Method and reagent for determination of an analyte via enzymatic means using a ferricyanide/ferric compound system

A composition useful in determining analytes, such as glucose or cholesterol, is disclosed. The composition contains an enzyme which specifically reacts with the analyte to be determined, a soluble ferricyanide compound which is reducible to form a ferrocyanide, a soluble ferric compound which provides ferric ions for reacting with ferrocyanide to form a reaction product, and a buffer which does not prevent formation of this reaction product. The composition has a pH of from about 3.0 to about 6.0.

FIELD OF THE INVENTION 
This invention relates to an enzymatic method for determining an analyte in 
a sample, and a reagent composition useful in this method. Of particular 
interests are methods and reagents useful in determining one of glucose 
and cholesterol. 
BACKGROUND AND PRIOR ART 
The central concern of clinical chemistry is the qualitative and 
quantitative determination of specific analytes in samples. Of special 
concern is the analysis of body fluid samples, such as blood, serum, 
urine, and so forth. Determination of the presence and/or amount of 
various analytes, followed by comparison to established parameters 
determines diagnosis of diseased or abnormal states. 
The literature on analytical determination of body fluid samples is an 
enormous one, as the art has investigated the determination of, e.g., 
glucose, cholesterol, creatine, sarcosine, urea, and other substances in 
samples of blood, serum, urine, and so forth. 
The early clinical literature taught various non-enzymometric methods for 
determining analytes. Exemplary of this are the early glucose 
determination tests taught by Kaplan and Pesce in Clinical Chemistry: 
Theory, Analysis And Correlation (Mosby, 1984), pages 1032-1042. Such 
tests include the reduction of copper ions, reaction of copper with 
molybdate, and so forth. As this reference points out, these methods are 
insufficiently accurate, due to poor specificity and interference by other 
analytes. One method described by Kaplan, et al. is the alkaline 
ferricyanide test. This method involves heating a solution containing 
glucose in the presence of ferricyanide, at alkaline conditions. The 
reaction: 
##STR1## 
is accompanied by a change in color from yellow to colorless. Either this 
decrease in color is measured or the reaction of the colorless 
ferrocyanide ion with a ferric ion to form the intensely colored 
precipitate "Prussian Blue" is measured. 
Formation of Prussian Blue is an essential part of the invention described 
herein, so this test will be referred to again, infra. 
These early "chelation" type tests became replaced by more specific assays 
as enzymology became a more developed science. Enzymes are known for their 
extreme specificity, so via the use of an appropriate enzyme, the skilled 
artisan could determine, rather easily, whether or not a particular 
analyte is present, and how much. These enzymatic systems must be combined 
with indicator systems which, in combination with the enzyme reaction, 
form a detectable signal. Kaplan describes a glucose-hexokinase system, as 
well as a glucose oxidase system, and these are fairly well known to the 
art. They are used in connection with indicator systems such as the 
"coupled indicators" known as Trinder reagents, or oxidizable indicators 
such as o-tolidine and 3,3',5,5'-tetramethylbenzidine. In such systems, 
reaction of the enzyme with its substrate yields a surplus of electrons 
carried by the enzyme, which are removed by the indicator systems. Color 
formation follows, indicating presence, absence, or amount of analyte in 
the sample. 
The patent literature is replete with discussions of such systems. A by no 
means exhaustive selection of such patents include 4,680,259, 4,212,938, 
4,144,129 and 3,925,164 (cholesterol oxidase); 4,672,029, 4,636,464, 
4,490,465 and 4,418,037 (glucose oxidase); and 4,614,714 (L-glutamic acid 
oxidase). All of these enzymatic systems "oxidize" their substrates (i.e., 
the analyte in question) in that they remove electrons therefrom. 
Once the analyte loses its electrons, it plays no further part in the 
determination reaction. As indicated, supra, the electrons may be 
transferred into a color forming system, such as the Trinder system 
described in U.S. Pat. No. 4,291,121, or a tetrazolium system, such as is 
described in, e.g., U.S. Pat. No. 4,576,913. 
