KR100681503B1 - Diagnostic strip treated with bead and method of the strip - Google Patents

Abstract

The present invention relates to a diagnostic strip widely used in the medical field, it is possible to remove a large molecular weight, such as a protein that interferes with the measurement without pre-treatment of the biological sample to improve the accuracy of the measurement results as well as reproducibility The superior, and in particular the purpose of enabling more accurate analysis by increasing the sensitivity of the reaction reagent during quantitative analysis, the present invention to achieve the above object is a diagnostic strip comprising a support and a membrane formed on the support In the diagnostic method using a bead comprising the step of providing a diagnostic strip using beads, characterized in that the membrane is treated with a bead and a color reagent, and forming a bead and a color reagent treated membrane on a support Provides a method of making a strip

Description

Diagnostic strip treated with beads and method for manufacturing the same {Diagnostic strip treated with bead and method of the strip}

1 is a schematic cross-sectional view of a diagnostic strip according to the invention.

Figure 2 is an enlarged view schematically showing the surface of the beaded membrane according to the invention.

3 is a graph showing the results of gepiti measurement according to the bead treatment of the membrane.

Figure 4 is a view showing the results of cholesterol measurement according to the bead treatment of the membrane.

Figure 5 shows the albumin measurement results according to the bead treatment of the membrane.

Figure 6 is a view showing the results of glucose measurement according to the bead treatment of the membrane.

7 is a graph showing the results of measuring the gepieti and geopi depending on the bead treatment of the membrane.

The present invention relates to a diagnostic strip that is widely used in medical fields, and more specifically, it is possible to remove substances having a large molecular weight such as proteins that interfere with the measurement without pretreatment of biological samples, thereby increasing the accuracy of the measurement results. In addition, the present invention relates to a diagnostic strip using beads and a method of manufacturing the same, which are excellent in reproducibility, and in particular, increase the sensitivity of the reaction reagent during quantitative analysis to enable more accurate analysis.

In recent years, biosensors that detect trace amounts of markers from biological samples such as blood or urine have been spotlighted in the field of point of care testing (POCT). In addition to AIDS, measurement items such as blood sugar, hepatitis B diagnosis, pregnancy diagnosis, and cholesterol have been diversified.

Such diagnostic strips are commonly used for the purpose of quantitative analysis as well as qualitative analysis of biological samples. Quantitative analysis uses an optical method or an electrochemical method. Among them, the quantitative analysis method using the optical method can be read by a transmission method using a conventional spectrophotometer, measured by reflectance using a conventional spectrophotometer, or the reaction results can be read as an image using a camera or the like. Various analysis methods are used, including post-processing and analysis.

In vitro diagnostic strips using this optical method are generally formed on the support is a membrane treated with a coloring reagent that is discolored in response to a biological sample. Therefore, when the biological sample is dropped, the sample enters through the micropores formed in the membrane and reacts with the color developing reagent, thereby discoloring, and analyzing the degree of discoloration by the above-described quantitative analysis method.

In this case, if the sample to be measured contains a protein having a large molecular weight, the protein should completely block the micropores of the membrane when the sample is loaded, thereby preventing the marker from penetrating into the sample. It interferes with it. For example, in the case of blood, red blood cells, platelets, white blood cells, etc. in addition to the plasma component containing the marker to be identified are those that interfere with the measurement. Herein, the micropores formed in the membrane of a conventional diagnostic strip have a size of 4 to 9 μm, and red blood cells have a size of about 7 μm, white blood cells have a size of about 9 μm, and platelets have a size of about 3 μm. When the blood cells, especially red blood cells or white blood cells penetrate into the micropores and block the pores, the plasma component containing the marker material does not pass through the micropores, thereby limiting the measurement.

In order to remove the disturbances of the above measurement, the blood collected in advance was operated in a centrifuge to remove the solid components of red blood cells, white blood cells, and platelets, and the plasma was separated and analyzed in a diagnostic kit. The disadvantage is that it requires equipment such as a centrifuge. In addition, there is a method of separating using a glass fiber filter paper of a certain density, which serves to filter the protein (blood cells), etc., but because it requires a separate filter paper, it is inconvenient during the manufacturing process of the diagnostic strip and additional costs are consumed. There are disadvantages. In addition to this, there is a method of adding a substance or a cell shrinkage agent to which the protein is well adsorbed to the glass fiber filter, but in this case, there is a disadvantage in inferior reproducibility.

