Calcium ion selective electrode

A calcium ion selective electrode having a sensitive membrane comprising an organic polymeric substance, a plasticizer and a calcium ion sensitive substance of neutral carrier type, wherein the dielectric constant of the plasticizer is 10 or less, thus remarkably improving the responsiveness and stability of the electrode.

BACKGROUND OF THE INVENTION 
The present invention relates to a calcium ion selective electrode of a 
polymeric support membrane type suitable for the measurement of the 
concentration of a calcium ion in a living body fluid such as blood. 
Ions which are cations contained in a living body fluid, particularly 
blood, and frequently measured in the field of clinical tests are sodium, 
potassium, lithium and calcium ions. Among them, the sodium ion which is 
contained in the highest concentration, having a normal value of 
concentration of 135 to 145 mmol/l. 
On the other hand, the normal value of calcium ion concentration is 1 to 2 
mmol/l, i.e., about two figures lower than that of the sodium ion. The 
measurement of the calcium ion has been regarded as clinically important 
for the diagnosis of thyroid gland function, never excitement and transfer 
function. From this point of view, it will be understood that whether or 
not the calcium ion can accurately be measured is an important key to the 
evaluation of the health condition based on the calcium ion concentration. 
One of the methods of measuring the ion concentration of a living body 
fluid is an ion selective electrode method, and in recent years this 
method has come into wide use in the field of clinical tests. However, in 
order to realize an accurate measurement, it is necessary to eliminate a 
primary factor of error based on the influence of an interfering 
substance, etc., on the electrode. 
If there is a coexistent ion, the relationship between the electrode output 
(E) and the activity (substantially equal to the concentration) when an 
intended ion is measured with an ion selective electrode can be expressed 
by the following equation (I): 
EQU E=E.sub.0 +(RT/Z.sub.i F) log[a.sub.i +.SIGMA.K.sub.ij 
(a.sub.j).sup.z.sbsp.i.sup./z.sbsp.j (I) 
where E.sub.0 is the standard potential, R is the gas constant, T is the 
absolute temperature, F is the Faraday constant, a and z are the activity 
and valence of the intended ion (i) and the interfering ion (j), 
respectively, and K.sub.ij is the selectivity factor. 
RT/z.sub.i F is called a slope sensitivity and serves as a measure of 
whether or not the ion selective electrode is satisfactory. 
The slope sensitivity (29.58 mV/dec. at 25.degree. C.) of a selective 
electrode (hereinafter referred to simply as the "electrode") which 
selects a calcium ion as a divalent ion and measures the ion concentration 
is one half of the slope sensitivity (59.16 mV/dec. at 25.degree. C.) of 
an electrode for a sodium ion as a monovalent ion. 
When the calcium ion concentration is measured by the ion selective 
electrode method, the selectivity of the electrode is lower than that of 
the electrode for the sodium ion and further as described above the 
concentration to be measured is very low, which makes it more difficult to 
conduct an accurate measurement. 
An ion sensitive membrane is essential to the ion selective electrode. This 
sensitive membrane comprises a sensitive substance selectively responsive 
to the intended ion, a plasticizer which plasticizes and serves also as a 
solvent for the membrane, and a matrix material comprising a polymeric 
substance such as polyvinyl chloride. The characteristics of the electrode 
depend mainly upon the sensitive substance and plasticizer. 
In order to realize an accurate measurement, it is necessary to: 
(1) lower the selectivity factor, K.sub.ij, of the ion selective electrode, 
(2) present the interference by a lipophilic anion as shown in the equation 
(II) described later, and 
(3) enhance the responsiveness of the electrode. 
Although the selectivity factor described in the above item (1) is 
substantially determined by the ratio of the stability constant in a 
complex forming reaction of the sensitive substance with the intended ion, 
it essentially depends upon the sensitive substance. On the other hand, 
the properties of the plasticizer affect the interference by a lipophilic 
anion and the responsiveness of the electrode. 
