Method of producing test strip for colorimetric detection of calcium content in body fluid using CA-OCPC complex

Abstract

The present invention provides a method of producing a test strip for measuring a calcium concentration in a body fluid test sample obtained from a living body (human or animal) by a colormetric change, and a test strip for measuring a calcium concentration in a human or animal body fluid test strip by a colormetric change.

Claims

1. A method of producing a test strip for measuring a calcium concentration in a sample obtained from a living body, comprising: (a) immersing paper in a first reagent and drying it; and (b) immersing the dried paper in Step (a) in a second reagent and drying it, wherein the first reagent is prepared by adding and mixing o-cresolphthalein complexone (OPCP), 8-hydroxyquinoline, sodium lauryl sulphate (SDS) and polyvinylpyrrolidone (PVP) in distilled water, and further adding an organic acid, and the second reagent is prepared by adding and mixing N-cyclohexyl-3-aminopropanesulfonic acid and N-methyl-D-glucamine in distilled water, and further adding Triton X-100 and triethylamine borate.

2. The method of claim 1, wherein the paper is Whatman paper.

3. The method of claim 1, wherein the drying in Step (a) or drying in Step (b) is performed at 55 to 65° C. for 10 to 20 minutes.

4. The method of claim 1, wherein the living body is a human or animal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

(2) FIG. 1 shows a test result for a calcium concentration previously adjusted to confirm the measurement efficiency of a test strip of the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(3) A recently known colorimetric method for measuring a calcium concentration is used for colorimetric measurement of calcium by chelating calcium with OCPC in the presence of an alkali buffer, and has been most widely used. However, since the pH of the prepared reagent itself is low, and it is difficult to control pH in many cases, a large measurement error is generated. In addition, the test strip produced according to the method results in false positive readings from the formation of a complex of magnesium and OCPC in a buffer solution with a pH 10.0 to 11.0. However, the method using this test strip includes instant color changes and provides the advantages of common calcium excretion testing, and thus is still widely used.

(4) The present invention provides a test strip having a low error in the measurement of a calcium concentration by an OCPC method using two types of solutions indicated as a first reagent and a second reagent, by drying the first reagent and immersing a test strip in the second reagent. The present invention uses a sample obtained from a living body, which is preferably a human or animal. In addition, the sample obtained from a living body is preferably urine.

(5) The method of producing a test strip according to the present invention is performed by two steps including immersing paper (preferably, Whatman paper) in a calcium dye reagent (first reagent) and drying it, and sequentially immersing the dried paper in a buffer solution (second reagent) and drying it.

(6) A calcium dye reagent, which is the first reagent, is prepared by adding and mixing o-cresolphthalein complexone (OPCP), 8-hydroxyquinoline, sodium lauryl sulphate (SDS) and polyvinylpyrrolidone (PVP) in distilled water, and further adding citric acid.

(7) The o-cresolphthalein complexone (OPCP) is a dye reacting with calcium to form a complex. Formula 1 below is a structural formula of o-cresolphthalein complexone.

(8) ##STR00001##

(9) As shown below, OPCP reacts with calcium at pH 10 to 11, exhibiting a violet color.

(10) ##STR00002##

(11) The 8-hydroxyquinoline is a component used to selectively mask magnesium causing false positives. In addition, 8-quinolinol sulfate or N-benzoyl-N-phenyl-hydroxylamine may also be used. The OCPC dye also reacts with magnesium within a pH range of 7 to 8, and as the pH increases, the reaction power is weakened. However, since the masking component can block interference caused by magnesium, it is recommended to use a component that selectively masks magnesium. By using a high pH (10 to 11) and a magnesium binder (8-hydroxyquinoline), magnesium acting as a source of interference in calcium measurement may be effectively removed.

(12) The SDS is a non-ionic surfactant, and not subjected to hydrolysis by an acidic or alkali aqueous solution. These surfactant reagents including Triton-X-100 used among the second reagent improve the color and concentration of a dye with color changes. In addition, the level of chromatic dispersion throughout a reactive pad, the smoothness of the surface of a test strip and a color change rate are improved. In addition, it was revealed that these surfactants improve the brightness of a changing color and increase the absorption of a test strip.

(13) The PVP is used as a stabilizer.

(14) The organic acid is used as a pH buffer. The organic acid that is able to be used as a pH buffer may be selected from the group consisting of citric acid, malonic acid, phosphoric acid, malic acid, succinic acid, phthalic acid and glutamic acid. However, the organic acid is preferably citric acid.

