WO2006016617A1

WO2006016617A1 - Method of quantifying substance and device for quantifying substance

Info

Publication number: WO2006016617A1
Application number: PCT/JP2005/014682
Authority: WO
Inventors: Eiichi Tamiya; Tatsurou Endou; Yoshihiro Murakami
Original assignee: Japan Science And Technology Agency; Japan Advanced Institute Of Science And Technology
Priority date: 2004-08-11
Filing date: 2005-08-10
Publication date: 2006-02-16
Also published as: JPWO2006016617A1; JP4253695B2

Abstract

[PROBLEMS] To enable the extremely highly sensitive quantification of a target substance. [MEANS FOR SOLVING PROBLEMS] A method of quantifying a target substance in a sample solution by using an enzyme immunoassay method which comprises providing one or more carrier beads, on which an antibody or an antigen binding specifically to the target substance has been immobilized, in a microchannel (in the case of using two or more beads, arranging the beads almost in a line in the width direction of the microchannel), feeding a mixture of a sample solution with a solution containing the enzyme-labeled target substance, and, while feeding a substrate solution containing a substrate, detecting the luminescence or color development in the vicinity of the carrier bead(s). After feeding the solution mixture, for example, a washing liquor is fed and then the substrate solution is fed. It is preferred that the solution mixture, the washing liquor and the substrate solution are continuously fed into the microchannel. It is also preferred that carrier beads are located in the microchannel while eliminating overlap in the height direction.

Description

Specification

Method of quantifying substance and device for quantifying substance

Technical field

The present invention relates to a method of quantifying a substance for quantifying a measurement target substance in a sample using an enzyme immunoassay, and a quantification device of the substance used in the method of quantification.

Background art

[0002] As a method for quantifying substances such as trace amounts of drugs and environmental pollutants contained in a solution, it is possible to use specific binding between an antigen and an antibody and, at the same time, for example, Enzyme-linked immunosorbent assay (ELISA: enzyme-linked immunosorbent assay) for quantifying a substance to be measured is known. For example, while analysis methods such as gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) require very complicated operations and long-time measurement, ELISA usually uses a 96-well microphone. By using the mouth plate etc., the measurement operation can be greatly simplified compared to the conventional analysis method, and high sensitivity detection can be realized.

[0003] A conventional ELISA, for example, a direct competition ELISA, which is a type of competitive method, comprises an antibody 102 immobilized on the surface of a carrier 101 such as a microplate well or test tube as shown in FIG. A sample or standard substance) 103 and an enzyme-labeled antigen 104 are added to cause a competition reaction, and after washing away the target substance 103 or labeled antigen 104 not bound to the antibody 102, an enzyme substrate 105 is added. Then, for example, fluorescent substance 106 is produced by enzyme reaction, that is, it is made to fluoresce, then the fluorescence intensity is measured with a colorimeter etc., and the concentration of the measurement object is measured by comparing with the fluorescence intensity of the standard of the measurement object It is a method to measure. The calibration characteristic by the direct competition method is a decay curve.

[0004] In addition to the above-mentioned ELISA, various methods for quantifying a very small amount of substance have been developed. For example, as an example of analysis of environmental hormones that require trace analysis, female hormone receptor immobilized on bead surface The body is made to compete with a known amount of fluorescently labeled female hormone and an unknown amount of environmental hormone, and the fluorescence intensity of fluorescently labeled female hormone bound to the female hormone receptor is measured for the amount of environmental hormone present. Methods are proposed (eg See, for example, Japanese Patent Application Laid-Open No. 2001-116753. ;). In JP 2001-116753 A, in particular, an example is described in which the column is packed with beads as a carrier and the flow is measured

[0005] As described above, although ELISA is known as a simpler and more sensitive quantitative method than conventional analysis methods, it is necessary to monitor, for example, blood levels of drugs, clinical examinations, environment, etc. In the field of quantifying very small amounts of substances, such as hormone analysis, there is a strong demand for the development of a concentration measurement method capable of high-sensitivity quantification.

[0006] In the invention described in the above-mentioned JP-A-2001-116753, the fluorescence labeling hormone power that is bound to the receptor is used to detect fluorescence. In this case, the fluorescence molecule of hormone bound to the receptor There is a problem that it is possible to obtain only low light emission intensity because the light emission intensity is extremely weak.

