CA1114723A - Glucose detection system free from fluoride-ion interference - Google Patents
Glucose detection system free from fluoride-ion interferenceInfo
- Publication number
- CA1114723A CA1114723A CA307,795A CA307795A CA1114723A CA 1114723 A CA1114723 A CA 1114723A CA 307795 A CA307795 A CA 307795A CA 1114723 A CA1114723 A CA 1114723A
- Authority
- CA
- Canada
- Prior art keywords
- glucose
- reagent
- fluoride
- layer
- elements
- 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
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- 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
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
- Y10T436/144444—Glucose
Abstract
IMPROVED GLUCOSE DETECTION SYSTEM
FREE FROM FLUORIDE-ION INTERFERENCE
Abstract of the Disclosure An improved multilayer element for detecting glucose in samples of biological fluid which may contain with fluoride ion as a contaminant or preservative is described. The improved element comprises a buffered Trinder's reagent, which reagent comprises glucose oxidase, peroxidase, 4-aminoantipyrine and a phenolic or naphtholic coupler, which is buffered to a pH between about 4.5 and about 6Ø Such a buffering reduces any interference which may be due to fluoride ion present in the sample under analysis.
FREE FROM FLUORIDE-ION INTERFERENCE
Abstract of the Disclosure An improved multilayer element for detecting glucose in samples of biological fluid which may contain with fluoride ion as a contaminant or preservative is described. The improved element comprises a buffered Trinder's reagent, which reagent comprises glucose oxidase, peroxidase, 4-aminoantipyrine and a phenolic or naphtholic coupler, which is buffered to a pH between about 4.5 and about 6Ø Such a buffering reduces any interference which may be due to fluoride ion present in the sample under analysis.
Description
The glucose oxldase method Or assaying ~or glucose used by Trinder ls well-known ln the art. Such method uses reagents, which comprise glucose oxidase, a substance havlng peroxidase activity, 4-aminoantipyrine and a phenollc cr naphtholic coupler. The method and reagents are descrlbed ln detail in: Trinder, P., Ann. Clln. Biochem., 6 (1969), 24~ and Trinder, P., J. Clin. Pathol., 22 (1969), 246.
Sod~um rluoride is used as a preservative in blood and blood serum samples whlch are to be held for an extended period Or time, i.e. up to ten days (Henry, Clinical Chemistry,
Sod~um rluoride is used as a preservative in blood and blood serum samples whlch are to be held for an extended period Or time, i.e. up to ten days (Henry, Clinical Chemistry,
2 Ed. p. 385). Such a preservative is routlnely used at a level of 250 mg/dl Or blood or blood serum. The addition of 250 mg/dl Or sodium fluoride to blood serum has been found to decrease the apparent glucose concentratlon, as measured -utllizing multilayer elements Or the type described ln U.S. Patent No. 3,992,158 by 20-30 mg/dl at a concentration Or 200 mg glucose/dl. ~his problem is compounded by the following factors: 1) the preservative 2G is not always used and 2) when the preservative is incorporated within blood withdrawal apparatus such as ~ound ~n commerclal ; embodlments, the concentration Or preservatlve is ~nown only when the container is ~llled to capaclty. Since the containers often are not filled to capacity (owing to loss of vacuum) the sodlum rluoride concentratlon may increase as much as 2-
3 fold over the 250 mg/dl level.
In view of the variability in sodlum ~luoride con-centration which may be encountered, it is clear that a need -exlsts for a multllayer element ror the analysls of glucose whlch exhibits reduced sensitlvity to ~odlum rluoride.
,. -1- ~k -, '. ', ." ' ' : : .'':,, , . . ' . ~, . ' ., 2~
The concentratlons of fluorlde lon used for this purpose have apparently not caused noticeable lnterference with prior-art methods ror analyzlng for glucose. ~his is probably due to dllution technlques commonly used ln prlor art solution quantitative assays and the seml-quantltative character Or prior art "dry" methods ~or glucose detection.
It has been foun~ however, ~hat, when a Trinder's reagent system as described above is lncorporated lnto a multllayer element of the type described ln U.S. Patent 3,992,1~8, the fluorlde lon used as a preservative contributes a substantlal negative bias. The presence and elimlnatlon or reduction Or this bias uslng the method described herein wlll be shown in the examples below.
U.S. Patent 3,992,158 descrlbes integral multilayer elements for the detection o~ varlous biological materials, lncluding glucose. Elements the type described therein are identical to those useful ln the successful practice of the preferred embodlments hereof, and the details for preparlng such elements are readily determinable therefrom.
