CA1073789A - Composition for glucose detection - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention provides a glucose indicating composition which comprises: glucose oxidase; horseradish per-oxidase; a compound which forms a color upon oxidation by hydro-gen peroxide; a compound selected from indoxyl sulfate; Uric acid; 3,4-dihydroxymandelic acid; 3-methyoxy-4-hydroxymandelic acid; 3,4-dihydroxyphenylacetic acid; 3-methoxy-4-hydroxy-phenyl-acetic acid; 3,4-dihydroxyphenylalanine; 3-methoxy-4-hydroxy-phenylalanine; 5-hydroxyindole-3-acetic acid; 5-hydroxyindole-2-carboxylic acid; 5-hydroxytryptophane; creatinine; 2,5-dihydroxy-phenylacetic acid; o-hydroxytyramine (dopamine); 5-hydroxytryp-tamine; and 2,5-dihydroxybenzoic acid; and a buffer composition which will maintain the pH of said glucose indicating composition within the range of from about 4 to about 6 in the presence of biological fluids.

Description

~Lo737~39 ~he present invention relates to devices for the quan-titative analysis of chemical substance concentrations in bio-logical fluids and more particularly concerns a disposable device useful for determining the glucose content of biological fluids.
The invention also concerns methods of making and using the devices of the invention and in particular a glucose indication composition for use with said devices.
This application is a divisional application of co-pending application No. 237,875 filed October 17, 1975.
Prior hereto, disposable devices have been known and commercially available which are useful for determining the `' concentration of glucose (and other substances) in liquids.
of particular importance for their simpllcity, low cost, ease of operation and widespread use are the relatively simple devices which comprise a strip of hibulous material or other support element bearing a reagent which yields a color indication upon contact with glucose. Representative of such devices are the devices described in U.S. Patents Nos. 2,865,718; 2,848,308;
~; 2,893,844; 2,981,606; 3,164,534; 3,212,855; and 3,791,988.
The devices described in the above prlor art patents all operate by immersion in e.g. a glucose containing solution for brief periods of time. On contact with glucose, the reagent element of the device undergoes a chemical change, . .

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:1~737i~9 which is indicated visually to the operator by a color change.
Generally, the degree of color change is a crude measure of the concentration of glucose in the immersion liquid. For example, the device of U.S. Patent No. 2,981,606 employs a reagent compo-sition which gives a color indication, ~epending on the concentra-tion of glucose in the immersion liquid, varying from tints of yellow to green to blue. An approximation of the glucose content of the immersion solution in terms of percentage is then obtained by comparing the color indication with color shades obtained by exposure of the reagent to solutions of known glucose content.
In practice, a secondary color comparison chart is used.
; Other devices of the prior art employ a single reagent which is sensitive to glucose above a specific concentration.
These latter devices, as represented by the devices disclosed in `~ U.S. Paten-t No. 2,893,844, also give the operator a visual indica-- tion by-color formed upon reaction of the reagent element with : . .
glucose. The devices do not indicate a range of concentrations, but only indicate if a certain predetermined level of glucose is i ` present or exceeded in the solution being tested.
The disposable devices of the prior art have not offered a means of obtaining a direct numerical reading of ` glucose or other substance content of the tested solutions.
- For this determination, either a comparison with a color chart must be made to convert the indicia registered by the device to ; a numerical value or multiple tests would have to be carried out using a separate indicator device for a number of different ;;~ minimum glucose level determinations. Both devices offer only - a crude approximation of glucose concentrations and are dependent , upon the ability of the user of the device to differentiate shades of colors.
A further disadvantage of the commercially available prior art devices resides in the fact that they must be employed . ' :
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~7378~

following closely defined operating rules. More particularly, ~he reagents employed continue to react after intial contact with the substance to be detected, such as glucose. The colors formed by the reagents continue to develop to the darkest shade possible and so the indicator must be read within a narrow and specific time period following immersion if any accuracy is to be obtained in the determination. If the operator fails to under-stand the criticality involved in reading the indicia registered within the prescribed time, or fails to do so accurately, an erroneous reading will be obtained. The indicators of my inven-tion are disposable, register an indicia of dissolved substance concentration which is directly readable in numerical terms and will indicate over any desired range of possible concentrations.
In addition, the indicators of my invention provide a registra-tion soon after brief immersion in the containing solution and do not have to be "read" within a critical time period following such immersion. The indicia registrations obtained are stable for reasonably long periods following exposure (particularly to glucose) so that a degree of freedom is obtained as to when the registration must be observed for an accurate reading.
Furthermore, instruments of my making, prepared accord-ing to the invention are comparatively insensitive to the effects of interfering substances in the test specimens. In the case of prior art glucose determinations in urine for example, the pre-sence of certain reducing substances such as 2,5-dihydroxy benzoic acid or ascorbic acid in the urine masks the presence of glucose;
see for example Feldman et al., Diabetes, Vol . 19 , No. 5 , Pps.
337-343.
This leads to the creation of potential gross errors in diagnosis and to the erroneous administration or withholding of certain therapeutic drugs. In using the instruments and reagçnt formulation of my invention, however the effects of such inter-73~

ferences are virtually eliminated, i.e. reduced to about 3% of the effect seen with prior art instrumen-ts.
Enzyme based reagent compositions for the determination of glucose in glucose containing solutions known prior to my invention arè represented by U.S. Patents Nos. 2,848,308;

