Porous polymer film calcium ion chemical sensor and method of using the same

A method of measuring calcium ions is disclosed wherein a calcium sensitive reagent, calcichrome, is immobilized on a porour polymer film. The reaction of the calcium sensitive reagent to the Ca(II) is then measured and concentration determined as a function of the reaction.

BACKGROUND OF THE INVENTION 
Chemical sensors are used to detect the presence of a metal cation in a 
substance and are useful in a variety of fields. Typically, a colorimetric 
reagent is immobilized on a support, such as optical fibers, and the 
sensor is applied in biomedical uses, process control, and environmental 
analysis. Detection of calcium ions (Ca(II)) is useful, for example, in 
detecting the amount of calcium in the blood of animals, or in detecting 
calcium in the food processing industry. The colorimetric reagent reacts 
with the calcium ion in a substance or solution so that there is a change 
in an optical property; for example, absorption, luminescence, or 
reflectance, and correlated with the concentration of the calcium ion. 
This is accomplished by correlating the concentration of the calcium ion 
to a calibration curve which relates the optical property to the 
concentration of the calcium. 
Sensors which have been developed to this point have several deficiencies, 
including a slow response time because of barriers to mass transport at 
the polymer support, a low sensitivity due to weak analytical signal, a 
low selectivity due to interferences, and a long term instability because 
of degradation of the immobilized reagent or its desorptive loss from the 
support. 
This invention relates to an improved sensor which provides for a rapid 
response time and high sensitivity through the use of a porous polymer 
film as the support material for the analytical reagent. High selectivity, 
and resistance to degradation are achieved by employing a calcium ion 
sensitive colorimetric reagent having sulfonic acid or carboxylic acid 
binding to the anion exchange polymer and calcium ion chelating groups. 
A primary object of the invention is to provide for an improved chemical 
sensor of calcium ions in a solution or substance. 
It is an objective of this invention to provide a chemical sensor for 
calcium ions which has a rapid response time. 
A further object of the invention is to provide for a highly sensitive 
chemical sensor of calcium ions. 
Yet a further object of the invention is to provide a chemical sensor with 
high selectivity for calcium ions. 
Another object of the invention is to provide a chemical sensor which is 
resistant to degradation. 
A still further object of the invention is to provide a chemical sensor in 
which the analytical reagent has strong binding to the underlying 
polymeric support. 
Further objects of the invention will become apparent in the following 
disclosure. 
SUMMARY OF THE INVENTION 
A porous anion exchange polymer film is provided which has immobilized on 
it a calcium ion sensitive colorimetric reagent, preferably calcichrome. 
The reaction of the calcium ion analyte may be measured using the 
colorimetric reagent.