Ex~mple 3 Or U.S. Patent 3,992,158 descrlbes an element for the detection of glucose uslng glucose oxldase, peroxidase and an lndlcator system of the type descrlbed herein in an element slmilar to that descrlbed ln the lnstant appllcatlon. However, no pH ls speclfied for the reagent -~ ' ~ ' , . :-.
layer containing the foregoing reagent system. A layer prepared accordlng to this example would have a pH Or above about 7Ø
U.S. Patent 3,983,005 de~cribes ln Example 3 the use of 4-aminoantipyrine and a naphthollc coupler ror the detection o~ hydrogen peroxide produced on contact with cholesterol oxidase ln an element of the type described herein. Accordlng to this example, the indicator reaction is carried out at a pH of 7Ø ;
~.S. Patent No. 3,~86,045 describes a test composltion for glucose assay comprlslng glucose oxidase, peroxldase, sodium or potassium ferrocyanide, an aminoantipyrine and a phenollc coupler. It is disclosed that such compositions,always lncluding the ferrocyanide, are useful when burrered at a pH of between about 5.5 and 8.o. There is no disclosure that compositions which omlt the ferrocyanide are useful throughout this pH range. This patent also includes an extensive discussion Or phenols useful ln analytical reagents of the type descrlbed hereln.
It has now been found that the aforementioned 6 interference of fluoride ion with glucose analyses perrormed ~ :
ln integral multilayer elements Or the type described in U.S. Patent No. 3,992,158 can be reduced or ellminated by bu~ferlng the reagent composition contained therein to a pH
between about 4.5 and 6Ø Although the mechanism ror this reduction or elimination Or ~luoride interference at this pH
- . -3-~. .
~.
': .
,. : ' 1$'~
is not completely understood, perrormance Or the assay reactlons within this pH range provldes the desired reduction or eliminatlon-Trinder's reagent for the detectlon Or glucose, which comprlses glucose oxldaseJ a substance havlng pero~ldatlve actlvlty, 4-amlnoantlpyrlne and a phenollc coupler, is well known ln the art and ls descrlbed ln detail ln the two Trinder publications rererred to above.
The detection Or glucose using Trlnder's reagent 10 is conventlonally carrled out at a pH of 7.0 or above.
However, at thls pH, fluorlde ion, whlch is used as a preservative ~or serum samples, acts as an lnterrerent when thls reagent is used ln an element Or the type described in U.S. Patent 3,992,158. Although the cause of thls phenomenon is not understood, we have discovered that the interference due to fluoride lon ln such multilayer elements can be reduced or ellmlnated by burfering the Trinder's - ~.
reagent system to a pH of between about 4~5 and 6Ø
Thus, the reagent composltions contalned in the 20 elements Or the lnstant invention comprlse Trinder's reagent . -or some modification thereof use~ul in the detection of glucose in an aqueous medium bu~ered to a pH of between about 4.5 and about 6.o.
Glucose oxidase and lts sources and preparation -~
are well known in the art and no ~urther discusslon thereor is neCessarY here. r 1, :
2~
Substances having peroxldative act~vlty ~hlch are useful in cllnical applications have been discussed at length in the prior art and are well known to the 6kllled artisan. One such discusslon is presented ln U.S.
Patent 3,884,764 at column 6, line 52, to column 7, llne 18. A prererred substance o~
this type is, of course, peroxidase, which has been used broadly in clinical analytical appllcations.
Aminoantipyrine, also ldentlfied as amlnophenazone, is an oxygen acceptor which, ln its oxldized state, couples with-a phenolic coupler. In the dry elements descrlbed herein, aminoa~tlpyrine is generally supplled ln the rorm Or an acid salt, ~or example, the hydrochlorlde salt. A discussion ~r aminoantipyrine ls presented in V.S. Patent No. 3,886,o45.
A preferred aminoantipyrlne ls 4-amlnoantlpyrlne.
A detailed discusslon o~ phenollc couplers includlng examples Or useful phenolic couplers ls presented by Mees, C. E. X., and James, T. H., The TheorY of the Photographlc Process, . . _ , .
Thlrd Ed., Macmillan Co., N.Y. (1966), p. 387fr and in the aforementioned U.S. Patent No. 3,886,o45. As ls apparent from the dlscusslons, phenollc couplers include both phenols and naphthols, both o~ which may be substltuted or unsubstltuted.
; Specifically, pre~erred from among such couplers is 7-hydroxy-l-naphthol. Thls coupler demonstrates outstandlng stabillty and reactivlty and provldes an indicator dye which absorbs at a convenient wavelength ror spectrophotometric measurement in the ~ormat indicated.
In order to provide an appropriate pH for the reaction to be carried out, a suitable bu~er must be lncluded . . .
, ` .
~L4 ~",f~i~
ln the reagent mixture. ~irtually any burrer that (1) buffers at a pH Or between about 4.5 and 6.o, (2) ls compatlble wlth the reagent co~posltlon andt3) ls sultable ~or lncorporatlon ln the elements descrlbed hereln according to the methods described ln arorementloned V.S. Patent 3,992,158 ls useful in the successful practlce of the lnstant lnvention. Such bufrers are described generally by Good, Biochemistry, ~, 467 (1966). Buffers that have been found useful lnclude dlmethylglutaric acids, succinic acld, mallc acid, potasslum acid phthalate and mixed phosphate-cltrate buffers. Speclfically preferred from among thls group is 3,3-dimethylglutarlc acid.
