CA1122889A - Reduction of detectable species migration in elements for the analysis of liquids - Google Patents
Reduction of detectable species migration in elements for the analysis of liquidsInfo
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- CA1122889A CA1122889A CA308,805A CA308805A CA1122889A CA 1122889 A CA1122889 A CA 1122889A CA 308805 A CA308805 A CA 308805A CA 1122889 A CA1122889 A CA 1122889A
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- 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
- G01N33/525—Multi-layer analytical elements
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Abstract
Abstract of the Disclosure An element for the analysis of liquids contains a radiation-transmissive, detectable species migration-inhibiting layer interposed between a porous radiation-blocking layer and a radiation-transmissive reagent layer. All three layers are permeable to a predetermined analyte. The reagent layer contains a composition that provides a detectable species such as a dye in proportion to the concentration of the analyte that diffuses into the reagent layer from the overlying porous radiation-blocking layer. The detectable species migration-inhibiting layer acts to reduce the migration of, for example, dye from the reagent layer into the porous radiation-blocking layer, where the optical density of the dye cannot easily be measured. Optionally, the above-described three layers can be carried on a radiation-transmissive support, and other layers such as spreading layers, registration layers, and subbing layers can also be present in the element.
Description
llZ~
It ls orten deslrable or necessary to determlne the presence and/or concentratlon o~ certain substances in liqulds such as water, foodstufrs, and blologlcal llquids.
A varlety of de~ices and methods have been empl~yed rOr such analyses.
Sophisticated elements are available for qu~ntitative diagnostic analyses Or blological llquids llke blood or ur~ne. When a llquid sample containing the analyte is brought into contact with these elements, they form the dye or other detectable change consistently and uniformly within the element in proportion to the concentratlon Or the analyte in the liquid sample. Analyte concentration can then be determined, for example, by spectrophotometric measurement of the optical density Or the dye ~ormed in the element.
Elements of this type are described in U.S. Patent No. 3,992,158. These elements can consist of two or more desirably discrete layers that are superposed and in substantially continuous intlmate contact with ad~acent layers. One such multilayer element comprises a -support layer havlng a reagent layer and an outermost spreading layer coated upon it. In this multilayer element, the liquid sample to be analyzed -~
ls placed on the spreading layer, which absorbs and transfers the liquid to the reagent layer. Preferably, as described in U.S. 3,992,158, the spreading layer is isotropically porous and transrers a uni~orm concentration (as measured across a per unit cross-sectional area of the spreading layer~ of the analyte contained in the liquid sample to the underlying reagent layer. The reagent layer has certain reagents uniformly distributed therein. ~ detectable species, such as ~ dye,ls ~ormed withln the reagent layer in an amount proportional to the concentration Or analyte in the , s llquid. Typlcally, the reagent and support layers are radiation-transmisslve so that a ~pectrophotometric measurement of the op~ical density Or the dye ~ormed ln the reagent ¦~1 layer can be made with the element remaining intact. 11 Additlonally, the spreadlng layer may comprlse a blushed ~¦
polymer and a pigment to provide both uniform transfer of the liquid sample to the reagent layer and an opaque, reflectlve il surface above the reagent layer to aid in a measurement of reflection density of the dye. With this element, however, some of the dye rormed in the reagent layer may migrate or wander lnto the opaque spreading layer where it would not be detected during the dye-density measurement, thereby reducing the sensitivity and the accuracy of the analysis.
Related elements are described in U.S. Patent No.
4,042,335. A registration layer and an opaque or radiation blocking layer are coated between the support layer and the reagent layer. During the analysis, a significant portion of the detectable species, e.g., a dye, formed ln the reagent layer ¦
will di~use through the radiation-blocking layer and into the -re~istration layer, where the dye density will be measured.
A mordant for the dye can be included ln the reglstration layer to insure that the dye that has dif~used into this layer -will be fixed there for easy detection and will not be allowed to diffuse or migrate out of the registration layer. Elements such as this are suggested for use where lt would not otherwise be practlcal to reliably measure the dye denslty within the reagent layer itself, ~or example, in analytical elements where other reagents and reaction products withln the reagent layer also provide density, thus preventing any accurate spectro- -photomet~ic measurement of the optical density ln this layer . .
Of only the dye. Such an elemen~ can provlde a reliable analysis. $
However, lt is obvlous that a signi~lcant portion of the dye formed during the analys~s can remaln in ~he reagent layer or mi6rate into and re~,ain in the radiatlon-blocklng layer.
The sensitivity and accuracy o~ the analytical element are thereby reduced, because the analyte-concentration determination must depend upon the measurement of the denslty of a smaller amount of dye than that which was actually formed. I
Other elements as described ln U.S. Patent No. ~i 10 3,585,112 and U.S. Patent No. 3j917,453 disclose means ~or overcoming these problems. Both of these patents suggest the use of mordants in the reaction zone or layer to provide a degree of immobility to the indicator dye formed. These elements, like others of the prior art, however, are susceptible to the additional problem of the mordant interfering with the formation of the dye or interferlng with any prerequis~te reactions leading to the formation of the dye. Such inter- ~¦
ference can make the analysis completely unreliable.
Accordingly, lt is deslrable to provide an analytical element that has all of the advantages of the elements described above, i.e., ease of use, low cost and quantitative results; _ and that also overcomes the problems inherent ln prior art elements, such as reduced sensitivity and accuracy of results caused by (a) migration of detectable species into porous radiation-blocking layers and (b) interference with the formation or release of the detectable species by mordants used to inhibit such migrations.
The elements of the present invention have -overcome the problems Or prior art analytical elements by providing for quantitative analyses which are highly accurate !
and sensltlv~ sbe pr~ent ~le~nts do ~o ~y 1nhibltingmigration of the detectable specles rrom the reagent layer ~o layers Or the element where such species could not easlly be measured, and by providing a means for avoiding lnterference with the reaction or reactions that result in detectable specles ~ormation in,or release ~rom,the reagent layer.
Elements according to thls invention can be used for diagnostlc purposes and include: a radiation-~ransmissive reagent layer, permeable to, and contalning a composition interactive with, a predetermined analyte (or reaction product thereof) to provide a radiometrically detectable species; a porous radiation-blocking layer permeable to the analyte; and the lmprovement of having a radiation-transmissive, detectable species migration-inhibiting layer, permeable to the analyte and interposed between the reagent layer and the radiation-blocking layer. This layer prevents a substantial amount o~ the detectable species which may dif~use out of the reagent layer from entering the porous radlation-blocking layer where it is not practically measurable, by fixing such migrating detectable species within the detectable species misration-inhibiting layer, where it ls easlly detectable. ~i Another advantage of the present lnvention is that the detectable species migratlon-inhibiting layer is separate ` ~rom the reagent layer, so that it does not interfere wlth the analytical interaction(s) taking place ln the reagent layer.
Optionally, analytical elements of the present lnvention can be carried on a radiatlon-transmissive supportS l ;
and other layers such as spreading layers, registration layers, and subbing layers can also be present in the element. Also~
:: , the porous radlatlon-blocklng layer can l~elr runction ~s a spreadlng layer in some embodlments Or the present ~nvent10n.
In the accompanylng drawln~s,each of Fig. l and Fig. 2 is an enlarged sectional vlew lllustratlng a preferred embodiment of an analytical element Or this lnvention.
The analytlcal elements o~ thls invent1on are mult1- -layered, consisting o~ three or more deslrably discrete layers that are superposed and in rluid contact with each other under condltions o~ use. These layers include a reagent laye~, a porous radiation-blocklng layer, and a detectable species migration-inhibiting layer. In certain embodiments of the lnventio~ the porous radiation-blocking layer can ~unction also as a spreading layer, or there can be a separate spreading layer ln addition to the porous radiation-blocking layer.
In other embodiments,the element can include a radiation-transmissive support layer in addition to the three layers described above. In still other embodiments,addltional radiation-transmissive layers~ e.g.~ subbing layers or registration layers, can also be included in the analytical element.
In the present invention,the layers are always arranged such that the detectable species migration-inhibiting !~
layer ls interposed between the porous radiation-blocking layer and the reagent layer. In those embodiments containing an additional layer to ~unction as a spreading layer, the porous radiation-blocking layer is interposed between the spreading layer and the detectable species migration-inhibiting ¦
layer. In those embodiments containing a radiation-transmissive ; -5- -- ,- . - . . . ' ,, ~ ' :. , , ~
: . . . . .
....
suppcrt layer, the reagent layer ls lnterposed between the deeectable specles migration~inhibitlng layer and the radiatlon-tr~nsmissive support layer. In those embodiments contalning additional radiation-transmlssive layers, such as subblng or re~istration layers, the additional subbing layers or registra-tion layers are interposed between the reagent layer and the optional radlation-transmissive support layer.
U.S. Patent No. 3,992,158 and U.S. Patent No.