[0007] Therefore, the present invention has been proposed in view of such conventional circumstances, and provides a method of quantifying a substance capable of quantifying a substance to be measured with extremely high sensitivity and a device for quantifying a substance. The purpose is to

Disclosure of the invention

Means to solve the problem

As a result of extensive investigations by the present inventors, in the ELISA method, the retention of the enzyme reaction product (luminescent substance or chromogenic substance) on the carrier surface has a great influence on the measurement of luminescence intensity or coloring intensity. In order to avoid this effect, it was found that combining ELISA and flow method quantification method was extremely effective.

The method for quantifying a substance according to the present invention is completed based on such findings, and is a method for quantifying a substance to be measured in a sample solution by enzyme immunoassay, which comprises: The bead carrier on which the specifically binding antibody or antigen is immobilized is arranged in the microchannel so as to be arranged in a row or in substantially one row in the width direction of the microchannel, and the sample solution and the enzyme labeled After sending a mixed solution in which the solution containing the substance to be measured is mixed, the luminescence or coloring in the vicinity of the bead carrier is detected while feeding the substrate solution containing the substrate.

[0010] In the method for quantifying a substance as described above, a substrate is included when detecting the degree of luminescence or color development. By continuing the flow of the solution onto the bead carrier surface, the luminescent material or the chromogenic substance continuously produced by the reaction of the enzyme and the substrate is rapidly removed from the bead carrier surface and allowed to flow downstream and stay on the bead carrier surface. I'm preventing. For this reason, the generation rate of the light emitting material or the coloring material which hardly receives the influence of the unnecessary light emitting material or the coloring material is almost exactly reflected on the light emission intensity or the coloring intensity.

Further, in the method for quantifying a substance according to the present invention, the number of beads arranged in the microchannel is small, such as arranging one bead or approximately one row in the width direction of the microchannel. The effect of luminescent or chromogenic substances generated in the vicinity of other beads is reduced as much as possible, and detection sensitivity is improved.

Furthermore, in the present invention, since the luminescent material or the coloring material is continuously generated by continuously supplying the enzyme substrate to the bead surface at the time of detection, the light number is sufficiently high even if the number of beads is small. The intensity or coloring intensity is secured to realize high sensitivity detection.

In addition, the device for quantifying a substance according to the present invention is a device for quantifying a substance to be measured in a sample solution by enzyme immunoassay, and is an antibody or an antigen that specifically binds to the substance to be measured. And a narrow portion provided in the middle of the minute channel for flowing the liquid and preventing the movement of the bead carrier downstream, in the narrow portion While the bead carrier is arranged in one or in substantially one row in the width direction of the microchannel, a mixed solution in which the sample solution and a solution containing the substance to be measured which is enzyme-labeled are mixed is delivered to the microchannel. After being liquidated, it is characterized in that luminescence or coloring in the vicinity of the bead carrier is detected while a substrate solution containing the substrate is fed.

When quantifying a measurement target substance using the above-described quantification device, it is unknown as a sample in a state where the bead carrier on which the antibody or the antigen is immobilized is disposed at the narrow portion in the middle of the minute channel. A substance to be measured at a concentration and an enzyme-labeled substance to be measured at a known concentration (competitive substance) are supplied to a microchannel, and the substance to be measured and the substance to be measured labeled with an enzyme are for example competitively reacted Combine. Next, a solution containing the enzyme substrate is supplied to the microchannel of the quantification device to detect luminescence or color development due to the reaction between the enzyme on the bead carrier surface and the substrate. [0015] When detecting the degree of luminescence or color development, by continuing the flow of the substrate solution to the microchannel, ie, the surface of the bead carrier, the luminescent substance or the color forming substance produced on the bead carrier surface by the reaction of the enzyme and the substrate It is quickly removed from the support surface and allowed to flow downstream. For this reason, the generation rate of the luminescent material or the chromogenic material is almost accurately reflected in the degree of luminescence or color development in the vicinity of the surface of the single bead, and the determination with extremely high sensitivity without being affected by unnecessary fluorescent material or luminescent material as much as possible. Is realized. In addition, by using a small number of bead carriers, the influence of luminescent substances or coloring substances generated in the vicinity of other bead carriers can be minimized, and quantification with further high sensitivity can be realized. Furthermore, when the measuring device of the present invention sequentially sends various solutions to the micro channel, it can be determined easily and easily by any convenient operation, which eliminates the need for complicated operations and measurement in a short time. Is possible! /, It also has the advantage. Effect of the invention

According to the present invention, by arranging one bead carrier or approximately one row in the width direction of the microchannel, it is possible to keep the substrate solution flowing on the bead carrier surface at the time of detection of luminescence or color development. It is possible to provide a method of quantifying a substance that can be quantified by sensitivity. Further, according to the present invention, it is possible to provide a quantitative device capable of easily and easily realizing high sensitivity measurement of a substance to be measured.