Accordingly, the present invention is to solve the above-mentioned problems, and even without a separate pre-treatment of the biological sample, it is possible to increase the accuracy of the measurement results by preventing the large molecular weight materials that interfere with the measurement to block the micropores of the membrane, reproducibility The purpose of the present invention is to provide a diagnostic strip using beads capable of more accurate analysis, as well as excellent sensitivity of the reaction reagent during quantitative analysis.

It is another object of the present invention to provide a method for manufacturing the diagnostic strip.

In order to achieve the above object, the present invention provides a diagnostic strip using beads, characterized in that in the diagnostic strip comprising a support and a membrane formed on the support, the membrane is treated with beads and a color reagent.

In another aspect, the present invention provides a method for producing a diagnostic strip using the beads comprising the step of forming a bead and a color reagent treated membrane on the support.

Hereinafter, the diagnostic strip according to the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic cross-sectional view of a diagnostic strip according to the invention, Figure 2 is an enlarged view schematically showing the surface of the beaded membrane according to the present invention, as shown in the diagnostic strip of the present invention is a support ( 10) and the bead 30 and the color-treated reagent membrane 20 formed on the support 10. Reference numeral 11 is a measurement sphere formed to confirm the degree of color development after dropping the biological sample on the membrane (20).

Here, the material selection of the support 10 and the membrane 20 and the formation of the membrane 20 on the support 10 can be easily formed by applying a known technique, which is common in the art Since those skilled in the art can easily form a detailed description thereof will be omitted. However, in the present invention, since the bead 30 and the membrane 20 made of the coloring reagent treatment are used instead of the conventional coloring reagent treated membrane, it will be described in more detail.

Membrane 20 to be applied to the diagnostic strip of the present invention uses the beads 30 and the color reagent treatment. As shown in FIG. 2, the membrane 20 treated as described above is fine as the beads 30 are infiltrated and fixed to the micropores 21 of the membrane 20 to block a part of the micropores 21. It serves to reduce the size of the hole (21). Therefore, when the biological sample is dropped into the membrane 20, proteins with a large molecular weight existing in the biological sample cannot be penetrated into the micropores 21 of the membrane 20, and only the labeling material and the coloring reagent in the biological sample are fine. It reacts while passing through the hole 21 and becomes colored. Therefore, it is possible to remove the protein that interferes with the measurement without pretreatment of the biological sample, thereby increasing the accuracy of the measurement result, and not only excellent reproducibility, but also the sensitivity of the reaction reagent in the quantitative analysis enables more accurate analysis. do.

At this time, as the bead treated in the membrane 20 may be used a variety of types, more preferably polystyrene (polystyrene), polymethyl methacrylate (polymethyl methacrylate), polyvinyl toluene (polyvinyltoluene), styrene butadiene polymer (styrene / butadien), styrene vinyl toluene polymer (styrene / vinyl toluene), latex (latex), etc. It is preferable to use resin beads or silica beads.

Preferably, the beads have a particle size of 1 to 3 μm. When the beads have a particle size of less than 1 μm, the effect of blocking the micropores 21 of the membrane 20 is inferior. As it passes through the hole 21 and is fixed, the accuracy of the measurement result is decreased, the reproducibility is lowered, the sensitivity of the reaction reagent is lowered, and the accuracy of the analysis is lowered. When the particle size of the beads exceeds 3 μm, the beads ( 30 increases the case where the micropore 21 of the membrane 20 is completely blocked, and thus, the labeling material and the coloring reagent in the biological sample do not pass through the micropore 21, and thus act as an obstacle to measurement. Since there is a problem of inferior accuracy, it is preferable to use beads having a particle size within the above range.

The beads 30 and the membrane 20 treated with the color reagent may be obtained through various methods.