2,981,606; 3,164,534; 3,721,607; and 3,791,988; and the article "Moxin et al, Clinical Chemistry, 10/9, 959-952 (1973)"; plus others too numerous to list. Although many of such compositions may be employed in fabricating the indicators of my invention, the preferred indicators employ glucose sensitive reagents which comprise novel compositions of the invention. The novel compo-sitions of the invention, hereinafter described more fully, are advantageously used in the fabrication of the indicators of the invent:ion. The novel compositions are particulàrly advantageous reagents for the quantitative determination of glucose, being rapid acting and of anticipated enhanced storage stability and - . :
result in a reduction of the magnitude of the effect due to inter-fering substances to about 3% of the effect seen in the use of prior art devices and compositions when used in an instrument of the invention as discussed above. Although the ratio of equiva~
lent proportions of reducing or antagonist compound to equivalent proportion of glucose to be detected may vary within a range of from about 1:1 to 5:1, it is preferred that the ratio be substan-tially stoichiometric. By stoichiometric I mean those amounts which are substantially in the same proportion as the chemically equivalent weight for the reacting species. When my compositions are used, this ratio is essentially stoichiometric, i.e. and therefore predictable and easily controlled in manufacture.
Furthermore, only when my -LCl 737~9 preEerred composi-tions are used do I observe the relative absence of interference from reducing substances that may occur in urine and which mask large amounts of glucose, in the case of prior art devices (see above).
The invention claimed in copending application No.
237,875 comprises an indicator for the measurement of substances dissolved in biological flulds, which comprises; a support member; and a plurality of indicating reagents for said sub-stances, each of which is located in a separate zone of said member and at least two of which indicate the presence of a different concentration of said substance in solution, by causing the registration of convenient symbols, including digits.
The invention of the copending application also com-prises a method of making the devices of the invention.
The invention of the copendlng application also com- ~;
prises a method of determining the concentration of a chemical compound in a solution/ which comprises; immersing an instrument ; ~of the invention in the compound containing solution, removing said instrument rom said solution and reading the indicated concentratlon of the chemical substance directly from the in-strument. The method is particularly advantageous for deter-mining th~ concentration of glucose in a solution.
' 737~3~

The present invention provides glucose indicating compo-sitions which comprise; glucose oxidase; horseradish peroxidase;
a compound which forms a colox upon oxidation by hydrogen peroxide in the presence of horseradish peroxidase; an antagonist compound selected from indoxyl sulfate; uric acid; 3,4-dihydroxymandelic acid; 3-methoxy-4-hydroxymandelic acid; 3,4-dihydroxyphenylacetic acid; 3-methoxy-4-hydroxyphenylacetic acid; 3,4-dihydroxyphenyl-alanine; 3-methoxy-4-hydroxyphenylalanine; 5-hydroxyindole-3-acetic acid; 5-hydroxyindole-2-carboxylic acid; 5-hydroxytrypto-phane; creatinine; 2,5-dihydroxyphenylacetic acid; o-hydroxy-tyramine (dopamine); 5-hydroxytryptamine; and 2,5-dihydroxybenzoic acid; and a buffer composition which will maintain the pH of said `glucose indicating composition within the range of from about 4 to about 6 in the presence of biological fluids such as blood, urine, tears, saliva and the like.
;The term "antagonist compound" as used throughout the -~specification and claims means a compound which will prevent in some manner the accumulation of oxidized indicator until such time as said compound has been completely consumed in such reac-tion. Preferred as the antagonist compound is 2,5-dihydroxy-:. .
benzoic acid. For convenience, the term "2,5-DHBA" will some-times be used hereinafter to mean 2,5-dihydroxybenzoic acid.

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~73789 The compositions of the invention are useful in fabri-cating the instruments of the invention.
The present invention will be further illustrated by way of the accompanying drawings in which:
Fig. 1 is an isometric view of an indicator embodiment of the invention, following use in a solution containing glucose~
Fig. 2 is an isometric view of an alternate indicator embodiment of the invention after use.
Fig. 3 is an isometric view of another embodiment of the invention after use.
Figs. 4a, 4b, 4c and 4d are isometric views of an alternate indicator embodiment of the invention showing the possible indicia registrations after immersion in various glu-cose containing solutions.
Fig. 5 is an isometric view of a preferred indicator ~ -embodiment of the invention shown after use in a glucose containing urine specimen.
Fig. 6 is a cross-sectional longitudinal view along lines 6-6 of Fig. 5.
Fig. 7 is an isometric view of an alternate embodi-ment of the invention.
The indicators of the invention are relatively simple devices which may be used once and thereafter disposed of. For this reason they may be termed "disposable" instruments.

