CA1295216C - Rainbow test device - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE Test compositions and test device are pro-vided which are capable of generating different hues at different analyte concentrations. The compositions are capable of generating a yellow hue in situ. Visual tests for clinically important analytes, such as glucose, are deter-mined by use of two independent catalytic systems which are reactive with reduced nicotinamide adenine dinucleotide to produce a range of hues; the particular hue produced depending on the concentration of the analyte. The invention provides a test device for the determination of analyte, e.g. glucose, in body fluid which exhibits a RAINBOW of hues, the particular final hue produced depending on the analyte concentration.

Description

~2~5~1~

RAINBOW TEST DEVICE

I~ Field of the Invention The invention relates to test compositions and test devices capable of generating different hues at different analyte concentrations. Visual tests for clinical analytes are the focus of the invention.
In particular, the invention provides a self-indicating test device for the determination of analytes, e~g. glucose, in body fluid which exhibits the colors of the RAINBOW.

II. Utility Colorimetric tests are conveniently used as visual tests with which relatively untrained personnel can routinely obtain results by simple comparison to an appropriate color chart. Visual tests are low cost and convenient since no instru-mentation is required. Presently, visual tests are used for routine screening of urine samples ~0 for a number of diagnostically important analytes, used by diabetics for home testing of urine or blood glucose and used in other fields, for example water testing for iron content.
However, currently available tests relate the intensity of a particular color to the ~k ;21~

concentration of analyte. For example, a test device may change from colorless to light blue to darker shades of blue with increasing concentra-tion of glucose. Greater visual discrimination, and therefore greater accuracy, is possible, when a range of colors is provided rather than dif-ferent shades of a single color. Therefore a test composition which exhibits different colors at different analyte concentrations would be easier to use and would provide more accurate visual results.
In order to allow visual differentiation of higher concentrations of glucose, many currently available products resort to the use of two reagent pads, one of which provides better color diferentiation at the higher concentration range.
Otherwise such products would exhibit only very slightly differing shades of dark blue (or dark green) above 150 mg/dL glucose. In contrast, a ~0 test device of this invention can produce dramatic color changes, blue to sea green to light yellow to red, over a range of 0 to 800 mg/dL.
This invention provides compositions which can be used to produce a range of colors, espe-cially for clinically important analytes. Inparticular, a test device, capable of generating the hues of a full spectrum RAINBOW~ for the determination of glucose in a body fluid, such as whole blood, is shown.

... . . .. .. .

;
, ;:

III. Information Disclosure U.S. Patent No. 4,490,465 discloses a test system for the determination of glucose having an extended range of measurement. The system in-volves at least one pyridine linked dehydrogenaseand one nonpyridine linked dehydrogenase. One example shows a determination of glucose with a coupled system of glucose dehydrogenase/nicotine adenine dinucleotide/diaphorase/tetrazolium salt and glucose dehydrogenase/dichlorophenolindo-phenol. A yellow component is introduced into the colors obtained by the use of a background dye.
DE 32 11 167 claims at least two enzyme systems each of which is independently capable of catalyzing the direct or indirect conversion of a substrate. The specification defines "independent of one another" to mean that, in the slmultaneous presence of the systems that react with the substrate, the reaction through the second system takes place only after the coenzyme of the first system has been largely consumed.
DE 32 47 894 discloses a test system and method for the determination of reduced nicotine adenine dinucleotide, which produces an enlarged measuring range for the determination of NAD(P)H
or substrates or enzymes reacting under the formation or consumption of NA~(P)H. The system is characterized in that it contains simulta-neously several substances with different electro-chemical potentials, functioning independently ofone another as electron acceptors for NAD(P)H.

..

.. .. .

~i9~i216 Particular examples of electron acceptors include dichlorophenolindophenol and INT, (2-(4-iodo-phenyl)-3-(4-nitrophenyl)-5-phenyltetra~olium chloride). The specification states that the test system can be impregnated into absorbent mate-rials. A yellow component is added to the color seen by incorporation of a background dye, titanium yellow.
Japanese Patent Application 59-106299 was published June 19, 1984. The application dis-closes a method for estimating NAD(P)H with oxidized glutathione in the presence of gluta-thione reductase and a color forming agent.
Examples of color forming agents given are 5,5'-dithiobis(2-nitrobenzoic acid), N-(l-anilino-naphthyl-4)maleimide, Beta-hydroxyethyl-2,4-dinitrophenyldisulfide, 2,2-dithiopyridine and benzimidazolyl maleimide.
European Patent ~pplication 0-153-872 dis-closes a method for the determination of thereducéd form of nicotinamide adenine dinucleotide (phosphate) which involves reacting NAD(P)H with (l) peroxidase or thiol oxide reductase and (2) diaphorase or an electron carrier in the presence of a chromogen and determining the pigment thus formed. These two reactions do not act on a common substrate.
None of these disclosures provides a system composed of two independent catalytic systems, one being a disulfide reductase system, which allows the production of a full spectrum RAINBOW, .

... . ......

: ~ .
,; ':
: : ' ' ' , ' including a yellow component generated in situ;
the particular final hue produced depending on the concentration of the analyte.

IV. Summary of the Invention The invention provides a test composition for the visual determination of the concentration of an analyte in a fluid sample, comprising:
(a) a catalytic system capable of generating reduced nicotinamide adenine dinucl~otide by reaction with the analyte of interest; (b) a first independent catalytic system capable of . generating a reduced first indicator by reaction with reduced nicotinamide adenine dinucleotide;
and (c) a second independent catalytic system capable of generating a change of hue of a second indicator component by reaction with reduced nicotinamide adenine dinucleotide, which second independent catalytic system includes (i) a disulfide reductase;
(ii) a disulfide substrate; and (iii)a thiol indicator, wherein the disulfide reductase is capable of catalyzing the reaction between reduced nicotinamide adenine dinucleotide and the disulfide substrate to produce a product which can interact with the thiol indicator to produce the change of hue of the second indicator;
whereby, the generation of the reduced first indicator and the change of hue of the second indicator can be controlled to provide a range of hues, the particular final hue produced by the - ~Z~5216 test composition depending on the concentration of the analyte.
Preferably one system produces a yellow hue.
In one preferred system, the thiol indica~or is reduced to produce a yellow second indicator.
The composition can be dissolved to provide a test solution or incorporated in a carrier matrix to provide a test device format.
A preferred embodiment is a glucose whole blood test device which is capable of generating a full spectrum rainbow, including a yellow hue generated in s~tu, the particular final hue of the test device depending on the concentration of glucose in the test sample.

V. Description of the Invention Visual color matching is convenient and it can provide an acceptably accurate determination of analyte concentration without the need for expensive instrumentation. Color can be broken into components such as saturation, lightness and hue. Hue is commonly referred to as "color" e.g., . whether something "looks" blue, red or yellow.
Throughout the specification, the term "hue~ is used.
Generally, one hue is associated ~it~ ~l ?
form of a single indicator. Ther~Ic~ d?^
to generate a range of hues, a number of different indicator molecules are required. The possible changes of hue are numerous. An indicator can change from one hue to another, from one hue to - ; ' , :, ~5~16 colorless or from colorless to a hue. It is even possible that the indicator itself will not change hue but that the format of a device incorporating the test composition is such that there is an apparent change in hue of the device when con-tacted with a test sample containing the analyte.
For example, the hue of the indicator could not be seen prior to the reaction of the test composition with the analyte but is visible after that reaction occurs. Because it is desirable that the change of final hue exhibited by the test device from one analyte concentration to another be as clear to the user as possible; the changes colorless to a hue and a hue to colorless have been preferred. When using two or more indicators, at least one indicator component is preferably changed from one hue to colorless.
Otherwise the final hue of the test composition or device will move toward black as more hues are ~0 produced.
It is particularly desirable to control the production and/or disappearance of indicators which in one form exhibit one of the primary hues:
red, yellow, blue. While a composition containing two indicator components can be used, the use of three indicator components has been found to be advantageous.
For example, consider a test composltion containing indicators which exhibit the following change in hue in the order shown.
indicator 1) blue to colorless .
. ' ~.

indicator 2) colorless to yellow indicator 3) colorless to red If the indicators react in sequence, the apparent hue of the test composition would be blue to r 5 colorless to orange (yellow + red).
However, if indicator 2 changed hue concurren~ly with the change in hue of indicator 1, the change in hue of indicator 3 occuring later, the apparent hue of the test composition would be blue to green (blue + yellow) to yellow to orange (yellow + red) to red. A similar example is:
indicator 1) red to colorless indicator 2) colorless to yellow indicator 3) colorless to blue If the indicators reacted in sequence, the ap-parent hue of the test composition would be red to colorless to yellow to green (blue + yellow). If the indicators reacted as described above, with ~0 two indicator changes being produced siT ltaneous-ly, the ~hird change being delayed, the apparent hue of the test composition would be red to orange (red + yellow) to yellow to green (yellow + blue) to blue.
Both of these schemes! containing simulta-neous hue changes, could produce a full spectrum RAINBOW. Such a RAINBOW would be desirable because it would provide the widest range of color visible to the eye which could be used to make the differentiation between levels of analyte easier to interpret.