A neutral carrier and an ion exchanger have been used as the sensitive 
substance for the calcium ion selective electrode. When the sensitive 
substance is the former one, ether compounds, such as o-nitrophenyl octyl 
ether (dielectric constant.apprxeq.24), have been used as the plasticizer 
[see Anal. chem. 1986, 58, 2282-2285 "Neutral Carrier Based Ca.sup.2+ 
--Selective Electrode with Detection Limit in the Sub-Nanomolar Range"], 
while when the sensitive substance is the latter one, plasticizers having 
relatively higher dielectric constants, such as esters of phosphoric acid 
such as di-n-octyl phenyl phosphate, have been used alone. 
The present inventors have made various studies on the conventional ether 
compounds and other plasticizers in connection with the plasticizer for 
the sensitive membrane of a calcium ion selective electrode wherein use is 
made of a neutral carrier type of sensitive substance and, as a result, 
have found that a calcium ion selective electrode wherein use is made of a 
neutral carrier type of sensitive substance is susceptible to the 
interference by a lipophilic anion, with a different extent of influence 
depending also upon the plasticizer, and has a responsiveness susceptible 
to the properties of the plasticizer. 
The above-described conventional sensitive membrane for a calcium ion 
selective electrode had problems with, for example, the sensitivity and 
responsiveness of the electrode. 
SUMMARY OF THE INVENTION 
An object of the present invention is to solve the above-described problems 
and to provide a calcium ion selective electrode which exhibits a high 
selectivity and excellent responsiveness and stability. 
In order to attain the above-described object, the present invention 
provides a calcium ion selective electrode of a polymeric support membrane 
type having a sensitive membrane comprising an organic polymeric 
substance, a plasticizer and a calcium ion selective substance of neutral 
carrier type, wherein the dielectric constant of the plasticizer is 10 or 
less. 
A plasticizer is essential to a sensitive membrane of an ion selective 
electrode. It plasticizes the sensitive membrane and serves also as a 
solvent for the membrane. According to the studies conducted by the 
present inventors, good results can be obtained when the dielectric 
constant of the plasticizer is 10 or less. 
Examples of the plasticizer having a dielectric constant of 10 or less 
include esters of adipic acid, such as dioctyl adipate, bis(1-butylpentyl) 
adipate, bis (2-ethylhexyl) adipate and 
bis(1-butylpentyl)-decane-1,10-diyldiglutarate, dialkyl sebacates as 
esters of sebacic acid wherein the alkyl group has 4 to 8 carbon atoms, 
such as dioctyl sebacate, dibutyl sebacate, dihexyl sebacate and diheptyl 
sebacate, esters of phthalic acid having 4 to 8 carbon atoms, such as 
dibutyl phthalate, dipentyl phthalate and dioctyl phthalate, esters of 
phosphoric acid, such as tris(2-ethylhexyl) phosphate and dioctyl phenyl 
phosphate, and ether compounds such as diphenyl ether. 
It is also possible to use alcohol compounds having 8 to 24 carbon atoms. 
The above-described plasticizers may be used alone. When they are used in 
the form of a mixture, it is also possible to use plasticizers having a 
dielectric constant exceeding 10, such as o-nitrophenyl octyl ether, 
acetophenone and nitrobenzene. In this case, however, they should be mixed 
with plasticizers having a dielectric constant of 10 or less so that the 
dielectric constant value of the mixture is 10 or less. 
It is desirable to add a salt of tetraphenylboric acid as an additive for 
preventing the interference by an anion. Examples of the salt of 
tetraphenylboric acid include sodium tetraphenylborate and potassium 
tetrakis-p-chlorophenylborate. 
The calcium ion contained in an aqueous sample forms a cationic complex 
with a calcium ion sensitive substance and combines with a hydroxide ion 
or the like to form a ternary complex which is extracted into a sensitive 
membrane. On the other hand, when living body fluids, such as blood or 
urine, are measured, the use of a compound having a high dielectric 
constant as the plasticizer causes a lipophilic anion contained in a large 
amount if a living body fluid to combine as a counter ion of the cationic 
complex, which facilitates the distribution within the sensitive membrane. 