4,042,335 disclose reagent layers, porous radlation-blocking layers, support layers, subbing layers, registratlon layers, and preferred types of isotropically porous spreading layers, that are useful in the practlce of the present invention. These materials also describe well known methods Or preparing these layers to form individual multilayer elements and describe the use of such elements for various quantitative analyses. -As used herein3 the term '~orous radiatlon-blocklng layer" defines a layer that is permeable to a predetermined analyte (or reaction product thereof) dissolved or dispersed ~0 in a liquid, and that reflects, or optionally absorbs, detecting radiation. "Detecting radiation" is radiation used together with the elements of the invention to facilitate result detection of the particular detectable species which is provided by the ¦
reasent layer. In other words, the porous radiation-blocking layer will allow the predetermined analyte to pass through it, and it is used together with suitable detecting radiation to racilitate result detection in the analytical ele~ents Or the invention,suGh as by reflection photometry. Because of the radiation-blocking properties o~ the porous radiatlon-blocking layer, the radiative properties, i.e.~ the partlcular ~ -emissive,;transmisslve, or absorptive properties, of any Or ' ' t~e detec~able specles w~ic~ ml~rates lnto ~hls layer can be I
substantially masked or hidden. Therefore, de~ectlng radlation used to determine the presence or absence Or detectable species formed in the rea~ent layer may be unable to detect accurately that portlon Or the detectable species which, although l provided in response to a &lven analyte, has ml~rated lnto I
the porous radiation~blockln6 layer.
As noted above, the analytical elements of the present invention can optionally contain a separate spreading layer ~n addition to the porous radiation-blocking layer~ or the porous radiation-blockln~ layer itsel~ can also function -as a spreading layer. Llke the porous radlation-blocking layer, a spreading layer must be permeable to a predetermlned analyte dissolved or dispersed ln a liquid. When liquid containing the analyte is brought into contact with the outermost surface Or a spreadlng layer, the spreading layer distributes the liquid within the spreading layer such that the concentra- l tion of the analyte provided at the surface of the spreading layer ~ -that races the reagent layer of the element ls regulated or 1, controlled. Pre~erably, but not necessarily, the spreading layer is isotropically porous and delivers a uniform concentra- -tion of analyte to the reagent layer. In one embodi~ent of the presen~ invention a separate spreading layer may be -lncluded ln addition to the porous radiation-blocking layer, as noted above, and in such case the spreading layer may be either radiation-transmissive or radiation-blocking. "Radiation-transmi5sive",as used herein, derines the ability to transmit ` detecting radiation used to determine the presence and/or concen-tration, optionally the absence, o~ the detectable species provided by the -reagent layer. Ir desired, one or more interactlve or reagent compositions may be incorporated ln the spreading layer or ~ ` -separate porous radiation-blocking layer to interact with the snalyte o~ choice, thereby rorming an analyte reaction product _7~
. ~ , ,, ' ' ~`' ' . ,'.
- ~
.
whlch c~n under~o further lnteractlon in the underlying rea~ent layer as described herelnarter.
In one preferred embodlment Or the present invention the porous radlation-blocking layer ltself fur,ctlons as an adequate spreading layer a~d comprises a blushed polymer and optlonally a finely-divided particulate material such as a pi~ment. Layers of this type are dlscussed in detail ln U.S~ Patents 3,992,158 and 4,042,335.
Useful blushed polymers include cellulose acetate, amides, ¦
and the like. Useful particulate materials lnclude pigments such as carbon, titanium dioxlde, barlum sulfate, and the like.
Reagent layers in the elements of this invention are radlation-transmissive. Preferably, the reagent -layer is uniformly permeable to the particular analyte to be measured. Within the reagent layer is distributed a material that can interact with the analyte or reaction product o~
the analyte. Such lnteraction causes the release of a preformed detectable species or the ~ormation of such a detectable species within the reagent layer, preferably, in proportion to the concentration of the analyte in the liquid sample bein6 analyzed. Such interaction is meant to refer to chemical activity, catalytic activity as in the formation of an enzyme-substrate complex, and any other form of chemical `
or physical interaction that can release, produce~ or otherwise provide within the reagent layer a species that is radio-metrically detectable, that is, by suitable measurement of llght or other energy~ Typically, the detectable species rormed or released from the reagent layer is a dye which is radiometrically detectable by rluorometric or colorimetric~
pre~erably colorimetri~ techniques. -.
',` "` ' , " , .
In additlon, lf nec~sary or deslrable, appropr1ate bu~er composltions may al~o be present ln the reagent l~yer.
Re~6ent layers Or the present lnventlon may also contaln one or more ~ydrophilic colloid~ lncluding natural c~lloids suc~ as 6elatln, agarose, polysaccharldes~ and the llke; and/or synthetic resins such as poly~lnyl alcohol)~ poly(vinyl pyrrolidone), polyacrylamides~ and the llke.
One appllcation Or the present invention comprlses an element ror the analysls of ~lucose ln llqulds,wherein the interactlve material ln the reagent layer preferably comprises ~lucose oxidase, perox~dase~ and an indlcator jl composition. A useful indicator c~mposltion comprlses 4- ¦~
amlnoantipyrene hydrochloride and 7~hydroxy-1-naphthol. In i the presence of glucose, the above interactive material effects the ~ormatlon o~ a dye in proport~on to the concen-tratlon Or glucose in the sample belng analyzed. Thls concen-tration can then be determlned by spectrophotometrlcally measurin~ the optlcal density o~ the dye rormed and performing an arithmetic calculation. Another embodiment Or the present lnvention comprises an element ror the analysis of calcium in llquids and includes a reagent layer containing an interactive -material which is an indicator ~or calcium and ~orms a ,l ~olored species ln the presence Or calc~um9 such as chloro- ~l phosphona~ III(i.e. sodium 3,6-bis(4-chloro-2-phosphonophenyla~o) 4, S~;~ ;
dihydroxy-2, 7-naphthalene di~ulfonate) or arsenazo III -~
(i.e. 1,8-dihydroxy naphthalene-3.6-disulfonic acid-2, 7-bis l(azo-2)~phenylarsonic acidJ). The use of arsenazo III as a calcium complexing agent is described in Anal. Chim. Acta., vol. 53 (1971), pp~ 194-198~ Other suitable indicator for calcium are known and may be found, for example, in Clinical Chemistry Princi~les and Technics,edited by Henry et al, 2nd.ed.~
.
.
_ g_ Elements Or the presenS inventlon are also useful ln the analysis of many other substances in liquids in additiOn to calcium or glucose as noted above.
As stated hereinabove, the elements of thls invention can also lnclude a radiatlon-transmlsslve support to support the other layers. Such a support transmits llght in the -9a-other layers. Such a support transmits light in the range Or the spectrum used to determlne the presence and/or absence of detectable specles provided by the reagent layer.
In the case where the detectable species ls a vlslbly colored material, e.g., a dye, this will allow the spectrophotometric measurement of the dye density to be per~ormed through the support layer with all layers of the element stlll lntact.
A useful support layer can comprise cellulose acetate~
polyethylene terephthalate, and the like.
Other optional layers mentioned herelnabo~e lnclude radiation-transmissive subbing and registratlon layers, whlch lf used, are located between the reagent layer and the optlonal -support layer. Subbing layers may also be lncluded between other layers to provide the requlred adheslon and rluid contact between such layers. Such optlonal re~istration and subbing layers are known ln the art and are described in U.S. Patent No. 3,992,158, and in U.S. Patent No. 4,042,335. ;
~:, The detectable species migration-inhibiting layer 20 of the present invention is interposed between the reagent i -~
layer and the porous radiation-blocking layer and is radiation-transmissive. The detectable species migratlon-inhibiting layer is permeable to the analyte, so that analyte can diffuse through it from the porous radiation-blocklng layer and into , the reagent layer. The detectable species migration-lnhibiting layer functions such that a significant portion of any detectable species, e.g., a dye, migrating into it from the reagent layer ~s fixed in place or otherwise prevented from further migrating into the porous radiation-blocking layer (and 1 30 ~urther into the separate spreading layer~ ir one is presen wherein it cannot easily be measured. Detectable species migration-inhibit~ng layers o~ a pre~erred embodIment Or th~
present i~vention comprise a hydrophilic collold and a mordant . .
for the partlcular detectable specles rormed in the reagent layer. Userul hydrophllic collolds lnclude those menkioned hereinabove as use~ul in rea~ent layers of the described elements. Use~ul mordants are chosen accordlng to the particular detectable specles formed in the reagent layer.
In the example Or an element for the analysis of glucose ln liquids, dlscussed above, one pre~erred mordant among others is a copolymer comprlsing recurring units Or styrene, N- ~!
vinylbenzyl-N,N-dimethylbenzylammonium chloride~ and divinyl benzene. It has been found that lf the mordant is placed directly in the reagent layer, it often unexpectedly lnter-reres with the reactions initiated by the presence of the analyte and prevents or significantly lnhlbits the forma~ion or release Or the detectable species.