~ . , .
- , ,,, . ', '' , ' , . ... . . .

, \
-` ~2~35~6 The problem is twofold: 1) to select the indicators having changes in hue induced indepen-dently to give the maximum change in hue, and 2 to ensure that these indicators react in an orderly fashion depending only on the concentra-tion of the analyte.
It has been found that a range of hues can be produced by using two indèpendent catalytic systems, each reactive with reduced nicotinamide adenine dinucleotide (NADH) to produce one or more changes in hue. The sys.tems react substantially simultaneously with NADH. The hue produced by a particular analyte concentration can be controlled by the relative increase or decrease of the concentrations of the components and catalysts in the test composition.
Each independent catalytic system is a system capable of interacting with one or more indicators in the presence of NADH to produce a change in hue of the indicator(s). One system contains at least an oxidized indicator and a catalyst capable of facilitating the reduction of the oxidized indica-tor in the presence of NADH. The other system contains at least a disulfide substrate, a thiol indicator and a disulfide reductase (the catalyst). The disulfide reductase system can function in three general ways: 1) the thiol indicator can interact with the product formed by the reaction of NADH and the disulfide substrate to produce the change of hue of the second indicator; 2) the thiol indicator can be reduced i;216 to form a reduced second indicator; and 3) that second indicator, in either case, can have a yellow hue. It has proven to be particularly difficult to generate a yellow hue in situ.
The terms catalyst and catalytic are used in their conventional sense herein. A "catalytic"
reaction is a reaction in which the rate is changed by the addition of a "catalyst" but the catalyst itself is unchanged. The catalytic reactions referred to herein are usually enæy-matic, but can also include nonenzymatic reactions such as those catalyzed with phenazine metho-sulfate. The term "independent" means that the oxidized indicator(s) being reduced in one cata-lytic system is not in equilibrium with the oxidized indicator(s) beins reduced in the other catalytic system during the reaction time in-volved.
The NADH is generated from the analyte of ~interest by a catalytic system which is usually enzymatic. Table 1 shows analytes of clinical interest and useful enzymatic systems which can produce NADH. These reactions and the reaction components required are well known and are pres-ently the basis for many diagnostic reactions which`generate changes in intensity of a single hue in response to analyte concentration. The enzyme system capable of generating N~DH from the analyte of interest is commonly a dehydrogenase system, although any system capable of generating NADH can be used.

.~ .
, . : .
' ' ~ - '~; ' ' ' ` , . .
. .
: . . .
: I ., . . .

, analyte enzyme glucose glucose dehydrogenase hexokinase/glucose-6-phosphate dehydrogenase cholesterol cholesterol dehydrogenase alcohol alcohol dehydrogenase lactate lactate dehydrogenase The reduced form of nicotinamide adenine di-nucleotide, NADH, has been found to be a particu-larly useful common substrate. Although this specification refers exclusively to nicotinamide adenine dinucleo,tide, NAD, and its reduced form NADH, it is to be understood that the disclosure applies equally to the phosphorylated forms NAD(P) and NAD(P)H.
Catalytic systems useful for the common substrate NADH, based on diaphorase or catalysts having diaphorase like activity, such as phenazine methosulphate and l-methoxyphenazine metho-sulphate, will now be described in detail.
The diaphorase system has been found to be useful as one pathway because two oxidized indica-tors are readily available which exhibit distinct changes in hue. DCIP, 2,6-dichloroindophenol, is a blue compound which is reduced to a colorless form in the presence of diaphorase and NADH. INT, 2-(4-iodophenyl)-3-, :

~2~ 6 (4-nitrophenyl~5-phenyltetrazolium chloride, is colorless in the oxidized form but becomes red when reduced in the presence of diaphorase and NADH. The two changes in hue are sequential.
Other indicators reactive with diaphorase and NADH
can be used. For instance, N-(2,3-dimethyl-5-oxo-l-phenyl-3-pyrazolin-4-yl)- 2-chloro-6-sulfo-4-iminobenzoquinone, referred to for convenience as TR-l, and p-nitroblue tetrazolium chloride, referred to herein as NBT, can be substituted for DCIP and INT. Other indophenols and related substituted alkyl, nitro, halogen and pseudo-halogen derivatives can be substituted for DCIP, as ~ell as other indicators of similar reduction potential capable of being reduced in the presence of NADH. Other tetrazoliums can be used in place of INT. Many are known in the art and have been catalogued in reviews such as "An Introduction to the Use of Tetrazolium Salts in Quantitative Enzyme Chemistry", F.P. Altman, Koch-light Labora-tories, Ltd., Colnbrook, England, 1972; and "The Chemistry of Formazans and Tetrazolium Salts", A.W. Nineham, Chem. Rev., 55:355 (1955).
A second parallel pathway is provided by the second independent catalytic system based on a disulfide reductase. A number of reductase systems are shown in Table 2 below.

- ` ' , ' ~: :

2:~6 _ disulfide substrate disulfide reductase L-cystine cystine reductase oxidi~ed glutathione glutathione reductase lipoamide dihydrolipoamide reductase (referred to as lipoamide dehydro-genase herein) protein-disulfide protein-disulfide reductase oxidi~ed thioredoxin thioredoxin reductase CoAS-Sglutathione CoAS-Sglutathione reductase asparagusate asparagusate reductase The disulfide reductase system has three components: a disulfide substrate, a disulfide reductase capable of facilitating the reaction between the disulfide substrate and NADH, and a thiol indicator which can interact with the product of the reaction between the disulfide substrate and NADH to produce the change of hue of the second indicator. A preferred disulfide substrate is lipoamide and analogs thereof which can be used with lipoamide dehydrogenase. The disulfide reductase system has been found to be particularly useful for introducing yellow into the range of hues generated by the test composition.

. ~ `" ' ~2~5;~6 The thiol indicator is any substance which will interact with a thiol (-SH compound) to give observable color. Prefered thiol indicators are colorless indicators which become yellow upon interaction. Commonly, the thiol indicator is an oxidized indicator which is reduced in the presence o the product of the reaction between the disulfide substrate and NADH to produce a reduced second indicator which can be, but need not be, yellow. However, other types of interaction which will produce color are also contemplated. For example, thiol indicators can be chelating agents such as nitroprusside, which would interact with the product of the disulfide substrate/NADH reaction to produce a red hue. A
palladiumcomplex can also be used to generate red.
A yellow hue can be generated by interaction of the thiol indicator with the product of the disulfide substrate/NADH reaction by alkylation if 2~ the product is cysteine. Alternatively a cysteine product could be reacted with noradrenochrome to produce a yellow hue.
Another general type of thiol indicator is a chromophore of the desired hue, preferably yellow, 2S immobilized behind an opaque barrier. On xeaction with the product of the disulfide substrate/NADH
reaction, the chromophore is released from its attachment and is free to diffuse through the opaque barrier to a position where it is visible to an observer.

.. . . :
~.
. . .

, g~Z~6 Commonly, the thiol indicators which undergo reduction are disulfide compounds. Especially preferred are analogs of 5,5'-dithiobis-(2-nitro-benzoic acid) referred to herein as DTNB, whose structure is shown below:

HO-C ~-OH

N2 ~} S--S ~N2 DTNB is commonly referred to as Ellman's Reagent.
Changes at the carboxylic acid hydroxyl group have been found to be useful. Analogs of DTNB, which are defined herein to include positional isomers of DTNB, of the structure shown below are pre-ferred:

R-~ -R
~-S-~
02N ~ 2 .
.

. ,,:, :
... , . . ... ~.
., .~ . . .. ,..... : .

.: ~

R can be many groups such as those providing an ester or amide linkage, e.g., -O(C2H4o)4H; -N N-CH3;

-NH-N(CH3)2; -N ;

While water solubilizing groups such as those shown above are preferred in gelatin matrix formats, water insoluble analogs can be used in compartmentalized formats,described later in the specificàtion.
A preferred water soluble analog compound is 3-N-(3-dimethylaminopropyljcarboxamido-4-nitrophe-lQ nyl disulfide, structure shown below, whosesynthesis is detailed in the examples.

(H3C)2N(C3H6)HN8 ~-NH(C3H6)N(cH3)2 02N ~ S - S ~ NO~

The compound is referred to subsequently in the specification as 3 ND for convenience. This compound is colorless in the oxidized form and 1~ becomes yellow in the presence of lipoamide, NADH
and lipoamide dehydroyenase.
Hues of particularly useful indicators are shown in Table 3 below.

~ .
,. :

' ' , ~ : .