Brief description of the drawings

[FIG. 1] FIG. 1 is a schematic view for explaining an example of a quantification method to which the present invention is applied.

[FIG. 2] FIG. 2 is a photograph showing a state in which a substrate supplied on the surface of a bead reacts with an enzyme and emits light according to the quantification method to which the present invention is applied.

[FIG. 3] FIG. 3 is a schematic perspective view showing an example of a micro flow chip to which the present invention is applied.

[FIG. 4] FIG. 4 is a schematic cross-sectional view along the length direction of the microchannel of the microflow chip shown in FIG.

[FIG. 5] FIG. 5 is a schematic cross-sectional view along the width direction of the microchannel of the microflow chip shown in FIG.

[Fig. 6] Fig. 6 is an enlarged photograph of the area of the narrow portion of the micro flow chip shown in Fig. 3 as viewed in plane. [FIG. 7] FIG. 7 is a schematic plan view showing an example provided with a plurality of microchannels, which is a micro flow chip to which the present invention is applied.

[Fig. 8] Fig. 8 is a schematic diagram for explaining the measurement principle by o-ferie diamine (OPD).

[FIG. 9] FIG. 9 is a schematic view for explaining the principle of detection of fluorescence using 10-acetonitrile 3 and 7-dihydroxydienoxazine.

[FIG. 10] FIG. 10 is a characteristic diagram for confirmation of antibody immobilization and determination of optimal FK-506 POD concentration.

[FIG. 11] FIG. 11 is a characteristic diagram showing a calibration curve when about 100 beads are used.

[FIG. 12] FIG. 12 is a characteristic diagram showing a change in calibration curve depending on the number of beads.

[FIG. 13] FIG. 13 is a characteristic diagram showing a calibration curve when 10 beads are used.

[FIG. 14] FIG. 14 is a photograph showing the luminescence of beads when 10 beads were used and a microflow antibody-type chip was used.

[FIG. 15] FIG. 15 is a characteristic diagram showing a calibration curve when using 10 beads and using a microflow antibody-type chip.

[FIG. 16] FIG. 16 is a characteristic diagram showing a calibration line when using one bead and using a microflow antibody type chip.

[FIG. 17] FIG. 17 is a photograph showing the luminescence of the bead when using one microbead and using a microflow antibody-type chip.

[FIG. 18] FIG. 18 is a diagram for explaining the principle of direct competition ELISA.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a method and device for quantifying substances to which the present invention is applied will be described in detail with reference to the drawings. First, the principle of the method for quantifying a substance to which the present invention is applied will be described.

The quantification method to which the present invention is applied is a method of quantifying a measurement target substance in a sample by applying an enzyme-linked immunosorbent assay (ELISA), which comprises one bead carrier or one microflow in a minute flow path. While arranging so as to be arranged in approximately one row in the width direction of the passage, while flowing a substrate solution containing a substrate onto the surface of the bead carrier to which the enzyme is bound, the surface of each bead carrier is in the vicinity of the surface. For example, the degree of fluorescence is detected by the reaction between the enzyme and the substrate. For example, when using a direct competition ELISA, first, a predetermined number of bead carriers on which an antibody that specifically binds to a substance to be measured is immobilized are arranged in a microchannel, and the target substance and the substance to be measured are placed on the bead carrier surface. An enzyme-labeled substance to be measured at a known concentration is supplied and reacted competitively. Next, while the substrate solution containing the substrate is continuously supplied to the bead carrier surface, the fluorescence near the bead carrier surface due to the reaction between the enzyme and the substrate is detected and measured based on the degree of the detected fluorescence. Determine the target substance.

The substance to be measured is not particularly limited, and any substance can be adopted such as, for example, a drug such as an immunosuppressant and various compounds such as a carcinogen such as dioxin.

A bead carrier for immobilizing an antibody that specifically binds to a substance to be measured has a large surface area, can immobilize a large amount of antibody etc., has a large contact area with a sample solution, and can be easily arranged in a microchannel. Certain things are also effective. The bead carrier is not particularly limited as long as it can immobilize an antibody or the like that specifically binds to the substance to be measured, and for example, glass, polystyrene or the like can be used. The bead carrier can also be reused by removing the immobilized antibody after measurement and washing.