For example, the beads 30 and the membrane 20 treated with the color reagent may be obtained by immersing the untreated membrane in a solution containing beads and drying the same. At this time, the solution containing the above-mentioned beads can be easily purchased and used from a commercially available thing.

When the non-treated membrane is immersed in the solution containing the beads and dried, beads are penetrated into the micropores of the membrane as described above. Then, when the coloring reagent is applied to the membrane again, the beads and the coloring reagent are treated. The membrane 20 can be obtained. Here, the application of the coloring reagent may be applied by using a known sample suitable for the item to be measured.

As another example, the bead 30 and the membrane 20 treated with the color reagent may be obtained by immersing the untreated membrane in a solution containing the beads to which the color reagent is bound and then drying. In addition, the membrane 30 treated with the beads 30 and the color developing reagent is dipped after the non-treated membrane is immersed in a solution containing the beads to which the enzyme necessary for color development is bound, followed by color development except for the enzyme on the surface of the membrane. It can be obtained by applying a reagent and drying it.

In general, when the coloring reagent is treated by applying the coloring reagent to the membrane, the binding force between the membrane and the coloring reagent is limited, so that the amount of the coloring reagent attached to the membrane is small, so that it is difficult to obtain accurate measurement results due to saturation at the time of diagnosis. However, as described above, when the coloring reagent is bound to the beads and treated with the membrane, or the enzyme is bound to the beads and then the remaining coloring reagent is treated on the membrane surface after the treatment with the membrane, the coloring reagent or enzyme is bound to the beads. As the amount of is increased, more accurate measurement results can be obtained.

Here, the beads combined with the coloring reagent or the enzyme involved in the color can be obtained by chemically or physically binding the coloring reagent or enzyme to the beads in various ways. For example, by adding a coloring reagent to the solution containing the beads and mixing the solids, or separating the solids by adding an enzyme to the solution containing the beads, and then separating the solids, Beads bound to enzymes can be obtained.

Here, the beads and the solution containing the beads may be selected from those described above, and the coloring reagents or enzymes can be easily applied through a known technique for the item to be measured.

The beads combined with the chromophore or the enzyme are obtained by storing them in a buffer solution, and after dipping the untreated membrane into a solution containing the beads with the chromophore bound, the beads are bound or dried. After immersing the untreated membrane in a solution, it is dried and coated with a coloring reagent except for an enzyme to the membrane, and then dried, it is possible to easily obtain a bead and the coloring reagent treated membrane 20.

When the beads 30 obtained as described above and the membrane 20 treated with the color reagent are formed on the support 10, a diagnostic strip may be manufactured, and the membrane 20 may be formed on the support 10. The post-treatment process may be further subjected to the following needs.

The diagnostic strip thus manufactured partially blocks the micropores 21 of the membrane 20 so that only the necessary components and reagent components of the biological sample except for proteins having high molecular weight can be moved into the beads 30. It is possible to derive the accurate measurement result of the analyte, and in particular, in the quantitative analysis, it prevents the color reaction product from spreading through the micropores 21 of the membrane 20 by the beads 30 to see a distinct color reaction. Can be.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

<Example 1>

Tris buffer (sigma) was added to PS05N (bangs laboratories) containing 10% latex beads with an average particle size of 2.93 µm to adjust the content of latex beads to 1%, and the nitro cellulose membrane was immersed therein. It was then diluted with Tris buffer and dried to prepare a beaded membrane. The reagent for measuring gephyti was diluted in Tris buffer solution on the surface of the beaded membrane and then applied and dried. At this time, the concentration of the reagent per cm ² of membrane in the dried strip was alpha ketoglutarate (a-ketoglutarate). 18 ug, alanine 320 ug, peroxidase (50 units), pyruvate oxidase 4.2 units, 160 ug of potassium hydrogen phosphate trihydrate (K ₂ HPO ₄ · 3 H ₂ O) and 20 ug of hydroxydiaryl imidazole. .