As used hereln, the term "integral element" refers to elements having at least two superposed layers, deslrably dlscrete, ln intimate contact. Preferably, such elements are formed prior to applicatlon of a llquid sample ~or analysis. Elements of thls lnvention are capable or perrorming internally a variety of sample-handling functions. They do not require expertise ln their use and can produce quantitative analytical results without the specialized spotting or other procedures, e.g. sample confinement, washing or removal o~ excess sample, typically needed for analyses made using ~nown elements. ~urther, the results ;~
produced by elements of thls lnvention are substantially ~ -conslstent and free from lnternal variatlons so that automated means of measurlng electromagnetlc radiation (radlometric technlques) can be used to detect such results with minlmal lnconsistency.
Stated more partlcularly, according to a preferred 3~ embodlment, tbe present lnventlon provldes lntegral analytlcal .
. , ~' ,, ' . .
elements for the detectlon of glucose, which elements are composed Or multiple, superposed layers whlch can provlde quickly within the element a hlghly quantltatlve dye productlon in response to the presence of fluoride-contalnlng solutions Or glucose applied to the element. Elements of thls lnventlon include a sample spreadlng layer ln fluld contact wlth a reagent l~yer. The sample spreading layer ls capable of dlstrlbutlng wlthln the layer glucose contalned ln a liquid sample applied to the element -to provide, at any given tlme, a unlrorm concentration of glucose at the surface Or the spreadlng layer raclng, l.e., closer to, the reagent layer. In varlous preferred embodiments, the spreading layer is isotropically porous; that is, it is porous in every direction within the layer. ~ererence herein to isotropic porosity identifies the fact Or substantial porosity in all directions withln the spreading layer. It ; will be understood that the degree Or such porosity may be variable, lr necessary or desirable, for example, regarding pore size, percentage Or vold volume or otherwlse. It shall be understood that the term "lsotropic poroslty" (or "isotroplcally porous") as used herein should not be con~used with the terms "isoporous" or "ionotroplc", o~ten used wlth reference to filter membranes to slgnlfy those membranes having pores that are continuous between membrane sur~aces. Likewlse, "lsotropic porosity" should not be conrused with the term "isotropic", used in contradistinction to the term "anisotropic", which signifies rilter membranes having a thln "skin" along at least onc sur~ace Or tùe membrane. See, ror example, :
. ' ~
~.
.
13L14'7~
Membrane Sclence and ~echnology, James Fllnn ed, Plenum Press, New York (1970).
The reagent layer ls a layer underlylng the spreadlng layer and containing the reagent compositions described herein, namely,glucose oxidase, a substance havlng peroxidative activity, amlnoantipyrine, a phenolic coupler and a burfer to maintain the pH of the layer between about 4.5 and 6.o when contacted with a test sample. 'l'he reagent layer is preferably of substantially uniform permeability to glucose contained in aqueous liquid applied to the spreadlng layer.
Uni~orm permeability Or the layer re~ers to permeability such that, when a homogeneous glucose solution ls provided uniformly to a surface of the layer, identical measurements of the co~centration Or glucose ln the solutlon within the layer, but made through different regions of a surface of the layer, will yield substantially equal results.
Reference herein to fluid contact between a spreading layer and a reagent layer ln an integral analytical element identi~ies the ability o~ a fluid, whether liquid or gaseous, to pass in such element between superposed regions of the spreading layer and the reagent layer. Stated in another manner, fluid contact re~ers to the ability to transport components of a fluid between the layers in fluid contact. Although such layers in fluld contact can be contiguous, they may also be separated by lntervening layers.
However, layers in the element that physically intervene a spreading layer and reagent layer ln mutual fluid contact wlll also be in fluid contact and will not prevent the passage of fluld between the fluid-contacting spreading and reagent la~ers.
:- : .. .
2~ :
The rollowing examples Or lntegral elements are provided to lllustrate further the practlce of the inventlon.
Examples 1-4: Reduced Fluoride Interference at Lower pH
Fvacuated blood containers, containlng either sodium fluorlde/oxalate or sodlum rluoride/EDTA as preservative, were partially or totally filled with blood samples; hence,the actual concentration of ~luoride ion present varied with each sample.
If the contalner was tota~y filled wlth a blood sample (l.e.g 7 cc), ;the amount of F present would be 250 mg/dl, if halr ~llled, 500 mg/dl. The glucose level of each sample was brought to ~350 mg/dl by spiking. The samples were then spotted (ln 10 ~1 allquots) onto test elements having the ~ollowing compositlon and which were prepared accordlng to the methods described in V.S. Patent No. 3,992,158.
Polyethylene terephthalate supports were coated with reagent layers comprislng deionized gelatin (21.5 g/m2), peroxidase (10,000 U/m2) glucose oxidase (24,400 U/m2), 4-aminoantlpyrine-HCl (0.86 g/m2), 7-hydroxy-1-naphthol (0.66 g/m2) and 3,3-dimethylglutaric acid (1.96 g/m ) (pH varied with each element as shown in Table 1). A
subblng layer comprising poly-n-isopropylacrylamlde and a :
In view of the variability in sodlum ~luoride con-centration which may be encountered, it is clear that a need -exlsts for a multllayer element ror the analysls of glucose whlch exhibits reduced sensitlvity to ~odlum rluoride.