[0022] In the quantification method of the present invention, since the fluorescence of the fluorescent substance produced one after another is detected by continuously supplying the substrate to the enzyme on the bead surface, even if only one bead, sufficient fluorescence is obtained. An intensity can be obtained and highly sensitive detection is possible. In addition, when detecting the fluorescence in the vicinity of the surface of each bead, the number of bead carriers can be generated in the vicinity of other beads by setting the number of bead carriers to one or a small number such as arranged in approximately one row in the width direction of the microchannel. Can reduce the influence of the Furthermore, by using a small number of bead carriers, the desired number of bead carriers can be simply and accurately disposed in the microchannel. Having one bead carrier is advantageous in that the error in analysis can be reduced.

The enzyme used to label the substance to be measured is not particularly limited, and enzymes such as peroxidase, galactosidase, and the like that generate a fluorescent substance by reacting with a substrate can be used. Further, as the substrate contained in the substrate solution, a fluorescent substrate which becomes a fluorescent substance such as, for example, resorufin by enzymatic reaction can be used. The use of fluorescent substrates is more sensitive than other luminescent or chromogenic substrates, such as luciferin, for example. It is preferable in the possible point.

Here, the principle of the present invention will be described in detail with reference to FIG. In the description of FIG. 1, the case where the concentration of the substance to be measured is measured using a direct competitive ELISA as an example is an antibody as a substance that specifically binds to the substance to be measured.

First, as shown in FIG. 1 (a), a bead 2 on which an antibody 1 that specifically binds to a substance to be measured is immobilized is prepared, and a minute flow such that the flow of the liquid interferes with the flow of the bead 2 Place beads 2 in the street. At this time, the number of beads 2 should be such that one bead or one bead or two beads will be arranged in substantially one row in the width direction of the microchannel.

Next, as shown in FIG. 1 (b), a solution containing a sample solution containing measurement target substance 3 of unknown concentration, and a solution containing labeled substance 4 (known concentration) obtained by labeling the measurement target substance (antigen) with an enzyme The mixed solution thereof is supplied to the surface of the beads 1 for 5 minutes, for example, to cause the substance 3 to be measured and the labeled substance 4 to compete with each other. When a mixed solution of a sample solution and a solution containing a substance to be measured for enzyme labeling is sent to a microchannel, the flow rate of the mixed solution should be 10 1 Z min or less from the viewpoint of reliably advancing the antigen-antibody reaction. preferable. However, if the flow rate of the mixed solution is too small, nonspecific adsorption may occur on the bead surface, so for example, 1 μ 1 Z min or more is preferable.

Next, as shown in FIG. 1 (c), buffer 1 etc. is supplied for 5 minutes, for example, and the substance to be measured 3 and the labeled substance 4 nonspecifically adsorbed on the surface of the beads 1 are washed and removed.

Finally, as shown in FIG. 1 (d), a substrate solution containing a substrate 5 that specifically reacts with the enzyme is supplied, and while flowing through the substrate solution, the fluorescence of the fluorescent substance generated by the enzyme reaction is measured. To detect. Then, the concentration of the substance to be measured 3 in the sample solution is determined based on the calibration curve prepared separately.

When the substrate solution is sent to the microchannel, it is preferable to increase the flow from the viewpoint of reducing the noise level, but if the flow is too high, it may make it difficult to detect the fluorescence. . Therefore, it is preferable to set the flow rate of the substrate solution to 10 1 Z minutes or less. However, if the flow rate of the substrate solution is too small, the fluorescent substance generated by the enzyme reaction will stay around the beads and there is a possibility that the background level will increase, so the flow rate of the substrate solution should be 1 IX 1Z min or more. It is preferable to do. The flow rate of the substrate solution is preferably set so that the substrate supply rate is higher than the reaction rate of the enzyme on the surface of the bead 1. If the substrate supply rate is smaller than the reaction rate of the enzyme, the reaction rate of the enzyme may be saturated, and accurate fluorescence intensity may not be measured.

A state in which the substrate supplied on the surface of the bead carrier is reacted with the enzyme by the above-described quantitative method is shown in FIG. As shown in FIG. 2, fluorescence due to the fluorescent substance generated by the enzyme reaction is observed along the surface of the bead carrier. In the present invention, by continuing the flow of the substrate solution, the fluorescent substance continuously generated by the reaction between the enzyme and the substrate is rapidly flowed downstream, and the retention of the fluorescent substance in the vicinity of the bead carrier is prevented. This makes it possible to reduce the background while securing a sufficient fluorescence intensity. In addition, by setting the number of bead carriers to one or a small number such that they are arranged in approximately one row in the width direction of the microchannel, fluorescent substances generated in the vicinity of other bead carriers interact with each other. Can be minimized. Therefore, according to the present invention, it is possible to accurately know the fluorescence intensity (generation rate of the fluorescent substance) in the vicinity of the surface of each bead carrier while securing a sufficient fluorescence intensity, and highly sensitive quantification of the measurement target substance is possible. It becomes possible.