The electromotive force (E) of the calcium ion selective electrode is 
expressed by the following equation: 
EQU E=E.sub.0 +(I-t.sub.x).multidot.Slog(Ac)-t.sub.x .multidot.S'log(Ax)(II) 
wherein 
E.sub.0 : standard potential, 
t.sub.x : transport number of anion, 
S,S': slope sensitivity, 
Ac: calcium ion concentration, and 
Ax: anion concentration. 
When a lipophilic anion is distributed within a sensitive membrane, the 
third term of the equation (II) becomes nonnegligible even when the 
calcium ion is present in the same concentration, which causes the 
lipophilic anion to affect the electrode potential. 
In view of the above, in order to decrease the influence of the lipophilic 
anion on the calcium ion selective electrode, measures should be taken to 
prevent the distribution of the lipophilic anion within the sensitive 
membrane. The plasticizer used for this purpose is preferably one having a 
low dielectric constant.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
FIG. 1 is a cross-sectional view of one embodiment of the calcium ion 
selective electrode according to the present invention. 
The form of the electrode is not limited to this only, and there is no 
particular limitation on the form of the electrode as far as use is made 
of a calcium ion sensitive membrane. 
An electrode barrel 1 accommodates an internal solution 2 containing 10 
mmol/l of calcium chloride, and an internal electrode 3 comprising 
silver/silver chloride is immersed in the internal solution 2. A sensitive 
membrane 4 is formed at the end of the electrode barrel 1. This sensitive 
membrane 4 comprises a polymeric substance, such as polyvinyl chloride, as 
a matrix material, and a calcium ion sensitive substance and a plasticizer 
and dispersed therein each in a suitable weight concentration. 
Embodiment 1 
1% by weight of 
[(-)-(R,R)--N,N'-bis[11-(ethoxycarbonyl)undecyl]-N,N'-4,5-tetramethyl-3,6- 
dioxaoctanediamide] (hereinafter referred to as "ligand A") as a calcium 
ion sensitive substance, 65.7% by weight of dioctyl sebacate (hereinafter 
referred to as "DOS"; dielectric constant: about 4) as a plasticizer, 33% 
by weight of polyvinyl chloride as a matrix material and 0.3% by weight of 
sodium tetraphenylborate as an additive for preventing the interference by 
cation were dissolved in tetrahydrofuran (THF). The resultant solution was 
cast within a glass ring having a diameter of 30 mm placed on a glass 
plate, and THF was evaporated to prepare a sensitive membrane of the 
present invention. 
The above-described sensitive membrane was cut into a size of about 5 mm in 
diameter and bonded to the end of the electrode barrel 1 made from 
polyvinyl chloride to form a calcium ion selective electrode. 
The selectivity for various ions was examined by the single solution 
method, and the results are given in FIG. 2. 
EXAMPLE 1 
A comparison was made on the performance of a calcium ion selective 
electrode having such an ion sensitive membrane composition such that only 
the plasticizer was different from that used in Embodiment 1. 
The plasticizer used was o-nitrophenyl octyl ether having a relatively high 
dielectric constant (hereinafter referred to as "o-NPOE"; dielectric 
constant: about 24). 
The calcium ion selective electrode was sufficiently conditioned with 10 
mmol/l of an aqueous CaCl.sub.2 solution, and the selectivity for various 
ions was examined by the single solution method according to JIS (K0122) 
to give the results of FIG. 2. 
The selectivity in the Embodiment 1 was substantially the same as that in 
the Example 1, except for the sodium, potassium and lithium ions. When a 
living body fluid is a sample to be measured, it appears that judging from 
the ion concentration of said solution, the difference in the selectivity 
between the calcium ion-selective electrodes of the Embodiment and the 
Example has substantially no effect on the measurements. 
Then, the responsiveness of the above-described calcium ion selective 
electrode was determined by the jet flow method. 
The jet flow method is an effective method for determining the 
responsiveness inherent in an ion selective electrode. 