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"- ' 0737~9 Although -their most important use may be or the determination of glucose levels in the urine and blood of mammals, they ara not restricted to this use and may be employed to determine the presence and concentration of any substan~e in any biological ~luid. For example, by the selection of appropriate reagents, the indicator devices of the invention may be employed to deter-mine the concentration of ketones, albumin, nitrogen, etc. in urine or urea, calcium, protein, albumin, cholesterol, triglycer-ides, alkaline phosphatase, bilirubin, uric acid, etc. in blood plasma or blood serum.
The indicators of the invention comprise a support mem-ber bearing a plurality of specific reagents. The support member is not critical in the sense that a specific material of construc-tion is required although several forms of preferred embodiments will be described hereinafter. In general, the support member may be of any material capable of bearing the reagent for exposure to the solution to be tested~ Specific examples of support mem-bers are webs, sticks, strips, splinters, sheets, rods and like forms of glass, metal, wood, paper; polymerics such as polyethylene, polypropylene, polyalkylene acetate, polycarbonates and the like;
textiles and the like. Pre~erred materials are the bibulous mat-erials which may be impregnated with solutions of reagent compo-sitions, such as filter papers, blotting papers and like bibulous materials.
The reagent components of the indicators of the inven-tion are preferably prepared in a liquid form for deposit upon the support member. Once placed on the support member, the reagent compositions in solution are dried to adhere the compositions to the support member. Generally, adhesion of the reagent compo-sitions to the support member is conveniently ef~ected when thesupport member is a bibulous material. Conventionally employed inert fillers, binders, surfactants and the like may be incorpor-, .-~ - - .

0~3789 ated into the reagent compositions when desired. Certain binders such as resin gums are advantageously incorporated into the reagent compositions to assist in adhering them to non-porous support members such as metal, glass or non-porous polymeric materials. For product elegance, it is desirable that the color -~ change in each indicator zone of the devices of the invention be clear, sharp, unequivocal and strongly positive. I have found that the degree of accuracy and precision with which the devices of the invention function in accord with this desire is related to the ratio of volume of reagent solution deposited on the support member to volume of test solution absorbed at the reagent site during immersion in the test solution or biological fluid.
The most advantageous device of the invention in terms of accur-acy and precisio~ obtainable are those wherein the above des-cribed ratio is substantially a 1:1 ratio. The further one departs from this optimum ratio, the greater will be the loss of accuracy and precision in the indicator devices.
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~ To obtain the above-described preferred ratio, it is ; necessary that a given zone of the preferred bibulous support mem-`~ 20 ber be fully saturated with the reagent in a solvent. The sol-vent is then evaporated to prepare an indicator device of the invention. Upon immersion in the biological solution being tested, the bibulous material at the site of reagent deposition ` will then generally take up a volume of the test solution which is equivalent to the volume of solvent evaporated resulting in ., .
the desired 1:1 ratio described above. However, unless the area of reagent impregnation is isolated ln a manner so as to provide - a site of precise and determinable volume, the reagent may be ` carried beyond the site of original deposition upon immersion in - 30 the test solution. This will result in dilution of the reagent in terms of its proportion in the absorbed test solution during ; the test procedure, and ultimately results in a variation from , . ~

' ~ ~73789 ' the desired 1:1 ratio described above. Therefore, preferred devices of the invention wherein the indica-tor reagent is carried on a bibulous material preférably have the plurality of indicator reagent sites separated from each other by a non-absorbent or hydrophobic material and each site preferably will absorb nearly identical volumes of the solvent employed in depositing reagent and of the biological fluid to be tested. The preferred device may be prepared, for example, by affixing a plurality of the reagent impregnated bibulous support members (each bearing a reagent of different sensitivity to the substance being tested for, as described more fully hereinafter) to a different zone of a hydrophobic support member such as a polymeric resin solid ; strip like polyethylene, polypropylene, etc.` Upon immersion of such a device in the test solution, a precise volume of the test fluid is absorbed at the site of the indicator reagént which is -then diluted accurately to substantially the same concentration ; at which it was deposited in the deposition solvent employed in preparing the indicator reagent site. Illustrative of such pre~
ferred indicator devices are those described more fully herein-after, particularly the embodiment of Figures 2 and 3.
; The reagent components of the indicators of the inven-tion may be any of the known reagents for the quantitative detec-tion of the substance being analyzed for, and which will give a visual indication when exposed to specific concentrations of such substance in a solution thereof. A plurality of such reagents are placed on the support member, each in a different zone of the member and at least two of said reagents providing an indication of different substance-in-solution concentration levels. Such reagents and the means for their preparation are generally known in the case of glucose testing. Exemplary of reagents which may be ~mployed for determining glucose levels and their method of preparation are the glucose indicating compositions disclosed in ~; ' ~` ' ' ' ' ' ' , U.S. Patent No. 2,893,8~. In general, such reagents comprise an enzyme having glucose oxidase activity, peroxidase, an indica-tor which undergoes a color change in the presence of hydrogen peroxide an~ peroxidase and a compound which prevents in some manner the accumulation of oxidized indicator until such time as said compound has been completely consumed in such reaction. Such a compound, conveniently, may be a reductant sufficiently reactive as to reduce any oxidized indicator formed in the enzymatic conver-sion described above. Examples of the latter compounds are well known and are illustrated by ascorbic acid and the like. An exten-sive list of such compounds may be found in U.S. Patent No. 2,893-844.
An alternative method of achieving the same effect is described in "Screening Method for Glucose o~ Blood Serum Utiliz-ing Glucose Oxidase and an Indophenol Indicator", Leonard Dobrick, ~ -~
J. Biol. Chem. 1958, pp. 403-409.
Preferred reagents for use in fabricating the indicators of the invention for use in detecting glucose concentrations are those constituting the above described compositions of the inven-tion. The compositions of the invention are prepared by admixing a glucose oxidase, peroxidase, an antagonist compound, a compound which forms a color upon oxidation by contact with hydrogen perox-ide in the presence of the peroxidase, and a buffer composition which will maintain the pH of the reagent composition within the range of from about ~ to about 6 in the presence of biological fluids.
The preferred glucose oxidase employed in preparing the compositions of the invention is a glucose oxidase obtained from molds. Such glucose oxidases are yenerally well known, see ;~
for example U.S~ Patent No. 2,981,606.
Peroxidase is also a generally well known class of enzyme, and any of the known peroxidases such as those described , ~:~737~9 ,:
in u.S. Patent No. 2,981,606 may be employed in preparing the ; compositions of the invention. ~orseradish peroxidase is a pre-ferred peroxidase for preparing the devices of the invention.
Compounds which form a color upon being oxidized by hydrogen peroxide are aiso generally well known, see for example U.S. Patent Nos. 2,981,606 and 3,721,607 describing for example color-forming substances which produce a color formation in the presence of hydrogen peroxide and peroxidase. Examples of such compounds which may be employed in the glucose indicators of the present invention include the following substances:
(l) Monoamines, such as anillne and its derivatives, ortho-toluidine, para-toluldine, etc.;
(2) Diamines, such as ortho-phenylenediamine, N,N~-dimethyl-para-phenylenediamine, N,N'-diethyl phenylenediamine, benzidine (which produces a blue or brown color), a dianisidine (turns green or brown), etc.;