-` ~Z~.52~

oxidized reduced DCIP blue colorless INT colorless red TR-l red colorless NBT colorless blue DTNB colorless yellow 3-ND colorless yellow EAl colorless yellow It has been found that a color retardant can be added to the composition. The color retardant has essentially no affect on the hue visible to the user, but its inclusion can delay the cata-lytic affect of the independent systems, delaying the reduction of the oxidized indicators and therefore changing the final hue produced for a particular concentration of analyte. Useful color retardants include potassium ferricyanide and l-N-ethyl-4-methylquinolinum iodide and analogs thereof or mixtures of these compounds, potassium ferricyanide being preferred. Color retardants effectively change the measurement range of the composition.
Other components such as buffers, surfactants and polymers can be added to the composition. The pH is generally chosen to give good performance and stability to the reagents and is controlled by use of buffers. The use of buffers is preferred since the enzymes function better within the pH

'~ :
' ' ' :

~L2~;216 range of about 6.0 to 8. Choice of a buffer is within the skill of the art. Useful buffers include, but are not limited to, N-2-hydroxyethyl-piperazine-N' 2-ethanesulfonic acid (HEPES), 2-[tris(hydroxymethyl)methyl]amino ethanesulfonic acid (TES), 2-[N-morpholino]-ethanesulfonic acid (MES) and [3-(N-morpholino)propanesulfonic acidj (MOPS). Surfactants and polymers can he particu-larly useful in formulations containing a cationic tetrazolium salt as an oxidized indicator since surfactants such as polyoxyethylene ether, avail-able under the trademark TRITON X-100 from Sigma Chemical Co., and polymers such as polyvinyl-alcohol and polyvinylpyrrolidone, available as PVP
K30 from Aldrich Chemical Co., appear to help solubilize the cationic indicator and prevent interaction with the other indicators in the test composition. Surfactants also improve wettability of the device in a dry phase formulation. Enzyme stabilizers~ such as bovine serum albumin, can also be added.
Test compositions of this invention can be used by dissolving the composition in a solution or they can be incorporated into a carriex matrix ~5 and affixed to a support member such as a poly-ester strip to provide dry reagent strips which are well known in diagnostics. These strips provide a format which is convenient to carry and store and which is particularly useful to home users such a diabetics. Preferred compositions of this invention generate a final hue, which can be . .. :. . ., ::

associated with a particular analyte concentra-tion, in less than about ten minutes.
The carrier matrix employed can be any of several known in the industry, as long as the matrix can be incorporated with the composition and it does not interfere with the reactions required for the production of color. These include paper and films such as those made from natural polymers, latexes, polyurethanes, silicones or combinations of these.
In order to obtain the clearest colors possible, a clear carrier matrix was preferred.
Since common analytes for this invention are water soluble compounds such as those found in body fluids, carrier matrices which can contain water, such as hydrophilic carriers, are preferred.
Suitable hydrophilic carriers include agarose, gelatin, poly(vinyljalcohol, poly(propylimine), carrageenan and alginic acid. Other carriers could be used. Mixed multilayer carriers composed of an absorbent opaque matrix, such as paper, and a hydrophilic (e.g., gelatin) carrier layer can be advantageously used when the test components are compartmentalized.
In a preferred embodiment, a solution of 1.25% carbodiimide was used to crosslink a multilayer gelatin carrier matrix . This provides a format suitable for a whole blood glucose test which allows the blood sample to be removed from the test device by wiping the surface. Other coating materials, well known in the art, can be , ~ , .

.' ' .

' '' ' , : .

2~S216 used to allow the device to be washed or wiped to remove a colored sample if necessary.
The hydrophilic carrier layer or layers are coated onto a rigid backing or support member such as polystyrene, polyester and the like. The backing can be opaque or transparent, although an opaque white backing is commonly preferred for visually read tests.
The number and types of components, which can be used in the independent catalytic systems described previously, is increased by the use of compartmentalization of possibly incompatible components in a test device format. Compart-mentalization can take on many forms. Components can be separated by placing some in a separate layer, by solubilizing within one phase of an emulsion, by precipitation, by encapsulation and so forth. Some of the available methods are described in detail in the Examples.
A multilayer gelatin carrier was preferred for compartmentalization. INT could be placed in one layer away from the other indicator compo-nents. It was also found that the position of the components in various layers could change the hue visible to the user at a particular analyte concentration and therefore~afforded another means of controlling the hue generated. The apparent hue of the device can be changed by changing~the order or thickness of layering.
A particular example showing the affect of compartmentalization of components in different .
~ MS-1444 .

. :

.: . . ~ : . .:

... ,. ~

;2~ E;

layers on the hue visible to the user will now be described in detail.
A rainbow test device for the determination of glucose can be prepared with the following in-dependent catalytic reactions:
NADH generating system:
glucose (analyte, from the sample) glucose dehydrogenase (enzyme) NAD (additional component) 1~ First independent catalytic system:
diaphorase (catalyst) DCIP (oxidized first indicator, blue) INT (oxidized third indicator, colorless) DCIP was reduced by NADH first. Therefore the first independent catalytic system produces a blue to colorless, then a colorless to red, change in the hue visible to the user.

Second independent catalytic system:
lipoamide (disulfide substrate) lipoamide dehydrogenase (disulfide reductase) DTNB (thiol indicator, oxidized second indicator, colorless) Compartmentalization of the oxidized indica-tors, e.g., placing the indicators in different ~elatin layers, can effectively change the hue seen at different glucose concentrations even though the concentrations of test components are the same. As an example, three films were made with the indicators arranged in different iayers, but keeping the concentrations of the components of the total test composition the same.

......

: . . .. .
.. ~.,. . : ~
: .

- ~2~5;~6 . .
DCIP, DTNB, Enzymes FILM A INT _ ~ INT
FILM B ~CIP, DTNB ! Enzyme¦

Enzyme FILM C INT
DCIP, DTNB

In gelatin, certain water soluble molecules, such as DNTB, are capable of diffusing through the gelatin after contact with an aqueous sample, while others, such as INT, do not readily migrate.
In Film A, glucose reacts with the enzymes, then the NADH produced reacts with the independent catalytic systems to reduce DCIP and DTNB.
However, reduction of INT is delayed~until the NADH can diffuse into the Iower layer of the film.
At 250 mg/dL glucose, Film A is yellow orange.
In Film B, the glucose must diffuse through the INT layer before reaching the enzymes to react ~ and produce NADH. With the production of NADH, the DCIP will be reduced but the~reduction of INT
is delayed since the NADH must diffuse back up onto the top layer before the INT can be reduced.
Therefore, at 250 mg/dL glucose, Film B is yellow-green. The compartmentalization of INT above theenzymes and other indicator components has changed the color observed at this particular concentra-tion of glucose.
In Film C, the glucose reacts with the enzymes in the top layer. As the NADH migrates through the INT layer, a small amount of INT
reduced causing a slight red color. As the NADH
migrates through to the bottom layer, the DCIP and the DTNB are reduced. Finally/ since not all the NADH has reacted due to the concentration of 1~ components chosen, the NADH will react with the INT. At 250 mg/dL glucose, Film C is a reddish orange.
The ultimate goal of the invention was to generate distinctly different hues at different analyte concentrations. It has been shown that this goal can be achieved with test compositions of this invention with a variety of methods as summarized below.
1) Choose oxidized indicators which will generate the desired hue or turn colorless upon reduction.
2) Choose indicators for reaction in a particular catalytic system which have differing reduction potentials which ~till control the ~5 sequence of reactions in tnat system.
3) Control the quantities of components in the independent catalytic systems so the compo-nents involved in one re~lction sequence are essentially exhausted at chosen analyte concentra-tions.

. , , :
.: : . .
, . ~,.

. , . .

2~ 6 ~ ) Increase (or decrease) the amount of catalyst in a system. This will increase (or decrease) the rate of interaction or reduction of the indicator components by that system and therefore change the apparent hue at a particular analyte concentration.
5) Compartmentalize the components of the reaction system in a test device. The advantages of compartmentalization can include the ability to: change the apparent final hue of the device even when component conoentrations are the same;
utilize incompatible indicators or water insoluble indicators; and utilize enzyme systems normally inhibited by thiol indicators.
6) Add a color retardant which will delay the reactions of the independent catalytic systems.

The methods suggested above can be combined.

~ ~, ~-2~52~;

The invention will now be illustrated, but is not intended to be limited, by the following examples:

VI. Examples Abbreviations MPMS 1-methoxyphenazine methosulphate PMS phenazine methosulphate DCIP 2,6-dichloroindophenol TR-1 N-t2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-2-chloro-6-sulfo-4-iminobenzoquinone (for preparation see Example lA) INT 2-(4-iodophenyl)-3-t4-nitro-phenyl)-5-phenyltetra-zolium chloride NBT p-nitroblue tetrazolium chloride DTNB 5,5-dithiobis~2-nitro-benzoic acid) 3-ND 3-N-(3-dimethylamino propyl) carboxamido-4-nitrop~.e~l di-sulfide tfor prepara-tion see Example lB) EAl Dibutyl-5,5'-dithiobis-(2-nitrobenzoate (for preparation see Example 2C) " :' `,' ~;; ' :
, .

