Unified test means for ion determination

A test means, test device and method for use for determining the presence of an ion in a test sample are disclosed. The test means comprises a substantially nonpolar, nonporous carrier matrix incorporated with an ionophore capable of forming a complex with the specific ion and reporter substance capable of interacting with the complex of ionophore and ion to produce a detectable response. The test means disclosed is particularly useful for serum potassium determinations. The test means forms the reagent layer in a multilayer format particularly suited to whole blood electrolyte determinations.

1. INTRODUCTION 
The present invention relates to the measurement of ions, in particular 
ions in aqueous solution, and to a test means or device for performing 
such measurements. The invention provides a convenient format for 
determining the presence and/or concentration of such ions whereby results 
are available shortly after contacting an aqueous test sample with the 
test means or device. Cumbersome, expensive electronic equipment such as 
ion-specific electrodes, flame photometers, atomic absorption 
spectrophotometers or the like is not needed. The present invention 
enables the user merely to contact the test sample with the test device or 
similar test means configuration according to the present invention, and 
determine any detectable response. 
The determination of aqueous ion concentration has application in numerous 
technologies. In the water purification art, calcium concentration must be 
carefully monitored to assess the degree of saturation of an ion exchange 
resin deionizer. Measurement of sodium and other ions in seawater is 
important in the preparation of drinking water aboard a ship at sea. 
Measurement of the potassium level in blood aids the physician in 
diagnosis of conditions leading to muscle irritability and excitatory 
changes in myocardial function and conditions such as oliguria, anuria, 
urinary obstruction and renal failure due to shock. The measurement of 
potassium in serum is particularly important clinically. Since the 
clinical range of serum potassium is only from about 2 to about 10 
millimolar with a normal range from about 3.5 to 5.5 mM, the measurement 
requires high sensitivity and precision. Measurement of lithium levels in 
the blood are also important since the toxic dose levels are only slightly 
higher than the therapeutic levels used in psychiatric treatment. 
Needless to say, a sensitive, convenient and inexpensive method for 
determining ion concentration would greatly enhance the state of these 
technologies, as well as any others where such rapid, accurate 
determinations would be beneficial. Thus, for example, if a medical 
laboratory technician could accurately measure the potassium or calcium 
ion level of a serum or whole blood sample in a matter of seconds or 
minutes, such rapid results would increase laboratory efficiency and aid 
the physician in diagnosis. 
2. INFORMATION DISCLOSURE 
Methods for determining ions in solution included flame photometry, atomic 
absorption photometry and ion-specific electrodes. Test strip formats have 
been disclosed in copending U.S. patent application Nos. 493,969, 493,983, 
493,982 and 583,127 assigned commonly herein. The use of certain compounds 
and compositions which selectively isolate ions from the sample solution 
has become popular in ion-specific electrodes. These compounds, known as 
ionophores, have the capability of transporting ions into an electrode 
membrane thereby causing a difference in potential which can be measured. 
Ion assays utilizing the ion/ionophore phenomenon include membrane 
electrodes, liquid/liquid partitioning, fluorescence and test strips. 
2.1 Ion-Specific Electrodes 
When two solutions having different concentrations of ions are separated by 
an electrically conductive membrane, an electrical potential (EMF) is 
generated. In membrane separation cells, the membrane can be a simple 
fritted glass barrier, allowing a small but measurable degree of ion 
diffusion from one solution to the other. Alternatively, a nonporous, 
electrically nonconductive film, such as polyvinyl chloride, impregnated 
with an ionophore can be employed. In the absence of the ionophore, the 
film is an insulator and no EMF can be measured; when blended with an 
ionophore, charged ions are bound to the film and a small, measurable 
current can be induced to flow. Because the ionophore is selective in its 
affinity, and thus will bind only certain specific ions, such cells are 
ion selective. Any measurable EMF is due solely to the presence of the 
bound ions. 
The current flowing across the membrane is so small that the actual 
quantity of the ion or its counterion transported is insignificant. 
Electrical neutrality of the membrane is maintained either by a reverse 
flow of hydrogen ions, or by a parallel flow of hydroxyl anions. This 
anion effect can reduce the specificity of the electrode towards the 
intended ion and is an interference to be minimized. 
A major difficulty in the use of such ion-selective electrodes has been the 
marked reduction of accuracy and speed of response over time. Further, 
small changes in ion concentration produce such small changes in EMF that 
sophisticated voltmeter equipment is required. 
It has been known that certain antibiotics, such as valinomycin, have an 
affect on the electrical properties of phospholipid bilayer membranes 
(biological membranes), such that these antibiotics solubilize cations 
within the membrane, in the form of mobile charged couples, thereby 
providing a "carrier" mechanism by which cations can cross the insulating 
hydrocarbon interior of the membrane. These complexes carry charge through 
the membrane such that a voltage differential can be determined between 
solutions on either side of the membrane. 
U.S. Pat. No. 3,562,129, issued to Simon, describes the use of porous 
membranes impregnated with macrocyclic derivatives of amino and oxy-acids 
in ion-sensitive electrodes. Materials used to form the membrane are glass 
frits and other porous membranes. Such electrodes are said to be effective 
in measuring ion activities. 
U.S. Pat. No. 4,053,381, issued to Hamblen, et al., discloses similar 
technology, and utilizes an ion specific membrane having ion mobility 
across it. 
2.2 Liquid/Liquid Partitioning 
Another known application of ionophores in ion determinations is through 
liquid/liquid partitioning. In this procedure, a hydrophobic ionophore is 
dissolved in an organic solvent immiscible with water. Eisenman et al., J. 
Membrane Biol., 1:294-345 (1969) disclose the selective extraction of 
cations from aqueous solutions into organic solvents by macrotetralide 
actin antibiotics. This technique involves shaking an organic solvent 
phase containing the antibiotics with aqueous solutions containing 
cationic salts of lipid-soluble colored anions, such as picrates and 
dinitrophenolates. The intensity of color developed in the organic phase 
is then measured spectrophotometrically to indicate how much salt has been 
extracted. Phase transfer has also been studied by Dix et al., Angew. 
Chem. Int. Ed. Engl., 17:857 (1978) and in reviews including Burgermeister 
et al., Top. Curr. Chem., 69:91 (1977); Yu et al., "Membrane Active 
Complexones," Elsevier, Amsterdam (1974); and Duncan, "Calcium in 
Biological Systems," Cambridge University Press (1976). 
Sumiyoshi, et al., Talanta, 24, 763-765 (1977) describes a method for 
determining potassium ion in serum. In this technique serum is 
deproteinated by trichloroacetic acid and an indicator dye is added and 
shaken with a solvent such as chloroform containing valinomycin. 
Partitioning of a compound between liquids is rapid and effective, as shown 
by Eisenman, because of the mobility of the ionophore carrier and ions, 
which allows the transported species to diffuse rapidly away from the 
interface. Such a mechanism is normally impossible in the solid phase, 
because of rigidity, immobility and essentially zero diffusion of 
materials in a solid phase. 
2.3 Fluorescent Anions 
Yet another approach to the measurement of ion activity in aqueous 
solutions utilizes fluorescent anions. Feinstein, et al., Proc. Nat. Acad. 
Sci. U.S.A., 68, 2037-2041 (1971). It is stated that the presence of 
cation/ionophore complexes in organic solvents is known, but that complex 
formation in purely aqueous media had theretofore not been detected. 
Feinstein, et al., demonstrated the existence of such complexes in water 
through the use of the fluorescent salts 1-anilino-8-naphthalene sulfonate 
and 2-p-toluidinyl sulfonate. It was found that interaction of the 
cation/ionophore complexes with the fluorescent dyes produced enhanced 
fluorescence emission, increased lifetime and polarization, and 
significant blue-shift at the emission maxima of the fluorescence 
spectrum. At constant concentrations of ionophore and fluorophore, the 
intensity of fluorescence emission was found to be a function of cation 
concentration. 
2.4 Chromophore-labeled Ionophore 
The ion assay disclosed in U.S. Pat. No. 4,367,072 is primarily directed 
toward the use of a a chromogenic ionophore, i.e., an ionophore covalently 
linked to a chromogen. A charged chromogen-ionophore complex, having the 
same charge as the ion to be determined, is also used. In use, the 
chromogenic ionophore or charged chromogen-ionophore complex is added to a 
liquid sample and the color of the solution is monitored 
spectrophotometrically. Mention is made of incorporating the ionophore 
into a carrier such as paper, synthetic resin film, silicon oxide, natural 
or synthetic fibers or metal. 
2.5 Multilayer Test Device 
Multilayer formats for the determination of analytes by chemical reaction 
of the analyte with the components in the reagent layer have been 
disclosed (see, for example, U.S. Pat. No. 3,992,158 to Przybylowicz et 
al). Kitajima et al. in U.S. Pat. No. 4,356,149 discloses a multilayer 
test device wherein the reagent layer is composed of a hydrophilic binder 
and fine hydrophobic particles dispersed in that binder, which particles 
contain the reagent capable of producing a color change with the component 
being analyzed. U.S. Pat. No. 4,356,149, to Kitajima et al, is directed 
toward eliminating layers made necessary by incompatible reagent 
components. U.S. Pat. No. 4,255,384, to Kitajima et al, concerns a 
multilayered integrated element for the chemical analysis of blood. 
2.7 Summary 
To summarize the background of technological developments leading up to the 
present invention, many methods are known for assaying ions in solution. 
Instrumental methods include such sophisticated techniques as ion-specific 
potentiometry, flame photometry and atomic absorption photometry. The use 
of ionophores which selectively complex with specific ions has led to five 
basic approaches: ion selective electrodes, liquid/liquid partitioning, 
fluorescence enhancement, chromophore-labeled ionophore conjugates and 
test strips. While multilayer test strips are known, none is useful for 
the determination of ions, as is the present invention.

