CA1255196A - Test device and a method for the detection of a component of a liquid sample - Google Patents
Test device and a method for the detection of a component of a liquid sampleInfo
- Publication number
- CA1255196A CA1255196A CA000475165A CA475165A CA1255196A CA 1255196 A CA1255196 A CA 1255196A CA 000475165 A CA000475165 A CA 000475165A CA 475165 A CA475165 A CA 475165A CA 1255196 A CA1255196 A CA 1255196A
- Authority
- CA
- Canada
- Prior art keywords
- membrane
- sample
- test device
- detection
- pore diameter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
Abstract
A B S T R A C T
Test device for the detection of a component in a liquid sample, the device comprising a support layer, a microporous polymer layer, where appropriate other layers and, incorporated in one or more of the layers, re-agents for the detection of the component to be determined, characterised in that the microporous polymer layer is a membrane which has an asymmetric pore struc-ture and is produced by the coagulation process, the pores narrow-ing toward the side to which the sample is applied, and in that the support layer is macroscopically smooth and preferably impermeable to the sample under the test conditions.
Test device for the detection of a component in a liquid sample, the device comprising a support layer, a microporous polymer layer, where appropriate other layers and, incorporated in one or more of the layers, re-agents for the detection of the component to be determined, characterised in that the microporous polymer layer is a membrane which has an asymmetric pore struc-ture and is produced by the coagulation process, the pores narrow-ing toward the side to which the sample is applied, and in that the support layer is macroscopically smooth and preferably impermeable to the sample under the test conditions.
Description
~5~5~
The present invention relates to an ;mproved ana-lyt;cal eLement for the spectrophotometric analysis of a component of a flu;d, ;n part;cular a body flu;d. The analyt;cal element according to the invention ;s sharac-ter;sed by hav;ng at least one asymmetric ~embrane pro-duced by the coagula~ion process.
The determ;nat;on of a component of a flu;d using "dry" reagents ~for example test str;ps) ;s becoming of ;ncreas;ng importance, ;n particular in clinical diagno-sis. Thus, the detection of certain components of urine,serum or blood, such as glucose, bilirubin, urea or pro-teins, is increas;ngly carried out using test strips.
Compared ~ith conventional ~et chemical methods, analyses of this type are more rapid, more straightfor~ard and more reasonably pr;ced.
In the test strips customarily used for liquid samples~ the reagents necessary for the determinat;on of the analyte sought are contained in a suitable fluid-absorbing support material. ~hen the l;quid sample is applied to this support, diffus;on of the fluid into the support ~reaction space) takes place, ~hereupon the detec-t;on reagents produce, w;th the sample component to be analysed, for example a spec;fic, concentrat;on dependent colorat;onO
The flu;d-absorb;ng support materials initially used ~ere simple papers~ and those used subsequently were chemically modified papers ~hich ~ere impregnated with the detection reagents. Ho~ever~ because of their lack of homogeneity in respect of layer thickness and compos;t;on~
papers are poorly su;ted for quantitat;ve determinations.
The use of polymer;c support materials by means of su;table coat;ng techniques represented an important advance in d;agnosis us;ng quant;tative test strips.
Thus, a multi-layer test device comprising a Le A 22 682 transparent support, a gel~t;n layer and a microporous cellulose acetate layer conta;ning f;ller is descr;bed ;n DE-AS (German Published Specification) 2,332,760. The gelat;n layer conta;ns the reagents and thus acts as the react;on and detect;on zone. The funct;on of the m;cro-porous cellulose acetate layer is the homogeneous distri-bution of the sample, the removal of the erythrocytes, and the reflection of the measurement radiation incident from the s;de of the support.
One advantage of test devices conta;ning gelat;n layers ;s that the coating techn;ques used for the;r pro-duct;on are kno~n from photography and are ;ndustr;ally ~ell developed~ A disadvantage of such systems, as it ;s for other test dev;ces ~hich conta;n polymers ~h;ch s~ell in ~ater tfor example agarose), that s~ell;ng processes take place ;n addition to the detection reaction so that the react;on does not come to a standst;ll unt;l after a lengthy period. Thus, for rapid analysis it ;s poss;ble to use only the kinet;cs of the react;on, not end-po;nt determination. In order to make quantitative analyses possible, the sample must be metered in. Moreover, gela-tin is not ideal because of the limited stability of the biochemical detection reagents in gelatin, and because of the limited stability of the coloration formed in the detection reaction (thus, delaying evaluation of the analysis for several days is not possible). In addition, gelatin is sensitive to proteases ~hich customarily occur as an impurity in the enzymes necessary for the detection reaction.
Another test strip system based on polymers for the determination of constituents in body fluids is des-cribed in European Patent Application 0,064,710. In this instance, the matrix used for the reagents is a porous polymer film produced by drying a latex ~for example an aqueous dispersion of polyYinyl propionate) in the pre-sence of an expanding agent ~for example silica gel~. The Le A 22 6~2 l;quid sa~ple to be anaLysed is applied directLy to the reagent layer ~hich is loc~ted on a PVC film. After a certain time, the excess sample and erythrocytes are ~iped off, and the colour reaction is observed or determined by reflectometry from the side facing away from the support.
An unsatisfactory feature of this system (and quite generally for systems in ~hich the sample ;s di-rectly applied to the reagent layer) is so-called bleed-ing. This means that detection reagents from the reagent layer, in particular ~ater-soluble ones (for example en-zymes)O can dissolve in the excess sample, and these are then removed from the system on ~iping, and this leads to falsification of the results. For this reason, it is also of interest to develop test strips in which evalua-tion via a transparen~ support is possible, so that it ispossible to dispense ~ith ~iping off the sample.
Other disadvantages of the system of European Pa-tent A 64~710 are that the sample must be allo~ed to ac~
for a relatively long time (for example 2 min~, and that i~ is difficult to produce specific, in particular rela-tively large, pores (in the ~m range). Large-pored sys-tems of this type ~ould be of part;cular interest for the detection of high molecular ueight analytes~
The aim of the present invention is to develop a ne~ test agent, in particular for components in ~hole blood, which is straightforward to manipulate and provides results ~hich are as quantitative as possible. Straight-for~ard ~anipulation is important to the extent that diag~
nosis using test strips is being increasingly employed by non-spec;alists for ~hom there are tifficulties uith, in particular, exact metering of the amount of sample or ~ip;ng off the excess sample after defined times.
}t should be possible straightfor~ardly to produce the test agent of constant quality~ and it should have the follo~ing propert;es, in particular:
- reproducible tependence of the colour reaction on the Le A 22 682 ~ ~ S ~ ~6 anaLyte concentration;
- ;ntensive ~oloration;
- rap;d react;on ~end-po;nt determ;nat;on);
- evaluation from the support side and (after ~iping) from the appl;cat;on s;de;
- measurement of ~hole blood possible;
- good stab;lity of the colours and the entire system;
- straightfor~ard production of spec;f;c pore sizes;
- analytical values independent of the sample volume applied;
- non-swelling polymer matr;x;
- no bleed;ng.
It has no~ been found, surpr;s;ngly, that test de~
v;ces having polymer layers for sample application can be produced by the method of membrane production by coagula-tion of polymer solutions, and these, alone or combined ~ith other elements~ meet all ~he requirements of clinical diagnosis ~ithout exhibit;ng the abovementioned de-f;c;enc;es. Thus, a large number of polymer systems of a ~ide variety of chemical const;tutions ~for example hydrophilic; hydrophobic; acid or basic ion exchanger groups), ~hich permit specific separations and are not biologically degradable~ are suitable for the production of membranes by the coagulation method. In addition, it is possible, using the production process, to produce mem-branes having a variety of defined pore sizes and pore volumes, and thus the specific separation of interfering constituents is made possi~le~ and the system acts in a self-metering fashion.
The present invention relates to a test device for the de~ection of a component in a liquid sample, in parti~
cular in a body fluid such as blood or urine, t`he device compris;ng a support layer, a microporous polymer layer, ~here appropriate other layers, and reagents, incorporated in one or more of the layers, for the detection of the components to be determined. The invent;on is Le A 22 6~2 `~ . , -" ` '` ' ~2~
characterised by, on the one hand, the use of a m;cro-porous polymer layer ~h;ch is a membrane produced by the coagulat;on method and has an asymmetric pore structure, the pores narro~;ng to~ard the s;de of the test deYice 5 ~hich ;s intended for application of the sample, and by, on the other hand, the use of a macroscop;caLly smooth support layer which ;s preferably ;mpermeable ~o the sample under the test cond;tions.
Microporous films composed of completely synthetic polymers ~hich can be produced ~ith defined pore volumes and variable pore s;zes are su;table and preferred accor-d;ng to the ;nvention. The completely synthetic nature of the polymers is important for the reason that, in con-trast to naeural or semi-synthetic polymers, they can be 1~ produced with high reproduc;b;l;ty and they perm;t straightfor~ard qual;ty control.
Microporous polyner films (membranes) ~h;ch are employed for the large-scale industrial separat;on of mo-lecular m1xtures by using an external pressure have been kno~n for so~e t;me and are commercially available. There are several processes for the product;on of membranes of this type, the so-called "phase~inversion method" having achieved the greatest ;mportance. Informat;on on the fundamentals of these techn;ques is to be found in, for Z5 example, H. Strathmann, "Trennungen von molekularen Mis-chungen mit Hilfe synthetischer Membranen" (separat;on of moLecuLar mixtures us;ng synthet;c membranes), Ste;nkopf-verlag Darmstadt (1979)~
There is a variety of variants ~;thin the phase-inversion process. In the coagulation process, the pro-cedure ;n pr;nc;ple ;s such that a sol;d support is coated ~ith a polymer solution (casting solut;on) of un;form thickness ~for example 100 ~m), where appropriate exposed to an atmosphere ~hich contains a non~solvent (preferably ~ater) for the polymer in the form of the vapour, until the solut;on has part;ally or completely gelled, and then Le A Z2 682 immersed ;n a coagulation bath, whereupon a solid, mirco-porous film ~;th an asymmetrical structure is produced.
If the membrane film remains adherent to the solid support during the coagulation twhen spec;ally porous polymer bonded ~ebs or polymer films are used), then supported membranes are obtainedA If the membrane film becomes de-tached from the solid support during the coagulation (for example when glass i~ used)~ then unsupported membranes are produced. The nature of the coagulation fluid is such that it is miscible with the solvent in the casting solu-tion, but is a precipitant for the polymer. A typical feature of this variant is that the coagulation (pore-formation~ essentially only takes place on immersion in the coagul~tion fluid, and that asymmetrical membrane structures are produced. In this context, asymmetrical denotes that the pores - regarded from the underside of the membrane ~support side) - narrow toward the surface of the membrane.
As a rule, the ratio of the mean pore diameter on the underside of the membrane to that on the surface of the membrane in these cases ~hich can be det~rmined by, for example, electron microscopic films) is greater than 5:1, preferably greater than 10:1, greatly preferably greater than 30:1. As has emergedr ~he use according to the invention of membranes of this type has the advantage that blockage of the pores is prevented when the product is applied to the surface of the membrane.
The majority of sommercially available membranes are produced by this process. It has already been pro-posed that membranes of this type be used directly as asupport matrix for detection systems (US Patent Specifi-cation 3,607,093) by subsequently impregnating the finished membranes with the test reagents. Ho~ever, the commercially available supported membranes do not meet the requirements of reproducibility ~hich are set in practice.
