JP2012098237A - Flow path design for making multi stage sensitizing operation one step in immunochromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a strip for chromatography analysis with which multi stage operation can be performed in one step.SOLUTION: The strip for chromatography analysis is formed of a membrane. Multiple flow paths in which development of solution requires different amounts of time are arranged in parallel. For example, a blocking pattern 2 is formed in the membrane 1 by an ink jet method to form multiple flow paths 3, 4 having different flow path length in one membrane 1.

Description

  The present invention relates to a strip for chromatographic analysis, and relates to a novel strip for chromatographic analysis capable of performing a multistage operation in one step.

  In immunochromatography, a protein having biochemical activity, such as an antibody, is immobilized on a membrane made of nitrocellulose or the like, and a sample containing a test substance (urine, saliva, blood, etc.) is absorbed into the membrane by capillary action. This is a method of detecting an analysis target by reacting with an antibody on a membrane.

  The above-mentioned immunochromatography has many advantages such as specificity and simplicity, low production cost, short analysis time, etc., for example, influenza test, HCV test, pregnancy test, allergy test, food poisoning test, residual It is widely used mainly in the field of medical tests such as pesticide testing, and its uses are expanding, such as providing test kits that can be used by general users.

  The general procedure of immunochromatography will be described. First, prior to analysis, a primary antibody (specific to a test substance and a specific substance) specifically binds to a test substance on a chromatographic analysis strip (a membrane made of nitrocellulose or the like). And a labeled substance for visualizing the binding site of the test substance [for example, a secondary antibody (different from the site recognized by the primary antibody of the test substance) A conjugation pad in which a labeled antibody (conjugate) in which a labeling substance such as colloidal gold is bound to a site specific antibody) is absorbed is joined to the end of the chromatographic strip on the development start side. .

  In the analysis, a test solution (sample) is dropped onto the conjugation pad. After dropping the test solution, it is sucked up to the strip for chromatographic analysis by capillary action. At this time, the test solution dissolves the conjugate contained in the conjugation pad, and the conjugate and the test substance specifically bind to each other. Wake up. The test solution is further sucked up on the chromatographic strip by capillary action, and when it reaches the fixed position of the primary antibody, it causes a specific binding reaction with the primary antibody to which the test substance to which the conjugate is bound is immobilized. Wake up. As a result, the test substance is locally bound to the fixed position of the primary antibody, and the gold colloid of the conjugate that is bound via the test substance accumulates and develops color.

  In the immunochromatography, if an unreacted conjugate is present in the vicinity of the fixed position of the primary antibody, it becomes a so-called background signal, which may hinder the identification of the original color development. Therefore, the conjugate remaining on the membrane is washed away without forming a specific bond by flowing the test solution beyond the amount necessary for the original analysis. This reduces the background signal due to the unreacted conjugate and provides a clear color development. By measuring the color development, the concentration of the test substance can be quantitatively measured.

  As described above, the above-mentioned immunochromatography has already been put to practical use in fields such as pregnancy tests and influenza kits. However, if further sensitivity can be achieved, it is more serious such as cancer. It is also expected to be applied to early diagnosis of various diseases and timely diagnosis (Point-of-care-testing; POCT) at the bedside.

  Under such circumstances, high sensitivity of immunoassay in immunochromatography has been studied, and sensitization methods using fluorescently labeled conjugates and enzyme conjugates have been reported. For example, Non-Patent Document 1 discloses that a conjugate of an antibody and an enzyme is reacted with an antibody on a chromatographic strip (membrane) as usual, and a test solution is sufficiently poured to wash away unbound conjugate. There has been reported a method for realizing a sensitization reaction by bringing a pad impregnated with a substrate that is colored by catalysis into contact with a membrane.

Enzyme-linked immuno-strip biosensor to detect Eschenchia O157: H7, Ultramicroscopy, 108 (2008) pp. 1348-1351

  By the way, in order to realize a sensitization reaction as described in non-patent literature, a multi-stage operation (in this case, a two-stage operation) is required. For example, in the method described in Non-Patent Document 1, first, a first pad containing an enzyme conjugate is bound to a chromatographic analysis strip, and a test solution is dropped on the first pad for development. . Then, after flowing the test solution, the substrate solution is soaked into a separately prepared second pad and brought into contact with the chromatographic analysis strip so that the substrate solution is absorbed by the chromatographic analysis strip.