On the other hand, when the fluorescence intensity is measured while the flow of the substrate solution is stopped, as in the conventional ELISA, for example, the fluorescence intensity significantly increases due to the retention of the fluorescent substance around the beads, and the fluorescence intensity increases. Accurate measurement of strength becomes difficult. In addition, when many bead carriers are used, for example, the bead carriers are arranged in two or more rows in the width direction of the microchannel, the fluorescence in the vicinity of the bead carriers mutually affects each other, again making accurate measurement of the fluorescence intensity difficult. Become.

[0033] A method for quantifying a substance to be measured by flowing a sample solution on the surface of a bead carrier by a flow is described in the above-mentioned Japanese Patent Application Laid-Open No. 2001-116753. The invention described in Japanese Patent Application Publication No. hei8-99 is to directly attach a fluorescent labeling substance to a substance (hormone receptor) on the surface of a bead carrier and observe fluorescence in this state, and after binding an enzyme to the surface of the bead carrier, the substrate is It is fundamentally different from the technique of the present invention which is introduced and lighted by the enzyme. In the invention described in JP 2001-116753 A, since the fluorescence directly bound to the bead carrier is detected, the bead carrier may be, for example, a small number such as arranged in a single row in the width direction of the flow path. In that case, weak fluorescence can not be obtained. In the present invention As described above, by using the enzyme and keeping the substrate solution flowing, fluorescence can be obtained even if the number of bead carriers used is small.

[0034] The method for quantifying the substance as described above is, for example, a quantitation device (microflow chip) in which the flow of the solution around the bead carrier is finely controlled by having an extremely narrow microchannel as described below. Can be performed reliably and simply. 3 is a perspective view of the microflow-type chip 11, FIG. 4 is a cross-sectional view along the microchannel of the chip 11, and FIG. 5 is a cross-sectional view of the microchannel in the width direction near the narrow portion. 6 is an enlarged photograph of the narrow area seen from the plane. A narrow micro channel 13 in the form of a groove is formed on the surface of a substrate 12 made of a transparent resin material such as polydimethyl siloxane (PDMS), for example, in the micro flow type chip 11. In 13 are placed beads 14 in which an antibody, an antigen, etc. are bound to a bead carrier. On the microchannel 13 is placed a lid 17 on which a hole for sample introduction (sample introduction part) 15 and a hole for sample discharge (sample discharge part) 16 are formed. A convex portion 18 is provided in the middle of the length direction of the microchannel 13 of the lid 17 to allow the flow of the solution in the microchannel 13 while preventing the downstream flow of the beads 14. Form the narrow part 19 of the When detecting the fluorescence, a detector 20 such as a colorimeter is disposed around the chip 11 so as to detect the fluorescence in the vicinity of the surface of the bead 14 staying on the upstream side of the narrowing portion 19.

The optimum shape and size of the microchannel 13 vary depending on the shape and size of the bead 14 to be placed, but the groove depth of the microchannel 13 is set in the microchannel 13 Preferably, the diameter of the bead 14 is greater than or equal to twice the diameter of the bead 14. By making the microchannel 13 of such a depth, the beads 14 are completely immersed in the solution, and only one bead 14 is arranged in the height direction, that is, the beads 14 are in the height direction. (The direction of detection) will not overlap. By making the beads 14 not to overlap in the height direction, it becomes easy to focus the device at the time of detecting fluorescence, and to measure the fluorescence in the vicinity of the surface of each bead 14, ie, the measurement object. The fluorescence in the vicinity of the bead surface can be easily spatially distinguished from other fluorescence and measured, and the intensity can be measured more accurately. Therefore, the S / N ratio is improved, and the sensitivity can be dramatically improved.

In the width direction of the groove of the micro flow channel 13, for example, a plurality of beads 14 may be micro flow channels 13. When arranged inside, these beads 14 are arranged in rows along the width direction of the microchannel 13.

The arrangement of the beads 14 in the minute channel 3 may be carried out manually with tweezers or the like, or may be carried out, for example, by liquid transfer using a syringe pump! The number of beads 14 arranged in the micro channel 13 is set to a small number from the viewpoint of high sensitivity measurement, and in the present invention, the number of beads 14 is set to one or approximately 1 row in the width direction of the micro channel. .