When a sample solution is sprayed on the surface of a sensitive membrane of 
an ion selective electrode at a linear velocity of 200 to 300 cm/sec, the 
responsiveness inherent in the ion selective electrode can be obtained, 
because the thickness of the diffusion layer provided on the surface of 
the sensitive membrane is negligible. When the ion selective electrode is 
immersed in a solution, a concentration gradient occurs between the 
surface of the electrode membrane and the solution bulk. This is called a 
diffusion layer. If this diffusion layer is not negligible, a time taken 
for the ion to diffuse into the diffusion layer and reach the surface of 
the electrode is unfavorably added to the response time inherent in the 
ion selective electrode. 
In order to make this diffusion layer negligible, there are a method 
wherein the solution is stirred by means of a stirrer, etc. and a jet flow 
method wherein the solution is sprayed on the surface of the ion selective 
electrode at a high velocity. The jet flow method is best suited when two 
solutions are used for determining the responsiveness. 
A mixed solution (A) comprising 140 mmol/l of sodium ions, 5 mmol/l of 
potassium ions, 105 mmol/l of chloride ions and 2 mmol/l of calcium ions 
and a mixed solution (B) comprising 140 mmol/l of sodium ions, 5 mmol/l 
potassium ions, 150 mmol/l chloride ions and 5 mmol/l of calcium ions were 
alternately flowed at a linear velocity of 250 cm/sec to compare the 
respective response times with each other. 
The response curves thus obtained are shown in FIGS. 3 (a) to (d). 
The 95% response time (FIGS. 3 (a) and (b)) from the time t.sub.1 to the 
time t.sub.2 of the calcium ion selective electrode of Embodiment 1 was 
about 0.5 sec on the average (when measured five times). 
On the other hand, the 95% response time (FIG. 3 (c) and (d)) from the time 
t.sub.1 to the time t.sub.2 of the calcium ion selective electrode of 
Example 1 was about 1.5 sec on the average (when measured five times). A 
significant difference exists between the responsiveness of both the 
electrodes, and it is apparent that DOS having a lower dielectric constant 
is more suitable for the plasticizer from the viewpoint of the 
responsiveness. 
Embodiment 2 
Use was made of the same calcium ion selective substance and matrix 
material as those of Embodiment 1, and dioctyl adipate (hereinafter 
referred to as "DOA"; dielectric constant: about 4) and potassium 
tetrakis-p-chlorophenylborate were added as the plasticizer and the 
additive for preventing the interference by an anion, respectively. The 
four raw materials for the sensitive membrane were used in a composition 
ration of 1.0% by weight, 64.0% by weight, 34.5% by weight and 0.5% by 
weight. 
EXAMPLE 2 
A comparison was made on the electrode performance through the use of the 
same calcium ion selective electrode as that of Example 2, except that 
o-NPOE was used as the plasticizer. The composition ratio of the four raw 
materials for the sensitive membrane was the same as that of Embodiment 2. 
The selectivity for sodium ion and potassium ion is important in the 
measurement of the concentration of a calcium ion in a living body fluid. 
The selectivity for sodium ion was 0.0005 in the case of Embodiment 2 and 
0.0001 in the case of Example 2. The selectivity for potassium ion was 
0.0003 in the case of Embodiment 2 and 0.001 in the case of Example 2. 
As with Embodiment 1, when o-NPOE was used as the plasticizer, the 
selectivity was slightly superior to the case where DOA was used as the 
plasticizer. However, the influence of the sodium ion or potassium ion in 
the living body fluid is one corresponding to that of the calcium ion of a 
concentration of 0.1 mmol/l or less, so that the difference in the 
selectivity between both of the electrodes brings about no problem from 
the viewpoint of practical use. 
On the other hand, a difference in the response to a lipophilic anion 
(SCN.sup.- as an example) was observed between both the electrodes. 
Two solutions (solutions C and D, respectively) prepared by adding each of 
CaCl.sub.2 and Ca(SCN).sub.2 to a mixed solution comprising 140 mmol/l of 
sodium ions, 5 mmol/l of potassium ions and 105 mmol/l of chloride ions in 
a final concentration of 3 mmol/l. The results of the measurement 
conducted by respective calcium ion selective electrodes are given in 
Table 1. 