This is because the membranes are located on Le A 22 682 , ~ ' ' .
porous bonded ~eb supports~ composed of~ for example, polyethylene, polypropylene or polyester, ~hich are not macroscopically smooth. Hence the membrane layer thick-ness, and thus the pore volume, is subject to relatively S large fluctuations uhich lead to variations in the detec-tion reaction. In addition, the consequence of the porous~ fluid-absorbing nature of the bonded steb support is that not just the pore volume of the membrane is re-sponsible for the amount of fluid absorbed but al~o the porous support which absorbs fluid because of capillary forces.
Unsupported asymmetrical membranes have not hitherto been used or proposed for the production of test devicesO However, they are less preferred according to the invention since they are difficult to manipulate and can, as a rule~ only be dried ~hen they are impregnated with preservatives. Thus, in the production of a test agent, they require another operation step, namely attach-ment to a support. It is possible to use adhesives for this purpose, but these may lead to further complications (for example part;al dissolution of the membrane; absorp-tion of fluid).
The incorporation of the detection reagents into the finished membranes is carried out by impregna~ion using the system described in US Patent 3,6û7~093. Since, as a rule, both organic and water-soluble reagents have to be incorporated, both impregnation in organic solution and impregnation in aqueous solution are necessary~
Thus, there is a restriction to membranes which are resistant to the appropriate organic solvent. In addition, systems of the type ~hich are merely impregnated ~ith the detection reagents tend to bleed.
Another variant of membrane production by the phase-inversion method is based on dissolving a polymer in a mixture of a good, readily vapourised solvent and 3 poor, higher boiling solvent. If a solution of this type Le A 22 ~82 ~2~5~36 is spread out to form a film, and heat is applied, then the good, lo~ boiling solvent evaporates fir~t, ~hile the poor solvent for the polymer accumulates and induces the system to coagulate. Then, if required to ~ash out the rema;nder of the high boil;ng solvent, the membrane can also be ;mmersed in a liquid bath ~hich, however, in con-trast to the coagulat;on ;n a precip;tat;on bath descr;bed above, does not essentiaLly contribute to the formation of the membrane.
Asymmetrical structures are not obta;ned by th;s method. In addition, in practice it is possible to use only those polymers ~or ~hich there exists a good, lo~
boil;ng solvent~ and this greatly l;mits the select;on of polymers. Thus, in pract;ce to date~ only the semi-syn-thetic cellulose derivat;ves tfor example cellulose ace-tateO cellulose n;trate), for ~h;ch acetone is a very good solvent, have been processed into membranes using this method. For example, the filter layers of cellulose ace-tate ("blush polymer layers") located on gelatin Layers, uhich ~ere used in accordance u;th DE-AS tGerman Publ;shed Spec;fication) 2~332,760, were produced in the solvent system acetone/toluene.
As described in DOS (German PubLished Specifica-tion) 2,6~2~975, this method has also already been used for the production of s;ngle-layer detect;on systems, the reagents being incorporated in the polymer solution.
Thus, using this processO porous membranes containing the detection reagents have beèn obtained directly. The test elements are produced in accordance ~ith DOS tGerman Pub lished Specification) 2,602,975 on a glass plate~ from ~hich, after drying, the membranes are detached and are glued to a plastic film. This results in the difficulties o~ unsupported membranes described above. It is also described in DOS (German Published Specification) 20602~975 that it is possible to produce the detection systems directly on an integral substrate by replacing the Le A 22 682 ..: ' :`;' ''' "'''' - -.~
., , :
. ;-. ~'' . , :~ .'' :, ~2~9i~
glass plate b~ a polymer film, ~hich needs to be of such a nature that it reacts chemically or physically ~ith the solvent system used, fusion of the membrane ~ith the sup-port film occurring. However, very fe~ variat;ons of this process are poss;ble, since the composition of the solvent and the evaporation t;me have to be selected to accord ~;th the des;red porosity of the membrane to be produced.
Accordingly, the effect on the support ~ill differ in its extent depending on ~hether ;t is des;red to produce mem-lQ branes w;th fine pores or ~ith coarse pores. As a rule,in the case of transparent supports, the per~eability ~o light is also impa;red due to the partial dissolution, and thus evaluation from the support s;de ;s d;fficult.
In add;t;on, as a rule polymer f;lms ~h;ch are attacked by solvents undergo irreversible changes, such as s~elling, shrinkage or irregularities, especially when the contact between the solvent and the polymeric support lasts a relatively long time, which is the case in the type of membrane production described there. Accordingly, th;s method ;s not su;table for the product;on of suppor-ted m;croporous polymer f;lms ~hich are ;ntended to meet the requ;rements for reagent supports.
Deta;ls of the product;on of microporous sheet-l;ke structures by the coagulation process to be used according to the invention are given in a large number of publications. Thus, in German Patent Specification 1,110~607, it is proposed for the coagulation of polyure-thanes based on polyethers that hygroscopic polyurethane solutions (an example of a solvent used for this is di-methylformamide) be exposed to the action of an atmospherecontaining ~ater ~apour, ~hich is, ~here appropriate, made to circulate and which has a relative humidity of 15 to 100X at a temperature of the dry thermometer of 10 to 38C. Because of the absorption of ~ater as a result of the hygroscopicity of the solvent~ the polyurethane starts to precipitate out of ~he solution from the Le A 22 ~82 ~2~ 6 direction of the surface, probably with preformation of the m;croporous structure. ~hen f;lms or coatin~s pre-~elled in this manner are placed in ~ater, the hygroscopic solvent ;s completely removed from the fil~, Mith coagu-lation of the solution~
DE~OS ~German Publ;shed Spec;f;cat;on) 1D4~4,163;nd;cates a some~hat modified process: the polyurethane solution is first, by addition of ~inor amounts of non-solvents (for example uater), converted into a state of incipient phase separation, that is to say in a slightly cloudy form resembling a dispersion, before it is coagu-lated ~after spreading out in the form of a sheet) di-rectly, that is to say Yithout pregelling in a moist at-mosphere, by ;mmersion ;n the non-solvent.
Another process is indicated in DE-OS ~6erman Pub-l;shed Specification) 1~444~165~ according to ~hich the polymer solution can be converted into microporous films, ~ithout pregelling, by indirect coagulation ;n a m;xture of non-solvent and solvent ~for example dimethylformamide/
20 H20 in a mixing ratio bet~een 10:90 and 95 5)a According to 3nother variant, which is described in Belgian Patent Specification ~24,25~o su~ficient non-solvent is added to the polymer solution for the poly~er to separate out as a gel. It is onl~ this ~el which is then spread onto a substrate and coagulated uith non-sol-vent (~ater) to give a microporous structure.
It is indicated, in DE-AS t6erman Published Speci-fication) 1,238,206, that direct coagulation of elastomer solutions leads to microporous structures ~hen the coated substrat~s are coagulated ;n baths ~hich are heated to the neighbourhood of the boiling point of the fluid in the bath~ for example in hot ~ater at 95C.
Improved results are obtained ~hen the pregelling is also carried out at elevated temperature. ~hus, 9E-OS
~German Published Specification) 2~025,616 describes a process for the production of microporous sheet-like Le A 22 682 _ ~ .
i~2~
structures ;n which a thin layer of a polyurethane soLu-tion is exposed to an atmosphere of ~ater v3pour ~ith a rela~ive humidity of at least 50X~ at temperatures above 65t5, and then the major amount o~ the solvent is removed in aqueous coagulation baths, and the product is then dried.
Accord;n0 to 9E-OS tGerman Publ;shed Specif;ca-tion) 2,125,908, ~ater ~apour at a temperature bet~een 101C and 190C is passed over a layer of a polyurethane solution until the content of organic solvent in the layer has decreased to less than 50X by ~e;ght, ancl the Layer has been converted ;nto a sol;d, mechan;cally stable microporous sheet-like structure~ Th;s process has the part;cular advantage that a m;croporous final product re-sults from a polyurethane solu~ion ;n a shor~ t;me and in a single process step.
It ;s possible in the processes mentioned to add certain coagulation aids to the polymer solutions ~ith the object of ;mprov;ng the coagulabil;ty. Thus, DE-AS (Ger-20 man Publ;shed Spec;fica~ion3 1~270,276, DE~OS (German Pub-l;shed Specif;cation~ 1,694,171 and DE-OS tGerman Pub-l;shed Specif;cation) 1,7~9,277 descr;be processes for ~he production of sheet-l;ke structures ~h;ch are permeable to ~ater vapour, in ~h;ch solutions of 90 to 70 parts by ~eight of polyurethanes or polyureas and 10 ~o 30 parts by weight of high ~olecular weight, essentially linear, cationic polyurethanes~ ~h;ch contain 0.5 to 2.0X by ~eight of quaternary ammon;um nitrogen atoms, are~ ~here appropr;ate after gelling in mois~ air, coagulated ~;th ~a~er or a mixture of water and solvent. In addition to the cationic polyure~hanes, these solutions can also ron-ta;n ~In;onic tanning agents as addit;onal coagulation regulators~
According to DE-OS ~German Published Specifica-35 t;on9 20427,274, in many cases it is possible to achieve a further improvement by the polyurethane solutions to be Le A 22 b82 ..~
coagulated c~ntai~ing certain cationic or anionic polyure-thane/urea suspensions alone orD preferably, simultane-ously cationic and anionic polyurethane ~urea)s in sal~
~orm. It is possible in this ~anner to control, in such a manner that sheet-l;ke structures of satisfactory microporosity are produced, ~he coagulation even of polyurethane soLut;ons ~hich are difficult to process.
Using the processes described in the printed mat-ter mentioned, it is possible to produce microporous mem branes from virtually every soluble polymer, it being pos-sible to achieve specific pore sizes by means of various parameters ~for example the concentration of the casting solu~ion, the temperature~ additives, the nature of the coagulation fluid) - ~here appropriate after a few pre-lininary tests~
Moreover~ the advantayes of the present inventionresult from these possibilities. Thus, it is possible in a straightfor~ard manner to adjust the composition and porosity of the polymeric ~embrane to suit the intended 2û detection system.
In most cases, it is preferred according to the invention that the coagulation is carried out directly ;n an aqueous coagulation bath, th~t is to say uithout spe-cial pretreatment.
The properties of the detection elements accor-ding So the invention (homogeneity and intensity of the colour react;on, bleedin~, stability o~ the detection sys-tem~ possibility of ~iping off erythrocytes, and rate of the detection reaction) are very dependent on the type of polymer system used.
In order to ~eet the requirements of straightfor ~ard production by machines~ defined pore volume and evaluation from the rear s;de of the support, the polymer casting solutions ~hich are preferably used according to the invention are such that the membrane can be produced directly on a macroscopically smooth, i~permeabLe support.
Le ~ 2~ 682 If the cast;n0 sol~tions of convent;onal membranes (for example celluLose acetate or polysulphone are applied to smooth, i~permeable fil~s, then the solid fil~ ~h;ch is produced during the coa0ulation detaches fro3 the support if the support is not attacked to a sufficient extent by the solvent that it fuses ~ith the membrane. Ho~ever, this leads to variation in the results of measurement.
In order ~o avoid this problem, it ;s necessary in the di-rect production of supported membranes to suit the system of casting solution and support to one another in such 3 manner that the cast;ng solution has a h;gh aff;n;ty for the support but does not attack it, that is to say dis-solve it.
