U.S. patent number RE28,575 [Application Number 05/459,126] was granted by the patent office on 1975-10-21 for indicator for detecting hydrogen peroxide and peroxidative compounds containing alpha naphthoflavone.
This patent grant is currently assigned to Miles Laboratories, Inc.. Invention is credited to Robert Bauer.
United States Patent |
RE28,575 |
Bauer |
October 21, 1975 |
Indicator for detecting hydrogen peroxide and peroxidative
compounds containing alpha naphthoflavone
Abstract
Alpha naphthoflavone is an excellent indicator for detecting
hydrogen peroxide and peroxidative compounds such as hemoglobin.
When formulated with either a peroxidative active compound or a
peroxide, this indicator provides a very sensitive chromogenic
response to the presence of said constituents in aqueous
fluids.
Inventors: |
Bauer; Robert (Bristol,
IN) |
Assignee: |
Miles Laboratories, Inc.
(Elkhart, IN)
|
Family
ID: |
26817874 |
Appl.
No.: |
05/459,126 |
Filed: |
April 8, 1974 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
119926 |
Mar 1, 1971 |
03654180 |
Apr 4, 1972 |
|
|
Current U.S.
Class: |
435/28; 422/428;
8/400; 8/401; 435/14; 436/66 |
Current CPC
Class: |
G01N
33/725 (20130101); C12Q 1/28 (20130101); G01N
31/228 (20130101); C12Q 1/54 (20130101); C12Q
2326/00 (20130101) |
Current International
Class: |
C12Q
1/28 (20060101); C12Q 1/54 (20060101); G01N
31/22 (20060101); G01N 33/72 (20060101); G01N
031/22 (); G01N 033/16 () |
Field of
Search: |
;252/408 ;8/1R
;23/23B,253TP ;195/13.5C |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Padgett; Benjamin R.
Assistant Examiner: Leland; David
Attorney, Agent or Firm: Klawitter; Andrew L.
Claims
What is claimed is:
1. In a composition for detecting hydrogen peroxide or peroxidative
active compounds utilizing the catalytic oxidation of an indicator
.Iadd.system.Iaddend..[.dyestuff.]. by hydrogen peroxide in the
presence of the peroxidative active compound, the improvement which
comprises the use of .alpha.-naphthoflavone .Iadd.in combination
with a water soluble iodide salt .Iaddend.as the indicator
.Iadd.system.Iaddend. .[.dyestuff..].
2. A composition as claimed in claim 1 in which
.alpha.-naphthoflavone is present in about 0.05% to 0.30% by weight
of said composition.
3. A composition as claimed in claim 1 in which the peroxidative
active compound is selected from the group consisting of
peroxidase, hemoglobin and molybdate.
4. A composition as claimed in claim 3 in which the molybdate is
present in about 0.1% to 1.5% by weight of said composition.
5. A composition as claimed in claim 3 in which the peroxidase is
present in about 0.01% to 0.05% by weight of said composition.
.[.6. A composition
as claimed in claim 1 which additionally contains iodide..]. 7. A
composition as claimed in claim .Iadd.1.Iaddend. .[.6.]. in which
the .Iadd.water soluble .Iaddend.iodide .Iadd.salt .Iaddend.is
present in
about 0.25% to 1.5% by weight of said composition. 8. A composition
for detecting glucose in aqueous fluids which comprises glucose
oxidase, a peroxidative active material, .Iadd.a water soluble
iodide salt, .Iaddend.and .alpha.-naphthoflavone.
Description
BACKGROUND OF THE INVENTION
The determination of glucose in urine is important since this test
is employed to detect diabetes. Procedures for the detection of
sugar in urine are well known in clinical chemistry. One such
procedure utilizes Benedict's copper reduction test, another
employs a self heating alkaline copper reduction test in tablet
form, while still another test depends solely on the action of
enzymes. The diagnostic composition in most glucose tests comprises
essentially glucose oxidase, peroxidase and an indicator which is
oxidized by hydrogen peroxide and undergoes a color reaction during
such oxidation. Typical indicators employed in the past include
o-tolidine, benzidine dianisidine and 27-diaminofluorene.
It is well known that glucose oxidase catalyzes the aerobic
oxidation of glucose to gluconic acid and hydrogen peroxide.
However in the presence of iodide, H.sub.2 O.sub.2 oxidizes the
iodide to free iodine which produces a color change in the
indicator. The color change produced is accurately indicative of
the amount of H.sub.2 O.sub.2 present as well as of the glucose
content of the fluid being tested. Molybdates merely accelerate the
oxidation of iodide to free iodine. Since some of the indicators
previously used are toxic it has spurred a search for more suitable
replacements which will give satisfactory results in detecting
H.sub.2 O.sub.2 generally and more specifically in detecting
glucose in urine or blood. If desired such indicators can be used
to detect peroxidase as well as peroxidative-active substances such
as hemoglobin in blood or urine.