As described above, the bead and the color reagent treated membrane were formed on a polyethylene terephthalate (PET) support, and then a standard sample was added thereto, and the result of measurement at a wavelength of 567 nm by light reflection measurement is shown in FIG. 3. Here, the standard sample was obtained by diluting the zipi enzyme in the blood at a concentration of 20 to 1000 units / L.

Comparative Example 1

After applying a gelatin measurement colorant to the surface of the bead-free nitro cellulose membrane and drying it, a membrane treated with colorant is formed on a PET support, and then dropping a standard sample on it, and then using a light reflectance method. The results measured at the wavelength of are shown in FIG. At this time, the colorimetric reagent and the standard sample for measuring zipi was treated in the same manner as in Example 1.

<Example 2>

Tris buffer (sigma) was added to PS05N (bangs laboratories) containing 10% latex beads with an average particle size of 2.93 µm to adjust the content of latex beads to 1%, and the nitro cellulose membrane was immersed therein. It was then diluted with Tris buffer and dried to prepare a beaded membrane.

On the surface of the bead-treated membrane, the reagent for measuring cholesterol was diluted in Tris buffer solution and then applied and dried. At this time, the concentration of the reagent per cm ² of membrane in the dried strip was cholesterol oxidase 0.1units /, cholesterol esterase 1.2 units /, peroxidase 1.1 units /, 33,3'5,5'-tetramethylbenzidine (3,3'5,5'-tetramethylbenzidine) 60ug.

As described above, the bead and the color reagent treated membrane were formed on a PET support, and then a standard sample was added thereto, followed by measurement at a wavelength of 642 nm by light reflection measurement. Here, the standard sample used cholesterol 100 ~ 600mg / dl.

Comparative Example 2

Applying a coloring reagent for measuring cholesterol to the surface of the bead-free nitro cellulose membrane and drying it, and then formed the membrane treated with the coloring reagent on the PET support, and then dropping a standard sample thereon and then using a light reflection measurement method of 642nm The results measured at the wavelength are shown in FIG. 4. At this time, the coloring reagent and the standard sample for measuring cholesterol were treated in the same manner as in Example 2.

<Example 3>

Tris buffer (sigma) was added to PS05N (bangs laboratories) containing 10% latex beads with an average particle size of 2.93 µm to adjust the content of latex beads to 1%, and the nitro cellulose membrane was immersed therein. It was then diluted with Tris buffer and dried to prepare a beaded membrane.

An albumin measurement color developing reagent was applied to the surface of the beaded membrane and then dried. At this time, the albumin measurement colorant was prepared by dissolving 1 mg of bromine cresol green in 5ml citric acid-NaOH (pH 3.1) buffer solution.

As described above, the beads and the color reagent treated membranes were formed on a PET support, and then a standard sample was added thereto, and the results of the measurement at the wavelength of 610 nm by light reflection measurement are shown in FIG. 5. Herein, standard serum human serum albumin (1-6 g / dl) was used.

Comparative Example 3

The albumin measurement coloration reagent was applied to the surface of the bead-free nitro cellulose membrane and dried. Then, the membrane treated with the coloration reagent was formed on the PET support, and a standard sample was added thereto, followed by light reflection measurement. The results measured at the wavelength are shown in FIG. 5. At this time, the coloring reagent and the standard sample for albumin measurement were treated in the same manner as in Example 3.

<Example 4>

Tris buffer (sigma) was added to PS05N (bangs laboratories) containing 10% latex beads with an average particle size of 2.93 μm to adjust the content of latex beads to 1%, and glucose oxidase to 100 ml of the tris buffer solution. 10 mg each of the peroxidase was added and stirred for 3 hours, followed by centrifugation to obtain a solid, which was then washed with Tris buffer solution and placed in Tris buffer solution containing 0.1% BSA and 0.1% PEG. It was made.

50 mg of 3-methyl-2-benzothiazolinone and 3-dimethylaminobenzoic acid in 100 ml of a mixture of Tris buffer solution and acetonitrile solution in a 10: 1 ratio 20 mg of the solution was mixed with 1 ml of a solution containing the above-described glucose oxidase and peroxidase-containing latex beads, which was then immersed in a nitro cellulose membrane, diluted in Tris buffer solution and dried to dry the beads. A color reagent treated membrane was prepared.