4. SUMMARY OF THE INVENTION 
The present invention resides in the discovery of a new test means for 
detecting the presence of a specific ion in an aqueous test sample and to 
determining its concentration. The test means comprises a substantially 
nonpolar, nonporous carrier matrix which is incorporated with an ionophore 
capable of selectively forming a complex with the ion under analysis. In 
addition, the carrier matrix is incorporated with a reporter substance 
which is capable of producing a detectable response such as change in, or 
appearance of, color or fluorescence. A test device is composed of a test 
means affixed to one flat side of an elongated support member, such as 
plastic film. In the multilayer format a transparent support member is 
used to prepare a multilayer test device wherein the detectable response 
is read through the support. 
A preferred embodiment is a multilayer test device particularly suited for 
whole blood determinations. The test means then forms the reagent layer of 
a multilayer device. 
In use the sample is contacted with the test means or multilayer test 
means, and the presence and/or concentration of the ion is then determined 
by observing any detectable response produced. 
The test means and device of the present invention provide rapid results, 
sufficient detectable response forming in most instances in at least a few 
minutes. The multilayer format is particularly useful, as the ion 
concentration of a whole blood sample can be determined without washing or 
wiping the device. 
5. DEFINITIONS 
The following definitions are provided to clarify the scope of the present 
invention and to enable its formulation and use. 
5.1 The term "ionophore" includes molecules capable of selectively forming 
a complex with a particular ion in a hydrophobic environment to the 
substantial exclusion of others. For example, the cyclic polyether 
2,3-naphtho-1,4,7,10,13-pentaoxacyclopentadeca-2-ene (sometimes known as 
2,3-naphtho-15-crown-5 and called Potassium Ionophore I herein) binds 
selectively to potassium ions in solution to form a cationic complex. Also 
included in the term are crown ethers, cryptands and podands. 
5.2 As used herein, "substantially nonpolar" is intended as meaning that 
quality of a substance not to exhibit a substantial dipole moment or 
electrical polarity. In particular, it includes nonionic substances and 
substances which are dielectric. 
5.3 The term "nonporous" is intended to mean substantially impervious to 
the flow of water. Thus, a nonporous carrier matrix is one which precludes 
the passage of water through it, one side to the other. For example, a 
polyvinyl chloride film would be considered for the purposes herein as 
being nonporous. 
5.4 A "reporter substance" is one which is capable of interacting with an 
ionophore/ion complex to produce a color change or other detectable 
response. A preferred reporter for the determination of a cation is a 
neutral compound such as a dye capable of interacting with the 
ionophore/cation complex causing the reporter to lose a proton and become 
charged, effecting a change in electron distribution. The change in 
electron distribution produces a detectable response. The expression 
"reporter substance" includes phenolic compounds, such as p-nitrophenol, 
which are relatively colorless in the nonionized state, but which color 
upon ionization and fluorescent compounds which produce more or less 
fluorescence upon a change in electron distribution. The reporter 
substance can also be one which can trigger a detectable response together 
with other components. For example, the change in electron distribution in 
the reporter substance caused by interaction with the complex can in turn 
facilitate the interaction of the reporter with another component which 
would then produce a detectable response. 
5.5 By "interacting" is meant any coaction between a reporter substance and 
an ionophore/ion complex which leads to a detectable response. An example 
of the reporter substance interacting with the complex is where the 
reporter is changed by the complex from a colorless to a colored state, 
such as in the case of p-nitrophenol. 
5.6 The expression "detectable response" is meant herein as a change in, or 
occurrence of, a parameter in a test means system which is capable of 
being perceived, either by direct observation or instrumentally, and which 
is a function of the presence of a specific ion in an aqueous test sample. 
Some detectable responses are the change in, or appearance of, color, 
fluorescence, reflectance, pH, chemiluminescence and infrared spectra. 
5.7 By the expression "intermediate alkyl" as used herein is meant an alkyl 
group having from about 5 to about 15 carbon atoms. It includes normal and 
branched isomers. It can be unsubstituted or substituted, provided any 
such substitution not interfere with the operation of the presently 
claimed test means. 
5.8 The expression "lower alkyl", as used in the present disclosure, is 
meant an alkyl moiety containing about 1 to 4 carbon atoms. Included in 
the meaning of lower alkyl are methyl, ethyl, n-propyl, isopropyl, 
n-butyl, sec-butyl and tert-butyl. These can be unsubstituted or 
substituted, provided any such substituents not interfere with the 
operation of the test means. 
5.9 By "pseudohalogen" is meant atoms or groups of atoms which, when 
attached to an unsaturated or aromatic ring system, affect the 
electrophilicity or nucleophilicity of the ring system, and/or have an 
ability to distribute an electrical charge through delocalization or 
resonance, in a fashion similar to the halogens. Thus, whereas halogen 
signifies Group VII atoms such as F, Cl, and I, pseudohalogens embrace 
such moieties as --CN, --SCN, --OCN, --N.sub.3, --COR, --COOR, --CONHR, 
--CF.sub.3, --CCl.sub.3, --NO.sub.2, --SO.sub.2 CF.sub.3, --SO.sub.2 
CH.sub.3, and --SO.sub.2 C.sub.6 H.sub.4 CH.sub.3, in which R is alkyl or 
aryl. 
6. TEST MEANS 
The test means comprises a substantially nonpolar, nonporous carrier matrix 
incorporated with an ionophore and a reporter substance. When an aqueous 
test sample contains an ion capable of specifically complexing with the 
ionophore, the ion can then enter the matrix forming an ionophore/ion 
complex, which can interact with the reporter substance to produce a 
detectable response. The test means can form the reagent layer of a 
multilayer test means. 
6.1 The Carrier Matrix 
In order for the test means or reagent layer to provide a detectable 
response solely as a result of the presence of a specific ion, it is 
necessary that other ions be substantially excluded from entering the 
carrier matrix. This is required because the ionophore/ion complex formed 
triggers the detectable response in conjunction with the reporter 
substance. Accordingly, the carrier matrix must be fabricated from a 
material which is both nonpolar and nonporous. Exemplary of such materials 
are films of such polymers as polyvinyl fluoride, polyvinyl chloride, 
vinyl chloride/vinyl acetate copolymer, vinyl chloride/vinylidene chloride 
copolymer, vinyl chloride/vinyl acetate/vinyl alcohol terpolymer, 
vinylidene chloride/acrylonitrile copolymer, and polyurethane. Of course, 
many other polymeric materials such as silicon polymers available from Dow 
Corning (e.g. Q3-9595) would be suitable for use in the present invention, 
and the identification of such materials would be well within the skill of 
the art, given the present disclosure. 
Preferably, incorporated within the polymer film as part of the nonpolar, 
nonporous carrier matrix are compounds known in the art as plasticizers 
which are nonvolatile, high boiling liquids, i.e., those having a boiling 
point of at least about 150.degree. C., ideally at least about 200.degree. 
C. As shown in the examples suitable liquids include diethylphthalate and 
dipentylphthalate. Other suitable liquids include tricresylphosphate, 
dioctylphthalate, tris-2-ethylhexylphosphate, di-2-ethylhexyl sebacate, 
n-butylacetyl-ricinoleate and nitrophenyl ethers such as 2-nitrophenyl 
octyl ether, 2-nitrophenyl butyl ether, dibenzyl ether and 
o-nitrophenyl-2-(1,3,3)-trimethyl-butyl-5,7,7-triethyl octyl ether. 
Mixtures of these liquids can be used. Such liquids are normally oxygen 
donors, containing functional groups such as ether, ester, amide and the 
like or combinations thereof. 
The carrier matrix must be nonporous and nonpolar because the ion must not 
be able to substantially penetrate the matrix unless it is the particular 
ion for which the ionophore has complexing affinity. The concept of a 
nonporous matrix, of course, does not exclude microscopic porosity. It is 
clear from the foregoing remarks as well as the very nature of the 
invention, that some porosity could be possible, provided the ion-analyte 
be precluded from permeation of the carrier matrix to a sufficient degree 
to cause the detectable response to occur in the absence of the ionophore. 
The composition of the carrier matrix in this invention is to be carefully 
distinguished over prior art carriers whereby porous materials such as 
paper were used. In that type of device, it is required that any test 
sample to which the device is exposed be capable of permeating the entire 
reagent area. Such test devices function on entirely different principles 
from the present one, and a paper carrier matrix is not considered as 
within the scope of the present invention unless such paper matrix be 
rendered substantially nonpolar and nonporous, i.e., such as by polymer or 
wax coating. 
Thus, the carrier matrix is one which substantially precludes penetration 
by the aqueous test sample. Moreover, it is intended that both the 
ionophore and reporter substance become virtually insoluble in the aqueous 
test sample due to their being entrapped with the carrier matrix. The 
requirement of nonporosity of the carrier matrix is to preclude 
dissolution or leaching of ionophore or the reporter substance, as well as 
to prevent permeation by test sample components other than the 
ion-analyte. 
6.2 Ionophores 
The ionophore element of the present invention is a concept which is broad 
in scope, as characterized by the definition of the term in paragraph 5.1, 
supra. It includes multidentate cyclic compounds which contain donor atoms 
in their cyclic chains. Such multidentate cyclic compounds can be 
monocyclic or polycyclic. Alternatively, the ionophore can be an open 
chain containing donor atoms. Thus, included in the term are monocyclic 
systems ion-specific compounds known as coronands; polycyclic ion-specific 
compounds known as cryptands; open chain ion specific compounds known as 
podands; and antibiotic type ionophores such as valinomycin and 
macrotetralide actins. 