. ~

NAD Nicotinamide-adenine dinucleotide, lithium salt HEPES buffer, N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid MES buffer, 2-[N-morpholino)-ethanesulfonic acid TAPSO buffer, 3-(N-tris(hydroxy-methyl)methylamino)-2-hydroxypropane-sulfonic acid TRIS buffer, Tris(hydrox~ethyl)-aminomethane Triton X-lO0 surfactant, polyoxyethyl-ene ether available from Sigma Chemical Co .
GDH glucose dehydrogenase (EC 1.1.147) capable of producing NADH
LipDH lipoamide dehydrogenase LDH lactate dehydrogenase U International Units, a . measure of enzyme activity (one U is the enzyme activity required ta catalyze the conversion of one micromole of sub-strate per minute under specified condi-tions of temperature and pH
PET polyethylene terephthalate FMN flavin mononucleotide ':

1 Z~ 1 6 PE 310 polyethylene coated paper BSA Bovine Serum Albumin PVP K30 polyvinylpyrrolidone, molecular weight 40,000, available from Aldrich Chemical Co.

dL deciliters . mL milliliters ~L microliters g grams mm Hg millimeters of mercury, pressure designation mp melting point ~ microns RT room temperature, usually 25C

.
.... . . . . : . . :
. ", ..
- .. , :
, ;: , .

a5Z~

Example 1: Preparation of Compounds A. N-(2,3-dimethyl-5 oxo-1-phenyl-3-pyrazolin-4-yl)-2-chloro-6-sulfo-4-iminobenzoquinone (TR-l) TR-l is an indicator which is red in the oxidized form and is colorless upon reduction. It has a reduction potential similar to DCIP and has a been used to produce a "reverse" RAINBOW. TR-l was prepared as follows:
Ammonium hydroxide (lN, 10mL) was added to a mixture of 0.4 g (1.9 mmol) of 4-aminoantipyrine, and 0.5 g (1.7 mmol) 2-hydroxy-3,5-dichlorobenzene sulfonic acid disodium salt in 50 mL of water.
After stirring briefly, 1.3 g (3.8 mmol) of potassium ferricyanide (K3Fe(CN)6) was added and the reaction allowed to stir at room temperature for one hour. The mixture was filtered to yield 0.2 g (21%) of a dark brown solid. The product was homogeneous on thin layer chromatography (silica gel, with 4:1 chloroform/methanol) and 2~ apparently was a mixture of alkali and ammonium salts.

.:

)52~6 Analysis: Calculated for C17HlOClN305Na: C, 47.54; H, 3.04; N, 9.72.
Found: C, 46.10; H, 3.61; N, 11.00 H NMR (D6DMSO) ~: 7.80-7.20 (m, 7H), 3O40 (s, 3H), 2.53 (s, 3H), IR (KCl): 1660, 1630, 1400 cm ~ I , : , ' :, -.

. .. .

, ~ 6 B. 3 N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl Disulfide (3-ND) A water soluble analog of 5,5'-dithiobis-(2-nitrobenzoic acid) was prepared for use as a thiol indicator. This is a preferred indicator for use with the disulfide reductase system which is colorless in the oxidized form and becomes yellow on reduction. The compound, 3-ND, was prepared as follows:
A suspension containing 7.93 g of 3-carboxy-4-nitrophenyl disulfide (20 mmol), 1.2 mL of dry N,N-dimethylformamide (1.55 mmol), 11.7 mL of thionyl chloride (60.3 mmol) and 400 mL of di-chloromethane (CH2C12) was refluxed for four hours and then was allowed to stir overnight at ambient temperature. The resulting clear solution was evaporated in vacuo (12 mm followed by 0.1 mm Hg) to a yellow solid. The residue was then placed under an argon atmosphere, dissolved in 200 mL of CH2C12, cooled to 0, and then treated with 10.1 mL of 3-dimethylaminopropylamine (80 mmol). The reaction mixture became a dark orange and a precipitate was formed. The resulting solution was allowed to warm to ambient temperature over-night. The reaction mixture was then successively extracted four times with 200 mL portions of 5%
sodium bicarbonate solution, twice with 200 mL of water, and once with brine. The organic layer was then dried over magnesium sulfate, filtered, and concentrated to give 9.39 g of a dark orange oil.
The mixture was then flash chromatographed on 500 ~ .

:

-' ~L29~

g of Si02-60 (70-230 mesh, available as Silica gel-60 from Merck & Co.) equilibrated and eluted with 50:10:1 dichloromethane/methanol/concentrated ammonium hydroxide solvent mixture. Fractions 110 to 205 containing the purified product were pooled and concentrated in vacuo to give 7.06 g of an orange solid. The crude product was recrystallized from ethyl acetate using a treatment with pulverized carbon black such as norite and a diatomaceous earth, a filtering aid available from Manville Products Corp., Denver, Colorado, ~nder the trademark Celite . Obtained was 5.47 g of light yellow crystals a~ter drying at 55, 0.1 mm. Yield = 48.4%. mp 152-156.

Analysis: Calculated for C24H32N606S2: C, 51.05;
H, 5.71; N, 14.88.
Found: C, 51.05; H, 5.56; N, 14.62.

PMR ~60MHz, CDCl3) ~:1.73 (quintet, J=6Hz, 4H);
2.17 (s, 12H); 2.45 (t, J=6Hz, 4H); 3.53 (q, ~0 J=6Hz, 4H); 7.57 (s, 2H); 7.65 (dd, J=7Hz, 2Hz, 2H); 8.02 (m, 2H, N-H); 8.03 (d, J=7Hz, 2H).

IR(KBr) cm : 3260, 3060, 2940, 2870, 2820, 2790, 1650, 1560, 1530, 1470, 1345.

Mass Spectrum (FAB) m/e: 565 (M~l, 13.6~).

.' ''' ' ' " `
.

~' , , ' ' ~., ~ , ~

~2~52~6 C. Dibutyl-5,5'-Dithiobis-(2-nitrobenzoate) (EA-l) This compound, which is referred to herein as EA-l, is a lipophilic analog of 5,5'-dithiobis-(2-nitrobenzoic acid). It was prepared for use as a thiol indicator as follows:
A suspension containing 6.85 g (17.25 mmol) of 5,5'-dithiobis-(2-nitrobenzoic acid), 3.46 mL
of thionyl chloride, 0.346 mL of N,N-dimethyl-l~ formamide, and 350 mL of dichloromethane wereheated to reflux for 3 hours. Additional thionyl chloride (3.46 mL) and N,N'-dimethylformamide were added and refluxing was continued for 2 hours. A
clear, light gxeen solution was obtained, indicat-~5 ing complete conversion to the bis-acid chloride.
The solvents were removed in vacuo and the result-ing light-green solid was placed under an Argon atmosphere. The residue was then suspended in 100 mL of pyridine and treated with 20 mL of ~ n-butanol. A mild exotherm and a darkened but homogeneous reaction was obtained, which was allowed to stir overnight. The reaction solvents were removed in vacuo and the residue was dissolved in 300 mL of chloroform. The organic layer was then successively extracted thrice with 200 mL of 0.1 N HCl, twice with 200 mL of 5% Na HCO3 solution, and with 200 mL of brine. Drying (MgSO4), filtration, and removal of solvent gave 12.79 g of an oil which was dissolved in ethyl acetate and adsorbed in vacuo onto a small amount of SiO2-60. The impregnated solid was then placed ~2~

atop a column of 500 g of SiO2-60 (230-400 mesh) which had been packed and equilibrated with 8:1 hexane-ethyl acetate. The column was then flash chromatographed using this solvent mixture with fractions of 25 mL collected. Fractions 190-270 were pooled and concentrated to give 8.07 g of product as an oil which solidified upon standing (92% yield).

Analysis: Calculated for: C, 51.96; H, 4.76; N, 5.08.
Found: C, 52.49; H, 4.88; N, 5.45.

PMR (60 MHz, CDCl3) ~: 0.97 (t, J=7Hz, 6H, CH3-CH2); 1.2-1.9 (m, 8H, CH2 _ 2 ) ~=6Hz, 4H, -O-CH2-CH2-) 7.77 (s, 2H, C6H); 7-83 (AB quartet, J=8Hz, 4H, C3H, C4H).

IR (KBr)cm : 1730 (Ar-C-O-CH2- stretch).

Mass Spectrum: E.I. (70 EV) m/e = 508 (37%, M ).

, .

.. . . . .
,, .

2~

D. Dimethyl 5,5'-Dithiobis-(2-nitrobenzoate) This compound is a lipophilic analog of 5,5'-dithiobis-(2-nitrobenzoic acid), which was prepared for use as a thiol indicator as follows:
A suspension containing 7.93 g (20 mmol) of 5,5'-dithiobis-(2-nitrobenzoic acid), 1.2 mL of N,N-dimethylformamide, 11.7 mL of thionyl chloride, and 400 mL of dichloromethane were refluxed for 4 hours until a clear light green solution was obtained. The solvents were then evaporated in vacuo to obtain a yellow solid which was then placed under an Argon atmosphere, dissolved in 200 mL of dichloromethane, and cooled to 0. The stirred mixture was then treated with 4~03 mL of dry pyridine (50 mmol) and 30 mL of methanol. The resulting mixture was then allowed `
to warm to ambient temperature overnight. The reaction mixture was then successively extracted thrice with 300 mL of 5% NaHCO3 solution, thrice with 300 mL of 1 M citric acid, and 300 mL of brine. Drying (MgSO4), filtration and removal of solvents gave 8.29 of a yellow solid which was recrystallized in two crops from toluene as a light yellow solid in 92% yield. mp 103-104.5.