,. -1- ~k -, '. ', ." ' ' : : .'':,, , . . ' . ~, . ' ., 2~
The concentratlons of fluorlde lon used for this purpose have apparently not caused noticeable lnterference with prior-art methods ror analyzlng for glucose. ~his is probably due to dllution technlques commonly used ln prlor art solution quantitative assays and the seml-quantltative character Or prior art "dry" methods ~or glucose detection.
It has been foun~ however, ~hat, when a Trinder's reagent system as described above is lncorporated lnto a multllayer element of the type described ln U.S. Patent 3,992,1~8, the fluorlde lon used as a preservative contributes a substantlal negative bias. The presence and elimlnatlon or reduction Or this bias uslng the method described herein wlll be shown in the examples below.
U.S. Patent 3,992,158 descrlbes integral multilayer elements for the detection o~ varlous biological materials, lncluding glucose. Elements the type described therein are identical to those useful ln the successful practice of the preferred embodlments hereof, and the details for preparlng such elements are readily determinable therefrom.
Ex~mple 3 Or U.S. Patent 3,992,158 descrlbes an element for the detection of glucose uslng glucose oxldase, peroxidase and an lndlcator system of the type descrlbed herein in an element slmilar to that descrlbed ln the lnstant appllcatlon. However, no pH ls speclfied for the reagent -~ ' ~ ' , . :-.
layer containing the foregoing reagent system. A layer prepared accordlng to this example would have a pH Or above about 7Ø
U.S. Patent 3,983,005 de~cribes ln Example 3 the use of 4-aminoantipyrine and a naphthollc coupler ror the detection o~ hydrogen peroxide produced on contact with cholesterol oxidase ln an element of the type described herein. Accordlng to this example, the indicator reaction is carried out at a pH of 7Ø ;
~.S. Patent No. 3,~86,045 describes a test composltion for glucose assay comprlslng glucose oxidase, peroxldase, sodium or potassium ferrocyanide, an aminoantipyrine and a phenollc coupler. It is disclosed that such compositions,always lncluding the ferrocyanide, are useful when burrered at a pH of between about 5.5 and 8.o. There is no disclosure that compositions which omlt the ferrocyanide are useful throughout this pH range. This patent also includes an extensive discussion Or phenols useful ln analytical reagents of the type descrlbed hereln.
It has now been found that the aforementioned 6 interference of fluoride ion with glucose analyses perrormed ~ :
ln integral multilayer elements Or the type described in U.S. Patent No. 3,992,158 can be reduced or ellminated by bu~ferlng the reagent composition contained therein to a pH
between about 4.5 and 6Ø Although the mechanism ror this reduction or elimination Or ~luoride interference at this pH
- . -3-~. .
~.
': .
,. : ' 1$'~
is not completely understood, perrormance Or the assay reactlons within this pH range provldes the desired reduction or eliminatlon-Trinder's reagent for the detectlon Or glucose, which comprlses glucose oxldaseJ a substance havlng pero~ldatlve actlvlty, 4-amlnoantlpyrlne and a phenollc coupler, is well known ln the art and ls descrlbed ln detail ln the two Trinder publications rererred to above.
The detection Or glucose using Trlnder's reagent 10 is conventlonally carrled out at a pH of 7.0 or above.
However, at thls pH, fluorlde ion, whlch is used as a preservative ~or serum samples, acts as an lnterrerent when thls reagent is used ln an element Or the type described in U.S. Patent 3,992,158. Although the cause of thls phenomenon is not understood, we have discovered that the interference due to fluoride lon ln such multilayer elements can be reduced or ellmlnated by burfering the Trinder's - ~.
reagent system to a pH of between about 4~5 and 6Ø
Thus, the reagent composltions contalned in the 20 elements Or the lnstant invention comprlse Trinder's reagent . -or some modification thereof use~ul in the detection of glucose in an aqueous medium bu~ered to a pH of between about 4.5 and about 6.o.
Glucose oxidase and lts sources and preparation -~
are well known in the art and no ~urther discusslon thereor is neCessarY here. r 1, :
2~
Substances having peroxldative act~vlty ~hlch are useful in cllnical applications have been discussed at length in the prior art and are well known to the 6kllled artisan. One such discusslon is presented ln U.S.
Patent 3,884,764 at column 6, line 52, to column 7, llne 18. A prererred substance o~
this type is, of course, peroxidase, which has been used broadly in clinical analytical appllcations.
Aminoantipyrine, also ldentlfied as amlnophenazone, is an oxygen acceptor which, ln its oxldized state, couples with-a phenolic coupler. In the dry elements descrlbed herein, aminoa~tlpyrine is generally supplled ln the rorm Or an acid salt, ~or example, the hydrochlorlde salt. A discussion ~r aminoantipyrine ls presented in V.S. Patent No. 3,886,o45.
