WO1988009824A1

WO1988009824A1 - Improvements in diagnostic test strips

Info

Publication number: WO1988009824A1
Application number: PCT/AU1988/000171
Authority: WO
Inventors: Anthony Brandon Bransgrove
Original assignee: National Diagnostic Products (Australia) Pty. Limi
Priority date: 1987-06-05
Filing date: 1988-06-06
Publication date: 1988-12-15

Abstract

A diagnostic test device for the detection and quantitation of analytes in the cell- and particle-free fraction of biological fluids, such as blood serum. The device comprises an absorbent carrier matrix impregnated with the reagents necessary to bring about a graduated response to specific analytes contained in the biological fluid. The matrix further serves as a support for an overlying porous membrane cast onto the upper surface of the matrix. The membrane serves as a filter and a robust wipe off surface. The reagent-containing matrix membrane composite is ideally attached to one end of a convenient length of an inert support base, such as a plastic strip, which can be used to handle the device.

Description

IMPROVEMENTS IX DIAGNOSTIC TEST STRIPS

TECHNICAL FIELD

The present invention relates to a diagnostic test strip device for the detection and quantitation of biochemical compounds (analytes) in the cell and particle free fraction of whole blood, otherwise known as plasma or serum.

Rapid diagnosis in clinical chemistry by means of test strips carrying dry reagents has assumed an ever increasing degree of importance, and in more recent times has come to be known as "Dry Chemical Pathology" . Compared with conventional methods dry chemistry tests offer the advantages of economy, speed and simplicity.

Test strips are also commonly used for the detection of substances in urine and other body fluids or may be used outside the medical field for monitoring industrial or agricultural substances. CURRENT TECHNOLOGY AND BACKGROUND ART

Present devices take the form of inert strips (but not exclusively so) to which are attached a pad of absorbent carrier or a water-stable film, either of which contain all the reagents necessary for the assay. The strip with its reagents is then brought into contact with the biological fluid being tested and a colour change develops proportional to the concentration of the analyte of interest.

In the case of blood this material may be washed or wiped clear to permit viewing of the colour change or it may be left at its site of application with the colour change viewed from the opposite side of the strip. In either case the intensity of the colour development can be read by comparison with standards or by the use of a reflectance colourimeter.

One great advantage of this method is that it allows the direct application of a small sample of blood, such as obtained from a finger prick, without the intermediate need for either measuring or carrying devices. This inherent simplicity is thus ideally suited for the layman or patients in need of self-monitoring facilities.

The reaction pad has commonly utilized paper as the absorbent carrier. This material however has deficiencies of layer thickness, surface regularity and homogeneity of composition, all of which are necessary for precision quantitative tests, and particularly so when using reflectance meters. Thus the course texture of a cellulose or glass fibre matrix will accentuate light scattering during reflectance measurements.

The introduction of water-stable films according to DE-1,598,153 overcame many of these problems. These films are applied as a polymer solution or dispersion to a substrate. They function primarily as a repository for the specific reagents however, they are also homogeneous and they have a regular and uniform surface which gives minimal light scattering. The surface is porous but also sufficiently durable to allow direct wipe off of excess blood.

Australian Patent No. 500976 discloses that a wipe off technique can also be achieved with a paper absorbent carrier by overlaying with a polymer strengthened by cross linkage. However, this patent does not disclose any means to overcome the disadvantages of the fibrous irregular surface.

The reagent-containing polymer films or absorbent pads are selectively permeable to small hydrophylic molecules absorbing an ultra filtrate (in the manner of a semi-permeable membrane). When blood is applied proteins, interfering coloured substances, red cells and other material fail to penetrate and can be wiped or washed from the surface. These strips are satisfactory for determinations such as blood glucose, however, they cannot be used for detection of large molecules such as enzymes or proteins and the many hydrophobic substances such as triglycerides or cholesterol which they transport. These molecules do not penetrate, or penetrate only poorly, into the film or pad and so precision assays are not possible.

Various inventions have attempted to address the problem of the failure of proteins to penetrate water-stable films. Thus in Australian Patent No. 513751, devices known as film openers have been used to increase the porosity of the film.

Although this is satisfactory for serum, it precludes a clean wipe off of whole blood and so interferes with assessment of colour change.