  In the sensitization reaction, the enzyme conjugate and the substrate solution cannot be reacted at the same time. When a general chromatographic strip is used, the enzyme conjugate and the test solution are developed in one direction. An operation such as washing away the excess enzyme conjugate and absorbing the substrate solution in a direction orthogonal thereto is necessary, and the analysis cannot be completed by a single operation.

  Therefore, simplicity, which is the greatest advantage of immunochromatography, is lost, and the range of use may be restricted. It is not difficult to imagine that the loss of convenience will hinder the spread of high-sensitivity immunochromatography.

  In the multi-stage immunochromatography, it is a big problem that the structure of the analysis kit is complicated. For example, it is necessary to place an absorption pad for the substrate separately from the chromatographic analysis strip (membrane). To achieve this, the structure of the analysis kit is complicated, and the pad is attached to the chromatographic analysis strip. You also need to be creative. The complicated structure of the analysis kit leads to an increase in manufacturing cost.

  The present invention has been proposed in view of such a conventional situation, and can perform a multi-step operation in chromatography in one step. For example, the simplicity of immunochromatography and the simplification of an analysis kit can be maintained. It is an object to provide a strip for chromatographic analysis.

  In order to achieve the above object, the chromatographic analysis strip of the present invention is a chromatographic analysis strip comprising a membrane, and a plurality of flow paths having different time required for developing a solution are arranged in parallel. Features.

  When the solution is developed from one end side of the chromatographic analysis strip of the present invention, the sucked-up solution permeates into the respective channels arranged in parallel. Here, since the time required for the development of the solution differs for each channel, a time difference is given to the time until the solution reaches the test line on which, for example, the primary antibody is immobilized, depending on which channel is passed. Is done. Therefore, in the strip for chromatographic analysis of the present invention, a multi-stage operation (reaction) can be realized only by a one-step development operation similar to that in normal chromatography.

  ADVANTAGE OF THE INVENTION According to this invention, it is possible to provide the strip for chromatographic analysis which can perform multistep operation in one step. By using the strip for chromatographic analysis of the present invention, it is possible to carry out highly sensitive immunochromatography while maintaining the simplicity that is the advantage of chromatography. Moreover, the chromatographic analysis strip of the present invention realizes the one-step operation only by forming a flow path, and it is not necessary to prepare a separate pad or the like, and does not complicate the structure of the analysis kit.

It is a top view which shows typically an example of the strip for chromatographic analysis to which this invention is applied. It is a top view which shows typically the other example of the strip for chromatographic analysis to which this invention is applied. It is a top view which shows typically the further another example of the strip for chromatographic analysis to which this invention is applied. It is a top view which shows typically the further another example of the strip for chromatographic analysis to which this invention is applied. It is sectional drawing which shows typically the further another example of the strip for chromatographic analysis to which this invention is applied. It is a top view which shows typically the flow path used for the confirmation experiment of a development speed. It is a characteristic view which shows the relationship between a flow path pattern and expansion | deployment delay time. It is a figure which shows the mode of expansion | deployment in the Example of immunochromatography. It is a characteristic view which shows the relationship between hCG density | concentration and coloring intensity.

  Hereinafter, embodiments of a strip for chromatographic analysis to which the present invention is applied will be described in detail with reference to the drawings.

  The strip for chromatographic analysis of the present invention is mainly composed of a membrane made of, for example, nitrocellulose, and is configured by arranging a plurality of flow paths in parallel, which have different time required for developing the solution, in parallel.

  There are various methods for forming the flow path. For example, the flow path can be easily formed by forming a blocking pattern that prevents penetration of the solution in the membrane. Specifically, by dividing the solution permeation region of the membrane by the blocking pattern and devising the pattern shape, it is possible to form a plurality of flow paths having different time required for developing the solution.

  FIG. 1 shows an example of a chromatographic analysis strip in which two flow paths having different lengths are formed by an ink jet printing method. In this example, a blocking pattern 2 that prevents the permeation of the solution as shown in FIG. 1 is formed on a single membrane 1, and a straight flow path 3 and a meandering flow path 4 are formed in parallel. The meandering flow path 4 has a longer flow path length than the straight flow path 3, and when the solution is simultaneously developed from the start end A of these flow paths 3, 4, the solution reaches the end B. The time is longer in the flow path 3.