A preferred aminoantipyrlne ls 4-amlnoantlpyrlne.
A detailed discusslon o~ phenollc couplers includlng examples Or useful phenolic couplers ls presented by Mees, C. E. X., and James, T. H., The TheorY of the Photographlc Process, . . _ , .
Thlrd Ed., Macmillan Co., N.Y. (1966), p. 387fr and in the aforementioned U.S. Patent No. 3,886,o45. As ls apparent from the dlscusslons, phenollc couplers include both phenols and naphthols, both o~ which may be substltuted or unsubstltuted.
; Specifically, pre~erred from among such couplers is 7-hydroxy-l-naphthol. Thls coupler demonstrates outstandlng stabillty and reactivlty and provldes an indicator dye which absorbs at a convenient wavelength ror spectrophotometric measurement in the ~ormat indicated.
In order to provide an appropriate pH for the reaction to be carried out, a suitable bu~er must be lncluded . . .
, ` .
~L4 ~",f~i~
ln the reagent mixture. ~irtually any burrer that (1) buffers at a pH Or between about 4.5 and 6.o, (2) ls compatlble wlth the reagent co~posltlon andt3) ls sultable ~or lncorporatlon ln the elements descrlbed hereln according to the methods described ln arorementloned V.S. Patent 3,992,158 ls useful in the successful practlce of the lnstant lnvention. Such bufrers are described generally by Good, Biochemistry, ~, 467 (1966). Buffers that have been found useful lnclude dlmethylglutaric acids, succinic acld, mallc acid, potasslum acid phthalate and mixed phosphate-cltrate buffers. Speclfically preferred from among thls group is 3,3-dimethylglutarlc acid.
As used hereln, the term "integral element" refers to elements having at least two superposed layers, deslrably dlscrete, ln intimate contact. Preferably, such elements are formed prior to applicatlon of a llquid sample ~or analysis. Elements of thls lnvention are capable or perrorming internally a variety of sample-handling functions. They do not require expertise ln their use and can produce quantitative analytical results without the specialized spotting or other procedures, e.g. sample confinement, washing or removal o~ excess sample, typically needed for analyses made using ~nown elements. ~urther, the results ;~
produced by elements of thls lnvention are substantially ~ -conslstent and free from lnternal variatlons so that automated means of measurlng electromagnetlc radiation (radlometric technlques) can be used to detect such results with minlmal lnconsistency.
Stated more partlcularly, according to a preferred 3~ embodlment, tbe present lnventlon provldes lntegral analytlcal .
. , ~' ,, ' . .
elements for the detectlon of glucose, which elements are composed Or multiple, superposed layers whlch can provlde quickly within the element a hlghly quantltatlve dye productlon in response to the presence of fluoride-contalnlng solutions Or glucose applied to the element. Elements of thls lnventlon include a sample spreadlng layer ln fluld contact wlth a reagent l~yer. The sample spreading layer ls capable of dlstrlbutlng wlthln the layer glucose contalned ln a liquid sample applied to the element -to provide, at any given tlme, a unlrorm concentration of glucose at the surface Or the spreadlng layer raclng, l.e., closer to, the reagent layer. In varlous preferred embodiments, the spreading layer is isotropically porous; that is, it is porous in every direction within the layer. ~ererence herein to isotropic porosity identifies the fact Or substantial porosity in all directions withln the spreading layer. It ; will be understood that the degree Or such porosity may be variable, lr necessary or desirable, for example, regarding pore size, percentage Or vold volume or otherwlse. It shall be understood that the term "lsotropic poroslty" (or "isotroplcally porous") as used herein should not be con~used with the terms "isoporous" or "ionotroplc", o~ten used wlth reference to filter membranes to slgnlfy those membranes having pores that are continuous between membrane sur~aces. Likewlse, "lsotropic porosity" should not be conrused with the term "isotropic", used in contradistinction to the term "anisotropic", which signifies rilter membranes having a thln "skin" along at least onc sur~ace Or tùe membrane. See, ror example, :
. ' ~
~.
.
13L14'7~
Membrane Sclence and ~echnology, James Fllnn ed, Plenum Press, New York (1970).
The reagent layer ls a layer underlylng the spreadlng layer and containing the reagent compositions described herein, namely,glucose oxidase, a substance havlng peroxidative activity, amlnoantipyrine, a phenolic coupler and a burfer to maintain the pH of the layer between about 4.5 and 6.o when contacted with a test sample. 'l'he reagent layer is preferably of substantially uniform permeability to glucose contained in aqueous liquid applied to the spreadlng layer.
Uni~orm permeability Or the layer re~ers to permeability such that, when a homogeneous glucose solution ls provided uniformly to a surface of the layer, identical measurements of the co~centration Or glucose ln the solutlon within the layer, but made through different regions of a surface of the layer, will yield substantially equal results.
Reference herein to fluid contact between a spreading layer and a reagent layer ln an integral analytical element identi~ies the ability o~ a fluid, whether liquid or gaseous, to pass in such element between superposed regions of the spreading layer and the reagent layer. Stated in another manner, fluid contact re~ers to the ability to transport components of a fluid between the layers in fluid contact. Although such layers in fluld contact can be contiguous, they may also be separated by lntervening layers.