Detergents have been proposed in Australian Patent No. 497687 as a means of separating the transported substances such a cholesterol from the larger carrier proteins. In all cases diffusion of the analyte of interest into the film is slow and imprecise.

In the case of polymer films, the small pores extend through the full thickness of the film which according to DE-1,598,153 is wet cast with a thickness of 400 microns. This diffusion distance is partly responsible for the slower rate of equilibration of water-stable films and hence the reaction time is longer when compared with the paper absorbent matrix. DISCLOSURE OF THE INVENTION

The present invention utilizes a much thinner film than heretofore and has thus preserved the regular surface (with its optical advantages) of water-stable films but with enhanced filtering capabilities. It has also transferred the function of reagent carrier to an underlying porous synthetic matrix.

By so separating the filtration function from the reagent repository site finely dispersed soluble material may be incorporated within the substance of the water-stable film at the time of casting, to be later removed by exposure to a suitable aqueous system which otherwise might be detrimental to the reagents. Subsequently the reagents may be applied to the matrix through the membrane.

This improvement of porosity serves the dual purpose of increasing filtration rate and in turn the reaction speed and allowing access to the matrix by a protein-containing filtrate more representative of blood serum.

Thus according to the present invention there is provided a diagnostic test device for the detection and quantitation of analytes in the cell and particle free fraction of biological fluids.

Such device comprising an absorbent carrier matrix impregnated with the reagents necessary to bring about a graduated response to a specific analyte. The matrix further serves as a supporting framework upon which is cast a thin membrane of a water-stable film-forming polymer.

The membrane serves as a filter and a robust wipe off surface. Its porosity is high by virtue of its thinness but this can be further augmented by the inclusion of substances in the casting composition which are soluble under aqueous conditions in which the polymer film is stable.

The reagent-containing matrix membrane composite on its substrate is ideally attached to one end of a convenient length of an inert support base, such as a plastic strip, which can be used to handle the device. SUMMARY OF THE INVENTION

The present invention therefore utilises a wipe off surface which is also a uniquely porous filtration mechanism. This generates a protein-containing filtrate of blood for direct mixing with the reagents. It is thus distinct from other similar test strips which employ an ultra-filtrate. In this manner the bias effect based on molecular size or solubility is minimised. Furthermore, the large pore size causes the filtrate to be imbibed more rapidly and thus the overall reaction speed is faster than in other test strips. Finally, because the filter membrane is integral to the whole pad it will not separate and allow red cell penetration and contamination along the edges as occurs with other devices. BEST MODES FOR CARRYING OUT THE INVENTION

The invention includes three systems:- The matrix; The reagent system; and The filter membrane, i) THE MATRIX

This is a thin layer of resilient porous material which by a wetting process moves the filtrate away from the site of blood application on the overlying filter.

The trabecula of this layer function as a substrate, upon which is first deposited the chromogen component of the reagent system, preferably as a poorly-soluble colloid, whilst the pores function as the repository for the analyte specific enzymes and other reagents .

Bibulous carriers with a more homogeneous structure than paper are known in the art. The novel advantage accrues when the benefits of this type of matrix are united with those of a water-stable emulsion or solution.

The Matrix is preferably made from a suspension of hydrophylic polymer - natural or synthetic - such as cellulose or polyamide. It may also include reagents, chromogen, and reflectants such as Barium salts. The suspension is spread directly onto an inert plastic substrate support in a layer preferably between about 25 microns and 250 microns thick.

The unique and novel property of this layer is the nature of its porosity. The pore size is sufficient to allow complete penetration with ease by the particles of the chromogen and the enzyme dispersion or solution. But it will impede penetration by the larger particles of the filter membrane emulsion.

The porosity of the Matrix is controlled by the casting process. The polymer is preferably soluble in an organic solvent from which it can be precipitated by a non solvent such as water. Addition of variable amounts of water under conditions of agitation just prior to casting will cause a nucleation process which then determines the pore size of the Matrix.

Several polymer systems have been found to satisfy this requirement and they can be cast by a dipping process, a chromatography spreader, or a Mayer Bar. ii) THE REAGENT SYSTEM

Generally, any reagent system may be employed provided it is able to react specifically with the analyte of interest and bring about a chromogen colour change.