6.2.1 Coronands 
The coronands are monocyclic compounds which contain donor atoms which are 
electron rich (or deficient) and which are capable of complexing with 
particular cations (or anions) because of their unique structures. 
Included in this term are the crown ethers in which the monocyclic chain 
contains oxygen as the donor atoms. Other coronands are compounds which 
contain an assortment of electron rich atoms such as oxygen, sulfur and 
nitrogen. Because of the unique sizes and geometries of particular 
coronands, they are adaptable to complexing with various ions. In so 
complexing, the electron rich atoms, such as the oxygens in a crown ether, 
orient towards the electron deficient cation. The carbon atom segments of 
the chain are simultaneously projected in a direction outwards from the 
ion. Thus, the resultant complex is charged in the center, but is 
hydrophobic at its perimeter. 
6.2.2 Cryptands 
The cryptands are the polycyclic analogues of the coronands. Accordingly, 
they include bicyclic and tricyclic multidentate compounds. In the 
cryptands, the cyclic arrangement of donor atoms is three dimensional in 
space, as opposed to the substantially planar configuration of the 
coronand. A cryptand is capable of virtually enveloping the ion in three 
dimensional fashion and, hence, is capable of strong bonds to the ion 
forming the complex. As with coronands, the donor atoms can include such 
atoms as oxygen, nitrogen and sulfur. 
6.2.3 Podands 
Ions can also be selectively complexed with noncyclic compounds. For 
example, a linear chain which contains a regular sequence of electron rich 
atoms such as oxygen has the capability of associating with positively 
charged ions to form complexes, not entirely unlike the coronands and 
cryptands. The main structural difference between podands and the other 
two types of ionophores is the openness of the structure. Thus, podands 
can be subcategorized into monopodands, dipodands, tripodands, and so 
forth. A monopodand, therefore, is a single organic chain containing donor 
atoms; a dipodand is two such chains attached to a central atoms or group 
of atoms is capable of variable spacial orientation; and a tripodand is 
three such chains attached to a central atom or group of atoms. Uncharged 
podands are preferred ionophores for the determination of sodium and 
calcium ions. 
Simon, et al. in U.S. Pat. No. 3,957,607 discloses dipodands particularly 
suited to the determination of calcium or barium ions. In the present 
invention, a preferred ionophore is the tripodand 
1,1,1-tris[1'-(2'-oxa-4'-oxo-5'-aza-5'-methyl) dodecanyl]propane referred 
to herein as Sodium Ionophore I, which was found to be particularly useful 
in a test means for the determination of sodium ion. In fact, Sodium 
Ionophore I is 90 times more selective for sodium ions than the dipodand, 
N,N'-dibenzyl-N,N'-diphenyl-1,2-phenylenedioxydiacetamide. [Guggi, M., 
Oehme, M., Pretsch, E. and Simon, W., Helv. Chim. Acta., 59:2417 (1976)]. 
6.2.4 Specific Ionophores 
Some of the ionophores which have been found to be especially useful with 
the instant invention are tabulated herein along with the cations with 
which they are capable of selectively complexing. 
Chemical names for preferred ionophores follow with their structures. 
Common names assigned for use herein are noted in brackets. 
______________________________________ 
Ionophore Cation 
______________________________________ 
1,1,1-tris[1'-(2'-oxa-4'-oxo-5'- 
Na.sup.+ 
aza-5'-methyl)dodecanyl]propane 
##STR1## 
[Sodium Ionophore I] 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
N,N'dibenzyl-N,N'diphenyl-1,2- 
Na.sup.+ 
phenylenedioxydiacetamide 
##STR2## 
[Sodium Ionophore II] 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
6,7,9,10,18,19-hexahydro-17-n-butyl 
Na.sup.+ 
dibenzo[b,k] [1,4,7,10,13]pentaoxa- 
cyclohexadecane-18-yl-oxyacetic acid 
##STR3## 
[Sodium Ionophore III] 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
2,3-naphtho-1,4,7,10,13-pentaoxa- 
K.sup.+ 
cyclopentadeca-2-ene 
##STR4## 
[Potassium Ionophore I] 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
N,N'diheptyl-N,N',5,5-tetramethyl- 
Li.sup.+ 
3,7-dioxanonane diamide 
##STR5## 
[Lithium Ionophore I] 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
N,N'diheptyl-5,5-dimethyl-N,N' 
Li.sup.+ 
di(3-oxapentyl)-3,7-dioxanane 
diamide 
##STR6## 
[Lithium Ionophore II] 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
cis-N,N,N',N'tetraisobutyl- 
Li.sup.+ 
1,2-cyclohexane dicarboxamide 
##STR7## 
______________________________________ 
______________________________________ 
Ionophore Cation 
______________________________________ 
diethyl-N,N'[(4R,5R)4,5-dimethyl- 
Ca.sup.++ 
1,8-dioxo-3,6-dioxaoctamethylene]- 
bis(12-methylaminododecanoate) 
##STR8## 
[Calcium Ionophore] 
______________________________________ 
Other ionophores which are useful in the present invention include those 
listed below: 
______________________________________ 
Ionophore Cation 
______________________________________ 
15-crown-5 Na.sup.+, K.sup.+ 
Valinomycin K.sup.+ 
4,7,13,16,21,24-hexaoxa-1,10-diaza- 
K.sup.+ 
bicylco [8,8,8]hexacosane 
(Kryptofix .RTM. 222) 
Dibenzo-18-crown-6 K.sup.+ 
Dicylcohexano-18-crown-6 K.sup.+ 
4,7,13,18-tetraoxa-1,10-diaza- 
Li.sup.+ 
bicylco[8,5,5]eicosane 
(Kryptofix .RTM. 211) 
12-crown-4 Li.sup.+ 
N,N'--diheptyl-N,N'--dimethyl 
Mg.sup.++ 
1,4-butanediamide 
______________________________________ 
Kryptofix.RTM. is a trademark of E. Merck, Darmstadt, West Germany. 
Although these specific ionophores were used advantageously in the test 
means of the present invention, other ionophores or mixtures thereof, can 
also be used. It has been shown that a 1:1 weight to weight ratio of 
valinomycin to Potassium Ionophore I exhibits an unexpectedly enhanced 
response as measured by the ratio of the slope of the dose response to the 
intercept (i.e., the blank). In particular, ionophores which contain 
ionizable groups, such as Sodium Ionophore III, can be substituted in the 
formulation, so long as they have sufficient ion-analyte specificity. 
6.3 The Reporter Substance 
Given the presence of the ion of interest in the test solution, it is the 
reporter substance which provides the detectable response as a result of 
its interacting with the ionophore/ion complex. The reporter substance can 
range in composition from a single compound which can ionize in response 
to the formation of the ionophore/ion complex, to a mixture of reactive 
species which produce a detectable product when their reaction chain is 
triggered by the complex. Thus, it can be seen that when no ion-analyte is 
present, the reporter substance remains dormant and no detectable response 
is observed. Alternatively, when the particular ion of interest is 
present, it is enabled by the ionophore to enter the carrier matrix to 
form a complex, which complex interacts with the reporter substance and 
induces it to undergo a detectable change. 
In the case where the reporter is a single compound, it can include a 
dissociable group such that upon dissociation the ionic species formed 
exhibits a color different from the undissociated species. For the 
determination of a cation, a particularly preferred reporter is a neutral 
compound having a dissociable proton such that upon interaction of the 
ionophore/cation complex with the reporter, the reporter loses a proton. 
This proton loss causes a change in, or appearance of, a detectable 
response in the matrix. Tetrabromophenolphthalein alkyl esters are useful 
reporters. Phenolic compounds such as p-nitrophenol, are relatively 
colorless in the nonionized state but are colored upon ionization can also 
be useful. Other compounds, such as those which produce more or less 
fluorescence upon a change in electron distribution, can also be used. 
Classes of fluorescent indicators and their derivatives which are useful 
in this invention include derivatives of fluorescein, especially 
fluorescein esters, 7-hydroxy coumarins, resorufins, pyrene-3-ols and 
flavones. 
The reporter substance can also be one which can trigger a detectable 
response together with other components. For example, a reaction sequence 
useful as the reporter substance is one which involves the dissociation of 
a proton from a phenol, thus initiating a coupling reaction to form a 
colored product. The so-called Gibbs Reaction is typical of such a 
reaction sequence, in which 
2,5-cyclohexadiene-1-one-2,6-dihalo-4-haloimine (I) couples with a phenol 
(II) to form a colored reaction product (III). 
##STR9## 
In this reaction sequence R, same or different, can be any 2, 3, 5 or 
6-position substitutent, or multiple substituents thereof, which will not 
hinder the overall reaction sequence, and n is 1 to 4. Thus R is H, lower 
or intermediate alkyl or aryl, or R can form a fused ring system at either 
the 2,3- or 5,6-positions. The X, same or different, is halogen such as F, 
Cl, Br and I, or X can be a pseudohalogen. It is preferred that 2 and 6 
position substituents be the same. The trichlorocompound is particularly 
preferred. This reporter substance can be utilized by incorporating 
compounds having the structures (I) and (II) directly with the carrier 
matrix. 
Still another utilization of the Gibbs chemistry involves compounds having 
a structure such as (III) in its nonionized form. The formation of the 
ionophore/ion complex results in an interaction such that reporter 
substance (III) yields observable color in and of itself. This phenomenon 
can be thought of as proceeding in accordance with the following reaction 
sequence and resonance structures: 
##STR10## 
in which each R, same or different, is lower alkyl or intermediate alkyl, 
aryl, or a fused ring system at the 2,3- or 5,6-positions, n is 0 to 4 and 
X is as defined above. Especially preferred is a compound having the 
structure 
##STR11## 
in which R' is H or lower alkyl and X is a halogen or pseudohalogen group 
as defined in sections 5.8 and 5.9, respectively. The case in which R' is 
methyl and each X is a chloro group has been found especially suitable to 
the present invention. 