A method of quantifying a substance to be measured using the chip 1 as described above will be described. First, beads 14 to which an antibody that specifically binds to the substance to be measured is immobilized are prepared, and a predetermined number of the beads 14 are arranged on the upstream side of the narrowing portion 19 of the microchannel 13.

Next, from a solution introduction tube (not shown) connected to the sample introduction unit 15, a sample containing the measurement target substance and the enzyme-labeled measurement target substance (competitive substance) using a syringe pump or the like The mixed solution is introduced into the microchannel 13 and supplied to the surface of the bead 14. As a result, the substance to be measured etc. is bound to the antibody on the surface of the bead 14.

Next, using a suitable buffer or the like, the non-bound or non-specifically bound measurement target substance and enzyme labeled substance are washed.

Next, the substrate solution is supplied from the solution introducing tube (not shown) connected to the sample introducing unit 15 to the microchannel using a syringe pump. Then, while supplying the substrate solution, the detector 20 detects fluorescence near the surface of the bead 14. At this time, since the substrate is kept flowing by the syringe pump, at the same time as the substrate is continuously supplied to the enzyme bound to the beads 14, the fluorescent substance generated by the enzyme reaction flows in the sample discharge portion by the flow of the solution. Continue to be discharged from 16. As a result, the fluorescent substance does not stagnate in the vicinity of the beads 14, and the influence of knock ground and the like is reduced, so that accurate fluorescence intensity can be measured. Therefore, by using the chip 1 of the present invention, the SN ratio can be improved, the detection sensitivity can be dramatically improved, and accurate determination can be performed.

In addition, since the chip 1 of the present invention is small, it has an advantage of being excellent in portability.

Furthermore, since the microchannel formed in the chip 1 is a very minute space, it is possible to suppress an increase in the amount of use of various solutions and shorten the measurement time.

By the way, a chip used in the quantification method of the present invention is a single chip as shown in FIGS. The present invention is not limited to the one in which the microchannel 13 is formed, but may be one in which a plurality of microchannels are formed. An example of such a multi-flow chip is shown in FIG. In the chip shown in FIG. 7, eleven microchannels 13 are arranged in parallel in a comb shape on the surface of a substrate 12 which is also a transparent material. In this chip, the sample introduction unit 15 is shared, and a sample discharge unit 16 for discharging the liquid after liquid feeding is individually formed in each of the microchannels 13. Ten of the microchannels 13 are microchannels for preparing a calibration curve, and the remaining one is a microchannel for sample solution.

When using this chip, all solution discharge tubes (not shown) respectively connected to the sample discharge unit 16 are closed in advance. Then, while opening the tube corresponding to any one of the microchannels 13, measurement is performed by flowing a standard substance solution of a predetermined concentration, for example, for preparation of a calibration curve from the sample introduction unit 15. This process is repeated for each microchannel 13 in the case of measurement of other calibration curve preparation solutions and sample solutions. By forming a plurality of minute channels 13 on the chip, it is possible to easily create a calibration curve and measure the concentration of the substance to be measured in the sample on one chip. Further, as shown in FIG. 7, by arranging the microchannels 13 in a comb shape, it is possible to suppress the bead carrier, the sample solution and the like from being mixed into the other microchannels 13.

Since it is low efficiency to manually arrange a predetermined number of bead carriers such as tweezers in the vicinity of the narrow portion of the plurality of microchannels 13, it is provided with the plurality of microchannels 13 as shown in FIG. In the case of a chip, it is preferable to place the bead carrier in each microchannel by, for example, liquid transfer using a syringe pump. As a result, the time for arranging the bead carrier in the microchannel is greatly shortened, and quantification can be performed more easily.

In the above description, the direct competition ELISA was described as an example, but the present invention is not limited to this, and can be applied to, for example, the indirect competition ELISA. Indirect competition E LISA is prepared by adding the substance to be measured and the primary antibody to the one obtained by immobilizing a hapten bound to the antigen used for the immunogen or protein different from the immunogen on the surface of the carrier such as beads, After washing away the substance to be measured and the primary antibody which did not bind to the immobilized antigen, the secondary antibody is added to bind to the primary antibody, and then the substrate is added to cause fluorescence reaction by the enzyme reaction. Measure the concentration of the substance to be measured by detecting this luminescence How to When the present invention is applied to the indirect competition ELISA, the same effect as the direct competition ELISA can be obtained. The present invention can also be applied to so-called sandwich ELISA. The above-mentioned competition method is suitable to be applied when the substance to be measured is a small molecule, whereas the sandwich method is suitable to be applied to cases where the molecular weight of the substance to be measured is relatively large.