As described above, the bead and the color reagent treated membrane were formed on a PET support, and then a standard sample was added thereto, and the results of measurement at a wavelength of 635 nm by light reflection measurement are shown in FIG. 6. Herein, the standard sample used plasma by glucose concentration.

<Comparative Example 4>

Applying a colorimetric reagent for measuring glucose to the surface of the bead-free nitro cellulose membrane and drying the formed, the membrane treated with the colorimetric reagent was formed on a PET support, and then dropped a standard sample on the 635nm by light reflection measurement method The results measured at the wavelength are shown in FIG. 6.

At this time, the colorimetric reagent for measuring glucose was 10mg of glucose oxidase and peroxidase in 3-ml mixture of Tris buffer solution and acetonitrile solution in 10: 1 and 3-methyl-2-benzothiazolinone (3-methyl-2- 50ug of benzothiazolinone and 20ug of 3-dimethylaminobenzoic acid were used, and a standard sample was treated with the same example as in Example 4.

Example 5

Tris buffer (sigma) was added to PS05N (bangs laboratories) containing 10% latex beads with an average particle size of 2.93 µm to adjust the content of latex beads to 1%, and the nitro cellulose membrane was immersed therein. It was then diluted with Tris buffer and dried to prepare a beaded membrane.

The gefiti measurement reagent and the geotyping reagent were diluted in Tris buffer solution on the surface of the beaded membrane, and then applied and dried at predetermined intervals, respectively, and the reagents per cm ² of membrane in the dried strip. The concentration of gefiti was the same as in Example 1, geothyme alpha-ketoglutarate (18 ug), alanine sulfinic acid (1.1 mg, alaninsulfinic acid), 50 units peroxidase, pyruvate oxidase 4.2 units, 160 ug of potassium hydrogen phosphate trihydrate (K ₂ HPO ₄ · 3H ₂ O), and 21 ug of hydroxydiaryl imidazole.

As described above, beads and color reagent treated membranes were formed on a PET support, and then a standard sample was added dropwise to each of them. Then, the lightness measurement method was used to measure Gfti and Giot at a wavelength of 567 nm, and the results are shown in FIG. 7. In this case, the standard sample is obtained by diluting the gefiti enzyme in the blood at the concentration of 20 to 1000 units / L in the case of the gephytimetry, and the dilute the blood in the blood by the concentration of 20 to 1000 units / L in the geiti measurement. Was used.

Comparative Example 5

After applying the reagent for measuring gephyti and the reagent for measuring geoti on the surface of the bead-free nitro cellulose membrane, they were dried and formed on the PET support, and then the standard sample was added dropwise thereto. The result of the measurement at the wavelength of 567 nm by the light reflection measurement method is shown in FIG. 7. At this time, the colorimetric reagent for measurement of geiti, colorimetric reagent and standard sample for measuring the geotti was treated in the same manner as in Example 5.

As shown in FIGS. 3 to 6, the strips of Examples 1 to 4 treated with beads in the membrane have a linearity (R ² ) as compared to the strips of Comparative Examples 1 to 4 treated without beads in the membrane. It can be seen that each item has been improved, which means that the accuracy and reproducibility of the measurement results are higher. This result is because the beads prevent the protein and the like from being absorbed into the micropores of the membrane and prevent the reagent from spreading.

In addition, as can be seen in Figures 3 to 6, the measurement results at the same wavelength can be seen that the digits of Examples 1 to 4 significantly dropped compared to the digits of Comparative Examples 1 to 4, which is reflected It means that the amount of light is reduced, indicating that the color reaction is greatly increased. This result is due to the fact that the beads prevent the absorption of proteins or the like, which cause errors in the measurement, and the color becomes thicker by preventing the reagent from spreading. Here, Examples 1 to 3 correspond to a case in which a coloring reagent is applied to the surface of the membrane after the beads are treated on the membrane, and Example 4 is first treated with a membrane by attaching an enzyme to the beads, and then applying the enzyme to the membrane. It can be confirmed that all excellent effects can be obtained as applicable to the case of applying the coloring reagent except.