(3) Phenols, such as phenol per se (producing a yellow color), thymol, ortho-, meta and para-cresols (producing a green-yellow color, a pink color and a milky suspension, respéctively), alpha-naphthol ~pro~ucing a magenta color), beta-naphthol (pro-ducing a white precipitate), etc.i

(4) Polyphenois, such as catechol, guaiacol (which forms an orange color), orcinol, pyrogallol (producing reddish ; or yellow color), p,p-dihydroxydiphenyl and phloroglucinol;
(S) Aromatic acids, such as sali~ylic, pyrocatechuic and gallic acids;
(6) Leuco dyes, such as leucomalachite green (to pro-duce malachite green) and leucophenolphthalein (des~rably - employed in~an alkaline medium);
(7) Colored dyes, such as 2,6-dichlorophenolindophenol;
(8) Various biological substances, such as epinephrine, the flavones, tyrosine, dihydroxyphenylalanine (producing an LCI 737~3g orange-reddish color) and -tryptophane; and (9) Other subs~ances, such as gum quaiac, guaiaconic acid, na~i reagent (producing a bluish color) potassium sodium, and other water soluble iodides; and bilirubin (producing a greenish color).
Buf f er compositions which may be employed in preparing the compositions of the invention are generally well known and include for example mixtures of sodium phosphate with sodium phos-phate monohydrate and mixtures o~ citric acid with tri-sodium citrate.
The components of the compositions of the invention are admixed employing conventional techniques, generally in the presence of a solvent such as water. The order of admixture is not critical, although it is generally preferred to add the antagonist compound component first. The solutions of novel compositions of the invention may then be used to deposit the com-positions of the invention may then be used to deposit the compo- `
sitions upon support members to fabricate the indicators of ~he invention.
Representative preferred compositions of the invention are those prepared using the following proportions of ingredients.
About 40 or more IU of glucose oxidase; about 60 or more purpuro-gallin units of peroxidase; from about 0.05 to about 15 mg. of the compound which ~orms a color upon oxidation and from about 0.01 to about 5.0 mg of the antagonist compound, all per 100 microliters of reagent. The buffer component is added in suf-ficient quantity to maintain the reagent composition at a pH
within the range of ~rom 4 to 6 in the presence of biological fluids.
The precise level or concentration of glucose in solu-tion which will be "indicated" by a color change occurring during reaction of the reagent compositions o~ the invention, is deter-10737~

mined by the proportion o~ antagonist compound employed in pre-paring the compositions of the invention. Indication of a mini-mum level of glucose occurs when a given proportion of antagonist compound is exhausted in reducing the indicator dye. By varying the proportion of antagonist compound, one may prepare reagent compositions which will "indicate" different minimum concentra-tions of glucose in solution. The proper proportion of antagon-ist compound required to obtain a reagent composition which will indicate the presence of a specific concentration of glucose in solution is readily calculated mathematically or may be determined by trial and error techniques, and is related to the amount of glucose reacting substantially in the usual stoichiometric manner.
The following examples describe the manner and process of making and using this invention and set forth the best mode contemplated by the inventor of carrying out the invention but are not to be construed as limiting. All parts are by weight unless otherwise indicated.
EXAMPLE I
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(A) To an appropriate vessel there is charged 300 microliters of a solution of glucose oxidase (Sigma Chemical Company, St.
Louis, Missouri, Catalog No. G-6500; glucose oxidase from molds having an activity of 1200 IU/ml), 150 microliters of a solution of horseradish peroxidase [prepared by dissolving 37.5 milligrams of horseradish peroxidase (Sigma, supra, Catalog No. P-8250) in 1.0 milliliters of O.OlM citrate buffer ~pH 5.5)], 300 microliters of 2.2'-azino-di-(3-ethyl-he~zothiazoline-6-sulphonic acid) in solution [prepared by dissolving 50 milligrams of 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulphonic acid) (soehringer Mannheim Corporation, New York, New York, product No. 15594) in 1.0 milli--liters of O.lM citrate buffer, pH 5.5~, 50 microliters of 1% work-ing solution of 2,5-DHBA (below), quantum sufficie~t to make a total of 1,250 microliters. The mixture is thoroughly admixed - 15 ~