Further, in the above description, although the case of detecting the fluorescence of a fluorescent substance such as resorphin generated by an enzyme reaction using a fluorescent substrate as a substrate has been described as an example, the present invention is limited thereto It is also possible to use luminescence other than fluorescent light, such as chemiluminescence or bioluminescence. Specifically, a substrate solution is prepared using a luminescent substrate other than a fluorescent substrate, specifically, a luminescent substrate such as luciferase, and an optimum enzyme is appropriately selected according to the substrate, and bioluminescence associated with the enzyme reaction is generated. (匕匕発光) can be used for detection.

Furthermore, in the present invention, not limited to the light emission, for example, color development may be used for detection. In this case, while preparing a substrate solution using a chromogenic substrate, an optimum enzyme may be appropriately selected depending on the substrate, and color development associated with the enzyme reaction may be detected. Examples of the chromogenic substrate include o-phenylenediamine (OPD) and tetramethylbenzidine (TMB).

Hereinafter, specific examples of the quantitative method and the quantitative device to which the present invention is applied will be described based on experimental results. The drug FK 506, which is used as an immunosuppressant at the time of organ transplantation, was used as the measurement target substance, and quantification was performed by ELISA using a short micro flow path microflow antibody-type chip in order to simplify the system. As a quantitative method, an FK506 antibody immobilized on polyethylene beads is placed in a micro channel, and a mixed solution of FK 506 and FK 506-peroxidase (POD) (horseradish peroxidase (HRP) conjugated FK 506) is added to the micro channel. The reaction was allowed to proceed, and the fluorescent substance produced by peroxidase and the substrate was measured using an optical detection system. In this example, a microflow antibody-type chip produced as follows was used.

In the following experiments, as reagents and materials, anti-FK506 mouse monoclonal antibody (Funakoshi Co., Ltd.), FK506, FK506-POD (provided by Fujisawa Pharmaceutical Co., Ltd.), o-Felen-diamine (OPD) ) (Manufactured by SIGMA CHEMICALS), Amplex (TM) re d ELIS A kit (Molecular probe), hydrogen peroxide (30% aqueous solution, for biochemistry, Wako Pure Chemical Industries), ushi serum albumin (SIGMA CHEMICALS), polydimethylsiloxane (PDMS: polydimethyl siloxane) 'Manufactured by Asia, Silpot 184 Silicon Elastmer kit', FEP tube (manufactured by BAS), polystyrene beads (manufactured by Polyscience, diameter 90 μm), glass plate (manufactured by MATSUNAMI), immuno module (strip and frame, F16, manufactured by NUNC) was used. The apparatus includes a stereo fluorescent microscope (manufactured by Leica, MZFL III), a CCD camera (manufactured by Hamamatsu Photonitas, ORCA-ER), a syringe pump (manufactured by HARVARD APPARATUS), a spectrophotometer (manufactured by JASCO, V530). Was used.

Quantification of FK506 with a microflow antibody-type chip was performed by direct competition ELISA. Direct competition ELISA is a method in which an antibody is coated (immobilized) on a microplate or test tube, etc., and a substance to be measured (sample or standard substance) and an antigen labeled with an enzyme are added for competition reaction to bind to the antibody. After washing away the substance to be measured and the labeled antigen, the enzyme substrate is added to make the enzyme react with light, and then the degree of luminescence is measured with a colorimeter etc. The luminescence degree of the standard substance of the substance to be measured This is a method of measuring the concentration of the substance to be measured by comparison.

In this example, the antibody was immobilized on the surface of a polystyrene bead, and placed in the microchannel of the microflow antibody type chip for measurement. Antibody immobilized beads were prepared by the following method.

First, a suspension of polystyrene beads about 90 μm in diameter was washed three times with phosphate buffer (PBS). to this,

It was immersed and immobilized on the bead surface by hydrophobic bonding. In order to ensure immobilization on the bead surface, the concentration of the antibody solution used for antibody immobilization was high. After fixation, the immobilized beads are washed with PBS, soaked in PBS (BT-PBS) containing 0.05% Tween 20, 0.5% sushi serum albumin (BSA) and soaked overnight. Blocking of the bead surface was performed. The obtained beads are washed three times with PBS containing 0.05% Tween 20 (T-PBS), and the T-PBS The medium was stored until use at a temperature of 4 ° C.

