fassati)

Solution 1: DHBSP ^ dichlorophenolsulfonic acid 13.22 g / i (buffer) 5 ml

Aminophenazone (4.15g / l buffer) 1ml

POD (at least 32 U / mg; 4.65 g / L buffer) 689μΙ

Phosphate buffer pH 7.0 (340 mmol / l) 14 ml solution 2: 6 ml solution 1 and 15μΙ uricase (100U / 1)

Solution 3: Scopacryl® LW300 1.50g

Ethylene glycol 1,25ml

NM1783 50,00μΙ

Aq. least. 6.25 ml

Blank:

50μΙ of the solution 1 are completely dried on the reagent carrier. Subsequently, 10 .mu.l of solution 3 are added and the reagents in turn are completely dried.

measurement:

50μΙ of the solution 2 are dried as in the determination of the blank on the reagent carrier and again 10μΙ of the solution 3 is added and dried.

Both samples are in turn transportable and storable under self-adhesive cover film.

The reagents are each in 170μΙ Aq. least. dissolved and in each case ηιίΐδμΙ of the test sample (preparation according to AB) is added.

The incubation time for the colorimetric measurement is between 30 and 40 min.

Claims (9)

1. reagent carrier for the photometric rapid determination of analytes in mainly biological fluids, are identical in the reaction and evaluation, characterized in that at least on the reaction and evaluation reagents are fixed in dry form, which stabilized by a non-influencing the detection reaction protective substance are. 2. Reagent carrier according to claim 1, characterized in that the reagents are applied in at least one layer, of which at least one contains the protective substance as an admixture. 3. Reagent carrier according to claim 1, characterized in that the protective substance is applied as a flowing adherent layer. 4. Reagent carrier according to claim 1 to 3, characterized in that aggressive or sensitive reagents are isolated from underlying layers in a separate layer. 5. Reagent carrier according to claim 1 and 2, characterized in that the protective substance is a Na salt of the polyacrylic acid with a concentration between 5% and 25%. 6. reagent carrier according to claim 1, 3 and 4, characterized in that the protective substance is a polyvinylpyrrolidone (MW 20000-350000), dissolved in ethanol or water, with a concentration between 5% and 25%, and is preferably used as a final adherent layer , 7. Reagent carrier according to claim 1 to 6, characterized in that the solutions of the protective substances additionally contain wetting agents in amounts of 0.05 to 1%. 8. reagent carrier according to claim 1 to 7, characterized in that the solutions of the protective substances additionally contain ethylene glycol between 50ml / l solution and 200ml / l. 9. Reagent carrier according to claim 6, characterized in that the solution of the protective substance contains phenol or phenol derivatives having a concentration of up to 15%.