~7371~9 to obtain a glucose indicating reagent solution.
(B~ In an appropriate vessel O . 5 grams of 2,5-dihydroxybenzoic acid is slurried in4 milliliters of waterO To -the slurry there is then added with stirring a solution of 10% sodium hydroxide in water until a clear solution is obtained. This is used as a stock 10% solution. A working 1% solution is prepared by appro-priate dilution with O.lM citrate buffer, pH 5.5.
(C) Four separate appropriate vessels are each charged with 75 microliters of the indicating reagent solution prepared in (A) above. In each ~essel there is then added with mixing t. VarlOUS
proportions of the 1% 2,5-dihydroxybenzoic acid solution prepared in (B), supra, and varying amoun~s of buffer as described in (A) ~ ~
above to obtain glucose indicating reagent solutions, each of which will indicate visually when exposed to various minimum con-centrations of glucose in solution, by a color change of from sub-stantially colorless to colored. The four vessels are identifiéd by the letters A through D inclusive. The concentration of 2,5-dihydroxybenzoic acid and buffer in each vessel and the minimum glucose concentration of a solution which each reagent composition A through D inclusive will indicate by a color change are shown in Table 1 below. `
TABLE I

Percent of Minimum Concen- Glucose Concentra-tration tion in Solution Buffer 2,5-DHBA of Required for Indi-Vessel (Microliters) (Microliters) 2,5-DHBA cation to Occur A 25 0 0.3~ 0.1%
B 21 4 0.75~ 0.25 C 13 12 ~...................... 5~ 0.5%
D 0 25 2.8~ 2.0%
Similarly, following the procedure of Example I, part A above, but replacing the 2,5-DHBA as used therein with an equi valent proportion of indoxyl sulfate; Uric acid; 3,4-dihydroxy-737l5 9 mandelic acid; 3-methoxy-4-hydroxymandelic acid; 3,4-dihydroxy-phenylacetic acid; 3-methoxy-4-hydroxyphenylacetic acld; 3,4-dihydroxyphenylalanine; 3-methoxy-4-hydroxyphenylalanine; 5-hydroxyindole-3-acetic acid; 5-hydroxyindole-2-carboxylic acid;

5-hydroxytryptophane; creatinine; 2,5-dlhydroxyphenylacetic acid;
o-hydroxytramine (do amine); and 5-hydroxytryptamine, respectively, glucose indicating reagent solutions are obtained which are use-ful in fabricating indicator devices of the invention.
EXAMPLE II
A series of five sets of circular discs are prepared, each set consisting of four discs and each disc being fashioned from ~52 Whatman filtex paper by cu-tting out a circle, 4mm, in diameter. Each disc within each set is then wetted with about 0.002 ml of a different one of the reagent solutions A, B, C and D, respectively, prepared in Example I, supra. The wet discs are allowed to air-dry. Each disc is calculated to contain, on drying, approximately 0.6 IU o glucose oxidase, 1.2 PU of peroxi-dase, 18 micrograms of citrate buffer, 24 micrograms of 2,2i-azine-di-(3-etnyl-benzothiazoline-6-sulphonic acid) and varying proportions of 2,5-dihydroxybenzoic acid. The dry discs of each set are then mounted on a single paper strip, and each disc labeled a, b, c or d to correspond to the particular reagent A, B, C and D, respectively, employed in its preparation. Four of the paper strips each bearing a separate set of the four di~cs a-d is then immersed in one of four aqueous solutions containing Q.l percent, 0.25 percent, 0.5 percent and 2.0 percent glucose respectively. The fifth strip is a control strip and is immersed in distilled water. Each strip is immersed in the solution for a period of about one second and then withdrawn. Within about one minute of immersion, each strip is observed for a change of color in the discs mounted thereon. The results are given in Table II

below, with the proportion of 2,5-dihydroxybenzoic acid calculated 1(~737~39 to be present in each disc.
TABLE I I

Proportion of ~ of Glucose 2,5-dihydroxy-Stripin Immersion benzoic Acid Color No. Solution Disc (mcg) per disc Change 1 0.1%
a 0.6 Yes b 1.4 No c 3.0 No d 5.6 No 2 0.25%
a 0.6 Yes b 1.4 Yes c 3.0 No d 5.6 No 3 0.5%
a 0.6 Yes b 1.4 Yes c 3.0 Yes - d 5.6 No ;

4 2.0 a 0. 6 Yes b 1.4 Yes c 3.0- Yes d 5.6 Yes 5 ~Control) 0.0%
a 0.6 No b 1.4 No c 3.0 No d 5.6 No The color changes obtained wexe a distinct darkening, strongly positive, and those discs which did not change showed no appreciable darkening. The registrations obtained remained stable for at least 72 hours. This is not possible with the commercially available glucose indicators of the prior art which must be read, usually within minutes.
The indicator of the invention and the method of the invention will now be further illustrated by reerence to the ~`
specific embodiments shown in the accompanying drawings.
In Fig. 1 there appears a perspective view of an indi-cator 20 of the invention. The indicator 20 comprises a support ~ ~8 -, .