Yet another preferred reporter substance is a compound having the structure 
##STR12## 
In which R* is an intermediate alkyl group, i.e., having 5 to 15 carbon 
atoms, and in which R' is H or lower alkyl and X is halogen or 
pseudohalogen. Compounds such as these have been found to be especially 
resistant to possible interference due to the presence of serum albumin in 
the test sample. Preferred among these type of reporter substances is 
7-(n-decyl)-2-methyl-4-(3',5' dichlorophen-4'- one)indonaphthol (referred 
to herein as 7-decyl-MEDPIN) in which R* is n-decyl, X is a chloro group 
and R' is methyl. More detailed information on the use and preparation of 
such compounds can be found in U.S. patent application Nos. 493,951 and 
431,981, both of which are assigned to the present assignee and are 
incorporated herein by reference. 
In general, neutral reporters which have a dissociable proton are preferred 
for the determination of a cation. 
6.4 Optional Components (Reagent Layer) 
The test means or reagent layer can optionally include photochemical 
stabilizers, thickeners, preservatives and so forth, provided they do not 
interefere with the production of the detectable response. Given the 
present disclosure, the choice of such components is well within the skill 
of those knowledgeable in the art. 
In the single layer format, when instrumental reading by reflectance is 
used, it is advantageous to incorporate light scattering centers with the 
carrier matrix. The use of such centers effectively increases the 
precision of the assay by reducing the effect of variations in film 
thickness. Light scattering centers can be produced by incorporating 
insoluble inorganic particles such as titanium dioxide particles or 
equivalents such as barium sulfate, calcium carbonate, aluminum oxide, 
magnesium oxide, zinc oxide, lead oxide, microcrystalline cellulose or 
talc. A working range for incorporation of titanium dioxide particles is 
up to about 40% (weight percent) of the coating emulsion; a preferred 
range is from about 0.5 to about 15 weight percent of coating emulsion. A 
particle size of less than one micron is preferred. 
7. CONCENTRATION RANGES OF TEST MEANS COMPONENTS 
The concentrations of the test means components are not critical to the 
invention provided that the concentrations of the ionophore and reporter 
substance are sufficient to produce the desired detectable response. For 
qualitative results neither the concentration of the ionophore nor the 
concentration of the reporter substance is tied to the concentration range 
of the ion-analyte to be determined. 
Determination of optimum concentrations is within the ability of one 
skilled in the art, given the present disclosure. However, the following 
guidelines are provided. It is preferable that the ionophore be present in 
molar excess over the reporter substance (i.e., greater than 1:1 molar 
ratio, ionophore:reporter substance). Working concentrations of the 
ionophore can range from 2 gm/L to saturation. 
The working and preferred concentration ranges for a test means responsive 
to potassium ion are given below. Preferred ranges are given for the 
determination of serum potassium by reflectance on an Ames SERALYZER.RTM. 
reflectance photometer. These concentrations, which can be used as a 
starting point for the determination of useful concentration ranges for 
the determination of other ions with other ionophores and reporters, are 
given below. The concentrations of ionophore and reporter refer to 
concentration in the volume of organic solvent used, other concentrations 
are defined as grams per 100 grams of solution. The film wet emulsion 
thickness for the test means (or reagent layer of a multilayer test 
device) can be from about 25 microns to about 500 microns with the 
preferred wet emulsion film thickness being from about 150 microns to 
about 200 microns. The preferred weight to weight ratio of polymer to 
plasticizer is from about 1:1.5 to 1:3. 
______________________________________ 
working preferred 
______________________________________ 
I. Test Means (Reagent Layer) 
Film thickness 
25-500 microns 150-200 
microns 
Ionophore 10-500 mM 80-160 mM 
Reporter 10-120 mM 30-60 mM 
Light Scattering 
0-40% 0.5-15% 
centers (optional) 
Polymer 5-25% w/w 5-25% w/w 
Plasticizers 8-50% w/w 8-50% w/w 
II. Optional Multilayers 
1. Reflecting layer 
Titanium dioxide 
5-40% 15-40% 
Gelatin 1-6% 2-8% 
Interferant removal 
0-30 gm/L 10-20 gm/L 
substance (optional) 
Buffer (optional) 
0-0.5 M 0.2-0.5 
M 
Sodium Chloride 
0-0.6 M 0.05-0.15 
M 
(optional) 
Wetting substance 
0-3 gm/L 1-2 gm/L 
(optional) 
Dry coating weight 
2.5-75 gm/m.sup.2 
10-25 gm/m.sup.2 
2. Opacifying layer 
Carbon black 3-30% 7-15% 
Gelatin 1.0-12% 1.5-6% 
Interferent removal 
0-30 gm/L 10-20 gm/L 
substance (optional) 
Buffer (optional) 
0-0.5 M 0.2-0.5 
M 
Sodium Chloride 
0-0.6 M 0.5-0.15 
M 
(optional) 
Wetting substance 
0-3 gm/L 1-2 gm/L 
(optional) 
Film thickness 
10-50 microns 15-35 microns 
(wet) 
______________________________________ 
Working and preferred ranges for additional layers used to prepare a 
multilayer test means or device particularly suited for whole blood 
electrolyte determinations are included in the above table for easy 
reference. The composition and use of such additional layers will be 
described more completely later in the specification. 
8. THE SINGLE LAYER TEST DEVICE 
The test means can be used by itself, prepared in a multilayer format 
and/or mounted at one end of an elongated support member The other end 
then serves as a handle. Such a test device can be held at the handle end, 
while the other end bearing the test means or multilayer test means is 
contacted with the test sample. 
Useful materials for the support member include films of a myriad of 
plastics or polymers. Examples include such polymeric materials such as 
cellulose acetate, polyethylene terephthalate, polycarbonates and 
polystyrene. The support can be opaque or it can transmit light or other 
energy. When the detectable response is fluorescence or when a coating is 
placed over the upper surface of the single layer test device to allow the 
sample to be wiped off, the test device can be read through the support 
material. Preferred supports include transparent materials capable of 
transmitting electromagnetic radiation of a wavelength in the range of 
about 200 nanometers (nm) to 900 nm. The support need not, of course, 
transmit over the entire 200-900 nm region, although for fluorometric 
detection of analytical results it is desirable that the support be 
transparent over a band wider than, or at least equal to, the absorption 
and emission spectra of the fluorescent materials used for detection. It 
may also be desirable to have a support that transmits one or more narrow 
wavelength bands and is opaque to adjacent wavelength bands. This could be 
accomplished, for example, by impregnating or coating the support with one 
or more colorants having suitable absorption characteristics. 
To prepare a single layer test device of the present invention, a solution 
of the requisite ingredients is prepared in a suitable organic solvent. 
Suitable organic solvents include cyclohexanone, tetrahydrofuran and 
acetone. Many others, of course, can be used. The solution is coated onto 
a support such as plastic and dried. The dried film can then either be 
peeled off, affixed to an elongated support and cut into a small rectangle 
member having a upper substantially flat face, such as an oblong piece of 
polystyrene film; or the coated film/plastic combination can be cut and 
affixed to a support member suitable for a handle. In either case the test 
means piece is affixed to the flat face at one end of the support, leaving 
the other end of the polystryene to serve as a convenient handle. 
The test means can be affixed by any means compatible with the intended 
use. A preferred method is by using a double faced adhesive tape between 
the test means square and the support member. Double sided adhesive tapes 
are available from 3M Company, St. Paul, Minn. 
10. USE OF THE SINGLE LAYER TEST DEVICE 
The test means and device of the present invention can be adapted for use 
in carrying out a wide variety of chemical analyses, not only in the field 
of clinical chemistry, but in chemical research and chemical process 
control laboratories. They are well suited for use in clinical testing of 
body fluids, such as blood, serum, cerebrospinal fluid and urine, since in 
this work a large number of repetitive tests are frequently conducted, and 
test results are often needed a very short time after the sample is taken. 
A preferred use is the testing of cations such as K.sup.+, Na.sup.+, 
Li.sup.+, or Mg.sup.++. In the field of blood analysis, for example, the 
invention can be adapted for use in carrying out quantitative analyses for 
many of the blood electrolytes of clinical interest. The present invention 
provides a single layer test particularly useful for the determination of 
serum potassium which requires measurements of high sensitivity and high 
precision. 
The test means (and test device) is used by contacting it with the test 
sample for a sufficient period of time. In the case of urine testing 
merely dipping the test means (or device) into the sample is sufficient. 
Although it is usually unnecessary to remove excess sample, in some cases, 
such as whole blood samples, it is desirable to remove any excess by 
wiping or blotting. 
If the ion under analysis is present in the test sample, the ionophore/ion 
complex will interact with the reporter substance, and a response will be 
detectable. Where the reporter substance is a dissociable substance 
producing a colored compound different from the undissociated compound, an 
observable color (change) will form in the test means which can be 
instrumentally monitored from either side of the device when a transparent 
support member is used. Where the reporter substance is a fluorophore such 
as fluorescein or its derivatives, a fluorescence spectrophotometer can be 
utilized to measure the detectable response formed in the test means 
(here, the appearance of or change in fluorescence). Other techniques 
useful in observing a detectable response include reflectance 
spectrophotometry, absorption spectrophotometry and light transmission 
measurements. 
Various calibration techniques are applicable as a control for the 
analysis. For example, a sample of analyte standard solution can be 
applied to a separate test means as a comparison or to permit the use of 
differential measurements in the analysis. Test means (or devices) can be 
formulated wich are suitable for semiquantitative visual determinations 
when an appropriate color chart is supplied. 