However, a preferred reagent system for the invention is one which provides for a redox reaction in which O- is the final electron acceptor and ^H-_?°2 -'•^s generated stoichiometrically by specific oxidases acting on the anlayte of interest. Highly sensitive colourimetric assays of H„0.-, based on the principle first described by Trinder (1969) can then effectively measure the analyte concentration.

A preferred system for glucose comprises glucose oxidase, peroxidase and a redox indicator such as Benzidine and its derivatives or Phenol/4 aminoantipyrine or others known in the art.

In cases where the analytes cannot themselves be oxidised they may first be degraded enzymically to oxidase-susceptible products. For instance, cholesterol esters to cholesterol, or triglycerides to glycerol.

In addition to enzymes and chromogen the reagent system may include other ingredients such as enzyme stabilizers, detergents and buffers.

A preferred reagent system of the present invention has some mutually incompatible components. In order to maintain their separation the reagents can be applied to the matrix as two separate solutions. The first containing the chromogen is dried before application of the second containing specific enzymes, buffer and other reagents.

The reagent system may also contain other ingredients, such as hydrophylic polymers, to aid imbibing of the sample fluid, or protective colloids to aid solubility of other reagents or polymers which aid the colour development and stability of the chromogen. iii) THE FILTER OR TOP LAYER

This may take many forms but it is essential that it blocks cellular elements and transfers the fluid component of blood. This requirement is satisfied in the invention by a novel device which incorporates a water-stable polymer film into the surface of the matrix. This has been done by exposing the matrix to a dilute solution of polymer emulsion with a particle size range between about 1 and 10 microns.

A novel advantage of the invention has been the incorporation of soluble inclusions in the polymer solution or dispersion which are later dissovled to leave a more porous membrane. These inclusions can take the form of any soluble or particulate matter which will not denature the film-forming polymer. Thus near saturated solutions of magnesium chloride will not disturb Propiofan but can be washed clear with ease after film formation.

The required porosity of the membrane filter will greatly influence the choice of film-forming polymer. The cold flow characteristics of many polymer films will quickly cause a closure of pores soon after they are ormed. It is therefore important for optimal conditions to use a polymer solution or dispersion with a high glassing temperature and a high film-forming temperature. This generally means a high molecular weight.

The following examples are preferred embodiments of the invention which are not to be construed as at all limiting. EXAMPLE 1

For the production of a test strip to measure blood glucose.

Matrix Preparation

20 g of polya ide (E poly-caprolactam. Aldwich Chemical Co.) were dissolved with the aid of mild heat and stirring, in 100 gm of 90% formic acid. When fully dissolved 14ml of water was injected rapidly into the vigorously stirred solution and it was immediately applied to a 100 micron thick sheet of polycarbonate film by a dipping process. The sheet of polycarbonate film was then hung vertically and allowed to harden under conditions of 60% relative humidity. After 24 hours the film was impregnated with the following solutions I and II with drying at 60°C after the first solution. Solution I

3,3 5,5 -tetramethybenzidine lOOmg

IN HC1 1.0ml

Water 1.5ml

6% hydrolysed Gantrez AN 149 (GAF) 2.5ml

Hydrolysed Gantrez AN 149 is a protective colloid with acid functions similar to sodium alginate, it has the added advantage of solubility at an acid pH.

Solution II

Glucose oxidase 116U/mg lO.O g

Horseradish peroxidase 300U/mg 6.0mg

5% polyvinyl pyrrolidone K90(GAF) 7.5ml

0.1M Sodium phosphate pH7.0 5.0ml

15% sodium lauryl sulphate 0.5ml

Polyvinyl pyrrolidone acts to enhance and stabilize the chromogen colour as does also sodium lauryl sulphate. After applying the second solution and before it dried solution III, the polymer dispersion for the filter layer, was applied in a 35 micron thick layer and subsequently dried with warm air.

Solution III s _*

50% polyvinylpropylene polyvinylchloride

(Propiofan 325D BASF) 10.0ml

0.1M Sodium phosphate pH7.0 12.0ml

10% dioctyl sodium sulphosuccinate 0.1ml

The sheet was then slit into 6mm wide strips which are bonded by double-sided transfer adhesive 3mm from one edge of a 75mm wide plastic base. This base with the glucose sensitive support was slit into 6mm wide strips.

When tested with glucose-containing blood a well graduated response was evident after 30 seconds of blood contact.

When tested with a reflectance meter and whole blood samples of various glucose content, the following results were obtained.