.

, :

~29~;21 6 .~

Analysis: Calculated for: C, 45.28; H, 2.86; N, 6.60.
Found: C, 45.74; H, 3.01; N, 6.25.

PMR (60 MHz, CDC13) ~: 3.93 (s, 6H, -0-~H3); 7-8 (s, 2H, C6H); 7.83 (AB quartet, J=8Hz, 4H, C3H, C4H).

IR (KRr) cm : 1740 (-C-O-CH3 stretch).

Mass Spectrum: EI (70EV~ m/e: 424.3 (M , 67.7~).

' : - :
': ~ '.

2952~

E. 3,6-Dioxaoctyl-5,5'-Dithio-(2-nitrobenzoate) This compound is a water soluble analog of 5,5'-dithiobis-(2-nitrobenzoic acid). It was prepared for use as a thiol indicator as follows:
A suspension containing 7.93 g (10 mmol) of 5,5'-dithio-(2-nitrobenzoic acid), 1.17 g of N,N-dimethylformamide, 11.7 mL of thionyl chloride, and 400 mL of dichloromethane was heated to re~lux with stirring for 2 hours to obtain a clear solution. The solvents were removed in vacuo to give a light green solid which was placed under Argon, cooled to 0, and suspended with stirring in 120 mL of dry pyridine. Carbitol~ (20 mL) was then added and the resulting mixture became an orangish-red homogeneous solution after l hour reaction time (Carbitol~ is a registered trademark of Dow Chemical Co. and is chemically named 2-(2-ethoxyethoxy)ethanol). The mixture was then allowed to come to ambient temperature ~ overnight. The sample was then evaporated in vacuo to a dark oil and flash chromatographed on 500 g of SiO2-60 (230-400 mesh) packed and eluted with a 0.5~ methanol-chloroform solvent mixture.
Fractions of 25 mL were collected. Fractions 150-186 were pooled and concentrated to give 7.86 g of a yellow oil. The sample was then dissolved in ether, treated with 4 g of Norit, filtered through Celite, and precipitated as a dense yellow oil with hexane. The solvents were then decanted and the residual solvents were removed in vacuo at . .

~, . ~

9~

40 (0.1 mm) for 1 hour to give 5.48 g Qf a viscous, yellow oil (44~ yield).

Analysis: Calculated for: C, 49.67; H, 5.13; N, 4.46.
Found: C, 49.41; H, 5.09; N, 4.37.

PMR (60 MHz, CDCl3) ~: 1.2 (t, J=7Hz, 6H, CH3-CH2-O-); 3.5 (q, J=7Hz, 4H, CH3-CH2-O-);
3.6 (s, 8H, -CH2-CH2-O-); 3.8 (m, 4H, O O
Il 11 C O CH2-CH2-); 4.55 (m, 4H, -C-CH2-CH2-O-);

7.8 (s, 2H, C6H); 7.82 tAB quartet, J=8Hz, 4H, C3H, C4H).

IR (CHCl3)cm : 1740 (-C-O-CH2- stretch).

, , .
:

, -:~.
"::

. . .

g~ 6 F. 3,6,9,12-Tetraoxadodecyl 5,5'-Dithio-(2-Nitrobenzoate) and 1,12-Cyclic Diester Both of these compound are water soluble analogs of 5,5'-dithiobis-(2-nitrobenzoic acid).
They were prepar~d for use as thiol indicators as follows:
A suspension containing 7.93 g (20 mmol) of 5,5'-Dithiobis-(2-nitrobenzoic acid), 11.7 mL of thionyl chloride, and 1.2 mL of N,N-dimethyl-formamide was heated to reflux for 2 hours toobtain a clear yellow solution. The solvents were removed in vacuo to give a light green solid, which was dissolved in a mixture of 50 mL of dichloromethane and 5 mL of pyridine. This solution was then added slowly to a 0 solution of 3~.5 mL (38.8 g, 200 mmol) of tetraethylene glycol in lO0 mL of dichloromethane under an Argon atmos-phere. The reaction mixture was allowed to come to ambient temperature overnight. Evaporation of ~ solvents in vacuo gave a brown oil which was partitioned between chloroform and water. The aqueous layer was extracted again with chloroform and the combined organic layers were washed with brine and dried tNa2so4)~ Filtration and evapora-tion of solvent in vacuo gave an orange oil whichwas flash chromatographed on 500 g of SiO2-60 (230-400 mesh) packed and eluted with a 5%
methanol-chloroform solvent mixture. Fractions of 25 mL were collected. Fractions 24-38 were~pooled and concentrated to give 2.21 g of a yellow glass.
This was identified as the 1,12-cyclic-3,6,9,12-;
.......
, ~ ..

. ~

tetraoxaundecyl ester of 5,5'-dithiobis-(2-nitro-benzoic acid). The yield of cyclic ester was 20%.

C NMR (22.5 MHz, CDC13) ~: 66.8, 68.3, 70.4 (all -O-CH2-CH2-O-); 128.8, 142.4, 146.3 (aryl C);

164.3 (-C-O-CH2).

IR (CHC13)cm : 1730 cm (-C-O-CH2- stretch).

Fractions 91-101 were pooled and concentrated to give 3.94 g of the expected 3,6,9,12-tetraoxa-dodecyl diester. (26% yield).

C NMR (22.5 MHz, CDC13) ~: 61.6, 65.6, 68.3, 70.2, 70.5, 72.4 (all -O-CH2-CH2-O-); 129.0, 142.4, 146.5 (aryl C);
1l 164.5 (-C-O-CH2-).

PMR (60 MHz, CDC13): integration ratio of aliphatic to aromatic protons =5.6.

IR (CHC13)cm : 3600-3350 (-CH2-OH stretch):
1l 1730 (-C-)-CH2- stretch).

`,`'' " -' .

-~ 12~

Example 2: Glucose Formulations Glu~ose test devices useful for testing whole blood can be prepared as follows. Reduced nicotinamide adenine dinucleotide was the common subst~rate and was generated from glucose by glucose dehydrogenase. The general chemistry for RAINBOW glucose formulations is shown schematically on a following page. The thiol indicator, DTNB, is commonly called Ellman's Reagent.

A. Pathway 1: Diaphorase/NBT/TR-1:
Pathway 2: LipDH/lipoamide/DTNB

Layer Component Quantity (g) 1 Gelatin }.13 Water 6.78 PVP K38 (20%) 1.69 Triton X-100 (4%)0.40 NBT 0.064 2 Gelatin 1.13 HEPES Buffer, 1 M, pH 7.5 5.42 Water 1.36 PVP K30 (20%) 1.69 K Fe(CN)6 0.073 D~NB 0.040 ~5 GDH (64.6 U/mg) a . 060 Diaphorase (118 U/mg) 0.005 Lipoamide 0.030 LipD+H (1350 U/~L) 200 ~L
NAD 0.060 Mutaro~ase (5060 U/mL) 100 ~L
Triton X-100 (4%) 0.4 ~2~%:1~

3 Gelatin 1.13 Water ~ 6.78 Triton X-100 (4%) 0.40 TR-1 0.060 4 carbodiimide 1.25%

; ~ :

: ,: ,., ,. : .
. ~ :

:. ~ .

~I.Z~5~ 6 ~RAtN~Q~ CHEMISTRY

Glucose NAD+
¦ GDH
BLUE Gluconote RED
(red) DC1~ (ox) + NADH + H+ + INT+ D~a~hornse , Formoz~n ¦Colorless -BLUEl ¦Colorless r RED ¦ ¦
Lipoomide (ox) \ Colorless \ L~po~m~ e lred) Green + Orange \ ~ Ellman's Reagent \ Yel lowYellow Adduct ~ /
Yellow ~35216 - Procedure: The components of each solution were combined at 40 in the order given. The solutions were degassed before coating. Layer 1 was spread onto a polyester backing and dried. Layer 2 was spread onto Layer 1 and dried and so on. The final device was made up of three gelatin layers on a polyester backing. The carbodiimide coating provided a hardened surface by crosslinking the gelatin, which allows wiping the surface of the device.

Dose Response: With increasing glucose concen-tration from 50 to 600 mg/dL, the color sequence was pale red to olive green to blue black.

','' .:

3LZ~i2~

B. Pathway 1: MPMS/DCIP/INT
Pathway 2: Glutathione Reductase/Gluta-thione/DTNB

Layer Component Quantity (g) 1 Gelatin 1.13 Water 6.78 PVP K30 (20%) 1.69 Triton~ X-100 (4%) 0.40 INT 0.064 2 Gelatin 1.13 HEPES Buffer, 1 M, pH 7.5 5.42 Water 1.36 PVP K30 (20%) 1.69 Triton~ X-100 (4%) 0.40 DCIP 0.0147 K Fe(CN) 0.073 D~NB 6 0.040 GDH (64.6 U/mg) 0.060 NAD 0.060 ~ MPMS 0.010 Glutathione Reductase (2320 U/mL) 230 ~L
Glutathione 0.089 Mutarotase (5060 U/mL) 100 ~L
3 Carbodiimide 1.25~

The procedure was used for the formulation and testing of the device that of Example 2A.