The polymer systems ~hich have proved to be favourable are those ~hich can be modified in respect of their hydrophilicity/hydrophobicity balance in a manner kno~n per se. In this connection, polymers having ionic groups are particularly advantageous, and these can, ~here appropriate, also be used as a component in a polymer mix-ture. ~he hydrophilic ~or hydrophobic) properties of the polymer membrane can be of importance ~or, for example, the optimisation of the colour reaction (the colour is frequently more intense ~hen the polymer contains ionic ~roups).
Examples of polymers from ~hich it is possible ~o prepare suitable casting solutions for the detection ele-ments claimed according to the invention are:
polyamides, polyamides having disulphimide groups Na~
-S02 N-S02-) tsee, for example, US Paten~ Specification 4,269,967~ po~yether carbonates (see, for example, DE-OS
(German Published Specification) Z,251~066), polyacrylo-nitrile and polyurethanes, as are described in, for example, DE-OS ~6erman Published Specification~ 2,427,274 and the printed material cited there.
Preferred casting solutions are obtained ~hen the Le ~ 22 682 "" , .
,. ,:.
~:~S5~6 - 1~
solu~ions of the poly~ers ~enti~ned are ~;xed ~;th aqueous polym~r dispersions kno~n per se~ uh;ch preferably have ion;c ~roupsO for example composed of polyvinyl compounds, vinyl copoly~ers, polysSyrenesulphon;c Dcids~ polyam;des or polyurethanes. Casting soLut;ons uhich consist of mix-tures of polyurethane solut;ons u;th aqueous polyurethane d;spers;ons ~see, for exa~ple, Ange~. MakromDl. Chem. 98 (1981) 133 and DE-OS (German Published Specif;cation)
The present invention relates to an ;mproved ana-lyt;cal eLement for the spectrophotometric analysis of a component of a flu;d, ;n part;cular a body flu;d. The analyt;cal element according to the invention ;s sharac-ter;sed by hav;ng at least one asymmetric ~embrane pro-duced by the coagula~ion process.
The determ;nat;on of a component of a flu;d using "dry" reagents ~for example test str;ps) ;s becoming of ;ncreas;ng importance, ;n particular in clinical diagno-sis. Thus, the detection of certain components of urine,serum or blood, such as glucose, bilirubin, urea or pro-teins, is increas;ngly carried out using test strips.
Compared ~ith conventional ~et chemical methods, analyses of this type are more rapid, more straightfor~ard and more reasonably pr;ced.
In the test strips customarily used for liquid samples~ the reagents necessary for the determinat;on of the analyte sought are contained in a suitable fluid-absorbing support material. ~hen the l;quid sample is applied to this support, diffus;on of the fluid into the support ~reaction space) takes place, ~hereupon the detec-t;on reagents produce, w;th the sample component to be analysed, for example a spec;fic, concentrat;on dependent colorat;onO
The flu;d-absorb;ng support materials initially used ~ere simple papers~ and those used subsequently were chemically modified papers ~hich ~ere impregnated with the detection reagents. Ho~ever~ because of their lack of homogeneity in respect of layer thickness and compos;t;on~
papers are poorly su;ted for quantitat;ve determinations.
The use of polymer;c support materials by means of su;table coat;ng techniques represented an important advance in d;agnosis us;ng quant;tative test strips.
Thus, a multi-layer test device comprising a Le A 22 682 transparent support, a gel~t;n layer and a microporous cellulose acetate layer conta;ning f;ller is descr;bed ;n DE-AS (German Published Specification) 2,332,760. The gelat;n layer conta;ns the reagents and thus acts as the react;on and detect;on zone. The funct;on of the m;cro-porous cellulose acetate layer is the homogeneous distri-bution of the sample, the removal of the erythrocytes, and the reflection of the measurement radiation incident from the s;de of the support.
One advantage of test devices conta;ning gelat;n layers ;s that the coating techn;ques used for the;r pro-duct;on are kno~n from photography and are ;ndustr;ally ~ell developed~ A disadvantage of such systems, as it ;s for other test dev;ces ~hich conta;n polymers ~h;ch s~ell in ~ater tfor example agarose), that s~ell;ng processes take place ;n addition to the detection reaction so that the react;on does not come to a standst;ll unt;l after a lengthy period. Thus, for rapid analysis it ;s poss;ble to use only the kinet;cs of the react;on, not end-po;nt determination. In order to make quantitative analyses possible, the sample must be metered in. Moreover, gela-tin is not ideal because of the limited stability of the biochemical detection reagents in gelatin, and because of the limited stability of the coloration formed in the detection reaction (thus, delaying evaluation of the analysis for several days is not possible). In addition, gelatin is sensitive to proteases ~hich customarily occur as an impurity in the enzymes necessary for the detection reaction.
Another test strip system based on polymers for the determination of constituents in body fluids is des-cribed in European Patent Application 0,064,710. In this instance, the matrix used for the reagents is a porous polymer film produced by drying a latex ~for example an aqueous dispersion of polyYinyl propionate) in the pre-sence of an expanding agent ~for example silica gel~. The Le A 22 6~2 l;quid sa~ple to be anaLysed is applied directLy to the reagent layer ~hich is loc~ted on a PVC film. After a certain time, the excess sample and erythrocytes are ~iped off, and the colour reaction is observed or determined by reflectometry from the side facing away from the support.
An unsatisfactory feature of this system (and quite generally for systems in ~hich the sample ;s di-rectly applied to the reagent layer) is so-called bleed-ing. This means that detection reagents from the reagent layer, in particular ~ater-soluble ones (for example en-zymes)O can dissolve in the excess sample, and these are then removed from the system on ~iping, and this leads to falsification of the results. For this reason, it is also of interest to develop test strips in which evalua-tion via a transparen~ support is possible, so that it ispossible to dispense ~ith ~iping off the sample.
Other disadvantages of the system of European Pa-tent A 64~710 are that the sample must be allo~ed to ac~
for a relatively long time (for example 2 min~, and that i~ is difficult to produce specific, in particular rela-tively large, pores (in the ~m range). Large-pored sys-tems of this type ~ould be of part;cular interest for the detection of high molecular ueight analytes~
The aim of the present invention is to develop a ne~ test agent, in particular for components in ~hole blood, which is straightforward to manipulate and provides results ~hich are as quantitative as possible. Straight-for~ard ~anipulation is important to the extent that diag~
nosis using test strips is being increasingly employed by non-spec;alists for ~hom there are tifficulties uith, in particular, exact metering of the amount of sample or ~ip;ng off the excess sample after defined times.
}t should be possible straightfor~ardly to produce the test agent of constant quality~ and it should have the follo~ing propert;es, in particular:
- reproducible tependence of the colour reaction on the Le A 22 682 ~ ~ S ~ ~6 anaLyte concentration;
- ;ntensive ~oloration;
- rap;d react;on ~end-po;nt determ;nat;on);
- evaluation from the support side and (after ~iping) from the appl;cat;on s;de;
- measurement of ~hole blood possible;
- good stab;lity of the colours and the entire system;
- straightfor~ard production of spec;f;c pore sizes;
- analytical values independent of the sample volume applied;
- non-swelling polymer matr;x;
- no bleed;ng.
It has no~ been found, surpr;s;ngly, that test de~
v;ces having polymer layers for sample application can be produced by the method of membrane production by coagula-tion of polymer solutions, and these, alone or combined ~ith other elements~ meet all ~he requirements of clinical diagnosis ~ithout exhibit;ng the abovementioned de-f;c;enc;es. Thus, a large number of polymer systems of a ~ide variety of chemical const;tutions ~for example hydrophilic; hydrophobic; acid or basic ion exchanger groups), ~hich permit specific separations and are not biologically degradable~ are suitable for the production of membranes by the coagulation method. In addition, it is possible, using the production process, to produce mem-branes having a variety of defined pore sizes and pore volumes, and thus the specific separation of interfering constituents is made possi~le~ and the system acts in a self-metering fashion.
The present invention relates to a test device for the de~ection of a component in a liquid sample, in parti~
cular in a body fluid such as blood or urine, t`he device compris;ng a support layer, a microporous polymer layer, ~here appropriate other layers, and reagents, incorporated in one or more of the layers, for the detection of the components to be determined. The invent;on is Le A 22 6~2 `~ . , -" ` '` ' ~2~
characterised by, on the one hand, the use of a m;cro-porous polymer layer ~h;ch is a membrane produced by the coagulat;on method and has an asymmetric pore structure, the pores narro~;ng to~ard the s;de of the test deYice 5 ~hich ;s intended for application of the sample, and by, on the other hand, the use of a macroscop;caLly smooth support layer which ;s preferably ;mpermeable ~o the sample under the test cond;tions.
Microporous films composed of completely synthetic polymers ~hich can be produced ~ith defined pore volumes and variable pore s;zes are su;table and preferred accor-d;ng to the ;nvention. The completely synthetic nature of the polymers is important for the reason that, in con-trast to naeural or semi-synthetic polymers, they can be 1~ produced with high reproduc;b;l;ty and they perm;t straightfor~ard qual;ty control.
Microporous polyner films (membranes) ~h;ch are employed for the large-scale industrial separat;on of mo-lecular m1xtures by using an external pressure have been kno~n for so~e t;me and are commercially available. There are several processes for the product;on of membranes of this type, the so-called "phase~inversion method" having achieved the greatest ;mportance. Informat;on on the fundamentals of these techn;ques is to be found in, for Z5 example, H. Strathmann, "Trennungen von molekularen Mis-chungen mit Hilfe synthetischer Membranen" (separat;on of moLecuLar mixtures us;ng synthet;c membranes), Ste;nkopf-verlag Darmstadt (1979)~
There is a variety of variants ~;thin the phase-inversion process. In the coagulation process, the pro-cedure ;n pr;nc;ple ;s such that a sol;d support is coated ~ith a polymer solution (casting solut;on) of un;form thickness ~for example 100 ~m), where appropriate exposed to an atmosphere ~hich contains a non~solvent (preferably ~ater) for the polymer in the form of the vapour, until the solut;on has part;ally or completely gelled, and then Le A Z2 682 immersed ;n a coagulation bath, whereupon a solid, mirco-porous film ~;th an asymmetrical structure is produced.
If the membrane film remains adherent to the solid support during the coagulation twhen spec;ally porous polymer bonded ~ebs or polymer films are used), then supported membranes are obtainedA If the membrane film becomes de-tached from the solid support during the coagulation (for example when glass i~ used)~ then unsupported membranes are produced. The nature of the coagulation fluid is such that it is miscible with the solvent in the casting solu-tion, but is a precipitant for the polymer. A typical feature of this variant is that the coagulation (pore-formation~ essentially only takes place on immersion in the coagul~tion fluid, and that asymmetrical membrane structures are produced. In this context, asymmetrical denotes that the pores - regarded from the underside of the membrane ~support side) - narrow toward the surface of the membrane.
As a rule, the ratio of the mean pore diameter on the underside of the membrane to that on the surface of the membrane in these cases ~hich can be det~rmined by, for example, electron microscopic films) is greater than 5:1, preferably greater than 10:1, greatly preferably greater than 30:1. As has emergedr ~he use according to the invention of membranes of this type has the advantage that blockage of the pores is prevented when the product is applied to the surface of the membrane.
The majority of sommercially available membranes are produced by this process. It has already been pro-posed that membranes of this type be used directly as asupport matrix for detection systems (US Patent Specifi-cation 3,607,093) by subsequently impregnating the finished membranes with the test reagents. Ho~ever, the commercially available supported membranes do not meet the requirements of reproducibility ~hich are set in practice.