373785~

member which i5 sho~m in Fig. 1 as a flat strip 18 and which pre-ferably is bibulous material such as an absorbent paper strip.
Disposed on the strip 18 by impregnation thereon in a manner so as to form the numerals 1, 2, 3 and ~ are reagents 11, 12, 13 and 14 respectively. Reagent 11 is a composition such as compo-sition A of Example I, supra, and will indicate by color change when a minimum glucose concentration of 0.1% is present in a solu-tion brought into contact with the reagent. Reagents 12, 13 and lg correspond to tne reagent compositions B, C and D respectively of Example I, supra. and will indicate the minimum glucose con-centrations of 0.25%, 0.5~ and 2.0% in glucose containing solu-tions, respectively. The disposition of the reagent compositions on the support member 18 may of course be in any convenient indicia.
For example, the minimum concentration indicated by a given rea-gent may be the form of disposition, i.e.; for example reagent 11 above described may be disposed in the configuration "0.1%''. The numerals 1, 2, 3 and 4 selected for illustration in Fig. 1 is especially useful when the indicator devlce is to be employed for determining the glucose concentration of urine specimens. Such - 20 indicia conforms to the customary practice of referring to a 0.1%
concentration of glucose in urine as a 'iplus 1" level, a 0.25%
concentration as "plus 2", a 0.5% concentration as "plus 3" and a 2.0~ concentration as ~Iplus 4".
Prior to use, the symbols 1, 2, 3 and 4 employed in the embodiment of Fig. 1 may be invisible to view or only slightly decipherable through a slight discoloration upon strip 18. ~he broken lines indicating the numerals 3 and 4 are intended to show that the numerals 3 and 4 are not clearly decipherable as originally formed on member 18. Upon exposure however to a glu-cose solution by immersion therein, the reagents react chemicallyto produce a color. If the glucose concentration in the tested solution exceeds the minimum glucose concentration for which the ~L~7378~

reagent will yive a color indication, such color change occurs.
In the Fig. 1, numerals 1 and 2 are shown in distinct dark tones and illustrate the indication obtained following immersion of indicator 20 in a solution of glucose containing a glucose con-centration of from between 0.25~ and 0.5~. Reagents ll and 12 have reacted to indicate a "plus 2" concentration, i.e.; the, highest number developed. The development of reagents ll and 12 to produce a clear and distinct color change forming distinct solid numerals gives a direct reading to the user of the minimum glucose concentration in the solution tested. Thus, the method of the invention is carried out by immersing that portion of indicator 20 which bears reagents 11, 12, 13 and 14, into a glu-cose containing solution momentarily, withdrawing it and visually observing the indicla registered. It will be observed that the operatox does not have to carry out a comparison of colors regis-tered, with standard charts to determine the glucose concentra-tion of the tested solution, as is necessary with prior art dis-posable devices~ When the preferred composition of the invention is employed as the reagents ll, 12, 13 and 14, clear and distinct indications of glucose concentration are obtained within about one minute. The indicia registered on the indicator are relatively stable and will last for a relatively long time so that at an interval of up to at least 72 hours after immersion and withdrawal of indicator 20, the user may refer back to rechec~ the test re-sults. This is not possible with prior art devices.
The embodiment illustrated in Fig. l may obviously be modified in many respects to meet specific needs. The degree of sensitivity of each reagent may be modifled to meet specific needs. -~Different ranges and sensitivities of the indicator devices may be obtained by selection of the reagent sensitivities and the numbex of different reagent compositions applied to the support member. Although the indicator shown in Fig. 1 is particularly : ' ' ' ' ' ';:.

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~LQ737~3~

well suited for testing urine for glucose content, appropriate selection of reagent compositions, range of indication and regis-trable indicia will yield indicators appropriately used for test-ing other biological fluids such as blood, sweat, urine and like biological liquids for glucose, alb~in, ketone and other sub-stances normally or abnormally found therein. The support member strip 18 may also be dyed a particular color to either blend with the reagent compositions prior to their indicating the presence of glucose or they may be dyed a color to provide a very sharp contrast when the reagent reacts to produce a registration.
As further examples of modifications which may be made to the basic indicator shown in Fig. 1, reference is made now to Fig. 2 which shows an isometric view of indlcator 22 which may also comprise a support member 18 as described above and upon which there have been mounted "plus-shaped" blocks 5 of a bibu-lous material. Each block 5 has been saturated with a solution of a reagent 11, 12, 13 and 14 respectively as described above in conjunction with Fig. 1 and then dried before fixation upon sup-port member 18. When indicator~22 is immersed briefly in a glu-cose containing solution the reagents carried on blocks 5 reactas described above, and if the glucose level exceeds the minimum indicating sensitivity of the particular reagent, a color change in the block 5 carrying that reagent is visually observable. By counting the "p~uses" which have changed color, the operator can read the device directly. In the device 22, as shown a "plus-2"
has been indicated, i.e. the device was exposed to a solution containing between 0.25 and 0.5% glucose.
- Figure 3 is an alternate embodiment similar to device 22 of Fig. 2, differing essentially in that the shape of the bibulous block 6 is a rectangle rather than a cross. This embodi-ment is operated in the same way as the device of Figure 2 and as shown here has been exposed to a solution of glucose in the "plus-~LC)73789 3" concentration range, i. e.; the solu~ion -tested had ~ ~lucose concentration of between 0.5~ to 2%.
The embodiment o~Figures 2 and 3 are particularly advan-tageous when ~he support rnember 18 is a hydrophobic material such as, for example, polyethylene. The blocks 5 and 6 will each then absorb a predetermined volume ~ th~ biological fluid being tested, upon immersion. When the indicator rea~ents 11, 12, 13 and 14 have been deposited on blocks 5 and 6 by saturation of said blocks 5 and 6 with the indicator reagents ll, 12, 13 and 14 in solution followed by evaporation of the reagent solvent, the volume of tes~ solution absorbed in blocks 5 and 6 on immersion in test solution will be substantially equal to the volume of solvent evaporated from the blocks 5 and 6 to deposit the indicator reagent. This is the preferred ratio of volume of reagent in solution to volume of test solution absorbed as previously des-cribed, and results in a highly accurate, precise indicator device of the invention~
Those skilled in the art will appreciate that the fabri-cation of the preferred indicator devices of the invention accord-ing to the embodiments of Figures 2 and 3 discussed above may be relatively costly due to the requirements of preparing a bibulous carrier of specific dimensions, saturating the carrier with a specific volume of reagent solution containlng a speclfic concen-tration of indicator reagent and drying the same on a supporting member. By a method of the invention, highly accurate and precise -~
indicator devices of the invention such as those illustrated in Figures 2 and 3 may be prepared obviating a number of the re~uire-ments as outlined above, therèby reducing the cost of manufacture and providing improved indicator products.
The improved method of manufacture comprises slurrying or dissolving the bibulous material, with the indicator reagent, depositing the slurry or solution on a hydrophobic support member .