'. '~
,.. ...

. . .

Dose Response:

Glucose (mg/dL) Color 110 Peacock Blue 140 Green-Blue 180 Dull Light Aqua 250 Dull Rose 300 Dull Rose 400 Rose 500 Rose 600 Dark Rose 800 Burgandy The hues, blue to rose to burgandy, can be easily distinguished visually.

. . .
. .-.. ,.. - . ~:

: ' ~
'~
.~:

;2~6 C. Pathway 1: MPMS/DCIP/INT -Pathway 2: LipDH/lipoamide/~TNB

Layer Component Quantity (g) 1 Gelatin 1.13 Water 6.78 PVP K30 (20%) 1.69 Triton X-100 (4~) 0.40 INT 0.064 2 Gelatin 1.13 HEPES Buffer, 1 M, pH 7.5 5.92 Water 1.86 PVP K30 (20%) 1.69 Triton X-100 (4~) 0.40 K Fe(CN)6 0.073 DTNB 0.040 GDH+(64.6 U/mg) 0.060 NAD 0.060 MPMS 0.010 DCIP 0.0147 Lipoamide 0.030 Mutarotase (5060 U/mL) 100 ~L
LipDH (1350 U/mL) 200 ~L
3 Carbodiimide 1.25%

The procedure used for the formulation and testing of the device was that of Example 2A

:. ' ~ ~ '` . ` :
.

.:
- `: ''~' ' ~

lZ95216 Dose Response:

Glucose (mg/dL) Color Peacock Blue Peacock Blue Aqua 110 Teal Green 140 Mint Green 180 Sea Green 250 Green Gold 300 Gold 400 Orange Gold 500 Orange 600 Dark Orange 800 Darker Orange The colors blue, green, gold, orange are visually distinct.

., . ~ .~ .:

.; ' , : ...
- ~ -.~ ,. :

~2~i2~6 D. Pathway 1: DCIP/INT/MPMS
Pathway 2: LipDH/lipoamide/3-ND

Layer Component Quantity (g) 1 Gelatin 1.13 Water 6.78 PVP K3~ (20%) 1.69 Triton X-100 (4~) 0.40 INT 0.064 2 Gelatin 1.13 HEPES Buffer, 1 M, pH 7.5 5.92 Water 1.86 PVP K3~ (20%) 1.69 Triton X-100 (4%~ 0.40 K Fe(CN)6 0.073 D~IP Q.0147 3-ND (20 mM) 1.25 mL
GDH+ 0.060 NAD 0.060 MPMS . 0.010 ~0 Lipoamide 0.030 Mutarotase (5060 U/mL) lO0 ~L
LipDH (1350 U/mL) 200 ~L
3 Carbodiimide 1.25~

The procedure used for the formulation and testing of the device was that of Example 2A.

' ~ '.. ' :;',' ~' ' .

21~

Dose Response:

Glucose (my/dL) Color Peacock Blue Teal Green 110 Mint Green 140 Light Mint 180 Sea Green 250 Tan 300 Dark Tan 400 Pale Brown 500 Rust 600 Rust 800 Dark Red With this formulation, using the DTNB derivative 3-ND, the generation of the final hu of the test device, visible to the user, was complete in 8 minutes.

, ;:

.. , - ' ,, :: ,... :

:
: :

21~

E. Pathway 1: MPMS/DCIP/INT
Pathway 2: LipDH/lipoamide/3-ND

Layer Component Quantity (g) 1 gelatin 1.13 water 6.78 PVP K30 (20%) 1.69 Tritono X-100 (4%) .40 INT .064 2 gelatin 1.13 HEPES Buffer, lM ph=7.5 5.92 water 1.86 PVP K30 (20%) 1.69 Triton X-100 (4~) .40 3-ND ~40 mM) 2.50 mL
DCIP 0.0147 GDH 0.060 NAD 0.060 MPMS 0.010 lipoamide 0.030 mutarotase ~5060 U/mL) 100 ~L
LipDH (1350 U/mL) 200 ~L
K3Fe(CN)6 0.055 3 carbodiimide 1.25%

.
.... ;, ' ', ~ .
.... . . . .
:` ' '~ .; : ,. , -~295;~1~

Dose Response:

Glucose mg/dL Hue generated o blue dark blue green blue green light green 110 yellow green 140 gold 180 gold orange 250 orange 400 red orange 800 deep red This device exhibits a full spectrum RAINBOW;
blue, green, gold, orange~red. The hue visible to the user depends on the concentration of glucose and is generated in 7 to 8 minutes.

MS-1444 ~

.

;, ':

: . . :, . .

~ 52~6 F, Whole blood glucose RAINBOW test device.
Pathway 1: Diaphorase/DCIP/INT
Pathway 2: LipDH/lipoamide/3-ND

Layer Component Quantity (g) 1 Gelatin 1.13 Water 6.78 PVP K30 (20%) 1.69 Triton X-100 (4~) .40 INT .064 2 Gelatin 1.13 MES buffer, lM tpH-6.5~ 5.92 Water 1.86 PVP K30 (20%) 1.69 Triton X-100 (4%) .40 3-ND (40 mM) 2.5 mL
GDH (64 U/mg) `.060 NAD+ .060 Diaphorase (118 U/mg) .010 ~ Mutarotase (5060 U/m) 100 ~L
LipDH (1350 U/mL) 200 ~L
DCIP 0.0147 K3Fe(CN)6 3 Carbodiimide 1.25%

~5 The procedure used for formulation of the device was that described in Example 2A. Testing was done with whole blood samples spiked to the desired glucose levels, analyzed with a YSI
glucose analyzer. The dose response data is given below. The test device, containing a test composition of the invention compartmentalized by layering in a gelatin carrier, exhibited a full spectrum RAINBOW over a glucose concentration of 0 to 800 mg/dL. The composition was designed to .

~2~52~6 produce a green hue covering the normal blood glucose range.

Dose Response:

Glucose mg/dL Hue Generated 0 blue light blue blue green 110 sea green 140 pale green 180 light yellow 250 yellow orange 400 orange red 800 red : MS-1444 , . .

1i~9~

G, Rainbow: Varying enzyme concentration.
The following example was designed to show how the final hue produced can be controlled by changing the concentrations of the ~nzymes.
S Pathway 1: Diaphorase/DCIP/INT
Pathway 2: LipDH/lipoamide/3-ND

.' . . ' . .; , '' 95;2~;

r o o ~ o o d~
O ~D ~) N ~D ~ O ~ ~D ~ N ~) ~r ~ ~ IS~
D ~ O ~1 ~ CO ~ ~r u ) o o o o o o 3 3 N
t~ ......... ........ .... -~1 ~ -1 N O O 0 ~1 N

~r o o o o o O~o ~ 00 ~ O ~ ~) N ~0 ~ O ~1 ~ ~
,1 1~ ~ ~r o ~1 a~ o o o o o o 3 3 N
_. C~
~ r-l ~D ~ ~1 IS) ~1 ~1 N O O O ~1 _ O O

~rooooo ~1 ~ ~ O ~ ~ N D ~ O ~1 ~
~1 1` ~D ~r o _~ ~ ~) ~ ~r u~ O O o o o o 3 3 N
~ m ..... ............
C~ ,t ~ .-, ~1 Is~ r--t ~ N O O O
O O
~I N
~`
r o o o o o dP
CO ~ O ~ ~ N ~ ~ O ~1 ~ ~
~1 1~ ~D ~r o ~1 ~ co ~ ) o o o o o o 3 3 N
~1 ~ ~1 I,r) ~1 ~I N O O O
O U') ~1 1~ o ~ D k CO o ~ o~ - o ~ ~ p ~P o 5~ oP o ~
o ~a.l o ,~ ~ - o ~ ..
Ul ~ I ~I N I E
--X ~1 -- X U~
O
O) ~:: ol3 ~ Q o~
1:: ~ O E~ O ~
O ~ s~ ~ o ~ tn ~ ~: o ~ ~ ~ ~ o o~ ~ a ~ + ~ o ~ a Q
E~ ~ 1 Z H m a o a~ z ~ ) a C.) t~ 3 ~ E~l H ~ 3 3 ~ a ~ z a ~ m ~ ~ N ~

M~1444 ~5~

The films were prepared and tested as des-cribed in example 2A. The results are shown in the following table.

.

.

:~ ~ R
R R R ~ tn X

~ .0 0 R R ~1 ~ Q ~ tJl ~ ~
t~ I ~ 3 3 0 ~: ~ C:
:~ ~ ~ O O tJ'~
O
E~ 1 ~ ~ O O 'a .. ..
o R ~1 ~ h ~ O n~
R R O~
ml ~ ~ o ~ ~

R R R
O

~3 ~ ~1 a O
o o o oc: o o o o o ~ ~ ~ ~ r oo In, O O
-I ~ ~1 ,~

MS'.1444 :

, 12~5216 Example 3: Paper RAINBOW

Although multilayered gelatin is a preferred matrix for a rainbow device, a device can be prepared using paper as a carrier. A solution of the following composition, made in the order shown was prepared.
, .
.