This is because the membranes are located on Le A 22 682 , ~ ' ' .
porous bonded ~eb supports~ composed of~ for example, polyethylene, polypropylene or polyester, ~hich are not macroscopically smooth. Hence the membrane layer thick-ness, and thus the pore volume, is subject to relatively S large fluctuations uhich lead to variations in the detec-tion reaction. In addition, the consequence of the porous~ fluid-absorbing nature of the bonded steb support is that not just the pore volume of the membrane is re-sponsible for the amount of fluid absorbed but al~o the porous support which absorbs fluid because of capillary forces.
Unsupported asymmetrical membranes have not hitherto been used or proposed for the production of test devicesO However, they are less preferred according to the invention since they are difficult to manipulate and can, as a rule~ only be dried ~hen they are impregnated with preservatives. Thus, in the production of a test agent, they require another operation step, namely attach-ment to a support. It is possible to use adhesives for this purpose, but these may lead to further complications (for example part;al dissolution of the membrane; absorp-tion of fluid).
The incorporation of the detection reagents into the finished membranes is carried out by impregna~ion using the system described in US Patent 3,6û7~093. Since, as a rule, both organic and water-soluble reagents have to be incorporated, both impregnation in organic solution and impregnation in aqueous solution are necessary~
Thus, there is a restriction to membranes which are resistant to the appropriate organic solvent. In addition, systems of the type ~hich are merely impregnated ~ith the detection reagents tend to bleed.
Another variant of membrane production by the phase-inversion method is based on dissolving a polymer in a mixture of a good, readily vapourised solvent and 3 poor, higher boiling solvent. If a solution of this type Le A 22 ~82 ~2~5~36 is spread out to form a film, and heat is applied, then the good, lo~ boiling solvent evaporates fir~t, ~hile the poor solvent for the polymer accumulates and induces the system to coagulate. Then, if required to ~ash out the rema;nder of the high boil;ng solvent, the membrane can also be ;mmersed in a liquid bath ~hich, however, in con-trast to the coagulat;on ;n a precip;tat;on bath descr;bed above, does not essentiaLly contribute to the formation of the membrane.
Asymmetrical structures are not obta;ned by th;s method. In addition, in practice it is possible to use only those polymers ~or ~hich there exists a good, lo~
boil;ng solvent~ and this greatly l;mits the select;on of polymers. Thus, in pract;ce to date~ only the semi-syn-thetic cellulose derivat;ves tfor example cellulose ace-tateO cellulose n;trate), for ~h;ch acetone is a very good solvent, have been processed into membranes using this method. For example, the filter layers of cellulose ace-tate ("blush polymer layers") located on gelatin Layers, uhich ~ere used in accordance u;th DE-AS tGerman Publ;shed Spec;fication) 2~332,760, were produced in the solvent system acetone/toluene.
As described in DOS (German PubLished Specifica-tion) 2,6~2~975, this method has also already been used for the production of s;ngle-layer detect;on systems, the reagents being incorporated in the polymer solution.
Thus, using this processO porous membranes containing the detection reagents have beèn obtained directly. The test elements are produced in accordance ~ith DOS tGerman Pub lished Specification) 2,602,975 on a glass plate~ from ~hich, after drying, the membranes are detached and are glued to a plastic film. This results in the difficulties o~ unsupported membranes described above. It is also described in DOS (German Published Specification) 20602~975 that it is possible to produce the detection systems directly on an integral substrate by replacing the Le A 22 682 ..: ' :`;' ''' "'''' - -.~
., , :
. ;-. ~'' . , :~ .'' :, ~2~9i~
glass plate b~ a polymer film, ~hich needs to be of such a nature that it reacts chemically or physically ~ith the solvent system used, fusion of the membrane ~ith the sup-port film occurring. However, very fe~ variat;ons of this process are poss;ble, since the composition of the solvent and the evaporation t;me have to be selected to accord ~;th the des;red porosity of the membrane to be produced.
Accordingly, the effect on the support ~ill differ in its extent depending on ~hether ;t is des;red to produce mem-lQ branes w;th fine pores or ~ith coarse pores. As a rule,in the case of transparent supports, the per~eability ~o light is also impa;red due to the partial dissolution, and thus evaluation from the support s;de ;s d;fficult.
In add;t;on, as a rule polymer f;lms ~h;ch are attacked by solvents undergo irreversible changes, such as s~elling, shrinkage or irregularities, especially when the contact between the solvent and the polymeric support lasts a relatively long time, which is the case in the type of membrane production described there. Accordingly, th;s method ;s not su;table for the product;on of suppor-ted m;croporous polymer f;lms ~hich are ;ntended to meet the requ;rements for reagent supports.
Deta;ls of the product;on of microporous sheet-l;ke structures by the coagulation process to be used according to the invention are given in a large number of publications. Thus, in German Patent Specification 1,110~607, it is proposed for the coagulation of polyure-thanes based on polyethers that hygroscopic polyurethane solutions (an example of a solvent used for this is di-methylformamide) be exposed to the action of an atmospherecontaining ~ater ~apour, ~hich is, ~here appropriate, made to circulate and which has a relative humidity of 15 to 100X at a temperature of the dry thermometer of 10 to 38C. Because of the absorption of ~ater as a result of the hygroscopicity of the solvent~ the polyurethane starts to precipitate out of ~he solution from the Le A 22 ~82 ~2~ 6 direction of the surface, probably with preformation of the m;croporous structure. ~hen f;lms or coatin~s pre-~elled in this manner are placed in ~ater, the hygroscopic solvent ;s completely removed from the fil~, Mith coagu-lation of the solution~
DE~OS ~German Publ;shed Spec;f;cat;on) 1D4~4,163;nd;cates a some~hat modified process: the polyurethane solution is first, by addition of ~inor amounts of non-solvents (for example uater), converted into a state of incipient phase separation, that is to say in a slightly cloudy form resembling a dispersion, before it is coagu-lated ~after spreading out in the form of a sheet) di-rectly, that is to say Yithout pregelling in a moist at-mosphere, by ;mmersion ;n the non-solvent.
Another process is indicated in DE-OS ~6erman Pub-l;shed Specification) 1~444~165~ according to ~hich the polymer solution can be converted into microporous films, ~ithout pregelling, by indirect coagulation ;n a m;xture of non-solvent and solvent ~for example dimethylformamide/
20 H20 in a mixing ratio bet~een 10:90 and 95 5)a According to 3nother variant, which is described in Belgian Patent Specification ~24,25~o su~ficient non-solvent is added to the polymer solution for the poly~er to separate out as a gel. It is onl~ this ~el which is then spread onto a substrate and coagulated uith non-sol-vent (~ater) to give a microporous structure.
It is indicated, in DE-AS t6erman Published Speci-fication) 1,238,206, that direct coagulation of elastomer solutions leads to microporous structures ~hen the coated substrat~s are coagulated ;n baths ~hich are heated to the neighbourhood of the boiling point of the fluid in the bath~ for example in hot ~ater at 95C.
Improved results are obtained ~hen the pregelling is also carried out at elevated temperature. ~hus, 9E-OS
~German Published Specification) 2~025,616 describes a process for the production of microporous sheet-like Le A 22 682 _ ~ .
i~2~
structures ;n which a thin layer of a polyurethane soLu-tion is exposed to an atmosphere of ~ater v3pour ~ith a rela~ive humidity of at least 50X~ at temperatures above 65t5, and then the major amount o~ the solvent is removed in aqueous coagulation baths, and the product is then dried.
Accord;n0 to 9E-OS tGerman Publ;shed Specif;ca-tion) 2,125,908, ~ater ~apour at a temperature bet~een 101C and 190C is passed over a layer of a polyurethane solution until the content of organic solvent in the layer has decreased to less than 50X by ~e;ght, ancl the Layer has been converted ;nto a sol;d, mechan;cally stable microporous sheet-like structure~ Th;s process has the part;cular advantage that a m;croporous final product re-sults from a polyurethane solu~ion ;n a shor~ t;me and in a single process step.
It ;s possible in the processes mentioned to add certain coagulation aids to the polymer solutions ~ith the object of ;mprov;ng the coagulabil;ty. Thus, DE-AS (Ger-20 man Publ;shed Spec;fica~ion3 1~270,276, DE~OS (German Pub-l;shed Specif;cation~ 1,694,171 and DE-OS tGerman Pub-l;shed Specif;cation) 1,7~9,277 descr;be processes for ~he production of sheet-l;ke structures ~h;ch are permeable to ~ater vapour, in ~h;ch solutions of 90 to 70 parts by ~eight of polyurethanes or polyureas and 10 ~o 30 parts by weight of high ~olecular weight, essentially linear, cationic polyurethanes~ ~h;ch contain 0.5 to 2.0X by ~eight of quaternary ammon;um nitrogen atoms, are~ ~here appropr;ate after gelling in mois~ air, coagulated ~;th ~a~er or a mixture of water and solvent. In addition to the cationic polyure~hanes, these solutions can also ron-ta;n ~In;onic tanning agents as addit;onal coagulation regulators~
According to DE-OS ~German Published Specifica-35 t;on9 20427,274, in many cases it is possible to achieve a further improvement by the polyurethane solutions to be Le A 22 b82 ..~
coagulated c~ntai~ing certain cationic or anionic polyure-thane/urea suspensions alone orD preferably, simultane-ously cationic and anionic polyurethane ~urea)s in sal~
~orm. It is possible in this ~anner to control, in such a manner that sheet-l;ke structures of satisfactory microporosity are produced, ~he coagulation even of polyurethane soLut;ons ~hich are difficult to process.
Using the processes described in the printed mat-ter mentioned, it is possible to produce microporous mem branes from virtually every soluble polymer, it being pos-sible to achieve specific pore sizes by means of various parameters ~for example the concentration of the casting solu~ion, the temperature~ additives, the nature of the coagulation fluid) - ~here appropriate after a few pre-lininary tests~
Moreover~ the advantayes of the present inventionresult from these possibilities. Thus, it is possible in a straightfor~ard manner to adjust the composition and porosity of the polymeric ~embrane to suit the intended 2û detection system.
In most cases, it is preferred according to the invention that the coagulation is carried out directly ;n an aqueous coagulation bath, th~t is to say uithout spe-cial pretreatment.
The properties of the detection elements accor-ding So the invention (homogeneity and intensity of the colour react;on, bleedin~, stability o~ the detection sys-tem~ possibility of ~iping off erythrocytes, and rate of the detection reaction) are very dependent on the type of polymer system used.
In order to ~eet the requirements of straightfor ~ard production by machines~ defined pore volume and evaluation from the rear s;de of the support, the polymer casting solutions ~hich are preferably used according to the invention are such that the membrane can be produced directly on a macroscopically smooth, i~permeabLe support.
Le ~ 2~ 682 If the cast;n0 sol~tions of convent;onal membranes (for example celluLose acetate or polysulphone are applied to smooth, i~permeable fil~s, then the solid fil~ ~h;ch is produced during the coa0ulation detaches fro3 the support if the support is not attacked to a sufficient extent by the solvent that it fuses ~ith the membrane. Ho~ever, this leads to variation in the results of measurement.
In order ~o avoid this problem, it ;s necessary in the di-rect production of supported membranes to suit the system of casting solution and support to one another in such 3 manner that the cast;ng solution has a h;gh aff;n;ty for the support but does not attack it, that is to say dis-solve it.