O~her mordants use~ul in the present lnvention include compounds of the structure:
I. Rl_~_R3 X~
R2 , wherein -Z~- is -N~- or -P~-;
each o~ Rl, R2 and R3, which may be the same or di~erent, ls selected ~rom alkyl, alkenyl, aralkyl, or aryl, each having less than eight carbon atoms, including cycloalkyls such as cyclohexyl, alkenyls such as allyl, aralkyls such as benzyl, and aryls such as phenyl and substituted phenyls;
R ls a ballasting group havlng more than ei~h~
carbon atoms such as alkyl, including substituted alkyl 30 and alkyl havlng hetero atoms or groups within or appended to the alkyllchaing aralkyl, and aryl as deflned above; and T
¦ X~ is an ~cid anion such as a hallde lon, e.g., ¦ chloride or bromide; nitra~; methosulrate; p-toluenesulronate;
etc.
One example of a u~eful mordant of Formula I above ls a compound having the structure:
II. CloH
H13C6-N ~C6H13 X~ ' ¦ Other mordants useful in the invention are polymeric mordants lncluding copolymers, e~g., terpolymers. A partial listing Or representatlve use~ul polymeric mordants includes polymers having recurring unlts derived from 70 to about 98 :
weight percent Or one or a mixture of hydrophobic monomers, : for example, styrene; and recurring units derived ~rom about 2 to 30 weight percent, preferably about 5 to 20 weight percent, of at least one ~ :
cationic monomer, such units typicPlly, but not necessarily, confo~
to the structure:
R7 R~
.
~ III. CH-C- ;~
'~ X4 wherein L is a chemlcal linking group between Q~ nd the atoms in the chain of the polymer backbone; ~.
n is 0 or li :
X is an acid anion as deflned above; and Q~ is 2 linear or heterocycllc ammonium, phosphonium, - or sulrur-conta1ning group Or the structure:
IVa. Rl_z~_R3, IVb. fD ~ IVc. ~D-~
R2 ~Z -CH, ~Z =CH, ~.
: -12- .
. . .
, -- , :
:
~2~
Rl S0 ,1 q ~ ., .
IYd. SW- J IVe. ~ N-H
R5 R6 jl whereln -Z~- is -N~- or -P~-;
each of Rl, R2, and R3, which may be the same or dirrer~nt~ is as defined above;
each of R5, R6, R7, and R8, which may be the same or di~ferent, represent H or R as defined above; and D is the atoms necessary to complete a heterocyclic ring. In addition to styrene other hydrophoblc monomers useful as recurring units in these polymer~c mordants include .
substituted styrenes~ alkyl acrylates and methacrylates~ di-runctional monomers such as divinylbenzene and ethylenedi-methacrylate J acrylamides, methacrylamides, and the like.
A partial listing Or representative cationic monomers useful ln preparing these polymeric mordants includes:
N-vinylbenzyl-N,N,N-trimethylammonium chloride, N-benzyl-N,N-dimethyl-N-vinylbenzylammonium chloride, N,N,N-trihexyl-N-vinylbenzylammonium chloride, 1l ' N-(3-maleimidopropyl)-N,N,N-trimethylammonium chloride~ l N-benzyl-N-(3-maleimidopropyl)-N,N-dimethyla~monium chloride, ¦ :
N-vinyloxycarbonylmethyl-N,N,N-trimethylammonium chloride, .
N-(3-acrylamido-3,3-dimethylpropyl)-N,N,N-trimethylammonium , methosulfate~
1,2-dimethyl-5-vinylpyridinium methosul~ate, N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylammonium chloride, N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylamrnonlum sulfate, -N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium iodide, : :1 N-(2-methacryloyloxyethyl)-N~N,N-trlmethylammonium p-toluene- -sul~onate, N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium methosulfate, ,` . . '~ '~.
8~3 3-methyl-1-vinyl1mldazol1um methosul~te~
I N-(2-methzcryloyloxyethyl)-N~N~N-trlmethylammonium acetate, ¦ N-~-methacryloyloxyethyl)-N~N~N trlmethylammonlum bromide, N-(2-methacryloyloxyethyl)-N~N~N~tr~methylammonium chlorlde, N-(2-methacryloyloxyethyl)-N,N,N-trimethylamrnonium rluorlde, N-(2-methacryloyloxyethyl)-N~N,N-trimethylammonium nltrate, and N-~2-methacryloyloxyethyl)-N,N,N-trlmethylammonlum phosphate.
An example of one suitable polymeric morda~t Or khe type desc~ibed ls that copolymer identified hereinbefore as use~ul ln an element for the analysis of glucose ln liquids.
In addition to the use o~ mordants to ~ormulate the detectable species migration~inhlbitlng layer used ln the present lnvention, one can also employ as the migratlon-inhibiting material an antibody for the detectable species provided by the reagent layer. Such antibodies can be prepared by conventional immunological techniques and, of course, can vary widely depending on the partlcular material to be used as the detectable species in a given element of the invention.
Typically, such antibodies are immobillzed in the detectable species migration-inhibiting layer.
Exemplary elements of this invention includes those illustrated in the accompanying drawings. Ih Figure 1 is represented an analytical element composed of a reagent layer 12, a detectable species migration-inhibiting layer 14, a porous radiation-blocking layer 16, and, optionally, a spreading layer 18. A11 of these layers are in substantially continuous lntimate contact with their ad~acent layers. In an alternative embodiment of the invention, shown ln Figure 2, the analytical element is composed of a support 20 on which is coated a reagent layer 24, a detectable species migration-inhibiting layer 26, and a porous radiation-blocklng layer 28, which in this case serves al~o as a spreading layer. Optionally, either or both subbing and registration layers 22 may also be included in the `
. .
.
an~lytlcal element. All Or the~e layers are in substantially -continuous ln~lmate contact wlth thelr adJacent layers.
In the practlce o~ this inventlon, a sample of a llquid to be analyzed is placed on the outermost surface layer Or the element 9 which in the case Or the element lllus-trated in ~lgure 2 is the porous~ radlatlon-blocking, spreading layer 28. Any predetermined analyte present ln thls llquld di~uses through the porous, radlat~on-blocklng layer 28 and the ,, detectable species mlgration-lnhibiting layer 26, and enters the f,, reasent layer 24. There, lnteraction wlth the test reagents causes the release of or the formatlon Or a detectable specles such as a dye. This dye either remains ln place or in part mi~rates out of the reagent layer 24, lnto the detectable species mlgration inhibiting layer 26, and also into any porous, , radiation-transmlssive layers underlying the reagent layer. -All or most o~ the dye enterlng the detectable species mi~ration-inhibiting layer 26 ls fixed in place and prevented ~rom ~urther migrating into the overlylng porous radiation-blocking layer or layers 28. ~he reflective density of all dye in th~ detectable species migration-inhibiting layer 26, the reagent layer 24, and any other underlying radiation-transmissive layers is then determined while the element is still intact by measuring this density spectrophotometrically through all of these radiation-transmissive layers at the same tlme. ,l The following examples are provided to further ~¦
illustrate certain embodiments o~ the present invention.
Example 1 - Element For The Analysis 0~ Glucose Two elements ~or the analysis of glucose in llquids were prepared in the following manner:
-15- ;
Polyethylene t~r~phthalate fllm supports were coated wlth reagent layers comprlsln~ peroxidase at 10,200 U/m2, (the symbol U refers to internatlonal unlts~ whlch are the well known and generally accepted unlts of measurement of enzyme activity), glucos2 oxldase at 24g400 U/m2, 7 hydroxy-l-naphthol at 0.66 g/m , and 4-amlnoantipyrene hydrochloride l .
at o.86 g/m2. The rea6ent layer Or control sample 1 ~urther comprised deionized gelatin at 21.5 g/m . The reagent layer o~ sample 2 also comprised deionized gelatln, but at 19.4 g/m2.
The second sample was then coated with a detectable species l migration-inhibitlng layer, ln this case a dye migration- ¦
inhibiting layer comprising deionized gelatin at 2.1 g/m2 and the mordant, poly(styrene-co-N-vinylbenzyl-N,N-dimethyl-ben ylammonium chloride-co-divinyl benzene) (weight ratlo 49.5:49.5:1.0) at 1.08 g/m2. All gelatin-containing layers -were buffered at pH 6.o with a disodium phosphate-potassium ~
phosphate buffer. Both samples were then overcoated with ¦ .
a subbing layer comprising n-isopropylacrylamide at 0.32 g/m2 and a blushed-polymer, radiatlon-blocking, spreading layer l .
comprising cellulose acetate at 9.4 g/m and titanium dioxide ¦
at 64.5 g/m2.
The two resulting elements were then contacted at the outermost surface of their spreading layers with 10 ~1 samples of glucose standards contalning various concentrations of glucose. After 7 minutes of contact at 37C the reflection densities Or the dye rormed were measured spectrophotometrlcallY
using a photomultipller unit and a Wratten 65 rilter. The following Table I illustrates the resu.~.ts, the control sample .
being representative Or elements Or the prior art.
! !
1` .
.
.. ... ,,_. . `--.,, ~C
~C~
~ H
o o o o u~
U ~J ~D O ~D O
. ~ ~ ~Y; ~
CC ~ a ~ a~ ~ c ~ ~
C
.,~
.,, ~ .