However, layers in the element that physically intervene a spreading layer and reagent layer ln mutual fluid contact wlll also be in fluid contact and will not prevent the passage of fluld between the fluid-contacting spreading and reagent la~ers.
:- : .. .
2~ :
The rollowing examples Or lntegral elements are provided to lllustrate further the practlce of the inventlon.
Examples 1-4: Reduced Fluoride Interference at Lower pH
Fvacuated blood containers, containlng either sodium fluorlde/oxalate or sodlum rluoride/EDTA as preservative, were partially or totally filled with blood samples; hence,the actual concentration of ~luoride ion present varied with each sample.
If the contalner was tota~y filled wlth a blood sample (l.e.g 7 cc), ;the amount of F present would be 250 mg/dl, if halr ~llled, 500 mg/dl. The glucose level of each sample was brought to ~350 mg/dl by spiking. The samples were then spotted (ln 10 ~1 allquots) onto test elements having the ~ollowing compositlon and which were prepared accordlng to the methods described in V.S. Patent No. 3,992,158.
Polyethylene terephthalate supports were coated with reagent layers comprislng deionized gelatin (21.5 g/m2), peroxidase (10,000 U/m2) glucose oxidase (24,400 U/m2), 4-aminoantlpyrine-HCl (0.86 g/m2), 7-hydroxy-1-naphthol (0.66 g/m2) and 3,3-dimethylglutaric acid (1.96 g/m ) (pH varied with each element as shown in Table 1). A
subblng layer comprising poly-n-isopropylacrylamlde and a :
4 ~ ~
spreading layer comprlslng cellulose acetate (6.6 g~m2) and TiO2 (46 g/m2) were then applled.
Arter 7 min. at 37~C., the reflectlon densltles produced were measured and a callbratlon curve Or reflectlon density vs. glucose concentratlon plotted ror each element. The rerlection densities obtained were then checked agalnst the derived calibration curves ànd the corresponding apparent glucose concentration was obtained. The difrerence between the glucose concentratlon Or the callbratlon curves and the glucose concentration as determined according to a conventional rererence method is shown in Table 1.
.
: . .,.:
.
,: ~ . -- ~
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0 1 0 1~ N
td ~:q 1*.1 J
E-~ ~
a C c~ o o o ~ Q~
L~ ~t ~ ~ 3 C ~ J~:J
~O ~ ~t~r _~ a~
a: .
O
~( ~:: 1~ ~ E
o E
0 ~ ....
~o ~ ~ o o C ~ ~ Lr~
r~
D
O
....
~1 ~ _I u~co m I ~ ~
~I ~ ~ ~
a~ 0 ~I _~
D 3 J~ a~
u~ ~ ~ oooo x c~
~ o s~ ~ ~ ~ ~u C) ~ Q) U~
~: a~ ~ ~ ~ ~ r~
~ _l ~ .
a~ ~ ~1 ~ 3 a ~1 .
1~ E E
~ --t ~ cr~
_I
~ M C~ II~C~
O ~ a ~
3 D ~ ~ ~.
~ :~ ~ . ' ' .
' :~ 1 o ~
. . .
-'::
.
O
Z : ::
O
_~ ~ )J
~ . ,~.
-: ' ' .. .. - .
. : ' ' : ' ' ' ' ~
.
.
.
Example 5: Effect Or ~H and Fluorlde Interrerence on Rate Or Develo~ment Elements prepared ln the same manner as ln Examples 1-4, also varying ln pH (5.0 and 7.0)~ were evaluated ln the manner described in Examples 1-4, using serum containlng no fluoride ion, plasma contalning 2.5 mg/dl fluoride/EDTA and plasma contalning ~5.0 mg/dl rluorlde/EDTA.
The rates of dye development (DR) vs. tlme Or the elements were compared in Figs. 1 and 2. The rates for each sample were much slower at pH 7.0, while at pH 5.0 they di~fered only in the early stages Or dye development.
Examples 6~ Effect of Serum_pH Upon Element Res~onse A series Or elements was prepared as in Examples 1-4, but using three dirferent burfer systems: dimethylglutarate (1.96 g/m2), succinlc acld (1.40 g/m2) or mallc acld (1.58 - g/m2) (pH is indicated in Table 2).
Two samples Or sera containing 100 mg/dl and 400 mg/dl glucose were each adJusted to a pH of 7.7 and 8.6.
The samples were then spotted onto the test elements and evaluated as in Example 1. Results shown ln Table 2 suggest that the elements, as prepared, have adequate bu~fering capacity and can tolerate fluctuations that may occur ln the pH Or serum.
.
.- .., . -' - !
- . .. .-' a~ l ~ o u~
~ O ~1 0 _I
.~ 1''' . .
bO Cl:l C~ C~ ) 3 E 11 ......
O G ~ ~1~ _I ~1 ~1 C .