.

.

`; ' ~' ~ ' `, ` ' '` . ` . ' ' ' %~

m o O cO
z ~ O
E~ Ln a ~ o ~9 p~ ~ o ~ ~ m ~
~ o a a~ o G~
~ I u~ o z o u~ ~ ~ ~, ~t~1 ~
~ ~.
o o O O ~D
o o ,~ ,, ,o, u~ o ~ o ~ u~ ~
O O d~ ~ ~1 ~1 ~ _~ o o r-.,~ I ,, o ~1 ~ ,~
~x ,,O. ~

u- ~
,~ o ~ ~ o o o r-o u~ . ~ . ~ Ln ,~ I`~1 ~1 ~ o o o .,1 ,~

~ o I ~ ~ o o u~ x o u~ ~ x 1~1 0 3 .C -- ~ 0 3 J~ O ~\ ,~ O ~ W
~ S~ I) ~ O ~ ~ a o a~
~ o _ ~ ~

al O E~ O ~Vl h h Cl O E3 0 ~; ~-- ~ 14 ~ O ~:; c,~ O

M~1444 ,, The buffer was TAPSO, pH 7.4. Both poly-vinylalcohol (PVA) and the order of reagent addition were critical in this example. A con-centration of 1 to 1.5% PVA prevents the copre-cipitation of DCIP and INT in a paper matrix.
Whatman 31 ET paper was impregnated with this solution and dried at 50C. The dried paper was cut and mounted on a plastic backing to form test strips. The test strips were assayed with NADH
solution and the results shown below.

... ~. . ...
.:
:

~L295i~

a~
,~

O
O

O O O
. S~ ~ ~
~17 R R R

_ O
.,, , R

o t~ ~ ~ ~
,1 ,1 _I
.q .q ~ ~ ~ ~ S
,1 _I _I

o ,1 ,1 ,~
R R R

_ ~ ~ _I ~ ~
~ C

MS ^1444 . .
...
,; .
.
, `........
. .... : - , . ~
,; .; - ... - .

~29~

Example 4: NADH Generating Systems Containing Enzymes Sensitive to Thiol Reagents A general problem with the described thiol detection reagent system is that thiol indicators can react with protein systems, frequently leading to inactivation o~ enzyme. Many such enzyme inhibitions by DTNB are described in the litera-ture, There are two general solutions, bo~h in-volving compartmentalizing the thiol reagent: (1) l~ sequester the thiol indicator in an organic phase or as insoluble particles; (2) physically separate the sensitive enzyme and the thiol reagent by immobilizing the latter.

A. Approach (1) - Sequestering the Reagent.
A water insoluble analog of DTNB, EAl was used (for preparation see example lC).
There are two approaches to sequestering the reagent: (a) EAl was dissolved in an oil and dispersed as an emulsion in a gelatin film.

~0 Organic Phase: EAl (300 mM) was dissolved in tricresyl phosphate with trioctylamine (1%).

: .

.. ~ , ~2~5216 Aqueous Phase Final Concentration Gelatin 10~
Potassium Phosphate, 50 mM
pH 7.5 DC~P 1.5 mM

The oil phase was emulsified with the aqueous phase in a Waring Blender to a final concentration of 5~. Lipoamide dehydrogenase (Sigma type III, 13.3 U/mL) was added to the emulsion. The emulsion was coated on a plastic support (170 ~) and dried at room temperature.
Treatment of the dried film with NADH/Lipo-amide (approximately lmM) at pH 8.5 (tris buffer) produced a very rapid bleaching of the blue hue of DCIP and formation of yellow by the EA1 reagent.
Without NADH there was no reaction.
The enzyme lactate dehydrogenase (LDH), which is inhibited by DTNB was tested with this film.
LDH, approximately 150 U/mL, and lipoamide (ap-~ proximately lmM) in about 250 mM lactate, pH 8.5,produced a yellow hue in about 10 minutes. The blank, without LDH~ was uncolored. The con~lusion is that the use of an oil soluble thiol indicator, if compartmentalized in an oil emulsion, permits the use of enzymes which are commonly believed to be sensitive to thiol reagents.

(b) - EAl deposited directly into paper.

.
,. . .

-` 3L2~Zl~

EAl was dissolved in toluene to a concen-tration of 5.7 mM. Whatman 31 ET paper was impregnated with this solution and dried at 50.
A second solution was prepared of the following constituents:
Final Concentration Triton X-100 0.1%
(tris)2 sulphate pH 8.5 0.2 M
PVA (98.5% hydrolys.ed) 0.62%
MPMS 0.25 ~M
Lipoamide 0.25 mM
LipDH 30 U/mL
NAD 0.5 mM
DCIP 0.8 mM

(Tris)2 sulphate is Tris buffer adjusted to the desired pH with sulfuric acid.
The dried paper was dipped into the second solution and dried again at 50C and affixed to a `20 ` plastic support.
Test solutions for LDH contained L-lactate (167 mM), (tris)2 sulphate, pH 8.5, (0.33 M) and LDH enzyme (rabbit muscle, Sigma type II) as shown in the following table with the test results.

~ .

~2~

S~ 3 O
. ~ ~

~: 3 ~q O
,1 '' 'I a Q R

~ ~D ~ ~ ~
~D
3 ~D .-1 _ o ~ ~ R
~ ~ a~
O ~

R ,5:1 ~ .
o O ~a t~ . .
m a) a) O
h 'Y

_ .
; , .

.

~ 5Z~;

Very good coior discrimination is seen between the various levels of LDH.

Test Solution for alcohol detection The ability of this compartmentalized film format to handle assays requiring the use of an enzyme which is sensitive to thiol reagents was further tested using alcohol dehydrogenase.
Test solutions were prepared which contained various concentrations of ethanol (t~is)2 sulphate buffer, 0.5 M, pH 8.5, and alcohol dehydrogenase (from Baker's yeast, Sigma Catalog #A7011, 22.5 U/mL).
The test strips prepared as described above were used. The results are shown in the following table.

-:: ,`'.: -, .
~ ;. ,.

z9~Z~6 U~ ~ O
.
_~ U~
,1 ~ ~

a .
~o a~
U~

,1 ~O I rq ~
_ ~1 ~1 _ ~ ~1 ~1 O
C~l . ~ ~ ~
~:
a ~ ~ ~ .
a~
O ~r ,1 _~
U~ U~
U~ ~Q

a) Q) l .Y
h _ a) ~
E~

MS `1444 .
. -~2~5;~6 Very good discrimination was seen between levels of alcohol.
From these experiments it was concluded that the RAINBOW system utilizing a thiol detection reagent can be used with thiol reagent sensitive enzymes.

~ ~ . , .
: ,:
.. ...

:

. " .,. ~
, ~:

B. Approach 2: Physical separation of thiol reagent sensitive enzyme and thiol reagent by immobilizing the latter.
DTNB can be covalently bound to a large molecule or can be physically trapped in a matrix, such as gelatin, which only allows penetration of small molecules. Therefore direct interaction between the enzyme and thiol reagent would not be expected.

Preparation of immobilized DTNB
DTNB was linked to human-serum albumin through the carboxyl function using a water soluble carbodiimide reagent.
A 30~ human serum albumin solution was made 0.2M in sodium phosphate and the pH adjusted to 4.57, It was then diluted to 4.8% albumin. DTNB
(10.7 mM) and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 10.7 mM) were dissolved in a small amount of ethanol and added, with vigorous stirring, to the albumin solution maintained at 0C. After 2 to 3 hours, the lightly gelled material was transferred to d,i~lysis tubing and dialysed extensively against water. Then it was lyophilized.
The crusty yellow solid was pulverized to a fine powder and dispersed in 10% gelatin to a concentration of 1%. A coating was made (170 ~), and dried at room témperature.

.

.: ,~.. . .

,' .~ ,.
.~ . .. . .. .
' ~ ' , ~ 2g5Z~6 The film was evaluated with the following test mixture:

Final Concentration lithium lactate 250 mM
potassium phosphate250 mM
buffer, pH 7.5 glutathione 0.5 mM
glutathione reductase9 U/mL
NAD 0.2 mM
LDH 5 U/mL

When contacted with this solution, the film produced a clear yellow color within one. minute.
A control solution, without LDH, produced no color.
The conclusion is that immobilizing the thiol reagent allows assay with thiol reagent sensitive enzymes.
The overall conclusion is that although many enzymes, such as alcohol dehydrogenase and choles-terol dehydrogenase are inhibited by DTNB accord-ing to the litarature, there are many ways of avoiding this inhibition. Consequently, the second catalytic system which involves thiol indicators is generally applicable to assay of NADH as an intermediate and could be used to determine alcohol or cholesteroI as analytes.