.~ E c R O ta Q E~
,~ U~ ~ U~
,C ~ ~ ~ ~r o HH C`l ~¢ 1~ --~ ~ ~ ~J
51:1l ~ ~ ~ ~ O ;~
.:1 C: ~1 U~ Q~ 1 O O ~O~ ~ O
~ I~ ~C
U W
~: a ~ o ' o C aJ C::
~ o3 Q~
u~
1~ ~ C~l O O O O
~ ~ I N
~`
I
~ .
, :
.
Example 2 - Element For The Analysis or Calclum Two elements, one wlth and the other without a detectable species migration-inhlbiting layer contalning a mordant, were prepared according to the followlng:
A tereph~halate r~ lm support was coated with a reagent layer comprising gelatin (4.3 g/m2), Triton X-lOOTM (0.17 g/m2)~
chlorophosphonazo III (0.21 g/m2), bis(vinylsulfonylmethyl) ether (0.04 g/m2) and 0.1 M 3,3-dimethylglutaric acid, pH 5.4; a detectable species (dye) migration-inhibiting layer comprising gelatin (4.3 g/m2), and poly(styrene-co-N-vinylbenzyl-N,N-dimeth-ylbenzylammonium chloride-co-divinylbenzene) (2.15 g/m2); a sub-bing layer comprising (poly-N-isopropylacrylamide) (0.32 g/m2);
and a blushed-polymer, radiation-blocking spreading layer com-prising TiO2 (50.4 g/m2), cellulose acetate (7.0 g/m2) and Triton X-405TM ~1.4 g/m2). Triton X-lOOTM and Triton X-405TM are alkyl-pheno~y polyethoxy ethanols, commercially available ~rom the Rohm and Haas Company.
A second control element ~outside the scope Or the present invention) was prep~red in the sa~e manner except without a detectable species migration-lnhibiting layer between the spreadlng layer and reagent layer.
The elements were evaluated as in Example l, using calcium standards containing 1 to 5 mM of calcium and readlng the reflection densities at 670 nm. Table II s~ows ~he improved results obtained with the element containing the detectable species migration-inhiblting layer, in thls case a dye migration-inhibiting layer.
The results o~ Examples l and 2 above indicate that a signi~icantly ~igher dye density was consistently measured wlth ~he element containing a detectable species mlgratlon-lnhiblting layer. The control element, having no such layer, allowed signi~lcant amounts o~ the dye to mlgrate lnto the blushed~polymer~ radiatlon-blocking, spreadlng layer where lt coul~ not b~ det~cted.
- - -- --- - -~ c~c C C
a 1~ ' ~ o o I CO U~ O ~ ~ ~
c ~ ~ ~ m c E ~ a O o O O O O
h ~ ~ J
: Q~ ~1 m u~ Q~
:~
CQ~ C
~ ,1~
~ E_~
R O I~ ~ C~
,C Q~ I .,~
C ~ U~ U~ Q~
H H aa ~
O O ~ ~ ~ ~ ~
., ~ U ~ U~
~ ~ ~~ ~0 o o o o o o E~ :~ c U c : .
:~ ~ ~ ~ ,~
O -~ ~:
o c u E E ~ ~:
~1 0 . ~ .:
~ u c~ ~ o ,~
~ ...
~ u --L) C
~ O : `
~ , ~ ;
~ :~
Example 3 Example No. 2 was repeated, except that the ~eagent layer contained as a calcium indicator 0.48 G/M2 arsenazo III, rather than chorophosphonazo III. The reagent layer was buffered to a ph of 5.6. The resulting element demonstated a dye density comparable to that of the test element of example 2.
It ls orten deslrable or necessary to determlne the presence and/or concentratlon o~ certain substances in liqulds such as water, foodstufrs, and blologlcal llquids.
A varlety of de~ices and methods have been empl~yed rOr such analyses.
Sophisticated elements are available for qu~ntitative diagnostic analyses Or blological llquids llke blood or ur~ne. When a llquid sample containing the analyte is brought into contact with these elements, they form the dye or other detectable change consistently and uniformly within the element in proportion to the concentratlon Or the analyte in the liquid sample. Analyte concentration can then be determined, for example, by spectrophotometric measurement of the optical density Or the dye ~ormed in the element.
Elements of this type are described in U.S. Patent No. 3,992,158. These elements can consist of two or more desirably discrete layers that are superposed and in substantially continuous intlmate contact with ad~acent layers. One such multilayer element comprises a -support layer havlng a reagent layer and an outermost spreading layer coated upon it. In this multilayer element, the liquid sample to be analyzed -~
ls placed on the spreading layer, which absorbs and transfers the liquid to the reagent layer. Preferably, as described in U.S. 3,992,158, the spreading layer is isotropically porous and transrers a uni~orm concentration (as measured across a per unit cross-sectional area of the spreading layer~ of the analyte contained in the liquid sample to the underlying reagent layer. The reagent layer has certain reagents uniformly distributed therein. ~ detectable species, such as ~ dye,ls ~ormed withln the reagent layer in an amount proportional to the concentration Or analyte in the , s llquid. Typlcally, the reagent and support layers are radiation-transmisslve so that a ~pectrophotometric measurement of the op~ical density Or the dye ~ormed ln the reagent ¦~1 layer can be made with the element remaining intact. 11 Additlonally, the spreadlng layer may comprlse a blushed ~¦
polymer and a pigment to provide both uniform transfer of the liquid sample to the reagent layer and an opaque, reflectlve il surface above the reagent layer to aid in a measurement of reflection density of the dye. With this element, however, some of the dye rormed in the reagent layer may migrate or wander lnto the opaque spreading layer where it would not be detected during the dye-density measurement, thereby reducing the sensitivity and the accuracy of the analysis.
Related elements are described in U.S. Patent No.
4,042,335. A registration layer and an opaque or radiation blocking layer are coated between the support layer and the reagent layer. During the analysis, a significant portion of the detectable species, e.g., a dye, formed ln the reagent layer ¦
will di~use through the radiation-blocking layer and into the -re~istration layer, where the dye density will be measured.
A mordant for the dye can be included ln the reglstration layer to insure that the dye that has dif~used into this layer -will be fixed there for easy detection and will not be allowed to diffuse or migrate out of the registration layer. Elements such as this are suggested for use where lt would not otherwise be practlcal to reliably measure the dye denslty within the reagent layer itself, ~or example, in analytical elements where other reagents and reaction products withln the reagent layer also provide density, thus preventing any accurate spectro- -photomet~ic measurement of the optical density ln this layer . .
Of only the dye. Such an elemen~ can provlde a reliable analysis. $
However, lt is obvlous that a signi~lcant portion of the dye formed during the analys~s can remaln in ~he reagent layer or mi6rate into and re~,ain in the radiatlon-blocklng layer.
The sensitivity and accuracy o~ the analytical element are thereby reduced, because the analyte-concentration determination must depend upon the measurement of the denslty of a smaller amount of dye than that which was actually formed. I
Other elements as described ln U.S. Patent No. ~i 10 3,585,112 and U.S. Patent No. 3j917,453 disclose means ~or overcoming these problems. Both of these patents suggest the use of mordants in the reaction zone or layer to provide a degree of immobility to the indicator dye formed. These elements, like others of the prior art, however, are susceptible to the additional problem of the mordant interfering with the formation of the dye or interferlng with any prerequis~te reactions leading to the formation of the dye. Such inter- ~¦
ference can make the analysis completely unreliable.
Accordingly, lt is deslrable to provide an analytical element that has all of the advantages of the elements described above, i.e., ease of use, low cost and quantitative results; _ and that also overcomes the problems inherent ln prior art elements, such as reduced sensitivity and accuracy of results caused by (a) migration of detectable species into porous radiation-blocking layers and (b) interference with the formation or release of the detectable species by mordants used to inhibit such migrations.
The elements of the present invention have -overcome the problems Or prior art analytical elements by providing for quantitative analyses which are highly accurate !
and sensltlv~ sbe pr~ent ~le~nts do ~o ~y 1nhibltingmigration of the detectable specles rrom the reagent layer ~o layers Or the element where such species could not easlly be measured, and by providing a means for avoiding lnterference with the reaction or reactions that result in detectable specles ~ormation in,or release ~rom,the reagent layer.
Elements according to thls invention can be used for diagnostlc purposes and include: a radiation-~ransmissive reagent layer, permeable to, and contalning a composition interactive with, a predetermined analyte (or reaction product thereof) to provide a radiometrically detectable species; a porous radiation-blocking layer permeable to the analyte; and the lmprovement of having a radiation-transmissive, detectable species migration-inhibiting layer, permeable to the analyte and interposed between the reagent layer and the radiation-blocking layer. This layer prevents a substantial amount o~ the detectable species which may dif~use out of the reagent layer from entering the porous radlation-blocking layer where it is not practically measurable, by fixing such migrating detectable species within the detectable species misration-inhibiting layer, where it ls easlly detectable. ~i Another advantage of the present lnvention is that the detectable species migratlon-inhibiting layer is separate ` ~rom the reagent layer, so that it does not interfere wlth the analytical interaction(s) taking place ln the reagent layer.