3.C173789 and evaporating the solvent to leave a residue of a dried mixture of indicator and bibulous material adhered to ~he support member.
The slurry of bibulous material and indicator reagent in solvent may be prepared by admixture of the bibulous material in finely diviaed form with the reagent in solvent using conventional appara-tus and technique. As an example, a slurry of cellulose powder or a solution of other bibulous material mixed with compositions of the invention dissolved in water may be prepared, deposited on a polyethylene stick and dried.
Figs. 4a, 4b, 4c and 4d illustrate still another embodi-ment of the invention. The indicator device is referred to gen-erally by the numeral 24 in these Figures. In Fig. 4a, indicator 24 is shown to comprise a support member 18 as described previously but bearing reagents 11, 12, 13 and 14 as previously described in a sequence so that reagent 11 is disposed in the form of a symbol 1, reagent 12 is disposed in the form of a symbol 2 and is also disposed in a zone surrounding the symbol 1 formed by a reagent 11. In a si~ilar manner reagent 13 isdisposed in the form of a symbol 3 and also in the zone surrounding the symbol 2. Reagent ` 20 14 is disposed in the form of symbol 4 and also in the zone sur-rounaing the symbol 3. In this embodiment, exposure to a glucose con~aining solution of a specific level will indicate numerically the minimum concentratlon of glucose in the tested solution and will obliterate numeral symbols for any lower concentrations of glucose. For example, Fig. 4a shows by broken lines the zones and disposition into indicia of the reagent compositions 12, 13 and 14 not registering the specific concentrations of glucose to which they are sensitive. The numeral 1 appears in bold outline as indicative of a ''plus 1" concentration of glucose, meaning the indicator 24 as shown in Fig. 4a has been exposed to a solution containing at least 0.1% of glucose.

Referring to Fig. 4b, there is seen the registration :

.. .. . . .

~7378~ :

obtained by immersion, briefly, of indicator device 24 in a test solution containing between 0.25% and 0~5~ glucose concentration.
The registration of the numeral 2 and obliteration o~ the also developed numeral 1. This is a clear indication to the user of a "plus 2" glucose concentration in the tested solution. Fig . 4c shows the indicia registered when indicator 24 was immersed in a -~
solution containing between 0.5% and 2.0% glucose and Fig. 4d shows the indicia registered upon immersion of indicator 24 in a solution containing more than 2.0~ glucose, i.e.; a "plus 4"
reading.
Fig. 5 is an isometric view of a preferred indicator of the invention shown after use by immersion in a solution con-taining between 0.25% and 0.5~ glucose. The preferred indicator 26 is particularly useful in urine analysis and comprises as a support member a bibulous material 30 which has been laminated to a strip 32 of stiff supporting material such as polymeric plastic for example a strip of polyethylene, polypropylene or like poly-meric material. Apertures 35, 36, 37 and 38 have been cut into supporting strip 32 in the form of positive signs in those areas overlying support member 30 which have been impregnated with the various reagents 11, 12, 13 and 14 previously described. In this -~
manner, the various zones of support member 30 bearing reagents 11, 12, 13 and 14 are viewed through the corresponding apertures 35, 36, 37 and 38 respectively. In addition, an area or zone of support member 30 has also been impregnated with a reagent 9 ~; which is sensitive to an acid pH. The reagent 9 impregnated zone of support member 30 is visible through aperture 39 cut into strip 32. The purpose of the zone impregnated with reagent 9 is to serve as a control. When the indicator 26 is immersed in urine - 30 to determine glucose content thereof, contact of the urine with reagent 9 registers a color change. The user is then assured that all of the reagent zones below reagent 9 on member 30 have ': ~

~ .
, . .