The polymer systems ~hich have proved to be favourable are those ~hich can be modified in respect of their hydrophilicity/hydrophobicity balance in a manner kno~n per se. In this connection, polymers having ionic groups are particularly advantageous, and these can, ~here appropriate, also be used as a component in a polymer mix-ture. ~he hydrophilic ~or hydrophobic) properties of the polymer membrane can be of importance ~or, for example, the optimisation of the colour reaction (the colour is frequently more intense ~hen the polymer contains ionic ~roups).
Examples of polymers from ~hich it is possible ~o prepare suitable casting solutions for the detection ele-ments claimed according to the invention are:
polyamides, polyamides having disulphimide groups Na~
-S02 N-S02-) tsee, for example, US Paten~ Specification 4,269,967~ po~yether carbonates (see, for example, DE-OS
(German Published Specification) Z,251~066), polyacrylo-nitrile and polyurethanes, as are described in, for example, DE-OS ~6erman Published Specification~ 2,427,274 and the printed material cited there.
Preferred casting solutions are obtained ~hen the Le ~ 22 682 "" , .
,. ,:.
~:~S5~6 - 1~
solu~ions of the poly~ers ~enti~ned are ~;xed ~;th aqueous polym~r dispersions kno~n per se~ uh;ch preferably have ion;c ~roupsO for example composed of polyvinyl compounds, vinyl copoly~ers, polysSyrenesulphon;c Dcids~ polyam;des or polyurethanes. Casting soLut;ons uhich consist of mix-tures of polyurethane solut;ons u;th aqueous polyurethane d;spers;ons ~see, for exa~ple, Ange~. MakromDl. Chem. 98 (1981) 133 and DE-OS (German Published Specif;cation)
2,427,27~, uhich ha~ already been mentioned, and the prin-ted material cited there) are very particularly suitable.The polyurethane dispersions can be non-;on;c orO prefer-ably9 ;onic, it being poss;ble for the ;on;c rad;cals to be, for exa~ple9 -S03 ~ -COO or -N~R3 groups.
Ion;c poly~er systems are particularly preferred since they show good adhesion to the support fi lm and ;m~obilisation of the enzymes necessary for the detect;on reactionr The detection systems accordin~ to the inven-tion ~hich are based on ionic polymers can be rinsed ~ith ~ater for several hours ~ithout the occurrence of any bleeding uorth mention;ng.
The solvents used for the preparation of the casting solutions are those customary for the parti-cular polymers~ such as, for exampleO d;methylformam;de, N-methylpyrrolidone or d;oxolane, it being possible~ uhere appropriate, for inorganic salts uhich are soluble in the casting solution, such as L;Cl~ C~Cl2 or Mg~ClO43z, to be added as expanding agents. It is also possible to add, as further additives~ substances wh;ch are ;nsoluble in the casting solution and ~hich serve as ~illers, such as SiO2~ T;02 (see Furopean Patent bpplication 0,077,509)~ BaS04, ZnO or cellulose or agarose pouder, on ~hich, ~here appropriate~ b;ologically active material is immobilised.
Supported microporous detection ele~ents accor-d;ng to the invention are produced by un;for~ly coatinga suitable support ~ith a casting solution of th;s ~ype Le A 22 682 5~5 (layer thickness about 50-1~0 ~m) ~nd then~ preferably i~mediatley, inmersing it in a coa0ulation bath, ~hereupon the ~icroporous supported polymer films are produced. The ~i~e be~een the coatin~ of the support and th2 coagula-tion is kept as short ~s possible ~about ~ ~e~ seconds)so that the support is not attacked by the solvent ;n the casting solution. ExampLes of suitabLe coagul~t-on fluids are water or aqueous buffer solutions which can~ where appropria~e, also contain plasticisers, for example glycerol.
The pore structure can be modif;ed in a stra;ght-for~ard manner by the nature of the coagulation bath.
ThusO from just one casting solution, increasing pore sizes on the membrane surf3ce can be achieYed by prec;pi-tation in ~ater at increasin~ temperatures, as can besho~n by SEM photo~raphs of ~icroporous polymer films uhich uere coagulated at roo~ temperature~ 45C and S0C.
Similar effects can also be achieved by using mixtures of ~ater with organic solvents~ for example water/dimethyL-2C formamide.
Supports Mhich are suitable for the detection ele-ments according to the invention are~ in principle, macro-scopically smooth polymer films on which the polymer sys-tem used shows good adhes;on during coagulation as ~ell as after drying, and ~hich undergo no shange by the poly-mer casting solution. Transparent poLymer films ~hich also perm;t evaluation of the colour reaction through the side of the support are particularly preferred. Trans-parent poLyethylene terephthalate films are very particu-~0 larly preferred~
The reagents necessary for the detection reactioncan be introduced into the microporous polymer films in a variety of ways, for example by stirriny the~ into the casting solution, by subsequent impre~nation of the porous films, or by a combination of these two processes.
According to a preferred variantr enzyme-cnntaining Le A 22 682 ... ; ,: .
~s~
reag~nt systems for the detect;on of ~n analyte, ~hich are kno~n per se, are introduc~d ;nt~ the test devices accor-ding to the invention.
~ straiyhtfor~ard ~ethod for the production of ~icroporou~ poly~er films loade~ ~ith detection reagents comprises, for example~ dissolvins the chro~o~en used (for example 3,3',5,5'-tetramethylbenzidine) in the cast;ng solution, and processing the latter by the coagulation process to give a chromogen-loaded microporous film ~hich is then impregnated ~ith the enzyme system (buffered ~here appropriate~.
The test agents according to ~he invention can also be designed as a ~ulti-layer system. This is an ad-vantage ~hen the detection re3gents have to be separated.
For example, a support film can be provided ~ith a polymer layer onto which is then applied the asymmetric membrane using the coagulation method described, or a separately produced, unsupported, finished membrane is la~inated on.
Where appropriate~ all or part of the detection reagents can be incorporated in the polymer layer located between the support and the membrane, the other part of the detect;on reagents be;ng located ;n ehe membrane loca-ted above~
Examples of suitable polymeric intermediate layers into ~hich the detection reagents can, ~here appropriate, be incorporated (reagent l~yer), are ~ilms kno~n per se and deriYed from aqueous dispersions belonging to the class sf polyYinyl compou`hds, of vinyl copolymersO of polystyrenesulphonic acids, of polyam;des or of polyure-thanes. Since ~he reagent-containing layer should prefer-ably be soluble~ or at least s~ellable, in water, polyure-thane dispersions which are~ ~here appropriate, ~ixed ~ith polymers ~hich ~re soluble in water or s~ellable ~i~h ~ater, such as polyvinyl alcohol~ polyethylene glycolO
cellulose ethers, polyacrylamide~ polyacrylic ~cid or polyvinylpyrrolidone, are particularly suitabler Yery Le A 22 682 - ~7 -particularly preferred reagent layers are obtained from mixtures of ionic polyurethanes dispersions with poly-vinylpyrrolidone.
After ~iping off the excess sample~ the colour re-action can be observed from the application side tmembranesurface) or, ~hen transparent supports are used~ from the support side~ ;n ~hich case the e~cess sample need not be iped off. In the latter case, it is preferable for the chromogen to be located in a reagent layer bet~een the mem-brane and the transparent support.
The detection elements according to the inventionare su;table for the quantitative determination of lo~ and high molecular ~eight components in liquid samples, in particular for the quant;tative spectrophoto~etric detec tion of constituents of body fluids, such as, for example, bilirubin~ ketones, triglycerides, urea or haemoglobin.
As th~ e~amples ~hich follo~ show, the detection systems according to the invention are very par~icularly suited for the quantitatiYe detection of glucose ;n ~hole blood, and for the detection of enzymes, such as glucose oxidase or chol;nesterase, and the detection of bilirubin or ke-tone bod;es.
In the examples, unless other~ise noted amounts indicated are to be understood to be parts by ~e;ght or percentages by ~eight~
Example 1 Detection of ~lucose Using a high speed stirrer tdissolver)D a casting solution of the follo~;ng composit;on ~as prepared:
8D 02 9 of disulphimide-polyamide 2.40 9 of CaCl2 43.02 ~ of dimethylformamide (DMF) 0.01 ~ of ascorbic acid 0.01 9 of sodium citrate 0.4D ~ of citric acid 0.4~ 9 f 3,3'~505'-tetramethylbenzidine Le A 22 682 ,' '''': , , ~ 18 -45.40 ~ of titanium dioxide D~13 ~ of peroxidase ~OD, 277 U/~a D.13 ~ of ~lucose oxidase (GOD~ 116 U/mg)~
Using this castin~ solution, a polyethylene tere-S phthalate film ~as uniformly coated in a spreadin0 thick-ness of 100 ~m using a doctor knife. This supported film was coagulated in a 30% stren~th aqueous glycerol ba~h for 10 minD The solid supported membrane thus produced ~as dried ~ith Yarm air S35C) and tested for its functioning ~ith uhole blood. The biood was applied to the membrane surface, and ~iped off after 1 minute. A homo0eneous green coloration had deveLoped on the membrane surface about 30 sec. after ~iping off.
The disulphimide-poLyamide is a poLycondensate, produced in a one-pot reaction in accordance ~ith US Pa-tent Sp~c~fication ~,269,9~7, of the follo~ing:
J ~ ~ ClCC ~ COCl \ ~ Na ~ ~H2 Exa~ple 2 Detection of ~lucose Casting soLution: ~3.73 ~ of poLyurethane 66.37 ~ of dimethylformamide 7.24 g of polyurethane dispersion in water/DMF
0.07 9 of sodium dioctyL sulpho~
succinate 11.01 ~ of titanium dioxide 0079 ~ of 3,3',5,5'-tetramethyL-benz;dine 0.79 ~ of ascorbic acid.
The polyurethane used is a ~hermoplastic material ~h;ch was obtained by reaction of 75 parts of a polyester Le ~ ?2_~82 of adip;c acid, 70 moleX of ethylene glycol and 30 mol-%
of 1,4-butanediol (MW ~ 2~000), 25 p2rts of a polyester of adipic acid and 1,4-butanediol ~M~ = 2~250), 25 parts of 1~4-butanediol and 85 parts of dimethyl~ethane diiso-S cyanate.
The polyurethane dispersion serves as a coagula-tion aid and is a cat;onic d;spers;on~ conta;ning no emuLsifier, of a reaction product of 200 parts of a poLyester of ad;pi~ ac;d, phthal;c ac;d and ethyLene glycoL (M~ = 1,700), 50 parts of toluylene diisocyanate, ZO parts of N-methyldiethanolam;ne and 6 parts of p-xylylene dichloride~
The supported, microporous polymer membrane was produced as in Example 1 uith the follo~ing modifications:
Support: polyethylene terephthalate film Coagulation bath: 1X strength solution of Na lauryl sul-phate.
After drying, the film ~as impregnated for 1 mi-nute ~ith a 1X strength solution of POD ~277 U/mg)/GODt116 V~m~) in citrate buffer ~pH 5.5) and ~as dried.
Test ~ith uhole blood: 10 sec. after application of the sample~ a homogeneous blue coloration ~as observ-able through the transparent supportD l;kew;se after ~iping off the excess sample on the membrane surface.