Indicator systems are not the only means by which the captured electrons 
may be measured, however. Free electrons produce an electrical current, 
which can be measured as an indication of analyte. Such systems are 
described by, e.g., 
Schlapfer, et al., Clin. Chim. Acta 57: 283-289 (1974). These systems 
employ substances known as "mediators" which remove the electrons from the 
enzymes. Eventually, the mediators release the electrons as well, 
producing a measurable current as a result. These mediators can either 
absorb one, or two electrons per molecule of mediator. Ferricyanide, the 
preferred mediator described in the Schlapfer reference, picks up one 
electron per molecule of ferricyanide. 
Electron mediators have been known and used in indicator systems in 
connection with so-called "dye molecules". The 4,576,913 patent, described 
supra, e.g., teaches the mediator phenazine methosulfate in combination 
with a tetrazolium salt. It is the latter which serves as the indicator. 
The use of these mediators enables one to proceed without oxygen. 
Normally, in a glucose determination reaction, oxygen is necessary to 
remove electrons from the reduced enzyme. This produces hydrogen peroxide: 
##STR2## 
with the hydrogen peroxide taking part, in the presence of peroxidase, in 
reactions leading to formation of a color. 
It is sometimes undesirable to use oxygen, or aerobic systems, because of 
various problems inherent in such systems. For example, in these 
reactions, the reaction is dependent on the partial pressure of O.sub.2 in 
the atmosphere. In addition, because the O.sub.2 must permeate throughout 
the entire test medium, the design of such media must be adapted to permit 
such permeatron. There is interest, then, in indicator systems which are 
anaerobic, such as those where a mediator is used in connection with the 
indicator, or electrochemical systems using the mediator alone. 
Electrochemical systems, while useful, are not always practical for 
frequent testing at the present time, and, in terms of home use, 
individuals who must measure glucose levels daily, are accustomed to 
systems where a color change is used. Therefore, there exists a need for 
anaerobic systems which utilize indicator reactions producing a detectable 
signal, such as a color. 
While indicator systems of the type described supra are available, there is 
a difficulty with these in that the indicator molecules themselves are 
frequently unstable and do not have long shelf lives. There is therefore 
an interest in systems which utilize stable molecules which can form a 
detectable signal. 
It will be recalled that Kaplan taught the formation of Prussian Blue in 
glucose determination, but dismissed it as a viable alternative because of 
the lack of specificity. Apart from this, the severe conditions under 
which the reaction are taught to take place are totally unsuitable for 
enzymatic assays. The reaction Kaplan teaches requires boiling the 
solution. Enzymes are protein molecules, and inactivation via denaturing 
is characteristic of what happens when proteins are boiled. Thus, the 
skilled artisan, seeing the heat parameters of Kaplan would avoid this 
teaching for enzymatic assays. 
Mention of the Prussian Blue system is found in the aforementioned U.S. 
Pat. No. 4,576,913. This patent teaches a glycerol dehydrogenase which 
operates in a fashion similar to oxidases in that it teaches removal of 
two electrons from its substrate molecule. Column 5 of the patent refers 
to the Prussian Blue system (referred to as "Berlin Blue") as an the 
indicator. 
This patent, however, must be read as a whole, and especially its teaching 
about the enzyme's operability. Enzymes are extremely pH sensitive, and 
the enzyme of the Adachi patent is said to operate in a pH range from 6.0 
to 10.0, and optimally at 7.0 to 8.5. The teachings, therefore, would 
suggest to the artisan that since the glycerol dehydrogenase operates at 
alkaline pHs, the adaptation of the Prussian Blue system to enzyme 
detection would be at alkaline pHs. However, ferric salts precipitate at 
alkaline pHs, which would eliminate them from participating in a reaction 
to form Prussian Blue under the conditions Adachi describes as necessary. 