Glucose mg/lOOml Meter reading (Microamps)

30 36.0

50 31.0

75 27.5

100 25.0

150 19.0

200 15.5

300 8.0

500 3.0

EXAMPLE 2

For the production of a test strip to measure blood cholesterol.

The matrix preparation according to Example 1 was moistened with distilled water and then layered with a 35 micron thick film of the polymer dispersion from Solution I aanndd tthheenn ddiried with warm air at 50°C. Solution I

50% polyvinylpropylene polyvinylchloride copolymer (Propiofan 200D BASF) 10.0ml

Freshly precipitated calcium carbonate 4.0 gm 0.1M Sodium phosphate pH7.0 12.0ml

10% dioctyl sodium sulphosuccinate 0.1ml

Propiofan 200D has a high molecular weight and a film forming temperature of about 30 C.

Following drying the matrix with its attached membrane was exposed to 0.1 N HC1 for 30 minutes after which it was rinsed for 60 minutes in distilled water and ^* dried. It was next impregnated with Solution I from E a- _ _.e 1 for 30 minutes and dried. Finally, it was impregnated with Solution II, again for 30 minutes and then dried. Solution II

Cholesterol esterase - 75 microns

Cholesterol oxidase 100 microns

Horseradish peroxidase 1800 microns

5% polyvinylpyrrolidone K90 (GAF) 7.5ml

0.1 M Sodium phosphate pH7.0 5.0ml

15% sodium lauryl sulphate 0.5ml

After application of blood this reactive film was wiped clear of red cells and after 60 seconds gave a well graduated response to cholesterol-containing blood.

Although the invention has been described above with reference to examples and to preferred embodiments, it will be appreciated that the invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The above description is therefore to be considered as in all respects, illustrative and not restrictive, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Claims

1. A diagnostic test device for the detection and quantitation of analytes in the cell- and particle-free fraction of biological fluids, said device comprising an outer porous membrane and an underlying absorbent carrier matrix containing predetermined reagents to bring about a graduated response to specific analyte(s).

2. A diagnostic test device according to Claim 1, wherein the membrane is formed from a water-stable film-forming polymer cast onto the underlying matrix which forms a support for the membrane.

3. A diagnostic test device according to Claim 2, wherein the film-forming polymer casting composition includes compounds which are soluble under aqueous conditions and may subsequently be dissolved out of the membrane.

4. A diagnostic test device according to any one of the preceding claims, wherein the matrix comprises a hydrophilic polymer, such as cellulose or polyamide .

5. A diagnostic test device according to Claim 4, wherein the matrix includes reagents, chromogen, and optionally reflectants.

6. A diagnostic test device according to any one of the preceding claims, containing reagents for the quantitation of glucose and/or cholesterol.

7. A diagnostic test device according to Claim 6, containing reagents for the quantitation of glucose, cholesterol and other hydrophilic or hydrophobic blood analytes.

8. A diagnostic test device according to Claim 5, wherein the chromogen includes a 3,3 5,5 - tetraalkylbenzidene.

9. A diagnostic test device according to any one of the preceding claims, contained on an inert support base.

10. A diagnostic test device according to Claim 9, wherein the support base is a plastic strip.

11. A diagnostic test device according to any one of the preceding claims, in which sample metering is an intrinsic capability.

12. A diagnostic test device according to any one of the preceding claims, wherein the substrate on which the matrix is cast is optically clear, and the monitoring of chromogen colour change is possible from the side of the device opposite to that on which the biological fluid is applied.

13. A diagnostic test device according to Claim 2 , wherein the film-forming polymer has a glassing temperature such that cold flow will not adversely affect porosity.

14. A method of manufacturing a diagnostic test device for the detection and quantitation of analytes in the cell- and particle-free fraction of biological fluids, comprising forming a porous matrix on a inert substrate or support, impregnating the matrix with reagents, chromogens and optionally reflectants and forming an outer membrane on the matrix from a water-stable film-forming polymer cast onto the upper surface of the matrix.

15. A method according to Claim 14 wherein the reagents, chromogens and optional reflectants are contained in the matrix when it formed on the substrate or support.

16. A method according to Claim 14 or Claim 15 wherein the film-forming polymer cast onto the underlying matrix includes compounds which are soluble under aqueous conditions and which may subsequently be dissolved out of the membrane.