Optionally, analytical elements of the present lnvention can be carried on a radiatlon-transmissive supportS l ;
and other layers such as spreading layers, registration layers, and subbing layers can also be present in the element. Also~
:: , the porous radlatlon-blocklng layer can l~elr runction ~s a spreadlng layer in some embodlments Or the present ~nvent10n.
In the accompanylng drawln~s,each of Fig. l and Fig. 2 is an enlarged sectional vlew lllustratlng a preferred embodiment of an analytical element Or this lnvention.
The analytlcal elements o~ thls invent1on are mult1- -layered, consisting o~ three or more deslrably discrete layers that are superposed and in rluid contact with each other under condltions o~ use. These layers include a reagent laye~, a porous radiation-blocklng layer, and a detectable species migration-inhibiting layer. In certain embodiments of the lnventio~ the porous radiation-blocking layer can ~unction also as a spreading layer, or there can be a separate spreading layer ln addition to the porous radiation-blocking layer.
In other embodiments,the element can include a radiation-transmissive support layer in addition to the three layers described above. In still other embodiments,addltional radiation-transmissive layers~ e.g.~ subbing layers or registration layers, can also be included in the analytical element.
In the present invention,the layers are always arranged such that the detectable species migration-inhibiting !~
layer ls interposed between the porous radiation-blocking layer and the reagent layer. In those embodiments containing an additional layer to ~unction as a spreading layer, the porous radiation-blocking layer is interposed between the spreading layer and the detectable species migration-inhibiting ¦
layer. In those embodiments containing a radiation-transmissive ; -5- -- ,- . - . . . ' ,, ~ ' :. , , ~
: . . . . .
....
suppcrt layer, the reagent layer ls lnterposed between the deeectable specles migration~inhibitlng layer and the radiatlon-tr~nsmissive support layer. In those embodiments contalning additional radiation-transmlssive layers, such as subblng or re~istration layers, the additional subbing layers or registra-tion layers are interposed between the reagent layer and the optional radlation-transmissive support layer.
U.S. Patent No. 3,992,158 and U.S. Patent No.
4,042,335 disclose reagent layers, porous radlation-blocking layers, support layers, subbing layers, registratlon layers, and preferred types of isotropically porous spreading layers, that are useful in the practlce of the present invention. These materials also describe well known methods Or preparing these layers to form individual multilayer elements and describe the use of such elements for various quantitative analyses. -As used herein3 the term '~orous radiatlon-blocklng layer" defines a layer that is permeable to a predetermined analyte (or reaction product thereof) dissolved or dispersed ~0 in a liquid, and that reflects, or optionally absorbs, detecting radiation. "Detecting radiation" is radiation used together with the elements of the invention to facilitate result detection of the particular detectable species which is provided by the ¦
reasent layer. In other words, the porous radiation-blocking layer will allow the predetermined analyte to pass through it, and it is used together with suitable detecting radiation to racilitate result detection in the analytical ele~ents Or the invention,suGh as by reflection photometry. Because of the radiation-blocking properties o~ the porous radiatlon-blocking layer, the radiative properties, i.e.~ the partlcular ~ -emissive,;transmisslve, or absorptive properties, of any Or ' ' t~e detec~able specles w~ic~ ml~rates lnto ~hls layer can be I
substantially masked or hidden. Therefore, de~ectlng radlation used to determine the presence or absence Or detectable species formed in the rea~ent layer may be unable to detect accurately that portlon Or the detectable species which, although l provided in response to a &lven analyte, has ml~rated lnto I
the porous radiation~blockln6 layer.
As noted above, the analytical elements of the present invention can optionally contain a separate spreading layer ~n addition to the porous radiation-blocking layer~ or the porous radiation-blockln~ layer itsel~ can also function -as a spreading layer. Llke the porous radlation-blocking layer, a spreading layer must be permeable to a predetermlned analyte dissolved or dispersed ln a liquid. When liquid containing the analyte is brought into contact with the outermost surface Or a spreadlng layer, the spreading layer distributes the liquid within the spreading layer such that the concentra- l tion of the analyte provided at the surface of the spreading layer ~ -that races the reagent layer of the element ls regulated or 1, controlled. Pre~erably, but not necessarily, the spreading layer is isotropically porous and delivers a uniform concentra- -tion of analyte to the reagent layer. In one embodi~ent of the presen~ invention a separate spreading layer may be -lncluded ln addition to the porous radiation-blocking layer, as noted above, and in such case the spreading layer may be either radiation-transmissive or radiation-blocking. "Radiation-transmi5sive",as used herein, derines the ability to transmit ` detecting radiation used to determine the presence and/or concen-tration, optionally the absence, o~ the detectable species provided by the -reagent layer. Ir desired, one or more interactlve or reagent compositions may be incorporated ln the spreading layer or ~ ` -separate porous radiation-blocking layer to interact with the snalyte o~ choice, thereby rorming an analyte reaction product _7~
. ~ , ,, ' ' ~`' ' . ,'.
- ~
.
whlch c~n under~o further lnteractlon in the underlying rea~ent layer as described herelnarter.
In one preferred embodlment Or the present invention the porous radlation-blocking layer ltself fur,ctlons as an adequate spreading layer a~d comprises a blushed polymer and optlonally a finely-divided particulate material such as a pi~ment. Layers of this type are dlscussed in detail ln U.S~ Patents 3,992,158 and 4,042,335.
Useful blushed polymers include cellulose acetate, amides, ¦
and the like. Useful particulate materials lnclude pigments such as carbon, titanium dioxlde, barlum sulfate, and the like.
Reagent layers in the elements of this invention are radlation-transmissive. Preferably, the reagent -layer is uniformly permeable to the particular analyte to be measured. Within the reagent layer is distributed a material that can interact with the analyte or reaction product o~
the analyte. Such lnteraction causes the release of a preformed detectable species or the ~ormation of such a detectable species within the reagent layer, preferably, in proportion to the concentration of the analyte in the liquid sample bein6 analyzed. Such interaction is meant to refer to chemical activity, catalytic activity as in the formation of an enzyme-substrate complex, and any other form of chemical `
or physical interaction that can release, produce~ or otherwise provide within the reagent layer a species that is radio-metrically detectable, that is, by suitable measurement of llght or other energy~ Typically, the detectable species rormed or released from the reagent layer is a dye which is radiometrically detectable by rluorometric or colorimetric~
pre~erably colorimetri~ techniques. -.
',` "` ' , " , .
In additlon, lf nec~sary or deslrable, appropr1ate bu~er composltions may al~o be present ln the reagent l~yer.
Re~6ent layers Or the present lnventlon may also contaln one or more ~ydrophilic colloid~ lncluding natural c~lloids suc~ as 6elatln, agarose, polysaccharldes~ and the llke; and/or synthetic resins such as poly~lnyl alcohol)~ poly(vinyl pyrrolidone), polyacrylamides~ and the llke.
One appllcation Or the present invention comprlses an element ror the analysls of ~lucose ln llqulds,wherein the interactlve material ln the reagent layer preferably comprises ~lucose oxidase, perox~dase~ and an indlcator jl composition. A useful indicator c~mposltion comprlses 4- ¦~
amlnoantipyrene hydrochloride and 7~hydroxy-1-naphthol. In i the presence of glucose, the above interactive material effects the ~ormatlon o~ a dye in proport~on to the concen-tratlon Or glucose in the sample belng analyzed. Thls concen-tration can then be determlned by spectrophotometrlcally measurin~ the optlcal density o~ the dye rormed and performing an arithmetic calculation. Another embodiment Or the present lnvention comprises an element ror the analysis of calcium in llquids and includes a reagent layer containing an interactive -material which is an indicator ~or calcium and ~orms a ,l ~olored species ln the presence Or calc~um9 such as chloro- ~l phosphona~ III(i.e. sodium 3,6-bis(4-chloro-2-phosphonophenyla~o) 4, S~;~ ;
dihydroxy-2, 7-naphthalene di~ulfonate) or arsenazo III -~
(i.e. 1,8-dihydroxy naphthalene-3.6-disulfonic acid-2, 7-bis l(azo-2)~phenylarsonic acidJ). The use of arsenazo III as a calcium complexing agent is described in Anal. Chim. Acta., vol. 53 (1971), pp~ 194-198~ Other suitable indicator for calcium are known and may be found, for example, in Clinical Chemistry Princi~les and Technics,edited by Henry et al, 2nd.ed.~
.
.
_ g_ Elements Or the presenS inventlon are also useful ln the analysis of many other substances in liquids in additiOn to calcium or glucose as noted above.
As stated hereinabove, the elements of thls invention can also lnclude a radiatlon-transmlsslve support to support the other layers. Such a support transmits llght in the -9a-other layers. Such a support transmits light in the range Or the spectrum used to determlne the presence and/or absence of detectable specles provided by the reagent layer.
In the case where the detectable species ls a vlslbly colored material, e.g., a dye, this will allow the spectrophotometric measurement of the dye density to be per~ormed through the support layer with all layers of the element stlll lntact.