~ ith 0.05, 0.1 and 0.5X s~rength glucose solu-tions, homogeneous blue colorat;ons ~ere produced ;~med-iately, and these sho~ed increasin~ intensities of colour tcolour gradations~ appropriate for the increasing glucose concentration.
Accordingly, graded reflection values ~ere mea-sured on evaluation by reflectromety of various glucose concentrations. In addition~ the measure~en~s showed ~hat the coloration is linearly dependent on the glucose con-centration in the range from 20 to 800 mg glucose~dl ~ater, and that the end point of the detection reaction Le A 22 682 -:' :
.: , ;s reached after 40 seconds at the ~ost. Compared wi~h kn~wn test dev;ces, th;s has to be regarded as being ex-t~emely rapid.
Examinations by electron microscopy tsEM) of ~h~
test strip syst~m descr;bed in Example 2 showed that the membrane ~as hi~hly porousO the mean pore size being n.s ~.
Example 2a Te~t str;pg for b;lirub;n Cast;ng solut;on and pr~duction of the membrane as ;n Exampl~ 2, but ~;thout TMB and ascorb;c acid.
The poly~er membrane ~as ;mpregnated ~;th ~.68 9 p-toluenesulphonic ac;d, 0,18 9 of disodium salt of naphthalene-1~5-disuLphonic ac;d, 2.00 9 of 7-(2,3 dihydroxypropyl)theophylline, 0.06 g of sod;um nitr;te and 0O1O ~ of saponine in 9 ml of distilled water and dried.
The test strips produced from the impregnated mem-brane develop bro~n colours of varying intensi~y after application ùf various concentrations of a bilirubin con-trol seru~.
Example ~b Test str;ps for ketone bod;es Ca~t;ng solution and production of the membrane as in Example 2~ but without TMB and ascorbic acid.
The polymer membrane ~as, after a pH of ~.4 had been set up, impregnated with 2 9 of sodium nitroprusside and 8.2 g of magnesium sulphate in 11 ml of distilled ~ater and dried.
After immersion of the cut test strips in ace~o-acetic acid solutions or urine, the test strips developed Le A 22 682 ~.
~5~
violet colours of vary;n~ ;ntensit;es depending on the concentrat;on of ketone bodies.
Exa~ple 3 Detection of glucose T~o-layer syseem A) Product;on of a supported rea~ent layer B.40 9 of aqueous polyurethane dispers;on
Ion;c poly~er systems are particularly preferred since they show good adhesion to the support fi lm and ;m~obilisation of the enzymes necessary for the detect;on reactionr The detection systems accordin~ to the inven-tion ~hich are based on ionic polymers can be rinsed ~ith ~ater for several hours ~ithout the occurrence of any bleeding uorth mention;ng.
The solvents used for the preparation of the casting solutions are those customary for the parti-cular polymers~ such as, for exampleO d;methylformam;de, N-methylpyrrolidone or d;oxolane, it being possible~ uhere appropriate, for inorganic salts uhich are soluble in the casting solution, such as L;Cl~ C~Cl2 or Mg~ClO43z, to be added as expanding agents. It is also possible to add, as further additives~ substances wh;ch are ;nsoluble in the casting solution and ~hich serve as ~illers, such as SiO2~ T;02 (see Furopean Patent bpplication 0,077,509)~ BaS04, ZnO or cellulose or agarose pouder, on ~hich, ~here appropriate~ b;ologically active material is immobilised.
Supported microporous detection ele~ents accor-d;ng to the invention are produced by un;for~ly coatinga suitable support ~ith a casting solution of th;s ~ype Le A 22 682 5~5 (layer thickness about 50-1~0 ~m) ~nd then~ preferably i~mediatley, inmersing it in a coa0ulation bath, ~hereupon the ~icroporous supported polymer films are produced. The ~i~e be~een the coatin~ of the support and th2 coagula-tion is kept as short ~s possible ~about ~ ~e~ seconds)so that the support is not attacked by the solvent ;n the casting solution. ExampLes of suitabLe coagul~t-on fluids are water or aqueous buffer solutions which can~ where appropria~e, also contain plasticisers, for example glycerol.
The pore structure can be modif;ed in a stra;ght-for~ard manner by the nature of the coagulation bath.
ThusO from just one casting solution, increasing pore sizes on the membrane surf3ce can be achieYed by prec;pi-tation in ~ater at increasin~ temperatures, as can besho~n by SEM photo~raphs of ~icroporous polymer films uhich uere coagulated at roo~ temperature~ 45C and S0C.
Similar effects can also be achieved by using mixtures of ~ater with organic solvents~ for example water/dimethyL-2C formamide.
Supports Mhich are suitable for the detection ele-ments according to the invention are~ in principle, macro-scopically smooth polymer films on which the polymer sys-tem used shows good adhes;on during coagulation as ~ell as after drying, and ~hich undergo no shange by the poly-mer casting solution. Transparent poLymer films ~hich also perm;t evaluation of the colour reaction through the side of the support are particularly preferred. Trans-parent poLyethylene terephthalate films are very particu-~0 larly preferred~
The reagents necessary for the detection reactioncan be introduced into the microporous polymer films in a variety of ways, for example by stirriny the~ into the casting solution, by subsequent impre~nation of the porous films, or by a combination of these two processes.
According to a preferred variantr enzyme-cnntaining Le A 22 682 ... ; ,: .
~s~
reag~nt systems for the detect;on of ~n analyte, ~hich are kno~n per se, are introduc~d ;nt~ the test devices accor-ding to the invention.
~ straiyhtfor~ard ~ethod for the production of ~icroporou~ poly~er films loade~ ~ith detection reagents comprises, for example~ dissolvins the chro~o~en used (for example 3,3',5,5'-tetramethylbenzidine) in the cast;ng solution, and processing the latter by the coagulation process to give a chromogen-loaded microporous film ~hich is then impregnated ~ith the enzyme system (buffered ~here appropriate~.
The test agents according to ~he invention can also be designed as a ~ulti-layer system. This is an ad-vantage ~hen the detection re3gents have to be separated.
For example, a support film can be provided ~ith a polymer layer onto which is then applied the asymmetric membrane using the coagulation method described, or a separately produced, unsupported, finished membrane is la~inated on.
Where appropriate~ all or part of the detection reagents can be incorporated in the polymer layer located between the support and the membrane, the other part of the detect;on reagents be;ng located ;n ehe membrane loca-ted above~
Examples of suitable polymeric intermediate layers into ~hich the detection reagents can, ~here appropriate, be incorporated (reagent l~yer), are ~ilms kno~n per se and deriYed from aqueous dispersions belonging to the class sf polyYinyl compou`hds, of vinyl copolymersO of polystyrenesulphonic acids, of polyam;des or of polyure-thanes. Since ~he reagent-containing layer should prefer-ably be soluble~ or at least s~ellable, in water, polyure-thane dispersions which are~ ~here appropriate, ~ixed ~ith polymers ~hich ~re soluble in water or s~ellable ~i~h ~ater, such as polyvinyl alcohol~ polyethylene glycolO
cellulose ethers, polyacrylamide~ polyacrylic ~cid or polyvinylpyrrolidone, are particularly suitabler Yery Le A 22 682 - ~7 -particularly preferred reagent layers are obtained from mixtures of ionic polyurethanes dispersions with poly-vinylpyrrolidone.
After ~iping off the excess sample~ the colour re-action can be observed from the application side tmembranesurface) or, ~hen transparent supports are used~ from the support side~ ;n ~hich case the e~cess sample need not be iped off. In the latter case, it is preferable for the chromogen to be located in a reagent layer bet~een the mem-brane and the transparent support.
The detection elements according to the inventionare su;table for the quantitative determination of lo~ and high molecular ~eight components in liquid samples, in particular for the quant;tative spectrophoto~etric detec tion of constituents of body fluids, such as, for example, bilirubin~ ketones, triglycerides, urea or haemoglobin.
As th~ e~amples ~hich follo~ show, the detection systems according to the invention are very par~icularly suited for the quantitatiYe detection of glucose ;n ~hole blood, and for the detection of enzymes, such as glucose oxidase or chol;nesterase, and the detection of bilirubin or ke-tone bod;es.
In the examples, unless other~ise noted amounts indicated are to be understood to be parts by ~e;ght or percentages by ~eight~
Example 1 Detection of ~lucose Using a high speed stirrer tdissolver)D a casting solution of the follo~;ng composit;on ~as prepared:
8D 02 9 of disulphimide-polyamide 2.40 9 of CaCl2 43.02 ~ of dimethylformamide (DMF) 0.01 ~ of ascorbic acid 0.01 9 of sodium citrate 0.4D ~ of citric acid 0.4~ 9 f 3,3'~505'-tetramethylbenzidine Le A 22 682 ,' '''': , , ~ 18 -45.40 ~ of titanium dioxide D~13 ~ of peroxidase ~OD, 277 U/~a D.13 ~ of ~lucose oxidase (GOD~ 116 U/mg)~
Using this castin~ solution, a polyethylene tere-S phthalate film ~as uniformly coated in a spreadin0 thick-ness of 100 ~m using a doctor knife. This supported film was coagulated in a 30% stren~th aqueous glycerol ba~h for 10 minD The solid supported membrane thus produced ~as dried ~ith Yarm air S35C) and tested for its functioning ~ith uhole blood. The biood was applied to the membrane surface, and ~iped off after 1 minute. A homo0eneous green coloration had deveLoped on the membrane surface about 30 sec. after ~iping off.
The disulphimide-poLyamide is a poLycondensate, produced in a one-pot reaction in accordance ~ith US Pa-tent Sp~c~fication ~,269,9~7, of the follo~ing:
J ~ ~ ClCC ~ COCl \ ~ Na ~ ~H2 Exa~ple 2 Detection of ~lucose Casting soLution: ~3.73 ~ of poLyurethane 66.37 ~ of dimethylformamide 7.24 g of polyurethane dispersion in water/DMF
0.07 9 of sodium dioctyL sulpho~
succinate 11.01 ~ of titanium dioxide 0079 ~ of 3,3',5,5'-tetramethyL-benz;dine 0.79 ~ of ascorbic acid.
The polyurethane used is a ~hermoplastic material ~h;ch was obtained by reaction of 75 parts of a polyester Le ~ ?2_~82 of adip;c acid, 70 moleX of ethylene glycol and 30 mol-%
of 1,4-butanediol (MW ~ 2~000), 25 p2rts of a polyester of adipic acid and 1,4-butanediol ~M~ = 2~250), 25 parts of 1~4-butanediol and 85 parts of dimethyl~ethane diiso-S cyanate.
The polyurethane dispersion serves as a coagula-tion aid and is a cat;onic d;spers;on~ conta;ning no emuLsifier, of a reaction product of 200 parts of a poLyester of ad;pi~ ac;d, phthal;c ac;d and ethyLene glycoL (M~ = 1,700), 50 parts of toluylene diisocyanate, ZO parts of N-methyldiethanolam;ne and 6 parts of p-xylylene dichloride~
The supported, microporous polymer membrane was produced as in Example 1 uith the follo~ing modifications:
Support: polyethylene terephthalate film Coagulation bath: 1X strength solution of Na lauryl sul-phate.
After drying, the film ~as impregnated for 1 mi-nute ~ith a 1X strength solution of POD ~277 U/mg)/GODt116 V~m~) in citrate buffer ~pH 5.5) and ~as dried.