  Therefore, for example, in high-sensitivity immunochromatography, it is possible to make a difference in the arrival time of the enzyme conjugate and the substrate to the test line. That is, in the chromatographic analysis strip shown in FIG. 1, a conjugate line 5 impregnated with an enzyme conjugate or the like on the downstream side from the junction of the channel 3 and the channel 4, a test line 6 to which an antibody or the like is fixed, and The control line 7 is formed and the substrate 8 is included in the flow path 4. The conjugate or substrate may be spotted and dried in advance, so that it can be redissolved by contact with the test solution.

  When the test solution is developed using such a strip for chromatographic analysis, the test solution is branched into the flow path 3 and the flow path 4 and sucked up through the flow paths 3 and 4. At this time, the test solution that has passed through the flow path 3 reaches the conjugate line 5 earlier than the test solution that has passed through the flow path 4, and reaches the test line 6 together with the enzyme conjugate contained in the conjugate line. On the other hand, the test solution that has passed through the flow path 4 arrives at the test line 6 with a delay along with the substrate 8 on the flow path 4, but during this time, the excess conjugate near the test line 6 has already been washed away. As a result, an appropriate enzyme substrate reaction in which the reaction with the unbound enzyme is eliminated occurs, and color development according to the concentration of the test substance occurs.

  Thus, by using the strip for chromatographic analysis shown in FIG. 1, it is possible to perform a two-step reaction (enzyme conjugate reaction and substrate reaction) in a one-step operation as in ordinary chromatography. It is.

  In order to produce the chromatographic analysis strip shown in FIG. 1, it is necessary to form a blocking pattern 2 for blocking the penetration of the solution. As a method for forming this blocking pattern 2, for example, printing such as ink jet printing is performed. The law can be mentioned. If a solvent capable of dissolving the membrane is printed in a predetermined pattern by ink jet printing, the membrane on which the solvent is printed is dissolved and the micropores of the membrane are closed. Thereby, the penetration of the solution is blocked.

  In the formation of a blocking pattern by ink jet printing, a polymer solution or the like can be used in addition to the solvent. The blocking pattern 2 can be similarly formed by closing the micropores of the membrane with the polymer. In addition, the formation method of the said interruption | blocking pattern is not restricted to the said inkjet printing, It is also possible to employ | adopt other general-purpose printing methods, such as screen printing. In addition, the blocking pattern 2 can also be formed by patterning with photolithography, a method of melting the membrane linearly by heat, a method of crushing the micropores of the membrane by pressing, or the like.

  Also, as the blocking pattern 2, various patterns can be exemplified, and any pattern may be used as long as a difference can be given to the developing speed of the solution.

  For example, FIG. 2 shows that a straight branching pattern 2a is formed on the membrane 1 to branch into a flow path 3 and a flow path 4, and a comb-tooth pattern 2b orthogonal to the branching pattern 2a is formed in the flow path 4. Is formed. In the flow path 4, the developed solution needs to permeate so as to wrap around the tip of the comb-like pattern 2 b, and by repeating this, it takes a long time to develop the solution compared to the flow path 3. become. In the chromatographic analysis strip shown in FIG. 2, the delay time can be changed by changing the number of the comb-like patterns 2b.

  In the example shown in FIG. 3, the linear flow path 3 is formed in the center, and two flow paths 4 that require a long time for developing the solution are formed on both sides thereof. The form of the blocking pattern 2 for forming the flow path 4 is the same as the chromatographic analysis strip shown in FIG. If each channel 4 is impregnated with, for example, the substrate 8, the substrate 8 can reach the test line 6 more reliably.

  The example shown in FIG. 4 is an example in which three flow paths 11, 12, and 13 are formed. In the example shown in FIG. 4, the test solution that has passed through the flow path 11 first reaches the test line 6. Next, the test solution that has passed through the flow path 12 formed so as to wrap around the flow path 11 reaches the test line 6. Finally, the test solution that has passed through the outside of the channel 12 and passed through the channel 13 bypassed by the blocking pattern 2c reaches the test line. Therefore, for example, if the enzyme conjugate is impregnated at a predetermined position in the flow path 12 and the substrate is impregnated at a predetermined position in the flow path 13, the enzyme conjugate and the substrate are placed in the test line 6 with a time difference. Can be reached. In addition, if the communication part 2d is formed by partially missing the pattern at the midway position of the branching pattern 2a that partitions the flow path 12 and the flow path 13, a part of the solution that permeates the flow path 12 is flown. It is also possible to prevent the test solution entering the channel 13 and passing through the channel 13 from entering the channel 12. As described above, it is possible to realize multi-stage operation of three or more stages by devising the flow path.