Both of the calcium ion selective electrodes have substantially the same 
measured value with each other for solution C, while the measured values 
for solution D were different from each other. 
It is apparent that the use of DOA as the plasticizer provides a more 
accurate measurement than that of the case where o-NPOE was used as the 
plasticizer. 
The reason why the measured value for solution D was lower than the 
theoretical value even when use was made of DOA as the plasticizer resides 
in that part of SCN.sup.- combines with CA.sup.2+ to thereby reduce the 
amount of calcium in the ionic form measurable by means of the calcium ion 
selective electrode. 
TABLE 1 
______________________________________ 
Solution C 
Solution D 
______________________________________ 
Embod. 2 2.97 mmol/l 
2.90 mmol/l 
Embod. 3 2.95 mmol/l 
2.88 mmol/l 
Ex. 2 2.96 mmol/l 
2.60 mmol/l 
______________________________________ 
The responsiveness of each of the calcium ion selective electrodes was 
determined by the jet flow method in the same manner as that of Embodiment 
1. 
Solutions A and B were alternately flowed at a linear velocity of 250 
cm/sec to compare the response time. 
The 95% response time of the calcium ion selective electrode of Embodiment 
2 was about 0.4 sec on the average (when measured five times). On the 
other hand, the 95% response time of the calcium ion selective electrode 
of Example 2 was about 1.1 sec on the average (when measured five times). 
That is, a significant difference in the responsiveness was observed 
between both of the electrodes. 
As is apparent from the relationship between the dielectric constant and 
the selectivity shown in FIG. 2, a plasticizer having a higher dielectric 
constant provides a higher selectivity but is influenced by the lipophilic 
anion to a greater extent, so that the use of a plasticizer having a 
smaller dielectric constant is preferred. 
Embodiment 3 
In the present Embodiment, the calcium ion sensitive substance, the 
additive and the matrix material were the same as those used in Embodiment 
2, i.e., ligand A, potassium tetrakis-p-chlorophenyl-borate and polyvinyl 
chloride, respectively, and a mixture of DOA with o-NPOE (5:1) was used as 
the plasticizer. 
The selectivity factor for sodium ion and potassium ion as determined by 
the single solution method were each 0.0001. The use of a mixed 
plasticizer rather than the single use of DOA as the plasticizer provided 
the same selectivity as that in the case where use was made of o-NPOE. 
The measured values for solutions C and D are given in Table 1. As is 
apparent from Table 1, the results were substantially the same as those in 
the case of the single use of DOA. 
Embodiment 4 
In the present Embodiment N,N,N', N'-tetracyclohexyl-3-oxapentanediamide 
and tris (2-ethylhexyl) phosphate (dielectric constant: about 8) were used 
as the calcium ion sensitive substance and the plasticizer, respectively, 
and the additive, matrix material and concentrations thereof were the same 
as those of Embodiment 2. 
The calcium ion selective electrodes of Embodiment 4 and Example 2 were 
immersed in 10 mmol/l of a CaCl.sub.2 solution for one month or longer, 
and then subjected to the determination of the selectivity. 
The results are shown in FIG. 4. 
As is apparent from FIG. 4, although the selectivity varied in both the 
calcium ion electrodes, the extent of variation was smaller in the calcium 
ion selective electrode of Embodiment 4 and the use of a plasticizer 
having a lower dielectric constant provided a better stability. 
The relationship between the dielectric constant of plasticizers (single 
and mixed systems) and the 95% response time and the measured value for 
sample solution D of the calcium ion selective electrode as determined by 
the jet flow method is shown in FIG. 5. 
When the dielectric constant exceeds 10, the 95% response time increases 
and the measured value for solution D begins to lower. 
The lowering in the measured value for solution D is thought to be because 
the calcium ion selective electrode is influenced by a thiocyanate ion 
although the calcium ion concentration of the solution D is constant. 
Further, the relationship between the dielectric constant of the 
plasticizer and the slope sensitivity is shown in FIG. 9. From this 
drawing, it is apparent that the slope sensitivity is high and the 
long-term stability is excellent when the dielectric constant is 10 or 
less. 