10. MULTILAYER FORMAT 
The test means can form the reagent layer containing the ionophore and 
reporter substance of a multilayer format for the determination of an ion 
in an aqueous fluid sample. 
A preferred multilayer device can be prepared by the addition of a 
reflecting layer disposed on the top of the reagent layer and optionally 
an opacifying layer disposed on top of the reflecting layer. The use of 
these additional layers is particularly useful for colored samples such as 
whole blood, as the reflective or opacifying layer(s) permit reading of 
ion concentration from beneath the reagent layer without interference by 
the inherent color of the sample. 
10.1 Reflecting Layer 
The reflecting layer contains a material or materials which act as light 
scattering centers providing a background to aid the user in determining 
the detectable response in the reagent layer of the device. In a preferred 
embodiment for whole blood determinations, the purpose of the reflecting 
layer is to screen the color of the red blood cells in the sample being 
tested from the color change to be observed by the user. The preferred 
material for "screening purposes" is titanium dioxide. However, other 
materials can be used, for example, barium sulfate, calcium carbonate, 
aluminum oxide, magnesium oxide, zinc oxide, lead oxide, talc and 
microcrystalline cellulose. Such a material is contained in the reflecting 
layer in an amount of 15 to 50 weight percent, preferably 18 to 40 weight 
percent, based on the total weight of the reflecting layer. Such materials 
generally have a particle size of less than one micron. The materials 
generally have a dry coating weight of 2.5 to 75 gm/m.sup.2, preferably 10 
to 25 gm/m.sup.2. 
In addition to the aforesaid materials, the reflecting layer usually 
contains a hydrophilic substance. Useful hydrophilic substances include 
water soluble polymers which in their dry state exhibit a marked 
wettability by aqueous media, such as gelatin, poly(vinyl alcohol), 
poly(propyleneimine), carrageen, copolymers of acrylic acid and alginic 
acid. A preferred substance is gelatin which is contained in an amount of 
2 to 8 weight percent, preferably 1 to 6 weight percent, based on the 
total weight of the reflecting layer. The remainder of the reflecting 
layer is usually water. 
Additionally, the reflecting layer can contain one or more wetting agents 
(detergents) and/or one or more suspending agents. A nonlimiting example 
of a wetting agent that can be employed in the reflecting layer is 
Triton.RTM. X-100. For example, in a test involving the determination of 
potassium, 0 to 0.5 weight percent Triton.RTM. X-100 can be utilized. 
Nonlimiting examples of suspending agents for use in the reflecting layer 
include gelatin, alginate and hydrophillic urethanes. 
The reflecting layer has a dry coating weight in the range of about 2.5 to 
75 grams per square meter, preferably 10 to 25 grams per square meter. 
To prepare a multilayered device, the reflecting layer is coated on top of 
the reagent layer and dried at about 40.degree. C. for about 10 minutes. 
9.2 Opacifying Layer 
In a preferred embodiment, an opacifying layer is deposed on top of the 
reflecting layer, i.e., the opacifying layer is optional. 
The opacifying layer has a wet layer thickness (thickness when applied) of 
10 to 50 microns, preferably 15 to 35 microns. The opacifying layer 
contains substantially inert particles for imparting an opaque appearance 
to the layer. Such particles can be, for example, carbon black particles. 
A multilayer device containing an opacifying layer is prepared by spreading 
the opacifying layer on top of the reflecting layer and drying the whole 
device again at about 40.degree. C. for about 10 minutes. 
9.3 Optional Components for Reflecting and/or Opacifying Layers 
The reflecting and/or opacifying layers can contain optional components 
such as a buffer, an interferant removal substance or sodium chloride. 
9.3.1 Buffer 
A buffer or combination of buffers can be incorporated with the reflecting 
and/or opacifying layer. Upon contact with an aqueous fluid sample, the 
buffer redissolves into the aqueous phase thus created, raising or 
lowering the pH to the desired level for the generation of the detectable 
response to proceed. With preferred reporters capable of losing a 
dissociable proton upon interaction with the ionophore/ion complex, the 
buffer maintains a suitable pH for the reaction to proceed. Prebuffering 
allows the multilayer test device to be used with unbuffered and undiluted 
samples such as sera or whole blood 
Suitable buffers include 
bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane;1,3-bis[tris-(hydroxym 
ethyl)methylamino]propane, N,N-bis-(2-hydroxyethyl)glycine, 
tris(hydroxymethyl)aminomethane, N-[2-acetamido]2-iminodiacetic acid; 
N-2-hydroxyethylpiperazine-N', 3-propanesulfonic acid; 
3[N-tris(hydroxymethyl)methylamino-2-hydroxypropanesulfonic acid; 
tetramethylammonium borate; 3-(cyclohexylamino)propane sulfonic acid and 
tetramethylammonium phosphate. 
The preferred pH range depends on the reporter substance. Therefore, the 
choice of the buffer is determined by the reporter substance used and to 
some extent by the desired detectable response. For example, when 7-decyl 
MEDPIN is used as the reporter, the preferred pH range is from 6 to 8.5. 
However, when a reporter substance having a higher pKa for the dissociable 
proton is used, a higher pH range will be preferred. Similarly when a 
reporter having a lower pKa for the dissociable proton is used, a lower pH 
range will be preferred. When the detectable response is a color change, 
the buffer can influence the degree of such detectable response, and a 
particular buffer can be chosen for color intensity optimization. For 
example, the useful pH range for the reporter, 7-decyl MEDPIN, occurs from 
about pH 6 to 8.5 where the color change is from orange to blue. A higher 
pH, pH 8.5-10, gives shades of dark blue which are difficult to 
distinguish visually, and a lower pH, pH 5-6, gives shades of pale yellow, 
also difficult to distinguish visually. Both pH extremes could be used 
with instrumental analysis, although the best instrumental precision 
occurs at the pH range of from about 6 to 8.5. Determination of a suitable 
pH is a routine laboratory experiment. 
9.3.2 Interferant Removal Substance 
Body fluids normally contain many cations, such as sodium ion (Na.sup.+), 
potassium ion (K.sup.+), calcium ion (Ca.sup.++) and magnesium on 
(Mg.sup.++). Although the ionophore will usually be chosen for its 
selectivity for the desired analyte-ion, in some cases the presence of 
other cations could interfere with the coaction of the ionophore with the 
desired analyte-ion. For example, Sodium Ionophore I will bind sodium ion 
in preference to calcium ion in a ratio of approximately 4 to 1. In 
samples where the ratio of sodium ion to calcium ion is less than 4 to 1, 
it may be necessary to prevent the interaction of calcium ion with the 
ionophore to ensure the proper relationship between sodium ion 
concentration and the detectable response. An interferant removal 
substance can be provided to obviate this problem. 
An interferant removal substance can be incorporated into the reflecting 
and/or opacifying layers. In a preferred embodiment, the removal substance 
is designed to interact with an interfering cation so as to keep it in the 
aqueous phase formed by contact with the aqueous fluid sample, or 
otherwise prevent cation interaction with the ionophore in the nonporous 
nonpolar matrix. For example, ethylenediamine tetraacetic acid (EDTA) and 
ethyleneglycol bis(aminoethyl)tetraacetic acid (EGTA) are water soluble 
compounds which form complexes with divalent cations, such as calcium ion. 
If EDTA is incorporated with the reflecting and/or opacifying layers of a 
multilayer test device for the determination of sodium ion, EDTA will 
preferentially bind calcium ion on contact with an aqueous sample 
containing sodium and calcium ions. The bound calcium ion will not 
substantially interfere with the formation of the ionophore/sodium ion 
complex in the nonpolar, nonporous reagent layer. In addition, ionophores 
can be used to remove interfering cations if they are specific for the 
interfering ion and are water soluble or are modified chemically to 
increase their water solubility without decreasing their ability to 
interact with the interferant. For example, Sodium Ionophore III can be 
modified by the addition of solubilizing groups, such as (-SO.sub.3 H) 
groups, to the benzene rings to increase its water solubility without 
decreasing its ability to interact with sodium ion. Other compounds such 
as uramildiacetic acid and 
trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid can also be used 
advantageously. 
9.3.3 Sodium Chloride Addition to Decrease Hematocrit Dependence 
Sodium Chloride can be incorporated with the reflecting and or opacifying 
layers. The addition of approximately 0.1 to 0.2M sodium chloride has been 
found to be sufficient to obviate the hematocrit dependence otherwise seen 
with whole blood potassium tests. The addition of salt is advantageous in 
the multilayer format preferred for whole blood electrolyte 
determinations. 
9.4 Description of Multilayer Test Device 
A multilayered device 12 in accordance with the present invention is 
depicted in FIG. IXa and IXb. FIG. IXb shows a two layered multilayered 
device 12, composed of a transparent support layer 14 on top of which is 
disposed a reagent layer 16. A reflecting layer 18 is disposed on top of 
the reagent layer 16. FIG. IXa shows a three layered device in which an 
opacifying layer 20 is disposed on top of reflecting layer 18. A sample 22 
is placed on top layer. The color development is read from the bottom of 
layer 14 by the human eye or using a device, e.g., a Glucometer.RTM. 
reflectance photometer which has been adapted for ion determinations. 
9.5 Use of Multilayer Device 
In using the multilayer device, a sample, such as whole blood, is applied 
on top of the uppermost layer, i.e., the reflecting layer if no opacifying 
layer is employed, or the opacifying layer and the detectable response is 
read from below through a transparent support. Reading can be accomplished 
either visually when a suitable color chart is supplied or instrumentally. 