The inventor has now found, quite surprisingly, that enzymatic assays can 
be performed using the formation of Prussian Blue. These assays do not 
involve the use of parameters which risk inactivation of the enzyme, such 
as high heat. The invention is based upon the discovery that, upon 
addition of sample to a reagent combination containing an enzyme, such as 
glucose oxidase or cholesterol oxidase, a ferricyanide salt, and a ferric 
salt buffered at a pH below which the enzyme is expected to be 
inactivated, the enzyme reactions nonetheless take place, and the buffer 
does not interfere with the reaction of ferrocyanide and Fe.sup.3+. 
Hence it is an object of this invention to provide a reagent composition 
useful in determining an analyte in a sample, which comprises an enzyme 
specific for the analyte to be determined, a ferricyanide compound, and a 
soluble ferric compound, wherein the reagent composition is at a pH below 
7. 
It is a further object of the invention to provide apparatus, such as test 
strips which can be used to determine an analyte which incorporate into 
reagent carriers the above described reagent composition. 
Yet another object of this invention is to provide a method for 
determination of an analyte, comprising contacting a sample to the reagent 
composition described supra, and measuring formation of Prussian Blue, 
i.e., the complex of ferrocyanide and ferric ions as a measure of said 
analyte. 
As has been described, this invention is based on the starting and 
unexpected finding that the ferricyanide/ferric salt system is operable at 
acidic pH, and under conditions where the enzyme being used would normally 
be inactive. 
How the aforementioned objects of this invention are achieved will be seen 
in the following Detailed Description of Preferred Embodiments. 
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
In its simplest form, the invention is a reagent composition for 
determining an analyte, which comprises an enzyme which specifically 
reacts with the analyte to be determined, a soluble ferricyanide ion 
containing compound which, in the presence of a free electron, is reduced 
to a ferrocyanide ion containing compound, a soluble ferric ion containing 
compound which reacts with the ferrocyanide ion containing compound to 
form a reaction product, and a buffer which does not prevent reaction 
between said ferrocyanide and ferric ions, wherein the composition has a 
pH from about 3.0 to about 6.0. Especially preferred is a pH of from about 
3.9 to about 4.6. 
By an enzyme "which specifically reacts with an analyte to be determined", 
the skilled artisan recognizes that the enzyme reacts with the analyte 
being determined, to the exclusion of others. For example, glucose oxidase 
specifically reacts with glucose. 
A "soluble ferricyanide containing compound" refers to a compound which is 
soluble in the fluid sample being analyzed, and which contains the well 
known ferricyanide ion. When dissolved in the sample, the ferricyanide ion 
become available for reduction by a free electron to form ferrocyanide 
ions. 
A "soluble ferric compound" similarly, means a compound which dissolves in 
the sample, freeing ferric ions. These ions then react with the above 
mentioned ferrocyanide ions to form a reaction product. 
The buffer "does not prevent" the reaction between ferrocyanide and ferric 
ions, meaning that, although it may indeed compete for reactions 
therewith, this competition is not sufficient to prevent formation of the 
reaction product referred to supra. 
If a quantitative test is desired, the ferricyanide containing compound 
should be present at least in an amount sufficient to accept all of the 
electrons donated by a substrate/analyte molecule in its reaction with the 
specific enzyme. Most analytes, in, e.g., biological samples, are present 
in a defined range. Knowledge of this range, which can be ascertained, 
e.g., from The Merck Manual or Diagnostic Tests Handbook or any of the 
numerous clinical chemistry references available to the artisan. One uses 
an amount of ferricyanide in excess of the amount necessary to react with 
all of the particular analyte at the top of the maximum range in a given 
sample. Similarly, the amount of ferric compound to be present can be 
calculated fairly easily, as the ferric and terrocyanide ions react 1:1 
molar basis. Precise amounts of reagents are not required, however, when 
one is performing an assay to determine a general range of analyte, or is 
performing a "yes/no" test. 
The choice of ferricyanide and ferric compounds available to the artisan is 
a wide one, the only constraint being that they be soluble in the test 
sample. Examples of the salts which may be used are potassium ferricyanide 
and ferric chloride, although others will be known to the skilled artisan, 
the only constraints upon the choice being those described supra. The pH 
range recited supra may be maintained, e.g., via the use of an appropriate 
buffer. The preferred buffer is 4-amino butyric acid, although as long as 
the buffer operates in the recited range, and does not chelate the ferric 
ion to a degree that reaction with ferrocyanide is inhibited, any buffer 
will be suitable. 