As is apparent from the foregoing results, the dielectric constant of the 
plasticizer should preferably be 10 or less. 
Embodiment 5 
In the raw material composition of the ion sensitive membrane of Embodiment 
2, the plasticizer was changed to 1-tetradecyl alcohol having a dielectric 
constant of 4.7. 
The selectivity factor for sodium ion was 0.0005 as determined by the 
single solution method. 
The above-mentioned alcohol compound should preferably be one having 8 to 
24 carbon atoms from the viewpoint of preventing the deterioration of the 
calcium ion selective electrode caused by leaching from the sensitive 
membrane. 
An alcohol compound having 25 or more carbon atoms is unfavorable because 
it precipitates in the sensitive membrane due to its high crystalline 
nature, which brings about a possibility of a lowering of the 
responsiveness of the calcium ion selective electrode or an increase of 
the electrode resistance. 
Although a monohydric alcohol compound was used here, it is also possible 
to use a dihydric or higher alcohol compound. 
Embodiment 6 
A field-effect transistor was prepared through the use of the calcium ion 
sensitive membrane of Embodiment 2. 
FIG. 6 is a schematic cross-sectional view of the structure of the 
field-effect transistor. 
An n-type source 5 and drain 6 were formed on a silicon substrate 7, and a 
SiO.sub.2 film 8 and a Si.sub.3 N.sub.4 insulating film 9 were formed 
thereon. Subsequently, the sensitive membrane 4 of Embodiment 2 was formed 
on the Si.sub.3 N.sub.4 insulating film 9 to prepare a field-effect 
transistor 10 for the measurement of calcium ion. 
FIG. 7 is a circuit for measurement wherein use is made of the 
above-described transistor as a measuring circuit. 
The transistor 10 is built in a measuring circuit comprising amplifiers 11 
and 11', a constant-current power source 12, etc., and output (Vout) is 
effected through the use of the electrode in combination with a reference 
electrode 13 in a coupled form. 
Embodiment 7 
FIG. 8 is a block diagram of a living body fluid analyzer wherein a calcium 
ion selective electrode using the calcium ion sensitive membrane of the 
present invention was built as a detector. 
A sample 14 for the measurement was sucked by means of a pump 15 into a 
flow cell 16 equipped with a calcium ion selective electrode and a 
reference electrode. An electromotive force corresponding to the calcium 
ion concentration occurs across the electrodes and amplified by means of 
the amplifier 11. Calculation is conducted by means of a calculator 17 
based on the amplified signal, and the calcium ion concentration is 
displayed on a display 18. 
It is also possible to use a living body analyzer for the measurement of a 
plurality of items wherein the ion selective electrodes for sodium and 
potassium ions were built as a detector, or gas electrodes, such as carbon 
dioxide electrode and oxygen electrode, were included in combination with 
the above-described ion selective electrodes. 
It is also possible to use a measurement electrode of the field-effect 
transistor described in Embodiment 6. 
The present Embodiment provides a living body fluid analyzer which exhibits 
a higher sensitivity than that of the conventional calcium ion selective 
electrode and excellent responsiveness and stability. 