~2~S;~6 Example 5: Diffusible/Nondiffusible Dye Reaction Another approach to generating a RAINBOW is where the hue of the test composition is made to appear as a result of diffusion. For instance, if an indicator is unable to diffuse due to being covalently linked to a matrix, and this matrix is covered with an opaque layer, then the indicator will be invisible from the top. If, as a result of a reaction, the covalent linkage of the indica-tor to the matrix is severed, then the indicatorcan diffuse up through the opaque covering and become visible.
As an example, a film was prepared as described in Example 4~, where DTNB was covalently bound to albumin which was then incorporated into a gelatin matrix. Whatman 31 ET was impregnated with a solution containing:

Final Concentratlon ~0 potassium phosphate, 160 mM
pH 7.5 glutathione 0.4 mM
gluthathione reductase 36 U/ML

The impregnated paper was dried at 50C.
Pieces of the dried paper were cut and laid on the gelatin film. The paper was quite opaque. An NADH solution was added to the muItilayer device prepared in this way and water to another device to be used as a control. Within approximately 20 ~Z95Z16 seconds, the device contacted with NADH began to turn yellow and rapidly developed to a deep yellow. The control pad had no color.
- An LDH assay was made with the following solution:

Final Concentration potassium phosphate, 250 mM
pH 7.5 lithium lactate 250 mM
NAD 0. 2 mM

LDH (about 5 U/mL) was added to this solution and 30 ~L of the solution was added to one of the multilayer paper/gelatin device described above.
After about 5 1/2 minutes, the LDH pad showed a faint but definite yellow color. The control, without LDH, had no color.
The conclusion is that indicators can indeed be made diffusible as a function of analyte concentration.
In this approach, the indicator molecule is a combination of a hue determining (chromophoric) part and reactive (cleavable) part. These can be electronically isolated such that cleavage of the anchoring linkage does not significantly change the hue of the indicator. The amount, or intensity, of the indicatox can be controlled by the activity of the catalytic system involved in the reductive cleavage and the hue which is ,:

~Z~3~iZ16 generated is determined independently by the chromophoric part of the molecule. This is a considerable advantage as it can be difficult to find indicators with a suitable combination of hue and reactivity to generate the desired final hue.
Other matrices to which a thiol indicator could be immobilized by oxidation are: thiol-agarose or any protein using a bifunctional reagent e.g., Lomant's reagent, dithiobis(suc-1~ cinimidyl propionate).

Many variations and modifications can be made from these examples without departing from the scope or spirit of the invention.

.. :. . ., . ~:

Claims (26)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A test composition for the visual deter-mination of analyte in a fluid sample, comprising:
(a) a catalytic system capable of generating reduced nicotinamide adenine dinucleotide from the analyte;
(b) a first independent catalytic system capable of generating a reduced first indicator by reaction with reduced nicotinamide adenine dinuclaotide; and (c) a second independent catalytic system capable of generating a change in hue of a second indicator component by reaction with reduced nicotinamide adenine dinucleotide, which second independent catalytic system includes:
(i) a disulfide reductase;
(ii) a disulfide substrate; and (iii) a thiol indicator; wherein the disulfide reductase is capable of catalyzing the reaction between reduced nicotinamide adenine dinucleotide and the disulfide substrate to produce a product which can interact with the thiol indicator to produce the change in hue of the second indicator;
whereby the generation of the reduced first indicator and the changed hue of the second indicator is controlled to provide a range of hues, the particular final hue produced by the test composition depending on the concentration of the analyte.

2. A test composition for the visual deter-mination of analyte in a fluid sample, comprising:
(a) a catalytic system capable of generating reduced nicotinamide adenine dinucleotide from the analyte;
(b) a first independent catalytic system capable of generating a reduced first indicator by reaction with reduced nicotinamide adenine dinucleotide; and (c) a second independent catalytic system capable of generating a reduced second indicator by reaction with reduced nicotinamide adenine di-nucleotide, which second independent catalytic system includes (i) a disulfide reductase;
(ii) a disulfide substrate; and (iii) a thiol indicator; wherein the disulfide reductase is capable of catalyzing the reaction between reduced nicotinamide adenine dinucleotide and the disulfide substrate to produce a product which can reduce the thiol indica-tor to produce the reduced second indicator;
whereby the generation of the reduced indicators is controlled to provide a range of hues, the particular final hue produced by the test composi-tion depending on the concentration of the analyte.

3. The test composition of claim 2 in which the generation of the reduced first and second indicators occurs substantially simultaneously.

4. The test composition of claim 2 in which the first or second independent catalytic system is capable of producing a reduced third indicator.

5. The test composition of claim 4 in which one indicator component reduction produces a change from a hue to colorless.

6. The test composition of claim 2 which additionally includes a color retardant.

7. The test composition of claim 2 in which the generation of the final hue of the test composition is essentially complete in less than about ten minutes.

8. The test composition of claim 2 in which the reduced second indicator is yellow.

9. The test composition of claim 2 in which the thiol indicator is a disulfide.

10. The test composition of claim 9 in which the disulfide is an analog of 5,5'-dithiobis-(2-nitrobenzoic acid).

11. A test composition for the visual determination of glucose in an aqueous fluid sample, comprising:
(a) a catalytic system capable of generating reduced nicotinamide adenine dinucleotide by reaction with glucose;
(b) a first catalytic system composed of a catalyst having diaphorase activity, an oxidized first indicator and an oxidized third indicator each capable of reacting with reduced nicotinamide adenine dinucleotide in the presence of the catalyst to produce a reduced first and third indicator; and (c) a second catalytic system composed of a disulfide reductase, a disulfide substrate and a thiol indicator, wherein the disulfide reductase is capable of catalyzing the reaction between reduced nicotinamide adenine dinucleotide and the disulfide substrate to produce a product which reduces the thiol indicator to produce a reduced second indicator;
whereby the generation of the reduced indicators is controlled to provide a range of hues, the final hue produced by the test composi-tion depending on the concentration of glucose in the sample.

12. The test composition of claim 11 in which the generation of the reduced first and second indicators occurs substantially simulta-neously.

13. The test composition of claim 11 in which the reduction of at least one of the first, third or thiol indicator produces a change from a hue to colorless.

14. The test composition of claim 13 in which the generation of the final hue of the test composition is essentially complete in less than about ten minutes.

15. A test device for the visual determina-tion of an analyte in a fluid sample, comprising:
(a) a carrier matrix; and (b) a test composition incorporated there-with, the test composition including: a catalytic system capable of generating reduced nicotinamide adenine dinucleotide from the analyte; a first independent catalytic system capable of generating a reduced first indicator by reaction with reduced nicotinamide adenine dinucleotide; and a second independent catalytic system capable of generating a yellow second indicator by reaction with reduced nicotinamide adenine dinucleotide, which second independent catalytic system includes (i) a disulfide reductase;
(ii) a disulfide substrate; and (iii) a thiol indicator; wherein the disulfide reductase is capable of catalyzing the reaction between reduced nicotinamide adenine dinucleotide and the disulfide substrate to produce a product which can interact with the thiol indicator to produce a yellow second indicator:
whereby the generation of the reduced and yellow indicators is controlled to provide a range of hues, the particular final hue produced by the test composition depending on the concentration of the analyte.

16. The test device of claim 15 in which the generation of the reduced first and second indicators occurs substantially simultaneously.

17. The test device of claim 16 in which the first or second independent catalytic system is capable of producing a reduced third indicator.

18. The test device of claim 17 in which the reduction of at least one of the first or third oxidized indicators produced a change in the hue of that indicator of from a hue to colorless.

19. The test device of claim 18 in which at least one of the oxidized indicators or the thiol indicator is compartmentalized within the carrier matrix.

20. A multilayer test device for the visual determination of glucose in a body fluid sample, (a) an inert support member, and in laminar relationship thereto;
(b) at least two layers composed of a hydrophilic carrier, one layer containing one of a first or third oxidized indicator component and the second layer containing (i) nicotinamide adenine dinucleotide, (ii) a glucose dehydrogenase capable of generating reduced nicotinamide adenine dinucleotide by reaction with glucose, (iii) a disulfide substrate, (iv) the other of the oxidized first indicator or the oxidized third indicator, (v) a catalyst having diaphorase activity capable of facilitating the reaction between the reduced form of nicotinamide adenine dinucleotide and the oxidized first and third indicators to produce reduced first and third indicators, (vi) a disulfide reductase; and (vii) a thiol indicator; wherein the disulfide reductase is capable of catalyzing the reaction between the generated reduced nicotinamide adenine dinucleotide and the disulfide substrate to produce a product which can reduce the thiol indicator;

whereby the device is capable of generating a range of hues, the particular final hue produced in the test device depending on the concentration of glucose in the sample.

21. The multilayer test device of claim 20 in which the hydrophilic carrier is gelatin and each layer is affixed by coating.

22. The multilayer test device of claim 20 in which the one layer additionally includes a color retardant.

23. The multilayer test device of claim 22 in which the color retardant is chosen from potassium ferricyanide, 1-N-ethyl-4-methyl-quinolinium iodide or analogs thereof.

24. The multilayer test device of claim 21 in which the thiol indicator is a water soluble disulfide.

25. The multilayer test device of claim 24 in which the reduction of the water soluble disulfide produces a yellow hue.

26. The test device of claim 25 in which the water soluble disulfide is a water soluble analogue of 5,5'-dithiobis-(2-nitrobenzoic acid).