(15) Meanwhile, the buffer solution, which is a second solution (.fwdarw.second reagent), is prepared by adding and mixing N-cyclohexyl-3-aminopropanesulfonic acid and N-methyl-D-glucamine in distilled water, and further adding Triton X-100 and triethylamine borate.

(16) The second reagent serves as a pH buffer for adjusting a pH to 10.0 to 11.0 in an environment for the reaction between calcium and OCPC. The buffer needs to maintain the pH of the sample within a pH range of 10 to 11, and at this high pH level, OCPC reacts with calcium ions, but does not highly react with magnesium.

(17) A buffer suitable for a reagent composition may be selected from amino methyl propanol (AMP) or 3-(cyclohexyl amino)-1-propanesulfonic acid (CAPS), N-methyl-D-glucamine, a carbonate buffer and sodium borate, or a mixture thereof. However, it is preferable that N-cyclohexyl-3-aminopropanesulfonic acid and N-methyl-D-glucamine are used by adjusting triethyl amine borate, and N-cyclohexyl-3-aminopropanesulfonic acid and N-methyl-D-glucamine are mixed in a ratio of 9:1. It is important that the pH buffer does not react with calcium ions in the competition with a dye, and the pH buffer having the above components does not compete with the dye for calcium ions. Here, it is preferable that the concentration of the pH buffer is approximately 0.1 to 0.5M.

(18) Meanwhile, the component Triton X-100 is a surfactant, whose role has been already described above.

(19) Hereinafter, the present invention will be described in further detail with reference to the following examples and experimental examples. However, the scope of the present invention is not limited to only the following examples and experimental examples, and includes modification of equivalent technical ideas thereof

Example 1: Production of Test Strip of the Present Invention

(20) According to the method of producing a test strip of the present invention, which is able to measure a calcium concentration in a biological sample, paper was sequentially immersed in a first reagent and a second reagent, and before transfer to the second reagent, characteristically, the paper was dried. That is, paper (Whatman paper) was immersed in a first reagent to be described below and then dried, and the dried paper was immersed in a second reagent and then dried, thereby producing a test strip of the present invention. Here, drying was performed at 60° C. for approximately 15 minutes.

(21) {circle around (1)} Preparation of First Reagent

(22) Distilled water was used as a solvent, and 0.5 g of OPCP (component for measuring a calcium content), 5 g of 8-hydroxyquinoline (magnesium-selective masking reagent), 2.5 g of sodium lauryl sulphate (non-ionic surfactant) and 3.5 g of PVP (stabilizer) were mixed. Here, the pH was adjusted to 10 to 11 by adding citric acid.

(23) {circle around (2)} Preparation of Second Reagent

(24) Distilled water was used as a solvent, and 3.3 g of N-cyclohexyl-3-aminopropanesulfonic acid (pH buffer) and 0.37 g of N-methyl-D-glucamine (pH buffer) were mixed. 0.2 g of Triton X-100 (surfactant) and 4 g of triethylamine borate (pH buffer) were added and mixed in this solution. In the final step, distilled water was added to make a final volume of 1 L.

(25) The reagent test strip was formed from the above-mentioned first and second reagents. A pad is sensitive to calcium, and reacts when contacting a liquid obtained from a living body (urine, etc.) to have a color change according to a calcium concentration in the sample. Colorimetric assay may be performed by visually comparing a color change with a reference color chart, or using an analyzer that measures a concentration by calculating RGB values from a changed color to obtain an exact value. As the calcium concentration in the body fluid increases, the intensity of the expressed color may quantitatively indicate reproducibility, sensitivity and accuracy.

Experimental Example 1: Effectiveness Test for Test Strip of the Present Invention

(26) The effectiveness of the test strip of the present invention prepared as above was tested. FIG. 1 shows the test result with pre-adjusted calcium concentrations to confirm the effectiveness of measuring the test strip of the present invention. As shown in FIG. 1, it can be confirmed that a good colorimetric change occurs according to a calcium concentration.

(27) The present invention provides a test strip which is able to measure a calcium concentration in a human or animal body fluid test sample by a colorimetric change, thereby easily and inexpensively monitoring a calcium excretion rate, and thus osteoporosis is able to be detected early.

(28) It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided they come within the scope of the appended claims and their equivalents.