V~ ~ ~D ~ ~ac~
~D U~ : ~ ~ ~1 ll N N N N N N
Q
QJ
C~
c a~;¦ o ~u~
U~ 00_100~1 P: . 1~++
D ~1 a~ ~
3 ~ ~D
~: bi~ o~ N O O ~ N Lr~
~ O E co o~ o c~ u~
t~ tr) ~3 Q) ::~ oo :~: ......
D :~ ~1 E-~ E 6 ~ _~
.: ~ ~ ~ ~.
Cq ~1 t-- N ~11~ ~ O~ ~1 a~ ~ o ~ Ir~ ' -~)~ t~
O ......
X
C~
C~
C~ t) a~ c~
_~ td ~ : :
C~ ~
m c~
E Cl 0 0 6 E
:~ I o U~ o U~ o U~
a~ . :
E ~ o ,1 K _I _I . - .
.
.. .
:
spreading layer comprlslng cellulose acetate (6.6 g~m2) and TiO2 (46 g/m2) were then applled.
Arter 7 min. at 37~C., the reflectlon densltles produced were measured and a callbratlon curve Or reflectlon density vs. glucose concentratlon plotted ror each element. The rerlection densities obtained were then checked agalnst the derived calibration curves ànd the corresponding apparent glucose concentration was obtained. The difrerence between the glucose concentratlon Or the callbratlon curves and the glucose concentration as determined according to a conventional rererence method is shown in Table 1.
.
: . .,.:
.
,: ~ . -- ~
~4'~
0 1 0 1~ N
td ~:q 1*.1 J
E-~ ~
a C c~ o o o ~ Q~
L~ ~t ~ ~ 3 C ~ J~:J
~O ~ ~t~r _~ a~
a: .
O
~( ~:: 1~ ~ E
o E
0 ~ ....
~o ~ ~ o o C ~ ~ Lr~
r~
D
O
....
~1 ~ _I u~co m I ~ ~
~I ~ ~ ~
a~ 0 ~I _~
D 3 J~ a~
u~ ~ ~ oooo x c~
~ o s~ ~ ~ ~ ~u C) ~ Q) U~
~: a~ ~ ~ ~ ~ r~
~ _l ~ .
a~ ~ ~1 ~ 3 a ~1 .
1~ E E
~ --t ~ cr~
_I
~ M C~ II~C~
O ~ a ~
3 D ~ ~ ~.
~ :~ ~ . ' ' .
' :~ 1 o ~
. . .
-'::
.
O
Z : ::
O
_~ ~ )J
~ . ,~.
-: ' ' .. .. - .
. : ' ' : ' ' ' ' ~
.
.
.
Example 5: Effect Or ~H and Fluorlde Interrerence on Rate Or Develo~ment Elements prepared ln the same manner as ln Examples 1-4, also varying ln pH (5.0 and 7.0)~ were evaluated ln the manner described in Examples 1-4, using serum containlng no fluoride ion, plasma contalning 2.5 mg/dl fluoride/EDTA and plasma contalning ~5.0 mg/dl rluorlde/EDTA.
The rates of dye development (DR) vs. tlme Or the elements were compared in Figs. 1 and 2. The rates for each sample were much slower at pH 7.0, while at pH 5.0 they di~fered only in the early stages Or dye development.
Examples 6~ Effect of Serum_pH Upon Element Res~onse A series Or elements was prepared as in Examples 1-4, but using three dirferent burfer systems: dimethylglutarate (1.96 g/m2), succinlc acld (1.40 g/m2) or mallc acld (1.58 - g/m2) (pH is indicated in Table 2).
Two samples Or sera containing 100 mg/dl and 400 mg/dl glucose were each adJusted to a pH of 7.7 and 8.6.
The samples were then spotted onto the test elements and evaluated as in Example 1. Results shown ln Table 2 suggest that the elements, as prepared, have adequate bu~fering capacity and can tolerate fluctuations that may occur ln the pH Or serum.
.
.- .., . -' - !
- . .. .-' a~ l ~ o u~
~ O ~1 0 _I
.~ 1''' . .
bO Cl:l C~ C~ ) 3 E 11 ......
O G ~ ~1~ _I ~1 ~1 C .
V~ ~ ~D ~ ~ac~
~D U~ : ~ ~ ~1 ll N N N N N N
Q
QJ
C~
c a~;¦ o ~u~
U~ 00_100~1 P: . 1~++
D ~1 a~ ~
3 ~ ~D
~: bi~ o~ N O O ~ N Lr~
~ O E co o~ o c~ u~
t~ tr) ~3 Q) ::~ oo :~: ......
D :~ ~1 E-~ E 6 ~ _~
.: ~ ~ ~ ~.
Cq ~1 t-- N ~11~ ~ O~ ~1 a~ ~ o ~ Ir~ ' -~)~ t~
O ......
X
C~
C~
C~ t) a~ c~
_~ td ~ : :
C~ ~
m c~
E Cl 0 0 6 E
:~ I o U~ o U~ o U~
a~ . :
E ~ o ,1 K _I _I . - .