  In each of the above examples, a plurality of channels having different effective lengths are formed on a single membrane by printing a blocking pattern by ink jet printing, but membranes having different lengths are bonded together. Thus, it is possible to form a plurality of flow paths having different time required for developing the solution.

  FIG. 5 shows an example of a chromatographic analysis strip in which a plurality of channels are formed by bonding membranes having different lengths. In the case of this example, the long second membrane 22 is bonded to the main first membrane 21. Since the second membrane 22 is longer than the first membrane 21, the midway portion is folded and joined to the first membrane 21 at both ends. Further, between the first membrane 21 and the second membrane 22 and between the folded second membrane 22, a blocking sheet 23 for preventing solution penetration is disposed.

  In the chromatographic analysis strip shown in FIG. 5, the test solution sucked from one end is branched and developed into the first membrane 21 and the second membrane 22. At this time, since the length of the second membrane 22 is longer, the time required for deployment also becomes longer. Therefore, a time difference can be given like the previous examples.

  In the above-described chromatographic analysis strip to which the present invention is applied, a multi-step operation can be realized by a one-step operation in which the test solution is immersed in one end of the membrane by the flow channel structure that realizes the time difference of the flow. Therefore, for example, an enzyme sensitization reaction or the like can be performed by a single operation, and can be applied to quantitative analysis of various test substances.

  As mentioned above, although embodiment of the strip for chromatographic analysis of this invention has been described, it cannot be overemphasized that this invention is not what is limited to the above-mentioned embodiment. For example, in the above-described embodiment, application to a sensitization method using an enzyme-labeled conjugate mainly in immunochromatography has been described. However, the present invention is not limited to this, and other immunochromatography, chromatography other than immunochromatography, etc. To provide a strip for chromatographic analysis that can be applied to all types of chromatography and can realize one-step operation in a system that requires multi-step operation (reaction) such as a two-component mixed system or a three-component mixed system. Is possible. For example, the present invention can be applied to agrochemical detection in a multi-liquid mixture system as disclosed in Japanese Patent Application Laid-Open No. 2007-68331.

  Regarding immunochromatography, in the above-described embodiment, the example in which the antibody relating to the test substance is immobilized on the strip for chromatographic analysis has been described. However, when the test substance is an antibody, the antigen may be immobilized. In addition, as described in, for example, Japanese Patent Application Laid-Open No. 2006-37077, No. 2007/116811, WO 2007/061098, etc., other substances that can recognize and specifically bind to a test substance Any combination of the above can be applied to any analysis.

  Hereinafter, specific examples of the present invention will be described in detail based on experimental results.

Formation of Blocking Pattern by Inkjet Printing Using a membrane made of nitrocellulose, a solution containing a polymer was inkjet printed to form a rectangular blocking pattern. In the inkjet printing, the amount of the polymer solution dropped was adjusted to form blocking patterns with different line widths, and the blocking performance against the solution was evaluated. In the evaluation, a dye solution was dropped on a region surrounded by a rectangular blocking pattern, and bleeding into the surroundings was observed. As a result, it was found that by setting the line width of the blocking pattern to 120 μm or more, a good blocking pattern without solution leakage can be formed.

Setting of delay time by blocking pattern The flow path was formed by changing the number of comb-like patterns by inkjet printing, and the difference in the development time of the solution was investigated. The produced flow path is as shown in FIG. The flow path is formed by the formation of the long blocking pattern 31. In FIG. 6A, the number of comb-like patterns 32 is zero, in FIG. 6B, the number of comb-like patterns 32 is three, and FIG. In FIG. 6C, the number of comb-like patterns 32 is five, in FIG. 6D, the number of comb-like patterns 32 is 7, and in FIG. 6E, the number of comb-like patterns 32 is nine. The channel width is 3 mm in all cases.

  The solution was developed from the lower end of these channels, and the time required to reach the upper end was examined. The results are shown in FIG. FIG. 7 shows the delay time in the other flow paths based on the development time in the flow path of FIG. As can be seen from FIG. 7, by providing the comb-like pattern 32, the development time is delayed, and the delay time becomes longer as the number of the comb-like patterns 32 increases. From this result, it is possible to provide a difference (delay) in the solution development time by providing the comb-like pattern 32 in one of the branched flow paths, and to delay by selecting the number of the comb-like patterns 32. It can be seen that the time can be adjusted.