The enzyme chosen will, of course, depend upon the analyte to be detected. 
Of particular interest are glucose determination tests, where the enzyme 
used is glucose oxidase. Other enzyme systems may be used, and it is well 
within the skill of the art to determine if, in fact they are operable 
within the recited pH range. 
The reagent composition may be used in the form of a solution, e.g., or a 
lyophilizate, a tablet, and so forth. Further, the reagent may be used in 
the form of a kit, where the individual components are kept in separate 
containers to be combined just prior to use. 
The reagent composition may also be applied to products such as test 
strips. In such situations, the reagents are impregnated or incorporated 
into carrier materials such as fleece, films, and so forth. A detailed 
roster of these is not given there, as the art is quite familiar with the 
test carrier literature and the many available options. 
The ferrocyanide/ferric ion complex, as described supra, is a heavy, 
intensely colored material which precipitates out of solution. In view of 
this, it is desirable, though not necessary, to incorporate into the 
reagents and test carriers components such as surface active agents which 
keep the precipitate in solution or additives which modify the color 
somewhat to ease its intensity. The inert white pigment TiO.sub.2 is 
especially preferred for this. 
Carrying out the method of this invention is quite simple. The test sample 
blood, serum, urine, etc., is mixed with the reagent, regardless of its 
form, and one observes formation of the ferrocyanide/ferric complex or 
lack thereof. 
The following examples demonstrate the operability of the invention 
although they are not to be read as being in any way limitative of the 
broad disclosure contained herein.

EXAMPLE 1 
A buffered solution was prepared via dissolving 60 mmoles of 4-amino 
butyric acid in 50 ml of water, together with 1.8 mmoles of ferric 
sulfate. The pH was adjusted to 4.0 using IN H.sub.2 SO.sub.4. 
Thirty kU of glucose oxidase was added, together with 20 mmoles of K.sub.3 
[Fe(CN).sub.6 ], and water to obtain a final volume of 100 ml. This 
solution is referred to hereafter as "solution A". 
One ml of solution A was then combined with 50 ul of one of (i) distilled 
water; (ii) 6 mM glucose solution or (iii) 600 mM glucose solution. Color 
formation was then observed as follows: 
TABLE 1 
______________________________________ 
Test Solution Solution A 
______________________________________ 
Water NONE 
6 mM glucose slight blue color 
600 mM glucose heavy blue color. 
______________________________________ 
EXAMPLE 2 
The effect of pH on the test systems of the invention was examined. To do 
so, solutions were prepared which contained 200 mM of 4-amino butyric 
acid, 20 mM ferric sulfate, 80 mM K.sub.3 [Fe(CN).sub.6 ], and 450 U 
glucosr oxidase/ml. The pH of the solutions were adjusted as indicated, 
using either 4N H.sub.2 SO.sub.4 or 4N KOH. 
To test the pH effect, a glucose solution was added in each solution with a 
different pH to give a final concentration of 7.1 mg/dl of glucose, and 
color formation was observed after 1 minute. This was compared to a 
control, which was read after fifteen minutes. The degree of color 
formation is indicated via the number of plus signs in the following Table 
2. 
TABLE 2 
______________________________________ 
pH Control Sample 
______________________________________ 
3.05 ++ + 
3.30 + +++++++ 
3.60 + +++++++ 
3.92 - +++++ 
4.32 - ++++ 
4.50 - ++ 
4.80 - + 
5.20 - + 
______________________________________ 
This experiment shows that the invention operates over a range of pHs from 
about 3.0 to about 6.0. The preferred range is a pH of from about 3.3 to 
about 4.5. In a particular embodiment of the invention, a pH of from about 
3.9 to about 4.0 is preferred. 