Embodiment 8 
In the present Embodiment, the calcium ion sensitive substance, 
plasticizer, additive and matrix material were the same as those used in 
Embodiment 2, i.e., ligand A, dioctyl adipate (DOA), potassium tetrakis-p 
chlorophenylborate (hereinafter referred to as "KTpClPB") and PVC, 
respectively. The concentrations of the respective sensitive membrane 
materials were such that the PVC concentration was 34.5% by weight and the 
KTpClPB concentration was constant at 70% by mole based on the ligand A, 
while the concentration of the ligand A was varied from 0.5% by weight to 
15% by weight. The balance was DOA as the plasticizer. A sensitive 
membrane was prepared in the above-described weight proportions, and the 
above-described calcium ion selective electrode was formed through the use 
of this sensitive membrane. The obtained calcium ion selective electrode 
were subjected to the measurement of the initial slope sensitivity and the 
selectivity factor for coexistent ions and then immersed in the serum to 
determine the variation of the electrode characteristics with time. For 
comparison, calcium ion selective electrodes equipped with the same 
sensitive membranes as those of the present Embodiment except for the 
variation in the concentration of ligand A from 0.5% by weight to 15% by 
weight with the use of o-NPOE as the plasticizer were immersed in the 
serum for the measurement of electrode characteristics in the same manner 
as that of the present Embodiment. As a result, it has been found in the 
Example that the immersion of the calcium ion selective electrodes in the 
serum for 5 days causes the slope sensitivity to rapidly lower from the 
initial value of 25 mV/dec. to 19 mV/dec. in all of the calcium ion 
selective electrodes, which renders the calcium ion selective electrodes 
substantially unusable. By contrast, the calcium ion selective electrode 
equipped with the sensitive membrane of the present Embodiment wherein use 
was made of DOA as the plasticizer had the same slope sensitivity as the 
initial value of 25 mV/dec. or more, that is, the electrode was stable, 
even after the serum immersion test for about 20 days. Therefore calcium 
ion selective electrodes were prepared while varying the dielectric 
constant of the plasticizer to confirm that the initial slope sensitivity 
was 25 to 27 mV/dec, and the electrodes were immersed in 10 mmol/l of an 
aqueous calcium chloride solution for three weeks. As a result, no 
significant variation of the slope sensitivity was observed relative to 
the initial value. On the other hand, the immersion of the electrode in 
the serum for five days followed by the determination of the slope 
sensitivity in the same manner as that described above gave the results 
shown in FIG. 9. Namely it has become apparent that the higher the 
dielectric constant of the plasticizer, particularly when the dielectric 
constant is 10 or higher, the more rapid the lowering in the slope 
sensitivity and the poorer the stability. Thus, it has been found that the 
calcium ion selective electrode wherein use is made of a plasticizer 
having a high dielectric constant has a substantially short electrode 
life. This is thought to be because when the dielectric constant of the 
plasticizer is high, the electrode is susceptible to the adsorption of 
proteins contained in the serum. 
Next the calcium ion selective electrode of the present Embodiment was 
immersed in the serum to determine the variation with time of the 
selectivity factor for sodium ion which is a cation present in the largest 
amount in the blood, and the results are shown in FIG. 10. The abscissa 
represents the concentration of ligand A, while the ordinates represents 
the ratio of the selectivity factor for sodium ion after the immersion of 
the electrode in the serum for 20 days to the selectivity factor for 
sodium ion before the immersion. The larger the value, the larger the 
variation of the selectivity factor for sodium ion. It has been found that 
the variation of the selectivity factor for sodium ion in the case of the 
immersion of the electrode in the serum tends to vary depending upon the 
concentration of the calcium ion sensitive substance. Specifically, an 
observed tendency is that the variation of the selectivity factor for 
sodium ion reduces as the concentration of ligand A increases. From the 
above results, it is apparent that in order to stably maintain the 
characteristics of the calcium ion selective electrode for a long period 
of time, it is effective to use a plasticizer having a low dielectric 
constant and, at the same time, to set the concentration of the calcium 
ion sensitive substance at 1 to 15%. However, since the ion sensitive 
substance is generally expensive, the concentration of the calcium ion 
sensitive substance is thought to be preferably 3 to 10% from the 
viewpoint of the electrode characteristics and profitability. In view of 
the above-described results, a calcium ion selective electrode as one 
example equipped with a sensitive membrane comprising 6.0% by weight of 
ligand A, 56.5% by weight of DOA, 3.0% by weight of KTpClpB and 34.5% by 
weight of PVC was used for the measurement on a blood sample by the 
non-dilution method. As a result, the slope sensitivity of the calcium ion 
selective electrode was found to be substantially the same as the initial 
value of 27 mV/dec. even after the measurement of 6000 specimens of a 
blood sample. The selectivity factor for sodium ion was 3.times.10.sup.-4. 
As described above, the calcium ion sensitive membrane of the present 
invention can provide a calcium ion selective electrode having a high 
reliability and a living body fluid analyzer using said electrode by 
virtue of its high sensitivity and excellent stability and responsiveness.