Test ~ith uhole blood: 10 sec. after application of the sample~ a homogeneous blue coloration ~as observ-able through the transparent supportD l;kew;se after ~iping off the excess sample on the membrane surface.
~ ith 0.05, 0.1 and 0.5X s~rength glucose solu-tions, homogeneous blue colorat;ons ~ere produced ;~med-iately, and these sho~ed increasin~ intensities of colour tcolour gradations~ appropriate for the increasing glucose concentration.
Accordingly, graded reflection values ~ere mea-sured on evaluation by reflectromety of various glucose concentrations. In addition~ the measure~en~s showed ~hat the coloration is linearly dependent on the glucose con-centration in the range from 20 to 800 mg glucose~dl ~ater, and that the end point of the detection reaction Le A 22 682 -:' :
.: , ;s reached after 40 seconds at the ~ost. Compared wi~h kn~wn test dev;ces, th;s has to be regarded as being ex-t~emely rapid.
Examinations by electron microscopy tsEM) of ~h~
test strip syst~m descr;bed in Example 2 showed that the membrane ~as hi~hly porousO the mean pore size being n.s ~.
Example 2a Te~t str;pg for b;lirub;n Cast;ng solut;on and pr~duction of the membrane as ;n Exampl~ 2, but ~;thout TMB and ascorb;c acid.
The poly~er membrane ~as ;mpregnated ~;th ~.68 9 p-toluenesulphonic ac;d, 0,18 9 of disodium salt of naphthalene-1~5-disuLphonic ac;d, 2.00 9 of 7-(2,3 dihydroxypropyl)theophylline, 0.06 g of sod;um nitr;te and 0O1O ~ of saponine in 9 ml of distilled water and dried.
The test strips produced from the impregnated mem-brane develop bro~n colours of varying intensi~y after application ùf various concentrations of a bilirubin con-trol seru~.
Example ~b Test str;ps for ketone bod;es Ca~t;ng solution and production of the membrane as in Example 2~ but without TMB and ascorbic acid.
The polymer membrane ~as, after a pH of ~.4 had been set up, impregnated with 2 9 of sodium nitroprusside and 8.2 g of magnesium sulphate in 11 ml of distilled ~ater and dried.
After immersion of the cut test strips in ace~o-acetic acid solutions or urine, the test strips developed Le A 22 682 ~.
~5~
violet colours of vary;n~ ;ntensit;es depending on the concentrat;on of ketone bodies.
Exa~ple 3 Detection of glucose T~o-layer syseem A) Product;on of a supported rea~ent layer B.40 9 of aqueous polyurethane dispers;on
3.00 9 of polyvinylpyrrolidone (M~ 350,000) 9.50 9 of tetramethylbenzidine (dissolved in 1 9 of ethyl acetate) 0.12 9 each of ~lucose oxidase ~116 U/mg) and peroxidase ~277 U/mg) and 0.025 9 of ascorbic acid are stirred together~ and coated onto a polyester film and dried ~ith uarm air, a supported reagent layer being obtained.
The polyurethane dispersion is a 40X strength aqueous dispersion of a reaction product of 82 parts of a polyester of adipic acid, hexanediol and 23 neopentyl glycol (MW z 1,700), 15 parts of hexamethylene diisocyanate, 2 parts of Na ethylened;amine ethanolsulpho-nate and 1 part of ethylenediamine.
~ith aqueous glucose solu~ions, these supportecl reagent layers developed~ about 20 sec~ after application of the sample~ homogeneous colorations ~hich ~ere graded uith different glucose concentrations tsee Example 2).
~) Applicatisn of an asymmetrir. phase-;nversion mem-brane a~ all detection reagents in the reagent layer The casting solut;on described in Example 2, but uhich did not contain the tetramethylbenzidine and ascor-bic acid described there, ~as coated onto the supported reagent layer described in Example 3A), 3nd was coagulated in 1X strength aqueous Na lauryl sulphate solution.
Le A 22 ~82 s~
~ 22 ~
After drying ~ith uarm air~ it ~as tested ~i~h aqueous ~lucose solutions and ~;th uhole blood. Homo-geneous, concentra~ion~dependent blue colorations ~ere observed from the support side about 30 seconds after application of ~he sa~ple~
h) Reagents in various layers A supported, enzyme-containing Layer was produced in analogy to Example 3A) from 8.~0 9 of aqueous polyurethane dispersion (see Example 3) 3.00 9 of polyvinylpyrrolidone ~MW 350,00û) and 0.12 9 each of glucose oxidase (116 U/mg) and perox;dase (227 U/mg).
The castin~ solution described in Example 2 ~as coated on-to this, and coagulated in 1X strength Na lauryl sulphate solution and dried. On testing ~ith uhole blood, a homo-geneous blue coloration ~as observed from the application s;de about 4û ser. after applicat;on of the sample and ~iping off the erythrocytes. Graded blue coLorations ~ere developed ~ith glucose solutions of different concentrations G
Example 4 Detection of ~lucose T~o-layer system ~ith rea~ent layer.
Casting solution for the reagent layer:
8~00 9 of the polyurethane dispersion from Example 3 1.00 9 of polyvinylpyrrolidone (M~ 10,000) 0.05 g of tetramethylben~,idine dissolved in 10 9 of ethyl acetate 0.10 ~ of gLucose oxidase t116 U/m~ dissolved in 2 ml of ~ater 0.10 9 of peroxidase (227 U/mg) 0.01 ml of a SX strength aqueous solution of ascorbic acid were stirred together~ coated on~o a poly~thylene tereph-thalate f;lm and dried ~ith ~ar~ air ~= supported reagent layer~O
Le A 22 682 A polyamide membrane produced by the phaseoinver~
s;on process from the folLo~ing castin0 solution ~as appl;ed to this supported reagent layer: -8.10 9 of polyamide (polycondensation product of hexa-S m~thyLenediamine and isophthal;c acid according to DE-OS (German Published Specificat;on~
Z,743,515) 2.40 9 of CaCl2 43.00 9 of dimethylformamide and 46~00 9 of Ti02 Coating thickness: 100 ~m Coagulation bath: 30% stren~th aqueous glycerol solution Test ~ith ~hole blood one drop of blood ffas applied to the membrane surface. About 10 sec. after application of the sample, a homogeneous blue coLoration was observed throu~h the transparent support. Graded blue colorations uere developed ~ith glucose solutions of various concen-trations (see Example 2).
Example 5 Detection ~f alucose Membrane ~ith reagent layer Casting solut;on for the membrane:
20.00 g of polysulphone (condensat;on product of bisphenoL
A and bis(chlorophenylsulphone); Udel P 1700;
commercial product of Union Carbide) was dis-solved in 80~00 9 of N-methylpyrrolidione.
The casting solution was applied ~;th a doc~or to a glass plate ~100 ~m) and immersed for coagulation in an aqueous 10X strength glyr,erol bath~ During this, the film detached from the glass support, and an unsupported, asymmetrical membrane ~as obtainedO
A~ter drying, the rear side ~side of the film which had been located on the support) of the poLysulphone membrane ~as coa~ed ~ith the rea~ent layer described in Le A 22 682 ' `'`~' ''' ::
~5;5~
Example 4.
Test ~;th ~hole blood: one drop of blood ~as ap-plied to the membrane surface. About 30 sec. after appl;~
cation of the sample, a ho~ogeneous blue csloration ~as observed in the reagent layer. 6raded blue colorations ~ere developed ~ith glucose solutions of various concen-trations.
Example 6 Enzyme detection The membrane described in Example 2 ~as, after bein~ dried, impregnated ~ith a lX strength aqueous glu-cose/POD t277 U/mg) sslut;on and dried.
In tests with dilute GOD solutions (20 U/ml;
40 U/ml; ~0 U/~l; 120 U/ml; 160 U/ml), immediately after application of the sample a homogeneous blue colorationO
which ~as graded in accordance ~ith the GOD concentration, uas observed on the membrane surface as well as through the transparent support.
~e~
_est strips for choLinesterase Casting solution and production of the membrane as in E~ample 2~ but ~ithout TMB and ascorbic acid~
The membrane ~as impregnated ~ith a solution of 40 mg of indoxyl acetate in 5 ml of ethyl acetate and 25 120 mg of 2-methoxy-4-morpholinobenzenediazonium chlo-ride . ~nCl2 in 5 ml of ~ethanolO and then again ~ith tris HCl buffer (0.4 M; pH 7.5)0 and uas dried and then processed to form test strips.
After application of cholinesterase sslutions and serum, the test strips developed blue colorations at different rates depending on the enzyme activity~ The coloration can be quan~itatively evaluated usin~ a re-flection measuring apparatus.
Le A 22 ~R2 . .
The polyurethane dispersion is a 40X strength aqueous dispersion of a reaction product of 82 parts of a polyester of adipic acid, hexanediol and 23 neopentyl glycol (MW z 1,700), 15 parts of hexamethylene diisocyanate, 2 parts of Na ethylened;amine ethanolsulpho-nate and 1 part of ethylenediamine.
~ith aqueous glucose solu~ions, these supportecl reagent layers developed~ about 20 sec~ after application of the sample~ homogeneous colorations ~hich ~ere graded uith different glucose concentrations tsee Example 2).
~) Applicatisn of an asymmetrir. phase-;nversion mem-brane a~ all detection reagents in the reagent layer The casting solut;on described in Example 2, but uhich did not contain the tetramethylbenzidine and ascor-bic acid described there, ~as coated onto the supported reagent layer described in Example 3A), 3nd was coagulated in 1X strength aqueous Na lauryl sulphate solution.
Le A 22 ~82 s~
~ 22 ~
After drying ~ith uarm air~ it ~as tested ~i~h aqueous ~lucose solutions and ~;th uhole blood. Homo-geneous, concentra~ion~dependent blue colorations ~ere observed from the support side about 30 seconds after application of ~he sa~ple~
h) Reagents in various layers A supported, enzyme-containing Layer was produced in analogy to Example 3A) from 8.~0 9 of aqueous polyurethane dispersion (see Example 3) 3.00 9 of polyvinylpyrrolidone ~MW 350,00û) and 0.12 9 each of glucose oxidase (116 U/mg) and perox;dase (227 U/mg).
The castin~ solution described in Example 2 ~as coated on-to this, and coagulated in 1X strength Na lauryl sulphate solution and dried. On testing ~ith uhole blood, a homo-geneous blue coloration ~as observed from the application s;de about 4û ser. after applicat;on of the sample and ~iping off the erythrocytes. Graded blue coLorations ~ere developed ~ith glucose solutions of different concentrations G
Example 4 Detection of ~lucose T~o-layer system ~ith rea~ent layer.
Casting solution for the reagent layer:
8~00 9 of the polyurethane dispersion from Example 3 1.00 9 of polyvinylpyrrolidone (M~ 10,000) 0.05 g of tetramethylben~,idine dissolved in 10 9 of ethyl acetate 0.10 ~ of gLucose oxidase t116 U/m~ dissolved in 2 ml of ~ater 0.10 9 of peroxidase (227 U/mg) 0.01 ml of a SX strength aqueous solution of ascorbic acid were stirred together~ coated on~o a poly~thylene tereph-thalate f;lm and dried ~ith ~ar~ air ~= supported reagent layer~O
Le A 22 682 A polyamide membrane produced by the phaseoinver~
s;on process from the folLo~ing castin0 solution ~as appl;ed to this supported reagent layer: -8.10 9 of polyamide (polycondensation product of hexa-S m~thyLenediamine and isophthal;c acid according to DE-OS (German Published Specificat;on~
Z,743,515) 2.40 9 of CaCl2 43.00 9 of dimethylformamide and 46~00 9 of Ti02 Coating thickness: 100 ~m Coagulation bath: 30% stren~th aqueous glycerol solution Test ~ith ~hole blood one drop of blood ffas applied to the membrane surface. About 10 sec. after application of the sample, a homogeneous blue coLoration was observed throu~h the transparent support. Graded blue colorations uere developed ~ith glucose solutions of various concen-trations (see Example 2).