.
.. .
:
Claims (4)
1. An integral element for the determination of glu-cose in aqueous liquids, said element comprising a spreading layer and a reagent layer in fluid contact under conditions of use and containing Trinder's reagent as the glucose-determining reagent composition, Trinder's reagent comprising a substance having peroxidase activity, 4-aminoantipyrine and a phenolic or naphtholic coupler, said element characterized by the inclusion of a buffer which maintains the pH of the reagent composition between about 4.5 and 6.0 under conditions of use.
2. The element of Claim 1 wherein said substance having peroxidative activity is peroxidase.
3. The element of Claim 1 wherein said coupler is 7-hydroxy-1-naphthol.
4. The element of Claim 1 wherein the buffer is selected from the group consisting of dimethylglutaric acid, succinic acid, potassium acid phthalate, malic acid and mixed phosphate-citrate buffer.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US821,028 | 1977-08-01 | ||
US05/821,028 US4098574A (en) | 1977-08-01 | 1977-08-01 | Glucose detection system free from fluoride-ion interference |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1114723A true CA1114723A (en) | 1981-12-22 |
Family
ID=25232322
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA307,795A Expired CA1114723A (en) | 1977-08-01 | 1978-07-20 | Glucose detection system free from fluoride-ion interference |
Country Status (6)
Country | Link |
---|---|
US (1) | US4098574A (en) |
JP (1) | JPS5426793A (en) |
CA (1) | CA1114723A (en) |
DE (1) | DE2833741A1 (en) |
FR (1) | FR2399662A1 (en) |
GB (1) | GB2001758B (en) |
Families Citing this family (102)
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DE2856487A1 (en) * | 1977-12-29 | 1979-07-12 | Wako Pure Chem Ind Ltd | PROCEDURE FOR DETERMINING PEROXID SUBSTANCES AND COMPOUNDS THAT CAN BE USED THEREFORE |
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DE2964377D1 (en) * | 1978-11-15 | 1983-01-27 | Battelle Memorial Institute | Analytical process and means for measuring the amount of hydrogen peroxide in aqueous media and of organic substrates generating hydrogen peroxide by enzymatic oxidation |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2982700A (en) * | 1961-05-02 | Diagnostic composition | ||
US2848308A (en) * | 1955-12-05 | 1958-08-19 | Miles Lab | Composition of matter |
US3104209A (en) * | 1960-02-02 | 1963-09-17 | Fermco Lab Inc | Composition for determination of glucose |
US3278394A (en) * | 1963-07-22 | 1966-10-11 | Miles Lab | Method and composition for diagnosing glucose |
CH548029A (en) * | 1971-03-30 | 1974-04-11 | Hoffmann La Roche | MEANS OF DETECTING GLUCOSE. |
IT986838B (en) * | 1972-05-12 | 1975-01-30 | Sclavo Inst Sieroterapeut | COMPLEX OF REAGENTS FOR THE ENZYMATIC DETER MINATION OF GLUCOSE GLUCOSE SYSTEM OXIDASE PEROXIDASE DASES WITH MANUAL AND AUTOMATED METHODS CO WITH READING AT TERM OR IN KINETICS |
DE2332760C3 (en) * | 1972-06-30 | 1982-03-04 | Eastman Kodak Co., 14650 Rochester, N.Y. | Material for the quantitative spectrophotometric analysis of a liquid |
US3992158A (en) * | 1973-08-16 | 1976-11-16 | Eastman Kodak Company | Integral analytical element |
US3884764A (en) * | 1974-03-25 | 1975-05-20 | Eastman Kodak Co | Method and composition for blood serum cholesterol analysis |
US3983005A (en) * | 1974-03-25 | 1976-09-28 | Eastman Kodak Company | Integral element for the analysis of cholesterol |
US3964870A (en) * | 1974-03-29 | 1976-06-22 | Boehringer Mannheim G.M.B.H. | Diagnostic composition for the determination of glucose |
-
1977
- 1977-08-01 US US05/821,028 patent/US4098574A/en not_active Expired - Lifetime
-
1978
- 1978-07-20 CA CA307,795A patent/CA1114723A/en not_active Expired
- 1978-07-26 JP JP9057678A patent/JPS5426793A/en active Granted
- 1978-07-31 FR FR7822591A patent/FR2399662A1/en active Granted
- 1978-08-01 GB GB787831821A patent/GB2001758B/en not_active Expired
- 1978-08-01 DE DE19782833741 patent/DE2833741A1/en active Granted
Also Published As
Publication number | Publication date |
---|---|
DE2833741A1 (en) | 1979-02-15 |
FR2399662B1 (en) | 1983-03-18 |
DE2833741C2 (en) | 1988-01-14 |
GB2001758B (en) | 1982-02-03 |
FR2399662A1 (en) | 1979-03-02 |
JPS5728277B2 (en) | 1982-06-15 |
JPS5426793A (en) | 1979-02-28 |
GB2001758A (en) | 1979-02-07 |
US4098574A (en) | 1978-07-04 |
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