SUMMARY OF THE INVENTION
This invention is predicated upon the discovery that
.alpha.-naphthoflavone can be used as an indicator dyestuff in
formulations containing either peroxide or a peroxidative active
compound when the peroxidative active compound catalyzes the
oxidation of the indicator by the peroxide. Said indicator has the
formula ##SPC1## and is known chemically as 2-phenyl-4H naphtho
[1,2-b]pyran-4-one.
Although the test system may comprise the reagent composition in
the form of a tablet, powder or solution, it is preferable to affix
said composition on bibulous base materials or carriers such as
strips of filter paper by dissolving the components in a suitable
solvent, impregnating the strips with the resulting solution and
drying the impregnated test strips. Details of the compositions
contemplated are set forth in the following examples.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1
A composition was prepared by mixing the following components in
the volumes indicated below:
Glucose oxidase--5 ml. of an aqueous solution containing 1000
International units per ml.
Peroxidase--1 ml. of an aqueous solution containing 2 mg./ml.
Potassium iodide--160 mg.
Polyvinylalcohol--2 ml. of a 10% aqueous solution.
Dioctyl sodium sulfosuccinate--1 ml. of a 1% aqueous solution.
Tris-glutamate buffer--2 ml. of a 0.2 M solution of pH 7.
.alpha.-naphthoflavone--6 ml. of a 0.5% ethanol solution.
Water--5 ml.
Example 2
Another composition was prepared employing no peroxidase as shown
below:
Glucose oxidase--3 ml. of an aqueous solution containing 1000
International units/ml.
Potassium iodide--80 mg.
Ammonium molybdate--160 mg.
Sodium phosphate buffer--3 ml. of a 1 M solution of pH 6.
.alpha.-naphthoflavone--1 ml. of a 0.5% ethanol solution.
Water--5 ml.
Porous paper strips about 1/2 inch wide and 3 inches long were
dipped into the above compositions respectively so that about
one-half inch of each strip at one end was completely impregnated.
The strips were then dried at 100.degree. C. for 10 minutes. If
desired, other porous materials such as wood or plastics can be
used as carriers. When contacted with urine containing glucose,
such test strips give a positive reaction in 1 minute or less as
evidenced by the change in color of the indicator from colorless to
blue. The formation of color depends upon the liberation of free
iodine from the iodide by the action of hydrogen peroxide. When
dipped into urine containing no glucose, the strips show no color
change. The intensity of the blue color is enhanced in the presence
of polyvinyl alcohol. When molybdates and iodides are substituted
for the peroxidase, the .alpha.-naphthoflavone minimizes the
inhibiting effects of acetoacetate which is also an iodine
receptor. It was found that when the strips containing the
composition of Example 2 were dipped into a 1% aqueous solution of
glucose containing 0.1% by weight of acetoacetate they began to
change from colorless to blue in 30 seconds and the blue color
gradually intensified, whereas strips containing o-tolidine started
to show a blue color at 1.5 minutes with very little increase in
color intensity even after 5 minutes' contact with the same glucose
solution.
Example 3
A first solution was prepared containing 1.5 grams of carrageen, 15
grams of polyvinylpyrrolidone, 15 ml. of ethanol and 192 ml. of
water.
A second solution was prepared containing 9.24 grams of citric
acid, 40.79 grams of sodium citrate and 124.8 ml. of water.
A third solution was prepared containing 4.5 grams of a maleic
anhydride-methylvinylether copolymer, 1.5 grams of sodium lauroyl
sarcosinate and 105 ml. of water.
Still, a fourth solution was prepared containing 0.5 gram of
peroxidase and 76 ml. of an aqueous solution of glucose oxidase
containing 1,000 international units per ml. of water.
A composition for detecting H.sub.2 O.sub.2 and glucose was
thereafter prepared containing 9 ml. of a 0.5% ethanol solution of
.alpha.-naphthoflavone, 0.73 gram of potassium iodide, 9 ml. of
ethanol, 5.5 ml. of water, 34.5 ml. of the first solution above,
20.8 ml. of the second solution above, 17.5 ml. of the third
solution above and 7.6 ml. of the fourth solution previously
prepared. Bibulous paper strips were dipped in said solution and
then dried for 10 minutes at 100.degree. C. These strips readily
turned from colorless to blue when contacted with urine containing
lucose and the blue color increased with the glucose concentration.
Such strips also change from colorless to blue when a drop of
blood-containing urine and a drop of 3% hydrogen peroxide solution
is applied thereto.
In addition to the compositions set forth in the foregoing
examples, it was found that the amount of indicator employed could
be varied from 0.05% to 0.30% by weight in such compositions
whereas the glucose oxidase concentration could vary from 40 to 300
International units per ml. of peroxidase from 0.01% to 0.05%, the
sodium iodide from 0.25% to 1.5%, the ammonium molybdate from 0.1%
to 1.5%, and the polyvinyl alcohol from 0.2% to 2% by weight of
ultimate mix at a pH of from 4 to 7.
* * * * *