7 is a result corresponding to the case of two reagents on one membrane, in the case of Example 5 according to the present invention compared to Comparative Example 5, even if the two samples for each item at the same time linearity (R ² We can see that the digit value for) and the same wavelength are far apart. These results indicate that even if several chromophores are placed on one membrane, the problem of discoloration due to the dispersing of chromophoric reagents can be resolved, so that a clear and dark color can be confirmed. It means possible.

As described above, according to the present invention, the bead is treated on the membrane used for the diagnostic strip, so that even if the biological sample is not pretreated, the molecules having a high molecular weight, which interfere with the measurement, do not pass through the micropores of the membrane, the accuracy of the measurement results It is possible to increase the sensitivity of the reaction reagents in the quantitative analysis, and to improve the reproducibility, and to increase the sensitivity of the reaction reagents, and to prevent the color spreading during the measurement process. There is a useful effect that can simultaneously measure multiple items.

Claims (16)

In the diagnostic strip comprising a support and a membrane formed on the support, Diagnostic strip using beads, characterized in that the membrane is treated with a bead and a color reagent. The method according to claim 1, The beads are selected from polystyrene, polymethyl methacrylate, polyvinyltoluene, styrene / butadien, styrene / vinyltoluene, and latex Diagnostic strip using beads, characterized in that the resin-based beads or silica beads. The method according to claim 1 or 2, The beads are diagnostic strips using beads, characterized in that the particle size of 1 ~ 3㎛. The method according to claim 3, The membrane is a diagnostic strip using beads, characterized in that the non-treated membrane is immersed in a solution containing beads and dried, and then coated with a coloring reagent on the surface and dried. The method according to claim 3, The membrane is a diagnostic strip using beads, characterized in that it is prepared by immersing the untreated membrane in a solution containing the beads combined with the coloring reagent and dried. The method according to claim 5, Beads combined with the coloring reagent is a diagnostic strip using beads, characterized in that the prepared by separating the solids after adding the coloring reagent to the solution containing the beads. The method according to claim 3, The membrane is a diagnostic strip using beads, characterized in that the membrane is prepared by immersing the untreated membrane in a solution containing the beads to which the enzyme is bound, and then applying a coloring reagent except for the enzyme to the surface of the membrane and dried. . The method according to claim 7, Beads in which the enzyme is combined is a diagnostic strip using beads, characterized in that prepared by separating the solids after adding the enzyme to the solution containing the beads. Preparing a bead and a colorant treated membrane; And Forming the beads and the color reagent treated membrane on a support; Diagnostic strip manufacturing method using a bead comprising a. The method according to claim 9, The beads are selected from polystyrene, polymethyl methacrylate, polyvinyltoluene, styrene / butadien, styrene / vinyltoluene, and latex Method for producing a diagnostic strip using the beads, characterized in that the resin-based beads or silica beads. The method according to claim 9 or 10, The beads have a particle diameter of 1 to 3㎛ method for producing a diagnostic strip using the beads. The process of claim 11, wherein the bead and color reagent treated membrane is prepared by: A method of manufacturing a diagnostic strip using beads, comprising the step of immersing an untreated membrane in a solution containing beads and drying the same, followed by applying a color developing reagent to the surface thereof. The process of claim 11, wherein the bead and color reagent treated membrane is prepared by: A method for producing a diagnostic strip using beads, comprising the step of immersing an untreated membrane in a solution containing beads combined with a coloring reagent and drying the same. The method according to claim 13, Beads in which the coloring reagent is bound is prepared by separating the solids after adding the coloring reagent to the solution containing the beads. The process of claim 11, wherein the bead and color reagent treated membrane is prepared by: Preparation of a diagnostic strip using beads, comprising the step of immersing the untreated membrane in a solution containing the beads bound to the enzyme, followed by drying, applying a coloring reagent except for the enzyme to the surface of the membrane and drying Way. The method according to claim 15, Beads in which the enzyme is combined is a method for producing a diagnostic strip using the beads, characterized in that prepared by separating the solids after adding the enzyme to the solution containing the beads.

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