Example 5 Detection ~f alucose Membrane ~ith reagent layer Casting solut;on for the membrane:
20.00 g of polysulphone (condensat;on product of bisphenoL
A and bis(chlorophenylsulphone); Udel P 1700;
commercial product of Union Carbide) was dis-solved in 80~00 9 of N-methylpyrrolidione.
The casting solution was applied ~;th a doc~or to a glass plate ~100 ~m) and immersed for coagulation in an aqueous 10X strength glyr,erol bath~ During this, the film detached from the glass support, and an unsupported, asymmetrical membrane ~as obtainedO
A~ter drying, the rear side ~side of the film which had been located on the support) of the poLysulphone membrane ~as coa~ed ~ith the rea~ent layer described in Le A 22 682 ' `'`~' ''' ::
~5;5~
Example 4.
Test ~;th ~hole blood: one drop of blood ~as ap-plied to the membrane surface. About 30 sec. after appl;~
cation of the sample, a ho~ogeneous blue csloration ~as observed in the reagent layer. 6raded blue colorations ~ere developed ~ith glucose solutions of various concen-trations.
Example 6 Enzyme detection The membrane described in Example 2 ~as, after bein~ dried, impregnated ~ith a lX strength aqueous glu-cose/POD t277 U/mg) sslut;on and dried.
In tests with dilute GOD solutions (20 U/ml;
40 U/ml; ~0 U/~l; 120 U/ml; 160 U/ml), immediately after application of the sample a homogeneous blue colorationO
which ~as graded in accordance ~ith the GOD concentration, uas observed on the membrane surface as well as through the transparent support.
~e~
_est strips for choLinesterase Casting solution and production of the membrane as in E~ample 2~ but ~ithout TMB and ascorbic acid~
The membrane ~as impregnated ~ith a solution of 40 mg of indoxyl acetate in 5 ml of ethyl acetate and 25 120 mg of 2-methoxy-4-morpholinobenzenediazonium chlo-ride . ~nCl2 in 5 ml of ~ethanolO and then again ~ith tris HCl buffer (0.4 M; pH 7.5)0 and uas dried and then processed to form test strips.
After application of cholinesterase sslutions and serum, the test strips developed blue colorations at different rates depending on the enzyme activity~ The coloration can be quan~itatively evaluated usin~ a re-flection measuring apparatus.
Le A 22 ~R2 . .
Claims (29)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A test device for the detection of a component in a liquid sample, the device comprising a support layer, a micro-porous polymer layer, where appropriate other layers and, incor-porated in one or more of the layers, reagents for the detection of the component to be determined, characterized in that the microporous polymer layer is a membrane which has an asymmetric pore structure and is produced by the coagulation process, the pores narrowing toward the side to which the sample is applied, and in that the support layer is macroscopically smooth.
2. A test device as claimed in claim 1, characterized in that the microporous polymer layer contains all the reagents for the detection of the component to be determined.
3. A test device as claimed in claim 2, characterized in that the microporous polymer layer is located immediately adjacent to the macroscopically smooth support layer.
4. A test device as claimed in claim 1, characterized in that a polymer film reagent layer is located between the support layer and the microporous polymer layer and contains a part or all of the reagents for the detection of the component to be determined.
5. A test device as claimed in claim 1, 2 or 3 characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1.
6. A test device as claimed in claim 4, characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1.
7. A test device as claimed in claim 4, characterised in that the polymer film serving as the reagent layer is soluble in water or swellable in water.
8. A test device as claimed in claim 1, 2 or 3, characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, and the polymer film serving as the reagent layer is soluble in water or swellable in water.
9. A test device as claimed in claim 1, 2 or 3, characterised in that the membrane is synthesized from a polymer chosen from the group comprising polyamides, polyether carbonates, polyacrylonitriles and polyurethanes.
10. A test device as claimed in claim 4 characterised in that the membrane is synthesized from a polymer chosen from the group comprising polyamides, polyether carbonates, polyacrylonitriles and polyurethanes.
11. A test device as claimed in claim 1, 2 or 3, characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, the membrane being synthesized from a polymer chosen from the group comprising polyamides, polyether carbonates, polyacrylonitriles and polyurethanes.
12. A test device as claimed in claim 4, characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, the membrane being synthesized from a polymer chosen from the group comprising polyamides, polyether carbonates, poly-acrylonitriles and polyurethanes.
13. A test device as claimed in claim 1, 2, 3 characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, and the polymer film serving as the reagent layer is soluble in water or swellable in water, the membrane being synthesized from a polymer chosen from the group comprising polyamides, polyether carbonates, polyacrylonitriles and polyurethanes.
14. A test device as claimed in claim 7, wherein the membrane is synthesized from a polymer chosen from the group comprising polyamides, polyether carbonates, polyacrylonitriles and polyurethanes.
15. A test device as claimed in claim 1, 2 or 3, wherein the membrane is synthesized from a polymer chosen from the group comprising polyamides which carry disulphide groups, polyether carbonates, polyacrylonitriles and polyurethanes which contain polymer dispersions and insoluble fillers.
16. A test device as claimed in claim A or 7 wherein the membrane is synthesized from a polymer chosen from the group comprising polyamides which carry disulphide groups, polyether carbonates, polyacrylonitriles and polyurethanes which contain polymer dispersions and insoluble fillers.
17. A test device as claimed in claim 1, 2 or 3, characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, wherein the membrane is synthesized from a polymer chosen from the group comprising polyamides which carry disulphide groups, polyether carbonates, polyacrylonitriles and polyurethanes which contain polymer dispersions and insoluble fillers.
18. A test device as claimed in claim 4, characterised in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, wherein the membrane is synthesized from a polymer chosen from the group comprising polyamides which carry disulphide groups, polyether carbonates, polyacrylonitriles and poly-urethanes which contain polymer dispersions and insoluble fillers.
19. A test device as claimed in claim 1, 2 or 3 character-ized in that the ratio of the mean pore diameter on the underside of the membrane to that on the membrane surface is greater than 10:1, and the polymer film serving as the reagent layer is soluble in water or swellable in water, wherein the membrane is synthesized from a polymer chosen from the group comprising polyamides which carry disulphide groups, polyether carbonates, polyacrylonitriles and polyurethanes which contain polymer dispersions and insoluble fillers.
20. A test device as claimed in claim 1, 2 or 3 character-ized in that the macroscopically smooth support layer is impermeable to the sample under the test conditions.
21. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 1, 2 or 3 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed.
22. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 4, 6 or 7 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed.
23. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 10, 12 or 14 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed.
24. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 1, 2 or 3 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed, wherein the sample is applied to the membrane surface and the detection reaction is observed from the application side.
25. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 4, 6 or 7 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed, wherein the sample is applied to the membrane surface and the detection reaction is observed from the application side.
26. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 10, 12 or 14 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed, wherein the sample is applied to the membrane surface and the detection reaction is observed from the application side.
27. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 1, 2 or 3 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed, wherein a test device having a transparent support layer is used, and the detection reaction is observed from the support side.
28. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 4, 6 or 7 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed, wherein a test device having a transparent support layer is used, and the detection reaction is observed from the support side.
29. An analytical method for the detection of a component in a liquid sample, characterized in that a test device as claimed in claim 10, 12 or 14 is brought into contact with the sample, the sample being applied in a manner such that the sample enters the membrane at the surface having the narrower pore diameter, and the detection reaction is followed, wherein a test device having a transparent support layer is used, and the detection reaction is observed from the support side.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3407359.0 | 1984-02-29 | ||
| DE19843407359 DE3407359A1 (en) | 1984-02-29 | 1984-02-29 | TEST DEVICE AND METHOD FOR DETECTING A COMPONENT OF A LIQUID SAMPLE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1255196A true CA1255196A (en) | 1989-06-06 |
Family
ID=6229158
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000475165A Expired CA1255196A (en) | 1984-02-29 | 1985-02-26 | Test device and a method for the detection of a component of a liquid sample |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4824639A (en) |
| EP (1) | EP0154839B1 (en) |
| JP (1) | JPH0623745B2 (en) |
| AT (1) | ATE68884T1 (en) |
| AU (1) | AU579137B2 (en) |
| CA (1) | CA1255196A (en) |
| DE (2) | DE3407359A1 (en) |
| DK (1) | DK161543C (en) |
| ES (1) | ES8607551A1 (en) |
| HU (1) | HU205209B (en) |
| IL (1) | IL74442A (en) |
| ZA (1) | ZA851528B (en) |
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- 1984-02-29 DE DE19843407359 patent/DE3407359A1/en active Pending
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- 1985-02-16 DE DE8585101727T patent/DE3584459D1/en not_active Expired - Lifetime
- 1985-02-16 AT AT85101727T patent/ATE68884T1/en not_active IP Right Cessation
- 1985-02-16 EP EP85101727A patent/EP0154839B1/en not_active Expired - Lifetime
- 1985-02-18 AU AU38927/85A patent/AU579137B2/en not_active Expired
- 1985-02-25 ES ES540670A patent/ES8607551A1/en not_active Expired
- 1985-02-26 CA CA000475165A patent/CA1255196A/en not_active Expired
- 1985-02-26 JP JP60035426A patent/JPH0623745B2/en not_active Expired - Lifetime
- 1985-02-26 IL IL74442A patent/IL74442A/en not_active IP Right Cessation
- 1985-02-27 DK DK088785A patent/DK161543C/en not_active IP Right Cessation
- 1985-02-28 ZA ZA851528A patent/ZA851528B/en unknown
- 1985-02-28 HU HU85748A patent/HU205209B/en unknown
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1988
- 1988-02-03 US US07/151,779 patent/US4824639A/en not_active Expired - Lifetime
Also Published As
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| ZA851528B (en) | 1985-11-27 |
| AU3892785A (en) | 1985-09-05 |
| US4824639A (en) | 1989-04-25 |
| DE3407359A1 (en) | 1985-08-29 |
| DK161543B (en) | 1991-07-15 |
| HU205209B (en) | 1992-03-30 |
| JPH0623745B2 (en) | 1994-03-30 |
| IL74442A0 (en) | 1985-05-31 |
| DE3584459D1 (en) | 1991-11-28 |
| JPS60209174A (en) | 1985-10-21 |
| DK88785D0 (en) | 1985-02-27 |
| EP0154839B1 (en) | 1991-10-23 |
| ATE68884T1 (en) | 1991-11-15 |
| AU579137B2 (en) | 1988-11-17 |
| DK88785A (en) | 1985-08-30 |
| ES540670A0 (en) | 1986-05-16 |
| EP0154839A2 (en) | 1985-09-18 |
| IL74442A (en) | 1988-11-30 |
| HUT38729A (en) | 1986-06-30 |
| DK161543C (en) | 1991-12-23 |
| ES8607551A1 (en) | 1986-05-16 |
| EP0154839A3 (en) | 1986-12-30 |
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