A useful support layer can comprise cellulose acetate~
polyethylene terephthalate, and the like.
Other optional layers mentioned herelnabo~e lnclude radiation-transmissive subbing and registratlon layers, whlch lf used, are located between the reagent layer and the optlonal -support layer. Subbing layers may also be lncluded between other layers to provide the requlred adheslon and rluid contact between such layers. Such optlonal re~istration and subbing layers are known ln the art and are described in U.S. Patent No. 3,992,158, and in U.S. Patent No. 4,042,335. ;
~:, The detectable species migration-inhibiting layer 20 of the present invention is interposed between the reagent i -~
layer and the porous radiation-blocking layer and is radiation-transmissive. The detectable species migratlon-inhibiting layer is permeable to the analyte, so that analyte can diffuse through it from the porous radiation-blocklng layer and into , the reagent layer. The detectable species migration-lnhibiting layer functions such that a significant portion of any detectable species, e.g., a dye, migrating into it from the reagent layer ~s fixed in place or otherwise prevented from further migrating into the porous radiation-blocking layer (and 1 30 ~urther into the separate spreading layer~ ir one is presen wherein it cannot easily be measured. Detectable species migration-inhibit~ng layers o~ a pre~erred embodIment Or th~
present i~vention comprise a hydrophilic collold and a mordant . .
for the partlcular detectable specles rormed in the reagent layer. Userul hydrophllic collolds lnclude those menkioned hereinabove as use~ul in rea~ent layers of the described elements. Use~ul mordants are chosen accordlng to the particular detectable specles formed in the reagent layer.
In the example Or an element for the analysis of glucose ln liquids, dlscussed above, one pre~erred mordant among others is a copolymer comprlsing recurring units Or styrene, N- ~!
vinylbenzyl-N,N-dimethylbenzylammonium chloride~ and divinyl benzene. It has been found that lf the mordant is placed directly in the reagent layer, it often unexpectedly lnter-reres with the reactions initiated by the presence of the analyte and prevents or significantly lnhlbits the forma~ion or release Or the detectable species.
O~her mordants use~ul in the present lnvention include compounds of the structure:
I. Rl_~_R3 X~
R2 , wherein -Z~- is -N~- or -P~-;
each o~ Rl, R2 and R3, which may be the same or di~erent, ls selected ~rom alkyl, alkenyl, aralkyl, or aryl, each having less than eight carbon atoms, including cycloalkyls such as cyclohexyl, alkenyls such as allyl, aralkyls such as benzyl, and aryls such as phenyl and substituted phenyls;
R ls a ballasting group havlng more than ei~h~
carbon atoms such as alkyl, including substituted alkyl 30 and alkyl havlng hetero atoms or groups within or appended to the alkyllchaing aralkyl, and aryl as deflned above; and T
¦ X~ is an ~cid anion such as a hallde lon, e.g., ¦ chloride or bromide; nitra~; methosulrate; p-toluenesulronate;
etc.
One example of a u~eful mordant of Formula I above ls a compound having the structure:
II. CloH
H13C6-N ~C6H13 X~ ' ¦ Other mordants useful in the invention are polymeric mordants lncluding copolymers, e~g., terpolymers. A partial listing Or representatlve use~ul polymeric mordants includes polymers having recurring unlts derived from 70 to about 98 :
weight percent Or one or a mixture of hydrophobic monomers, : for example, styrene; and recurring units derived ~rom about 2 to 30 weight percent, preferably about 5 to 20 weight percent, of at least one ~ :
cationic monomer, such units typicPlly, but not necessarily, confo~
to the structure:
R7 R~
.
~ III. CH-C- ;~
'~ X4 wherein L is a chemlcal linking group between Q~ nd the atoms in the chain of the polymer backbone; ~.
n is 0 or li :
X is an acid anion as deflned above; and Q~ is 2 linear or heterocycllc ammonium, phosphonium, - or sulrur-conta1ning group Or the structure:
IVa. Rl_z~_R3, IVb. fD ~ IVc. ~D-~
R2 ~Z -CH, ~Z =CH, ~.
: -12- .
. . .
, -- , :
:
~2~
Rl S0 ,1 q ~ ., .
IYd. SW- J IVe. ~ N-H
R5 R6 jl whereln -Z~- is -N~- or -P~-;
each of Rl, R2, and R3, which may be the same or dirrer~nt~ is as defined above;
each of R5, R6, R7, and R8, which may be the same or di~ferent, represent H or R as defined above; and D is the atoms necessary to complete a heterocyclic ring. In addition to styrene other hydrophoblc monomers useful as recurring units in these polymer~c mordants include .
substituted styrenes~ alkyl acrylates and methacrylates~ di-runctional monomers such as divinylbenzene and ethylenedi-methacrylate J acrylamides, methacrylamides, and the like.
A partial listing Or representative cationic monomers useful ln preparing these polymeric mordants includes:
N-vinylbenzyl-N,N,N-trimethylammonium chloride, N-benzyl-N,N-dimethyl-N-vinylbenzylammonium chloride, N,N,N-trihexyl-N-vinylbenzylammonium chloride, 1l ' N-(3-maleimidopropyl)-N,N,N-trimethylammonium chloride~ l N-benzyl-N-(3-maleimidopropyl)-N,N-dimethyla~monium chloride, ¦ :
N-vinyloxycarbonylmethyl-N,N,N-trimethylammonium chloride, .
N-(3-acrylamido-3,3-dimethylpropyl)-N,N,N-trimethylammonium , methosulfate~
1,2-dimethyl-5-vinylpyridinium methosul~ate, N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylammonium chloride, N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylamrnonlum sulfate, -N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium iodide, : :1 N-(2-methacryloyloxyethyl)-N~N,N-trlmethylammonium p-toluene- -sul~onate, N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium methosulfate, ,` . . '~ '~.
8~3 3-methyl-1-vinyl1mldazol1um methosul~te~
I N-(2-methzcryloyloxyethyl)-N~N~N-trlmethylammonium acetate, ¦ N-~-methacryloyloxyethyl)-N~N~N trlmethylammonlum bromide, N-(2-methacryloyloxyethyl)-N~N~N~tr~methylammonium chlorlde, N-(2-methacryloyloxyethyl)-N,N,N-trimethylamrnonium rluorlde, N-(2-methacryloyloxyethyl)-N~N,N-trimethylammonium nltrate, and N-~2-methacryloyloxyethyl)-N,N,N-trlmethylammonlum phosphate.
An example of one suitable polymeric morda~t Or khe type desc~ibed ls that copolymer identified hereinbefore as use~ul ln an element for the analysis of glucose ln liquids.
In addition to the use o~ mordants to ~ormulate the detectable species migration~inhlbitlng layer used ln the present lnvention, one can also employ as the migratlon-inhibiting material an antibody for the detectable species provided by the reagent layer. Such antibodies can be prepared by conventional immunological techniques and, of course, can vary widely depending on the partlcular material to be used as the detectable species in a given element of the invention.
Typically, such antibodies are immobillzed in the detectable species migration-inhibiting layer.
Exemplary elements of this invention includes those illustrated in the accompanying drawings. Ih Figure 1 is represented an analytical element composed of a reagent layer 12, a detectable species migration-inhibiting layer 14, a porous radiation-blocking layer 16, and, optionally, a spreading layer 18. A11 of these layers are in substantially continuous lntimate contact with their ad~acent layers. In an alternative embodiment of the invention, shown ln Figure 2, the analytical element is composed of a support 20 on which is coated a reagent layer 24, a detectable species migration-inhibiting layer 26, and a porous radiation-blocklng layer 28, which in this case serves al~o as a spreading layer. Optionally, either or both subbing and registration layers 22 may also be included in the `
. .
.
an~lytlcal element. All Or the~e layers are in substantially -continuous ln~lmate contact wlth thelr adJacent layers.
In the practlce o~ this inventlon, a sample of a llquid to be analyzed is placed on the outermost surface layer Or the element 9 which in the case Or the element lllus-trated in ~lgure 2 is the porous~ radlatlon-blocking, spreading layer 28. Any predetermined analyte present ln thls llquld di~uses through the porous, radlat~on-blocklng layer 28 and the ,, detectable species mlgration-lnhibiting layer 26, and enters the f,, reasent layer 24. There, lnteraction wlth the test reagents causes the release of or the formatlon Or a detectable specles such as a dye. This dye either remains ln place or in part mi~rates out of the reagent layer 24, lnto the detectable species mlgration inhibiting layer 26, and also into any porous, , radiation-transmlssive layers underlying the reagent layer. -All or most o~ the dye enterlng the detectable species mi~ration-inhibiting layer 26 ls fixed in place and prevented ~rom ~urther migrating into the overlylng porous radiation-blocking layer or layers 28. ~he reflective density of all dye in th~ detectable species migration-inhibiting layer 26, the reagent layer 24, and any other underlying radiation-transmissive layers is then determined while the element is still intact by measuring this density spectrophotometrically through all of these radiation-transmissive layers at the same tlme. ,l The following examples are provided to further ~¦
illustrate certain embodiments o~ the present invention.