Immunochromatography The strip for immunochromatographic analysis shown in FIG. 8 was prepared, and a test solution containing hCG (Human chorionic gonadotropin) as an analysis target was developed. The strip for immunochromatographic analysis shown in FIG. 8 is obtained by forming a flow path by inkjet printing a polymer solution on a membrane 41 made of nitrocellulose. The polymer solution used was obtained by dissolving 20% of two types of modified acrylic resins as polymers using dipropylene glycol monomethyl ether acetate (DPMA) as a solvent. A delay channel 44 is formed at the intermediate position of the formed channel by the formation of the branching pattern 42 and the comb-like pattern 43, and the normal channel 45 is formed on both sides thereof.

  Further, a test line 46 and a control line 47 are formed on the downstream side of the flow paths 44 and 45, a substrate 48 is spotted on the delay flow path 44, and a conjugate 49 is spotted on the normal flow path 45. Yes.

The test line 46, control line 47, substrate 48, and conjugate 49 are as follows.
Test line: mAb to hCG [anti-hCG (human chorionic gonadotropin α subunit) mouse monoclonal antibody] (trade name 6601 SPR-5, manufactured by medixMAB)
Control line: pAb to Anti-hCG-ALP (anti-mouse IgG rabbit polyclonal antibody) (Dako cytomation, trade name Z0259)
Conjugate: Anti-hCG-ALP (ALP-labeled anti-hCG mouse monoclonal antibody)
Prepared by conjugating ALP to anti-hCG mouse monoclonal antibody (medixMAB, trade name 5008 SP-5) by alkaline phosphatase labeling kit (Dojindo molecular technologies, trade name LK12). Substrate: BCIP-NBT Staining kit (trade name 03937-60, manufactured by Nakakaraitesque)

  The immunochromatography was performed by a one-step operation in which a test solution 50 containing hCG (human chorionic gonadotropin, manufactured by Rohto Pharmaceutical, trade name R-506) was developed from the lower end of the immunochromatographic analysis strip. Each test solution 50 having a different hCG concentration was developed on the immunochromatographic analysis strip, and the color development state on the test line of each immunochromatographic analysis strip after development was image-analyzed, and the hCG concentration and color development intensity (subtracted from the background). I investigated the relationship. The results are shown in FIG.

  As is apparent from FIG. 9, color development corresponding to the concentration of the test substance is observed on the test line, and the sensitizing method using the enzyme-labeled conjugate is performed in one step by using the immunochromatographic analysis strip of this example. It can be seen that can be done.

  In this experiment, BCIP-NBT, which is a substrate, generates a black-purple insoluble substance by an enzymatic reaction. Since the enzyme reaction product is insoluble while the substrate is soluble, the dye produced in the test line and control line becomes particles, and a clear spot is obtained by depositing on the pores of nitrocellulose. By applying such a system to the strip for chromatographic analysis of the present invention, it is effective to increase the intensity of the signal.

1, 2, 22, 41 Membrane, 2, 31 Blocking pattern, 2a, 42 Branching pattern, 2b, 32, 43 Comb-shaped pattern, 3, 4, 11, 12, 13 Flow path, 5 Conjugate line, 6 , 46 Test line, 7, 47 Control line, 8 Substrate, 23 Block sheet, 44 Delay flow path, 45 Normal flow path

Claims (8)

  A strip for chromatographic analysis comprising a membrane, wherein a plurality of flow paths having different time required for development of a solution are arranged in parallel.   The strip for chromatographic analysis according to claim 1, wherein a blocking pattern is formed on the membrane by a printing method, and a plurality of channels having different channel lengths are formed on one membrane.   3. The chromatographic strip according to claim 2, wherein the printing method is ink jet printing.   The strip for chromatographic analysis according to claim 1, wherein a plurality of membranes having different lengths are combined.   The chromatographic analysis strip according to any one of claims 1 to 4, wherein a reagent is disposed at a predetermined position of at least one of the flow paths.   The chromatographic analysis strip according to claim 5, wherein different reagents are arranged at predetermined positions of the plurality of flow paths.   The chromatographic analysis strip according to any one of claims 1 to 6, wherein the chromatographic analysis strip is an immunochromatographic strip for detecting a test substance by an antigen-antibody reaction. 8. The strip for chromatographic analysis according to claim 7, wherein a substrate is disposed at a predetermined position of at least one flow path, and a labeling reagent is disposed at a predetermined position of at least one other flow path.

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