The particular examples disclosed are designed to be read by reflectance. 
The presence of the added reflecting layer or reflecting and opacifying 
layers effectively seal the reagent layer from the outside environment. 
Accordingly, the multilayer device protects the reagent in that without 
the added layer(s), the reagent would be exposed and, therefore, 
susceptible to damage due to mechanical contact. The presence of the added 
layer(s) also serve to prolong the stability of the generated color. 
In addition, in the case where potassium is sought to be detected (for 
example, with valinomycin as the ionophore), the presence of the 
additional layer(s) allow for improved visual discrimination between 
different concentration levels. 
The use of the reflecting layer or reflecting and opacifying layers also 
provides a blocking mechanism to cells, proteins and other macromolecules, 
thus permitting the use of whole blood in tests. In addition due to the 
filtration capabilities of these layers, a multilayer test device for 
calcium measures only ionized calcium (unbound calcium) as bound calcium 
does not reach the reagent layer. Single and multilayer calcium tests can 
be used to provide information on total versus ionized calcium. 
11. EXAMPLES 
Abbreviations used in the examples are as follows: 
Square brackets, [ ], are used to designate ion concentration in millimoles 
per liter (mM) in the linear regression equations. All percent 
concentrations are given in weight per deciliter unless otherwise 
indicated. 
______________________________________ 
Temperature: .degree.C. 
degrees Centigrade 
Length: cm centimeters 
Thickness: mil 1 mil is equal to 25.4 microns 
Weight: 
gm gram 
mg milligram 
Volume: 
dL deciliter 
mL milliliter 
.mu.L microliter 
L liter 
Concentration: 
mM millimolar (millimoles per liter) 
M molar (moles per liter) 
% w/v percent weight per deciliter 
% v/v percent volume per deciliter 
Ions: 
Na.sup.+ sodium ion 
K.sup.+ potassium ion 
Li.sup.+ lithium ion 
Ca.sup.++ calcium ion 
Mg.sup.++ magnesium ion 
______________________________________ 
Abbreviations for chemical components used are given below. The ionophore 
designations are assigned for convenience only. The name is usually based 
on the principal ion the ionophore was used to determine. However, the 
ionophores commonly respond, to varying lesser degrees, to other ions. 
(Structures of preferred ionophores are given in Section 6.3.4); 
______________________________________ 
Ionophores 
Sodium Ionophore I 
1,1,1-tris[1'-(2'- 
oxa-4'-oxo-5'-aza- 
5'-methyl)-do- 
decanyl]propane 
Sodium Ionophore II 
N,N'--dibenzyl-N,N'-- 
diphenyl-1,2-phenyl- 
enedioxydiacetamide 
Sodium Ionophore III 
6,7,9,10,18,19-hexa- 
hydro-17-n-butyl-di- 
benzo[b,k] [1,4,7,10,13] 
pentaoxacycloxadecane 
18-yl-oxyacetic acid 
Potassium Ionophore I 
2,3-naphtho-1,4,7,10,13- 
pentaoxacyclopentadeca- 
2-ene 
Lithium Ionophore I 
N,N'--diheptyl-N,N'-- 
5,5-tetramethyl-3,7- 
dioxanonane diamide 
Lithium Ionophore II 
N,N'--diheptyl-5,5- 
dimethyl-N,N'--di- 
(3-oxapentyl)-3,7- 
dioxanonane diamide 
CDA cis-N,N,N',N'--tetra- 
isobutyl-1,2-cyclo- 
hexane dicarboxamide 
Calcium Ionophore diethyl-N,N'--[(4R,5R)-- 
4,5-dimethyl-1,8- 
dioxo-3,6-dioxaocta- 
methylene]bis(12-methyl- 
aminododecanoate) 
Hydrophobic Substance 
NPOE 2-nitrophenyl octyl 
ether 
NPBE 2-nitrophenyl butyl 
ether 
CDA cis-N,N,N',N'--tetra- 
isobutyl-1,2-cyclo- 
hexane dicarboxamide 
[Those below all obtained from Aldrich Chemical Co., 
Milwaukee, WI. unless otherwise noted] 
PVC polyvinyl chloride 
(low MW) low molecular weight 
(very low MW) very low molecular 
weight 
VdC/VC vinylidene chloride/- 
vinyl chloride co- 
polymer (Scientific 
Polymer Products, Inc., 
Ontario, N.Y.) 
VdC/AN vinylidene chloride/- 
acrylonitrile co- 
polymer (Scientific 
Polymer Products, 
Inc. Ontario, N.Y.) 
PC - I polycarbonate 
(molecular weight 
20,000 to 25,000) 
PC - II polycarbonate 
(molecular weight 
33,800) 
PC - III polycarbonate 
(molecular weight 
38,100) 
Reporter Substance 
7-(n-decyl)-2-methyl- 
7-decyl MEDPIN 4-(3',5'-dichlorophen- 
4'-one)-indonaphthol 
Buffering Substance -Bis-Tris 
bis[2-hydroxyethyl]- 
imino-tris(hydroxy- 
methyl)methane 
Bis-Tris propane 1,3-bis[tris(hydroxy- 
methyl)methylamino]- 
propane 
Tris tris(hydroxymethyl)- 
aminomethane 
Tris-Cl tris(hydroxymethyl)- 
aminomethane hydro- 
chloride 
ADA N--[2-acetamido]-2- 
iminodiacetic acid 
HEPPS N--2-hydroxyethylpiper- 
azine-N',3-propane- 
sulfonic acid 
Bicine N,N--bis[2-hydroxyethyl] 
glycine 
TMA borate tetramethylammonium 
borate 
TMA phosphate tetramethylammonium 
phosphate 
TAPSO 3[N--tris(hydroxy- 
methyl)methylamino]-2- 
hydroxypropane sulfonic 
acid (obtained from P.L. - Biochemicals, Inc., 
Milwaukee, WI.) 
CAPS 3-(cyclohexylamino)- 
propane sulfonic acid 
Miscellaneous 
THF tetrahydrofuran 
EDTA ethylenediamine 
tetraacetic acid 
EGTA ethylene glycol- 
bis(aminoethyl)- 
tetraacetic acid 
(G. Fredrick Smith 
Chemical O., Columbus 
OH) 
Triton .RTM. X-100 
polyethylene glycol-p- 
isooctylphenyl ether 
(Sigma Chemical Co., 
St. Louis, MO.) 
Zonyl .RTM. FSK 2-O--acetoxy-3-(perfluoro- 
alkyl)-N--carboxymethyl- 
N,N--dimethylpropyl- 
amine (Dupont Chemical 
Co., Wilmington, Del.) 
Zonyl .RTM. FSN polyethylene glycol- 
1-(2-perfluoroalkyl)- 
ethyl ether (DuPont 
Chemical Co., Wilmington 
Del.) 
Zonyl .RTM. FSB N--perfluoroalkyl-N-- 
carboxyethyl-N,N-- 
dimethylamine (DuPont 
Chemical Co., Wilmington 
Del.) 
Zwittergent .RTM. 3-10 
n-decyl-N,N--dimethyl- 
3-ammonio-1-propane- 
sulfonate (Calbiochem- 
Behring, San Diego, CA.) 
Brij .RTM. 358P polyoxyethylene ethers 
of fatty alcohols 
(ICI United States, Inc., 
Wilmington, Del.) 
Na DDBS Sodium dodecyl benzene 
sulfonate 
______________________________________ 
This invention will now be illustrated, but is not intended to be limited, 
by the following examples. 
11.1 Preparation of 
7-(.eta.-Decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)-indonaphthol 
The captioned compound (hereafter 7-decyl-MEDPIN) was prepared for use as a 
reporter substance in the present test means and test device. The reaction 
pathway is depicted in the following sequence, in which R* is .eta.-decyl. 
##STR13## 
Preparation of .beta.-(p-.eta.-Decylbenzoyl)-propionic Acid 
A mixture of 26.2 grams (gm) phenyl-.eta.-decane (1.2 mole), 120 gm 
succinic anhydride (1.2 mole) and 360 mililiters (mL) nitroethane in an 5 
liter (L) three-necked flask equipped with HCl outlet and mechanical 
stirrer was cooled to 0.degree. C. in an ice-bath. To this mixture 360 gm 
AlCl.sub.3 (2.7 moles) was added slowly over 1/2 hour with stirring. 
Evolution of HCl was observed when about half of the AlCl.sub.3 was added. 
After the addition, the ice bath was removed, the reaction mixture was 
allowed to stand at room temperature (RT) for 5 minutes. The mixture was 
then heated over a steam bath. The heating and stirring was continued 
until the vigorous evolution of HCl ceased (about 30 minutes). The 
reaction was cooled in an ice bath while 2 L of ice water was added 
followed by 600 mL of concentrated HCl. This was stirred at RT for 2 hours 
until all the dark brown solid was hydrolyzed. The insoluble product was 
recovered by filtration. The solid was then recrystallized twice with 
acetic acid (250 mL each time) to give about 320 gm (85% yield) of product 
(dried in vaccum over KOH). TLC: Rf 0.43 in 1:1 (v/v) ethylacetate:toluene 
(silica gel plate). 
Analysis: Calculated for C.sub.20 H.sub.30 O.sub.3 : C, 75.42; H, 9.50. 
Found: C, 76.02; H, 9.89. 
Preparation of 4-(.rho.-.eta.-decyl-phenyl)-butyric acid 
Twenty grams of Pd/C (palladium-saturated carbon obtained from Aldrich 
Chemical Co., catalogue No. 20,569-9) and 
.beta.-(.rho.-.eta.-decyl-benzoyl)-propionic acid (150 gm 0.47 moles) were 
mixed with acetic acid (350 mL) in a 1 liter Paar bomb. The reaction was 
started at 50 psi (pounds per square inch) H.sub.2 pressure and 50.degree. 