Example 1 - Element For The Analysis 0~ Glucose Two elements ~or the analysis of glucose in llquids were prepared in the following manner:
-15- ;
Polyethylene t~r~phthalate fllm supports were coated wlth reagent layers comprlsln~ peroxidase at 10,200 U/m2, (the symbol U refers to internatlonal unlts~ whlch are the well known and generally accepted unlts of measurement of enzyme activity), glucos2 oxldase at 24g400 U/m2, 7 hydroxy-l-naphthol at 0.66 g/m , and 4-amlnoantipyrene hydrochloride l .
at o.86 g/m2. The rea6ent layer Or control sample 1 ~urther comprised deionized gelatin at 21.5 g/m . The reagent layer o~ sample 2 also comprised deionized gelatln, but at 19.4 g/m2.
The second sample was then coated with a detectable species l migration-inhibitlng layer, ln this case a dye migration- ¦
inhibiting layer comprising deionized gelatin at 2.1 g/m2 and the mordant, poly(styrene-co-N-vinylbenzyl-N,N-dimethyl-ben ylammonium chloride-co-divinyl benzene) (weight ratlo 49.5:49.5:1.0) at 1.08 g/m2. All gelatin-containing layers -were buffered at pH 6.o with a disodium phosphate-potassium ~
phosphate buffer. Both samples were then overcoated with ¦ .
a subbing layer comprising n-isopropylacrylamide at 0.32 g/m2 and a blushed-polymer, radiatlon-blocking, spreading layer l .
comprising cellulose acetate at 9.4 g/m and titanium dioxide ¦
at 64.5 g/m2.
The two resulting elements were then contacted at the outermost surface of their spreading layers with 10 ~1 samples of glucose standards contalning various concentrations of glucose. After 7 minutes of contact at 37C the reflection densities Or the dye rormed were measured spectrophotometrlcallY
using a photomultipller unit and a Wratten 65 rilter. The following Table I illustrates the resu.~.ts, the control sample .
being representative Or elements Or the prior art.
! !
1` .
.
.. ... ,,_. . `--.,, ~C
~C~
~ H
o o o o u~
U ~J ~D O ~D O
. ~ ~ ~Y; ~
CC ~ a ~ a~ ~ c ~ ~
C
.,~
.,, ~ .
.~ E c R O ta Q E~
,~ U~ ~ U~
,C ~ ~ ~ ~r o HH C`l ~¢ 1~ --~ ~ ~ ~J
51:1l ~ ~ ~ ~ O ;~
.:1 C: ~1 U~ Q~ 1 O O ~O~ ~ O
~ I~ ~C
U W
~: a ~ o ' o C aJ C::
~ o3 Q~
u~
1~ ~ C~l O O O O
~ ~ I N
~`
I
~ .
, :
.
Example 2 - Element For The Analysis or Calclum Two elements, one wlth and the other without a detectable species migration-inhlbiting layer contalning a mordant, were prepared according to the followlng:
A tereph~halate r~ lm support was coated with a reagent layer comprising gelatin (4.3 g/m2), Triton X-lOOTM (0.17 g/m2)~
chlorophosphonazo III (0.21 g/m2), bis(vinylsulfonylmethyl) ether (0.04 g/m2) and 0.1 M 3,3-dimethylglutaric acid, pH 5.4; a detectable species (dye) migration-inhibiting layer comprising gelatin (4.3 g/m2), and poly(styrene-co-N-vinylbenzyl-N,N-dimeth-ylbenzylammonium chloride-co-divinylbenzene) (2.15 g/m2); a sub-bing layer comprising (poly-N-isopropylacrylamide) (0.32 g/m2);
and a blushed-polymer, radiation-blocking spreading layer com-prising TiO2 (50.4 g/m2), cellulose acetate (7.0 g/m2) and Triton X-405TM ~1.4 g/m2). Triton X-lOOTM and Triton X-405TM are alkyl-pheno~y polyethoxy ethanols, commercially available ~rom the Rohm and Haas Company.
A second control element ~outside the scope Or the present invention) was prep~red in the sa~e manner except without a detectable species migration-lnhibiting layer between the spreadlng layer and reagent layer.
The elements were evaluated as in Example l, using calcium standards containing 1 to 5 mM of calcium and readlng the reflection densities at 670 nm. Table II s~ows ~he improved results obtained with the element containing the detectable species migration-inhiblting layer, in thls case a dye migration-inhibiting layer.
The results o~ Examples l and 2 above indicate that a signi~icantly ~igher dye density was consistently measured wlth ~he element containing a detectable species mlgratlon-lnhiblting layer. The control element, having no such layer, allowed signi~lcant amounts o~ the dye to mlgrate lnto the blushed~polymer~ radiatlon-blocking, spreadlng layer where lt coul~ not b~ det~cted.
- - -- --- - -~ c~c C C
a 1~ ' ~ o o I CO U~ O ~ ~ ~
c ~ ~ ~ m c E ~ a O o O O O O
h ~ ~ J
: Q~ ~1 m u~ Q~
:~
CQ~ C
~ ,1~
~ E_~
R O I~ ~ C~
,C Q~ I .,~
C ~ U~ U~ Q~
H H aa ~
O O ~ ~ ~ ~ ~
., ~ U ~ U~
~ ~ ~~ ~0 o o o o o o E~ :~ c U c : .
:~ ~ ~ ~ ,~
O -~ ~:
o c u E E ~ ~:
~1 0 . ~ .:
~ u c~ ~ o ,~
~ ...
~ u --L) C
~ O : `
~ , ~ ;
~ :~
Example 3 Example No. 2 was repeated, except that the ~eagent layer contained as a calcium indicator 0.48 G/M2 arsenazo III, rather than chorophosphonazo III. The reagent layer was buffered to a ph of 5.6. The resulting element demonstated a dye density comparable to that of the test element of example 2.
Claims (17)
1. An element for the analysis of liquids, said element comprising a radiation-transmissive reagent layer permeable to a predetermined analyte, which layer comprises a composition that is interactive in the presence of said analyte to provide a radiometrically detectable species, and a porous radiation-blocking layer permeable to said analyte;
characterized by a radiation-transmissive, detectable species migration-inhibiting layer interposed between the reagent layer and the porous radiation-blocking layer, said detectable species migration-inhibiting layer being permeable to said analyte and inhibiting the migration of said radiometrically detectable species to said porous radiation-blocking layer upon contact of said element with the liquid under analysis.
characterized by a radiation-transmissive, detectable species migration-inhibiting layer interposed between the reagent layer and the porous radiation-blocking layer, said detectable species migration-inhibiting layer being permeable to said analyte and inhibiting the migration of said radiometrically detectable species to said porous radiation-blocking layer upon contact of said element with the liquid under analysis.
2. An element as described in Claim 1 wherein said detectable species migration-inhibiting layer comprises an immobilized antibody for said radiometrically detectable spe-cies.
3. An element as described in Claim 1 wherein the detectable species is a dye upon contact of said element with the liquid under analysis.
4. An element as described in Claim 1 further com-prising a spreading layer permeable to said analyte and wherein the porous radiation-blocking layer, permeable to said analyte is interposed between said reagent layer and said spreading layer upon contact of said element with the liquid under analy-sis.
2. An element as described in Claim 1 wherein said detectable species migration inhibiting layer comprises an immobilized antibody for said radiometrically detectable spe-cies.
3, An element as described in Claim 1 wherein the detectable species is a dye upon contact of said element with the liquid under analysis.
4. An element as described in Claim 1 further com-prising a spreading layer permeable to said analyte and wherein the porous radiation-blocking layer, permeable to said analyte is interposed between said reagent layer and said spreading layer upon contact of said element with the liquid under analy-sis.
2. An element as described in Claim 1 wherein said detectable species migration inhibiting layer comprises an immobilized antibody for said radiometrically detectable spe-cies.
3, An element as described in Claim 1 wherein the detectable species is a dye upon contact of said element with the liquid under analysis.
4. An element as described in Claim 1 further com-prising a spreading layer permeable to said analyte and wherein the porous radiation-blocking layer, permeable to said Analyte is interposed between said reagent layer and said spreading layer upon contact of said element with the liquid under analy-sis.
5. An element for the analysis of liquids, said ele-ment comprising a radiation-transmissive support having thereon a radiation-transmissive reagent layer permeable to a predetermined analyte, which layer comprises a composition that is interactive in the presence of said analyte to provide a dye, and an outer-most radiation-blocking spreading layer permeable to said analyte upon contact of said element with the liquid under analysis.
characterized by a radiation-transmissive, dye migration-inhibiting layer interposed between the reagent layer and the radiation-blocking spreading layer, said dye migration-inhibiting layer being permeable to said analyte and inhibiting the migra-tion of said dye to said radiation-blocking spreading layer upon contact of said element with the liquid under analysis.
characterized by a radiation-transmissive, dye migration-inhibiting layer interposed between the reagent layer and the radiation-blocking spreading layer, said dye migration-inhibiting layer being permeable to said analyte and inhibiting the migra-tion of said dye to said radiation-blocking spreading layer upon contact of said element with the liquid under analysis.