The excellent results obtained at the low pHs are quite surprising in view 
of the teaching of the art regarding the operability of enzymes at 
different pHs. Bergmeyer, Methods of Enzymatic Analysis, Vol VI, at page 
180, for example, teaches the pH optimum of glucose oxidase is 5.6. 
EXAMPLE 3 
Test strips were prepared and used in order to show the use of the 
invention in reflectance assays. 
A coating mass was prepared which contained, per kilogram of the coating 
mass, 30 g. 4-amino butyric acid, 3.9g of ferric sulfate, 36g of 
ferricyanide salt, and 1000 ku of glucose oxidase. Nonreactive ingredients 
which made up the remainder of the mass included TiO.sub.2 as a white 
pigment, TWEEN-20, (nonionic surfactant), PROPIOFAN-70D (film former) and 
NATROSOL (swelling agent). The pH of this mass was 4.1. 
The mass was coated onto a clear, polyester foil of 100 um thickness, and 
was allowed to form a coating. The thickness of the coating was 150 um. 
Glucose solution of various concentrations were applied to the films, which 
were evaluated one minute thereafter in a reflectance photometer 
(Macbeth), following standard protocols well known to the art. The 
percentage of reflectance for each solution was measured at 660 
nanometers. The results are presented in Table 3: 
TABLE 3 
______________________________________ 
GLUCOSE CONCENTRATION 
(mg/dl) % RELFECTANCE 
______________________________________ 
0 78.46 
50 63.93 
100 54.03 
150 48.40 
230 41.82 
300 37.63 
400 33.72 
______________________________________ 
The values obtained show that the percentage of reflectance decreases as 
the concentration of the analyte being measured (glucose) increases. As 
such, the system is functional in test strip form, using reflectance 
photometry. 
EXAMPLE 4 
Experiments were carried out to test the invention in connection with the 
enzyme cholesterol oxidase. 
A test solution was prepared which contained 50 mM K.sub.3 [Fe(CN).sub.6 ], 
2.5 mM FeCl.sub.3, and 50 mM 4-aminobutyric acid per liter, and which had 
a pH of 4.0. 
Cholesterol oxidase was then added to a control sample, and a sample to the 
above test solution which also contained 100 mg/dl of cholesterol. The 
cholesterol oxidase was present at a concentration of 2 u/ml of sample. 
The samples were then evaluated in a photometer to determine if there was a 
change in absorbance, and if so, how much. The values obtained are 
presented in Table 4. 
TABLE 4 
______________________________________ 
Cholesterol 
concentration absorbance change 
(mg/dl) after 1 minute 
______________________________________ 
0 0.012 
100 0.355 
______________________________________ 
The control value is used for calibrating the assay, as will be recognized 
by the skilled artisan. The change in absorbance with the cholesterol 
concentration shows that the invention can be used with cholesterol 
oxidase. 
The foregoing examples show the operation of the invention at various pHs 
and reagent formulations, using diverse enzymes. It has been shown that 
formulations including solutions and test strips can be used to carry out 
the invention. The skilled artisan will note the easy adaptability of the 
system to include, e.g., powdered formulations or formulations where, 
e.g., the enzyme is immobilized on an inert bead or other type of carrier, 
with contact by the other components o& the reagent formulation. 
The skilled artisan will note as well that formulations such as reagent 
kits can also be prepared. Reagent kits contain the elements of the 
invention, e.g., the enzyme, the ferricyanide and ferric salts and the 
buffer in separated containers. These containers are in turn encompassed 
by a container means which holds all of them, such as a box or rack. The 
separated components can be presented as solutions, powders, 
lyophilizates, impregnated on carriers, and so forth, or in any of the 
other reagent formulations recognized by the art. 
In practice, the invention is of course used by contacting a liquid sample, 
such as blood, serum, urine, and so forth to the reagent composition. 
Formation of the Prussian Blue complex is measured as an indication of the 
analyte being assayed. This methodology does not vary, regardless of what 
form the composition takes, which includes it formulation as an analytical 
element or test strip, as described herein. 
It will be understood that the specification and examples are illustrative 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.