~0~737~9 made contact with the urine solution, and therefore reagents 11, -12, 13 and 14 have been exposed to the urine specimen.
Fig. 6 is a cross-sectional view along lines 6-6 of Fig. 5 and shows in greater detail the mounting of support member 30 beneath the cutaway zones 35, 36, 37, 38 and 39 in rigid supporting strip 32. This embodiment, as illustrated in Figs.
5 and 6, immediately provides the user, upon immersion in a urine specimen, with a visual indication of glucose level regis- -~
tered in terms of plus units which are readily calculated by counting the darkened indicia. Thus the illustrated device of Figures 5 and 6 has registered a "plus 2l value of glucose con-centration, in the illustration.
Fig. 7 is a view in perspective of another indicator ~mbodiment of the invention which is particularly useful for precise determinations of the concentration of a substance dis-solved in liquids, which potentially may have a broad range of possible concentrations. For example, the indicator shown gener-; ally by the numeral 70 is useful in measuring the glucose con-centration of mammalian blood specimens. Indicator 70 comprises a transparent or highly translucent support member 72 such as a sheet of transparent cellulose acetate, a film of polyethylene, ;~
transparentized paper or like materials. Disposed on member 72 ` are a variety of reagents, such as reagents 11, 12, 13 and 14 previously described, with reagent compositions of intermediate sensitivities disposed between them as illustrated in the figure.
Prior to exposure to a solution of glucose, for example blood .~ serum, the zones covered by the various reagent compositions are relatively colorless. A sample of blood serum is wiped cver the surface of the reagent bearing member 72, causing those reagents which indicate at the level of glucose concentration found in the blood speci~en to undergo a visible color change. The indicator -~
70 is "read" by a hand held optical aid or by inserting it in a :, .

~C~73789 light projector with a reticule 75. Reticule 75 projects a refer-ence grid, which is superimposed by projection over the indicator 70, whereby the indication registered on 70 is readily observed in a magnified and projected image. of course when the indicator i5 to be read directly without the aid of a light pro~ector, there is no nee`d for a transparent support member 72.
The embodiment of the invention described above in relation to Fig. 7 is representative of another facet of the invention which comprises the use of reading aids, such as opti-cal and electromechanical devlces to assist in reading the indi-cators of the invention. More specifically the indica~ors of the invention function basically by the dévelopment of a color change, darkening or opacifying of a zone upon the support member component of the indicator device. Reading nf the device is dependent upon an observation of a particular quantity of reacted or indicated zones The actual "reading" of the indicators can therefore be performed mechanically by instruments designed to count symbols through the use of lenses, mirrors, filters, pro-~.
jectors, detectors and the like which can discriminate by means of light transmission, light reflectance, light scattering, -~
electrical conductivity, radioactivity, etc. Thus, the reading of the indicator devices of the inventor may be carried out mech-anically with the aid of photo scanners and like devices. This facet of the invention is particularly advantageous for use of the indicator devices of the invention bearing a large number of diferent reagent zones, each zone being of a micro-dot dimension or some dimension not readily observable with the naked eye.
Such devices are particularly advantageous in that they use a r minimum of reagent material, are fairly compact and can provide readings of great accuracy over wide ranges of possible substance concentrations in the solutions being tested.
The indicators of the invention may be used in ways not ~0737~39 limited to those described in the foregoing. For example, before or after exposure to the test specimen, they may be exposed to reagents not previously incorporated in the reactive zones, or to a drying process, e.g. application of heat, or other chemical or physical agents sel~ected for the purpose of developing or enhanc-ing the appearance of visual or otherwise detectable indications.

'~ ' ' .` ~.' .

, ~ .- , .

Claims (4)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A glucose indicating composition which comprises:
glucose oxidase; horseradish peroxidase; a compound which forms a color upon oxidation by hydrogen peroxide; a compound selected from indoxyl sulfate; Uric acid; 3,4-dihydroxymandelic acid;
3-methoxy-4-hydroxymandelic acid; 3,4-dihydroxyphenylacetic acid; 3-methoxy-4-hydroxy-phenylacetic acid; 3,4-dihydroxyphenyl-alanine; 3-methoxy-4-hydroxyphenylalanine; 5-hydroxyindole-3-acetic acid; 5-hydroxyindole-2-carboxylic acid; 5-hydroxytrypto-phane; creatinine; 2,5-dihydroxyphenylacetic acid, o-hydroxytry-amine (dopamine); 5-hydroxytryptamine; and 2,5-dihydroxybenzoic acid; and a buffer composition which will maintain the pH of said glucose indicating composition within the range of from about 4 to about 6 in the presence of biological fluids.

2. A composition according to claim 1, wherein said selected compound is 2,5-dihydroxybenzoic acid.

3. A composition according to claim 1, wherein the proportions of components is from 40 IU or more of glucose oxidase; 60 PU or more of peroxidase; 0.05 to 15 mg. of said color forming compound; and from 0.01 to 5.0 mg. of a compound selected from indoxyl sulfate; Uric acid; 3,4-dihydroxymandelic acid; 3-methoxy-4-hydroxymandelic acid; 3,4-dihydroxyphenylacetic acid; 3-methoxy-4-hydroxyphenylacetic acid; 3,4-dihydroxyphenyl-alanine; 3-methoxy-4-hydroxyphenylalanine; 5-hydroxyindole-3-acetic acid, 5-hydroxyindole-2-carboxylic acid; 5-hydroxytrypto-phane; creatinine; 2,5-dihydroxyphenylacetic acid; o-hydroxy-tyramine (dopamine); 5-hydroxytryptamine; and 2,5-dihydroxyben-zoic acid; per 100 microliters of said buffer.

4. A composition according to claim 3, wherein said selected compound is 2,5-dihydroxybenzoic acid.