6. An element as described in Claim 5 which further comprises at least one radiation-transmissive registration layer interposed between said reagent layer and said support.
7. An element as described in Claim 5 which further comprises a radiation-transmissive subbing layer interposed between said reagent layer and said support.
8. An element as described in Claim 5 wherein the dye migration-inhibiting layer comprises a mordant for said dye.
9. An element as described in Claim 5 wherein the dye migration-inhibiting layer comprises a mordant for the dye, said mordant having the structure wherein is or ;
each of R1, R2 and R3, which may be the same or different, is selected from alkyl, alkenyl, aralkyl, or aryl, each having less than eight carbon atoms;
R4 is a ballasting group having more than eight carbon atoms; and X- is an acid anion.
each of R1, R2 and R3, which may be the same or different, is selected from alkyl, alkenyl, aralkyl, or aryl, each having less than eight carbon atoms;
R4 is a ballasting group having more than eight carbon atoms; and X- is an acid anion.
10. An element as described in Claim 5 wherein the dye migration-inhibiting layer comprises a mordant for the dye, said mordant comprising a polymer having recurring units derived from 70 to about 98 weight percent of one or a mixture of hydro-phobic monomers and recurring units derived from about 2 to 30 weight percent of at least one cationic monomer conforming to the structure:
wherein L is a linking group between Q and the atoms in the chain of the polymer backbone;
n is 0 or 1;
X? is an acid anion; and Q? is a linear or heterocyclic ammonium, phosphonium, or sulfur-containing group having one of the following structures:
, , , , wherein is or ;
each of R1, R2, and R3, which may be the same or different, is selected from alkyl; alkenyl, aralkyl, or aryl, each having less than eight carbon atoms;
each of R5, R6, R7, and R8, which may be the same or different, represent H or R1 as defined above; and D is the atoms necessary to complete a heterocyclic ring.
wherein L is a linking group between Q and the atoms in the chain of the polymer backbone;
n is 0 or 1;
X? is an acid anion; and Q? is a linear or heterocyclic ammonium, phosphonium, or sulfur-containing group having one of the following structures:
, , , , wherein is or ;
each of R1, R2, and R3, which may be the same or different, is selected from alkyl; alkenyl, aralkyl, or aryl, each having less than eight carbon atoms;
each of R5, R6, R7, and R8, which may be the same or different, represent H or R1 as defined above; and D is the atoms necessary to complete a heterocyclic ring.
11. An element as described in Claim 5 wherein the dye migration-inhibiting layer comprises a hydrophilic
12. In an element for the analysis of liquids, said element comprising a radiation-transmissive support having thereon a radiation-transmissive reagent layer permeable to a predetermined analyte, which layer comprises a composition that is interactive in the presence of said analyte to provide a dye, and an outermost radiation-blocking spreading layer permeable to said analyte;
the improvement comprising a radiation-transmissive, dye migration-inhibiting layer interposed between the reagent layer and the radiation-blocking spreading layer, said dye migration-inhibiting layer being permeable to said analyte and comprising a hydrophilic colloid and a mordant for said dye.
the improvement comprising a radiation-transmissive, dye migration-inhibiting layer interposed between the reagent layer and the radiation-blocking spreading layer, said dye migration-inhibiting layer being permeable to said analyte and comprising a hydrophilic colloid and a mordant for said dye.
13. An element as described in Claim 12 wherein the radiation-blocking spreading layer comprises a blushed polymer and a pigment.
14. An element as described in Claim 12 wherein the reagent layer comprises a hydrophilic colloid having siad interactive composition distributed therein.
15. In an element for the analysis of liquids, said element comprising a radiation-transmissive support having thereon a radiation-transmissive reagnet layer permeable to a predetermined analyte, which layer comprises a hydrophilic colloid, said colloid having distributed therein a composition that is interactive in the presence of said analyte to provide a dye; and an outermost radiation-blocking spreading layer, permeable to said analyte, comprising a finely-divided particulate pigment and a blushed polymer;
the improvement comprising a radiation-transmissive dye migration-inhibiting layer, interposed between the reagent layer and the radiation-blocking spreading layer, said dye migration-inhibiting layer being permeable to said analyte and comprising a mordant for said dye and a hydrophilic colloid.
the improvement comprising a radiation-transmissive dye migration-inhibiting layer, interposed between the reagent layer and the radiation-blocking spreading layer, said dye migration-inhibiting layer being permeable to said analyte and comprising a mordant for said dye and a hydrophilic colloid.
16. An element as described in Claim 15 wherein the reagent layer is permeable to glucose and wherein the inter-active composition in the reagent layer comprises glucose oxidase, peroxidase, and an indicator composition comprising a compound oxidizable in the presence of hydrogen peroxide and peroxidase to effect formation of said dye.
17. An element as described in Claim 15 wherein the reagent layer is permeable to calcium and wherein the interactive composition in the reagent layer comprises an indicator for calcium.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US82298777A | 1977-08-08 | 1977-08-08 | |
| US822,987 | 1977-08-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1122889A true CA1122889A (en) | 1982-05-04 |
Family
ID=25237486
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA308,805A Expired CA1122889A (en) | 1977-08-08 | 1978-08-04 | Reduction of detectable species migration in elements for the analysis of liquids |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5429700A (en) |
| CA (1) | CA1122889A (en) |
| DE (1) | DE2834713C2 (en) |
| FR (1) | FR2400202A1 (en) |
| GB (1) | GB2002514B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5647761A (en) * | 1979-09-27 | 1981-04-30 | Fuji Photo Film Co Ltd | Laminated analyzing piece and immunity analyzing method using the same |
| US4303408A (en) * | 1980-02-05 | 1981-12-01 | Eastman Kodak Company | Removal of interferents in analytical assays in a two phase interferent-removal zone |
| JPS57142562A (en) * | 1981-02-27 | 1982-09-03 | Fuji Photo Film Co Ltd | Quantitative analysis film and colorimetric quantitative analysis |
| JPS57208997A (en) * | 1981-06-17 | 1982-12-22 | Fuji Photo Film Co Ltd | Liquid analyzing material for oxidase enzyme reaction system |
| US4390343A (en) * | 1981-07-06 | 1983-06-28 | Miles Laboratories, Inc. | Multilayer analytical element having an impermeable radiation diffusing and blocking layer |
| JPS5818167A (en) * | 1981-07-24 | 1983-02-02 | Fuji Photo Film Co Ltd | Analyzing film and analysis using said film |
| US4459358A (en) * | 1982-12-29 | 1984-07-10 | Polaroid Corporation | Multilayer element for analysis |
| JPS6014141A (en) * | 1983-07-06 | 1985-01-24 | Kyoto Daiichi Kagaku:Kk | Monolithically multilayered analyzing implement |
| JPS62138198A (en) * | 1985-12-10 | 1987-06-20 | Fuji Photo Film Co Ltd | Dry type liquid analysis element containing self-developing substrate |
| US5063081A (en) * | 1988-11-14 | 1991-11-05 | I-Stat Corporation | Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor |
| ES2118062T3 (en) * | 1989-12-15 | 1998-09-16 | Hoffmann La Roche | REACTIVE COMPOSITIONS, METHODS AND REAGENTS FOR THE QUANTITATIVE ASSESSMENT OF MAGNESIUM OR CALCIUM AND MAGNESIUM. |
| US7288414B2 (en) * | 2005-04-19 | 2007-10-30 | Specialty Assays, Inc. | Use of phosphonazo III for the measurement of calcium, magnesium and sodium in analytical samples |
| JP6191217B2 (en) * | 2013-04-24 | 2017-09-06 | 凸版印刷株式会社 | Oxygen indicator |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2280081A1 (en) * | 1974-07-23 | 1976-02-20 | Kodak Pathe | UNIT COMPOSITE PRODUCT FOR CHEMICAL OR BIOLOGICAL ANALYSIS |
| CA1095819A (en) * | 1977-01-14 | 1981-02-17 | Eastman Kodak Company | Element for analysis of liquids |
-
1978
- 1978-08-04 CA CA308,805A patent/CA1122889A/en not_active Expired
- 1978-08-08 FR FR7823308A patent/FR2400202A1/en active Granted
- 1978-08-08 JP JP9588778A patent/JPS5429700A/en active Granted
- 1978-08-08 DE DE19782834713 patent/DE2834713C2/en not_active Expired
- 1978-08-08 GB GB7832636A patent/GB2002514B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB2002514A (en) | 1979-02-21 |
| JPS6129460B2 (en) | 1986-07-07 |
| DE2834713A1 (en) | 1979-02-22 |
| GB2002514B (en) | 1982-01-20 |
| JPS5429700A (en) | 1979-03-05 |
| DE2834713C2 (en) | 1985-09-19 |
| FR2400202B1 (en) | 1980-08-29 |
| FR2400202A1 (en) | 1979-03-09 |
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