C. A sudden increase in temperature accompanied by a drop in H.sub.2 
volume was observed. Thin layer chromatography on the reaction mixture 
indicated complete reaction. The catalyst was removed by glass fiber 
filtration while hot. The filtrate was allowed to crystallize at room 
temperature. The crystalline product was removed by filtration. Product 
which formed after the filtrate was chilled was also recovered. The total 
yield was 100 gm (68%) after drying under a vacuum over KOH. The melting 
point was 67.degree.-69.degree. C. 
TLC: Rf 0.68 in 1:1 (v/v) 
ethylacetate:toluene (silica gel plate) 
Analysis Calculated for: C.sub.20 H.sub.32 O.sub.2 : C, 78.90; H, 10.50. 
Found: C, 78.39; H, 10.70. 
Preparation of 7-.eta.-Decyl-1-tetralone 
A mixture of .rho.-decyl-phenyl butyric acid (30 gm, 98.7 mmoles) and 
polyphosphoric acid (150 gm) was heated in an oil bath until all solid was 
melted. The heating was elevated to an internal temperature of 150.degree. 
C. and the mixture was stirred vigorously for 30 minutes. The reaction was 
then cooled to room temperature and treated with 300 mL ice water and 150 
mL ethyl ether. After the mixture was stirred for 30 minutes at room 
temperature, the aqueous layer was separated and washed twice with 150 mL 
ethyl ether. The combined organic phases were washed with 150 mL saturated 
aqueous sodium chloride. Ether was removed by evaporation and the residue 
was distilled on a Kugelrohr distillation apparatus (Aldrich Chemical Co., 
Milwaukee, Wis.). The product has a boiling point of 
190.degree.-200.degree. C./0.1 mm Hg. The yield was 11 gm (39%) of pale 
yellow oil. 
TLC: Rf - 0.34 in toluene (silica gel plates) 
Analysis Calculated for: C.sub.20 H.sub.30 O: C, 83.90; H, 10.70. Found: C, 
85.63 H, 10.83. 
Preparation of 2-Hydroxymethylene-7-n-decyl-1-tetralone 
A mixture of sodium methoxide (5.4 gm, 40.5 moles), ethyl formate (7.4 gm, 
100 mmoles) and 100 mL dry toluene was cooled in an ice bath under inert 
atomosphere nitrogen. A solution of the 7-decyl-tetralone (11.5 gm, 40 
mmoles) in 100 mL dry toluene was added with rapid stirring. The ice bath 
was removed and the reaction was stirred at room temperature for 4 hours. 
The reaction mixture was treated with 100 mL water and 100 mL 6N HCl. The 
organic layer was separated and washed twice with 50 mL saturated sodium 
chloride, dried over anhydrous Na.sub.2 SO.sub.4, filtered and evaporated 
to remove all the toluene. The oily residue was used for the next reaction 
without further purification. 
TLC: Rf=0.56 in toluene (silica gel plates), the spot turned dark-brown 
after spray with 5% FeCl.sub.3 solution. 
Preparation of 2-Benzoyloxymethylene-7-.eta.-decyl-1-tetralone 
The oily residue from the previous reaction step was mixed with dry 
pyridine (120 mL). The solution was stirred under nitrogen at 0.degree. C. 
(ice bath). The solution was treated with 30 mL of benzoyl chloride. After 
the addition of the benzoyl chloride, insoluble pyridinium chloride was 
observed in the mixture. The reaction was stirred at room temperature for 
two hours. The product was poured into ice water (400 mL) with vigorous 
stirring. The light cream color solid was recovered by filtration and 
washed well with cold water. The slightly wet solid was recrystallized 
from hot absolute ethanol (120 mL). White solid (14 gm, 87% yield based on 
the 7-decyl-1-tetralone) was recovered. The melting point was 
64.degree.-66.degree. C. 
TLC: Rf=0.40 in toluene (silica gel plates) 
Analysis Calculated for: C.sub.28 H.sub.34 O.sub.3 : C, 80.34; H, 8.19. 
Found: C, 80.05; H, 8.27. 
Preparation of 7-.eta.-decyl-2-methyl-1-naphthol 
Cyclohexane (175 ml) was added to a mixture of 
2-benzoyloxymethylene-7-(.eta.-decyl)-1-tetralone (14 gm, 33.5 mmoles) and 
Pd/C (3.5 gm) under inert atmosphere. The mixture was heated to reflux 
while maintaining the inert atmosphere. The conversion of starting 
material to product was determined by thin layer chromotography after 3 
hours. After all the starting material reacted, the mixture was cooled 
down to room temperature. The catalyst was removed by filtration and 
washed twice with 50 mL hot toluene. The combined filtrate was evaporated 
to a small volume. The product was purified with a Prep-500 silica gel 
column (a high pressure silica gel preparative column, obtained from 
Waters Association, Milford, Mass.). Toluene was used as the mobile phase. 
The product fractions were pooled and evaporated to dryness under vacuum 
overnight. Cream white solid (9.0 gm, 90% yield) was recovered: The 
melting point was 65.degree.-67.degree. C. 
TLC: Rf=0.65 in toluene (silica gel plates). 
Pink color developed when the product spot was irradiated with short UV 
light. 
Analysis Calculated for: C.sub.21 H.sub.30 O: C, 84.51; H, 10.13. Found: C, 
84.49; H, 10.72. 
Preparation of 
7-(.eta.-Decyl)-2-methyl-4-(3',5'-dichlorophen-4'-one)-indonaphthol 
7-Decylmethyl-1-naphthol (4.5 gm, 15.1 mmoles) and 
2,6-dichloroquinone-4-chloroimide (3.0 gm, 14.3 millimoles) were dissolved 
in acetone (150 mL). The solution was treated with 700 mL potassium 
carbonate solution 0.1 M, pH=10.0). The solution was stirred vigorously at 
room temperature for 10 min. The pH of the reaction mixture was adjusted 
to 2.8 with HCl (1.0 N). The mixture was stirred for 15 minutes. The red 
solid was recovered by filtration and washed well with water. The solid 
was dissolved in toluene and filtered with glass fiber paper to remove any 
insoluble materials. The filtrate was concentrated and purified with 
Prep-500 silica gel column, using toluene as the mobile phase. Product 
fractions were pooled and evaporated to dryness. The residue was 
crystallized with .eta.-hexane (100 mL) to give product 3.9 gm, 58% 
yield). 
TLC: Rf=0.26 in toluene (silica gel plates). 
Brown color spot, turn purple-blue after treated with 0.1 N NaOH on the 
plates. 
Analysis Calculated for: C.sub.27 H.sub.31 NO.sub.2 Cl.sub.2 : C, 68.64; H, 
6.57; N, 2.97. Found: C, 68.88, H, 6.85; N, 2.97. 
This product, 7-decyl-MEDPIN, was used in the following experiments as the 
reporter substance. 
11.2 Potassium Ionophore I as Ionophore 
An acetone mixture was prepared containing 10.8 mg (milligrams) 
7-decyl-MEDPIN and 24 mg Potassium Ionophore I. Solvent was removed under 
a stream of nitrogen gas. Then dried solids were combined with 4 gm of a 
film solution comprising 70% by weight cyclohexanone, 12% by weight vinyl 
chloride/vinylidene copolymer, 18% by weight diethylphthalate, and 60 
.mu.L Triton.RTM. X-100 (a 1% by weight solution of nonionic detergent in 
acetone; available from Rohm and Haas, Co.). The mixture was homogenized 
on a vortex mixer, and then spread into a thin film on a piece of 
KODAR.RTM. A150 cyclohexylene/dimethylene terephthalate copolymer (Lustro 
Co.) plastic, using a doctor blade having a 10 mil (0.01 inch or 254 
microns) gap. The dried film has a thickness of about 3 mils (76.2 
microns). 
The test means was evaluated with aqueous test samples containing various 
potassium chloride concentrations. Each sample had 15.56 mM sodium 
chloride and 88.89 MM CAPS buffer [3-(cyclohexylamino)-propanesulfonic 
acid] and pH was adjusted to 10 with lithium hydroxide. The respective 
potassium chloride concentrations were 0, 0.33, 0.67 and 1.0 mM. 
The evaluations were conducted by innoculating a section of the test means 
film with 40 microliters (.mu.L) of test sample and the change in 
reflectance monitored for one minute in a SERALYZER.RTM. reflectance 
Photometer (Ames Division of Miles Laboratories, Inc.). 
The reflectance values (R) were converted to (K/S) in accordance with 
##EQU1## 
in which R is the fraction of reflectance from the test device, K is a 
constant, and S is the light scattering coefficient of the particular 
reflecting medium. The above equation is a simplified form of the 
well-known Kubelka-Munk equation (See Gustav Kortum, "Reflectance 
Spectroscopy", pp. 106-111, Springer Verlaz, New York (1969). The data is 
tabulated below as (K/S) with respect to time. 
______________________________________ 
[K.sup.+ ] mM 
(K/S) second.sup.-1 
______________________________________ 
0 0.001151 
0.33 0.007679 
0.67 0.01295 
1.0 0.01909 
______________________________________ 
As can be seen from the table, rate of change in (K/S) with time varies in 
accordance with potassium concentration. The data is shown graphically in 
FIG. I, and demonstrates easy differentiation of various potassium ion 
concentration levels. 