DETAILED DESCRIPTION OF THE INVENTION 
The anion exchange polymer film used in this invention is well known to 
those skilled in the art, however, a critical aspect is that the film must 
be porous. Enhanced porosity insures rapid response, and the large number 
of binding sites offered by a polymeric support provides a strong 
analytical signal. Such a porous anion exchange polymer film is available 
commercially and can be obtained, for example, from RAI Research 
Corporation in Hauppauge, N.Y., under the classification "N0030". 
Quaternized amines are used for this embodiment of the polymer film. The 
positive charge from the quaternized amines allows the calcichrome to 
stick to the film, and porosity assists in the improved response time. 
While quaternized amine polymers are preferred with the film, a polymer 
film which is permselective provides the desired properties. Such a film 
is permeable only to small molecules, and prohibits the permeation of 
macromolecules. 
The degree of porosity will depend upon what is being analyzed. The film 
should be porous enough to allow the analyte, calcium, to pass through 
while preventing permeation of contaminants. In using the film for blood 
serum analysis, for example, the smallest degree of porosity preferred 
would be about 5,000 angstroms, and the largest 10 microns. The preferred 
range would be 0.5 microns up to 10 microns, with 10 microns the most 
preferred. 
Immobilized on the film is a Ca(II) sensitive colorimetric reagent. Any 
agent with sulfonic acid or carboxylic acids that bind to the anion 
exchange polymer, and also contain Ca(II)-chelating groups provides the 
satisfactory response of the invention. However, it is necessary that the 
reagent retain its Ca(II)-chelating properties after being immobilized on 
the support. The effective reagent discovered in this invention is 
calcichrome, also referred to as calcion. Calcichrome is 
2,8,8-trihydroxyl-1,1-azonaphthalene-3,6,3,6,tetrasulfonicacid. 
Calcichrome is applied to the support in a concentration of 
5.times.10.sup.-4 mM/g to 2.times.10.sup.-2 mM/g of dry films. The 
preferred concentration is 1.times.10.sup.-2 mM/g of dry film. 
Several methods are available for trapping the reagent on the film and 
include covalent binding, electrostatic binding, adsorption, or trapping 
the analyte in a solution reservoir behind a membrane. With electrostatic 
binding calcichrome negative charges are attracted to the amine positive 
charges. In adsorption, a strong chemical bond is not employed, but a 
variety of interactions retain the reagent on the film. The calcichrome 
can be trapped behind a membrane, such as Teflon or cellulose, so that the 
calcium can diffuse through and react with the calcichrome. The specific 
method employed for trapping the reagent is not critical. 
If impregnating the calcichrome onto the film, the timing is not critical, 
and satisfactory results are obtained when a time period from about five 
minutes to overnight is employed. Optionally, the film may be air-dried 
for storage or used immediately. When used in the optical sensing of 
Ca(II), it is preferable that the sample solution be at or about pH 12. 
Once the calcichrome is immobilized on the film, the optical and/or 
chemical properties of the sensor are then evaluated as the function of 
the Ca(II). An example of a method of evaluating such properties is 
through the use of diffuse reflectance spectroscopy. A standard is used to 
which the results may be compared in order to determine the concentration 
of the Ca(II). 
By using the invention disclosed, it is possible to obtain a reaction time 
in a much shorter time period than is obtained with other methods. 
Usually, with present methods, one must wait five to ten minutes in order 
to get a reaction. Here, the results are obtained in far less than one 
minute and can be obtained in as short a time as fifteen seconds or less. 
The following is presented as a means of illustrating the invention, and is 
not intended to limit the process. Variations will be evident to one 
skilled in the art as falling within the scope of the invention. 
EXAMPLE 
Calcichrome, obtained as its tetra-sodium salt (Pfalz & Bauer, Inc., 
Waterbury, Conn.) was immobilized at the porous anion exchange polymer 
film (RAI Research Corporation, Hauppauge, N.Y.) by immersing a film in an 
aqueous calcichrome solution at room temperature for several hours. After 
removal from the solution, excess reagent was washed from the substrate 
with deionized water. 
Both optode and flow cell for the chemical sensor were constructed to use 
in testing the chemical sensor feasibility. Optical fibers (400 m 
diameter, Ensign-Bickford Optics Co., Avon, Conn.) were used as light 
transmission lines in both designs. Incoming light was transmitted by the 
first array of optical fibers and the diffused reflected light at the 
sensor film was collected by the second array of optical fibers and 
transmitted to a monochromator. A Xenon arc lamp (Oriel Corporation, 
Stanford, Conn.) was used as a light source; the transmitted radiation was 
dispersed with a 0.22 m grating monochromator and monitored with a digital 
photometer (Spex DPC-2, SPEX, Inc., Edison, N.J.) which was operated in an 
analog mode. 
The optical response of calcichrome immobilized at a porous anion exchange 
polymer film in the absence or in the presence of Ca(II) of pH 12.1 was 
then determined. The resulting calibration curve plotting relative 
reflectance to Ca(II) concentrations is shown in FIG. 1. This enabled the 
concentration of Ca(II) to be determined. The limit of detection of the 
sensor obtained under the stated conditions is in the order of mM. The 
transient optical response of the sensor was measured by injecting a 
Ca(II) solution at pH 12.1 through the flow cell. Equilibration (99% 
completion) was achieved in about 13 seconds; the response reaches 63% 
(1-1/e) of its maximum reflectance value in about 3.7 seconds. 
The stability of the sensor response was examined by measuring the diffuse 
reflected light intensity before and after prolonged exposure of incident 
light, and between successive days. No significant difference in intensity 
was observed. 
The interferences of other metal ions were examined using Mg(II), Ba(II) 
and Sr(II) as the test ions. The optical response was measured in pH 12.1 
buffer and in saturated solutions of these ions at pH 12.1. The 
reflectance spectra obtained were almost indistinguishable. 
Thus it can be seen that the invention accomplishes at least all of its 
objectives.