11.3 Potassium Test Means Using Potassium Ionophore I and Valinomycin as 
Ionophores 
A test means film was prepared and evaluated as in Example 11.2 except that 
6 mg of Potassium Ionophore I was replaced by 6 mg valinomycin. The data 
is reported in the following table: 
______________________________________ 
[K.sup.+ ] mM 
(K/S) second.sup.-1 
______________________________________ 
0 0.001182 
0.33 0.007554 
0.67 0.01331 
1.0 0.01857 
______________________________________ 
The data shows a direct correlation between potassium ion concentration and 
rate of change of (K/S), as is clearly depicted by the graphical depiction 
of the data in FIG. II. 
11.4 Potassium Test Means Using Equal Amounts of Potassium Ionophore and 
Valinomycin as Ionophores 
A test means film was prepared and evaluated as in Example 11.2 except that 
12 mg of Potassium Ionophore was replaced by 12 mg valinomycin. The data 
is reported in the following table. 
______________________________________ 
[K.sup.+ ] mM 
(K/S) second.sup.-1 
______________________________________ 
0 0.0007449 
0.33 0.008314 
0.67 0.01251 
1.0 0.01898 
______________________________________ 
The data shows a direct correlation between potassium concentration and 
rate of change of (K/S). This is clearly shown by the graphical plot of 
the data in FIG. III. 
11.5 Potassium Test Means Using Valinomycin as Ionophore 
A test means film was prepared and evaluated as in Example 11.2 except that 
the amount of 7-decyl-MEDPIN was 5.4 mg, the ratio of vinyl 
chloride/vinylidene chloride copolymer to diethylphthalate was adjusted to 
8.55:21.45 by weight and the amount of ionophore was replaced by 12 mg 
valinomycin. 
The aqueous test samples contained potassium chloride at concentrations of 
0, 0.33, 0.67, 1.0, 2.0 and 3.0 mM. In addition, each solution contained 
46.67 mM sodium chloride, 66.67 mM CAPS and was titrated to pH 10 with 
lithium hydroxide. 
The reflectance data is reported in the following table: 
______________________________________ 
[K.sup.+ ] mM 
(K/S) second.sup.-1 
______________________________________ 
0 0.001008 
0.33 0.01090 
0.67 0.01787 
1.0 0.02872 
2.0 0.04321 
3.0 0.05330 
______________________________________ 
As can be seen from the data, the test means exhibited a direct correlation 
between potassium ion concentration and the rate of change of (K/S) with 
time. As the plot of the data in FIG. IV shows, easy differentiation 
between potassium ion concentration levels was obtained. 
11.6 Potassium Test Means Using Dipentyl Phthalate as Plasticizer 
A test means film was prepared and evaluated as in Example 11.5 except that 
the diethylphthalate was replaced by an equal weight of dipentyl 
phthalate. 
Aqueous test samples were as in Example 11.5 and contained potassium 
chloride as indicated in the table of data below: 
______________________________________ 
[K.sup.+ ] mM 
(K/S) second.sup.-1 
______________________________________ 
0 0.0005070 
0.33 0.004041 
0.67 0.007020 
1.5 0.01391 
3.0 0.02195 
______________________________________ 
The data shows a direct correlation between potassium concentration and the 
rate of change of (K/S) with time. The data is plotted in FIG. V, which 
portrays the ease of differentiation of various potassium levels using the 
film test means of the present example. 
11.7 Sodium Test Means 
A solution of 10.8 mg 7-decyl-MEDPIN in acetone and a solution of 5 mg 
Sodium Ionophore I in tetrahydrofuran (THF) were mixed and the solvents 
removed under a stream of nitrogen. To the dried solids was added 0.5 gm 
of a film solution. The latter was 70% by weight cyclohexanone, 8.55% by 
weight of vinylchloride/vinylidene chloride copolymer, and 21.45% by 
weight dipentylphthalate. The mixture was homogenized on a vortex mixer 
and the homogenate spread into a film on a piece of KODAR.RTM. A150 
plastic using a 10 mil (254 microns) doctor blade. The dried film had a 
thickness of about 3 mils (76.2 microns). 
Aqueous sodium test samples were prepared for evaluating the test means. 
Each contained 88.98 mM CAPS and potassium hydroxide was added to adjust 
the pH to 10. Samples were prepared containing 11.11 mM and 22.22 mM 
sodium chloride, respectively. 
To evaluate the ability of the test means to detect sodium, 40 .mu.L of a 
test sample was applied to a section of the test means film and 
reflectance at 640 nm was monitored over 2 minutes in a SERALYZER.RTM. 
reflectance photometer. Reflectance values were converted to (K/S) values 
as in Example 11.2. The rate of change of (K/S) with time and respective 
sodium concentrations are tabulated below: 
______________________________________ 
[Na.sup.+ ] mM 
(K/S) second.sup.-1 
______________________________________ 
0 0.001459 
11.11 0.003148 
22.22 0.004354 
______________________________________ 
The data shows a direct correlation between sodium ion concentration and 
the rate of change of (K/S) with time, as portrayed graphically in FIG. 
VI. 
11.8 Tetrabromophenolphthalein Ethyl Ester Used as a Reporter Substance in 
a Test Means for Detecting Potassium 
A solution was prepared containing 1.8 mM valinomycin, 5 mM 
tetrabromophenolphthalein ethyl ester (TBEE), 5% by weight 
polyvinylchloride ("high molecular weight", Aldrich Chemical Co. Catalogue 
No. 18,956-1) and 13.1% by weight dipentyl phthalate in tetrahydrofuran. 
This solution was spread onto a polyester film using a 10 mil (254 
microns) doctor blade, and dried. 
The test means was evaluated using aqueous solutions (test samples) 
containing potassium chloride at concentrations of 0, 0.222, 0.556 and 
1.111 mM, respectively. Each solution contained 10 mM sodium citrate 
(pH=5.3). The reflectance response of the test means to each test sample 
solution was monitored at 37.degree. C. and 520 nm using a SERALYZER.RTM. 
reflectance photometer. The results are tabulated below. 
______________________________________ 
[K.sup.+ ] mM 
(K/S) second.sup.-1 .times. 10.sup.+2 
______________________________________ 
0 -0.0108 
0.222 0.990 
0.556 2.013 
1.111 4.101 
______________________________________ 
The data shows a direct correlation between rate of change of (K/S) per 
unit time and potassium concentration. FIG. VII presents this data 
graphically, and demonstrates a linear relationship between actual 
potassium ion concentration and (K/S). 
11.9 Mixed Ionophores for the Determination of Potassium 
It has been found that a critical ratio of 1:1 by weight, of valinomycin 
and Potassium Ionophore I provides a more precise test means for the 
determination of serum potassium. The mixed ionophore provides a test with 
the same reactivity but with a lower blank reaction which increases the 
precision of the assay. Precision is particularly important with a serum 
potassium assay since the concentration range of interest is both low and 
narrow. 
A test means was prepared with a stock solution containing 12 gram 
PVC/PVdC, 18 gram DEP and 70 gram cyclohexanone. The film composition was 
as follows: 
______________________________________ 
Film stock solution 
1 gm 
7-decyl MEDPIN 10.8 mg 
Ionophore (total) 
24 mg 
Triton X-100 60 .mu.L (1% v:v in acetone) 
______________________________________ 
The film was drawn to a wet film thickness of 10 mil (254 microns) and 
dried at 70.degree. C. for 5 to 10 minutes. To assay, aqueous potassium 
chloride was diluted 9-fold with 10 mM CAPS buffer, pH 10 (i.e., 1 part 
potassium chloride to 8 parts buffer) and applied to the film. The rate of 
color development at 640 nm was measured for 1 minute with an Ames 
SERALYZER.RTM.. 
Results of various ratios of valinomycin to Potassium Ionophore I are shown 
in the table below: 
__________________________________________________________________________ 
Valinomycin: 
Potassium Ionophore I 
(w:w) 0:1 0:1 1:3 1:3 1:1 1:1 1:0 
__________________________________________________________________________ 
Slope of dose-response 
0.001983 
0.001964 
0.001910 
0.001918 
0.001963 
0.001983 
0.001911 
##STR14## 
Correlation 
0.9995 
1.000 
0.9987 
1.00 0.9946 
1.000 
0.9999 
Blank [(K/S)/sec] 
0.001151 
0.001226 
0.001063 
0.001078 
0.0007449 
0.0007626 
0.001101 
Ratio of dose slope 
1.72 1.60 1.79 1.78 2.63 2.60 1.74 
to blank 
__________________________________________________________________________ 
The results show that the maximum precision and minimal blank reaction 
occur at a 1:1 weight ratio. 
11.10 Serum Potassium Correlation Study 
Test means were prepared as follows. A film stock solution was prepared 
containing 8.55 gm PVC/PVdC, 21.45 gm diethylphthalate and 70 gm 
cyclohexanone. The film was prepared containing: 
______________________________________ 
Film stock solution 
1 gm 
7-decyl MEDPIN 5.4 mg 
Potassium Ionophore I 
6 mg 
Triton X-100 0.06 mL (1% in acetone) 
______________________________________ 
The film was spread with a doctor blade to a wet film thickness of about 10 
mil (254 cm) and dried at 70.degree. C. for 5 to 10 minutes. Serum samples 
were diluted three-fold (1 part serum to 2 parts CAPS buffer pH 10, 100 
mM) and applied to the film. The rate of color development at 640 nm was 
measured for 1 minute with an Ames SERALYZER.RTM. reflectance photometer. 
FIG. VIII shows the correlation of the reactivity of the potassium film to 
the potassium ion concentration in millimoles per liter before 3 fold 
dilution as determined by flame emission spectrophotometry. The regression 
coefficient of the correlation line is 0.9817 indicating very good 
correlation of the test means results with serum potassium in the 
concentration range (3 mM to about 7.5 mM) of clinical interest. 
Obviously many other modifications and variations can be made without 
departing from the spirit and scope of the invention.