[0054] Confirmation of Antibody Fixation and Examination of FK506 POD Optimal Concentration>

Next, while confirming the immobilization of the antibody on the bead surface, the necessary amount of FK506-POD was examined. Bead solution 10 1 (approximately 100 beads) were washed with PBS, FK506- POD (5 000-fold dilution) diluted solution, respectively it further nine stages in the BT- PBS (1 × to 10 ⁷ times) In addition, it was allowed to soak overnight to carry out an antigen-antibody reaction. Wash with T-PBS three times, react with substrate solution (OPD 0.5 mg / ml, 0.03% hydrogen peroxide, phosphoric acid citrate buffer (pH = 5. 4) 200 1 and react for 20 minutes at room temperature Then, the enzyme reaction was stopped by adding 2 M sulfuric acid (501) to the reaction solution, aliquoted each 200 1 to a 96-well plate, and the absorbance at 490 nm was measured. Show.

A 10 / z 1 Z ml methanol solution of FK506 was prepared, and this was diluted with methanol to make a 0.1 ng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml, 100 ng / ml solution, 100 ng / ml solution. Each one of them was placed in a glass tube and concentrated to dryness under a stream of nitrogen. FK506-POD was diluted 10-fold with BT-PBS, and 2001 each was added for dissolution. The obtained sample solution was put into an Epppen tube in 180 μl aliquots, 101 of bead solution (about 100 beads) was added thereto, and an antigen-antibody reaction was carried out at room temperature for 2 hours. The resulting reaction solution was washed three times with T-PBS, and then a substrate solution (OPD 0.5 mg Z ml, 0.03% hydrogen peroxide, phosphoric acid monobasic buffer (pH = 5. 4)) 200 1 was added and allowed to react at room temperature for 20 minutes. The enzyme reaction was then stopped by adding 2 M sulfuric acid (501). The reaction solution was dispensed in 200 1 aliquots into 96-well plates, and the absorbance at 490 nm was measured.

A solution of FK506 in ΙΟ / z lml was prepared, and these were diluted with methanol to prepare a solution of 0.1 ngz ml, 0.1 lng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml, 100 ng / ml. Each 50 1 was added to a glass tube and concentrated to dryness under a nitrogen stream. FK 506-POD was diluted 3-fold with BT-PBS, and each 1001 was added and dissolved. The resulting sample solution was placed in an aliquot of 90 μl each into an Eppendorf tube, and 5 μl of the bead solution was added thereto to carry out an antigen-antibody reaction for 2 hours at room temperature. The resulting reaction solution was washed nine times with T-PBS. After that, 1 bead, 3 beads and 10 beads were placed in duplicate on a 96-well plate. 200 μl of a substrate solution (OPD 0.5 mg / ml, 0.03% hydrogen peroxide, phosphoric acid buffer (pH = 5. 4)) was added and allowed to react at room temperature for 20 minutes. Next, 2% sulfuric acid (50 μl) was added to stop the enzyme reaction. The reaction solution was divided into 200 μl aliquots in a 96-well plate, and the absorbance at 490 nm was measured.

Make ΙΟ / z lZmL methanol solution of FK506 and dilute them with methanol to obtain 0.0 l ng / mU 0. 001 ng / ml, 0.1 ng / ml, 0.1 ng / ml, lng / ml, lOng / ml A 100 ng / ml solution was prepared. Each 50 1 was added to a glass tube and concentrated to dryness under a nitrogen stream. FK506-POD was diluted 3-fold with BT-PBS, and each 1001 was added to dissolve. The resulting sample solution was placed in an aliquot of 90 μl per well, and the bead solution 51 was added thereto to carry out an antigen-antibody reaction for 2 hours at room temperature. The resulting reaction solution was washed 9 times with T-PBS, and then 10 beads were placed on a 96-well plate, respectively. Substrate solution (OPD 0.5 mg Z ml, 0.03% hydrogen peroxide, phosphoric acid buffer (pH = 5. 4)) 200 1 was added and reacted at room temperature for 20 minutes. The enzyme reaction was then stopped by adding 2 M sulfuric acid (501). The reaction solution was divided into 200 aliquots in a 96-well plate, and the absorbance at 490 nm was measured.

The preparation procedure of the micro flow type antibody chip used in this research is as follows. The silicon wafer was cut into 40 mm × 30 mm with a diamond cutter and cleaned. This cleaning was performed by ultrasonic cleaning with pure water and boiling in acetone, and then immersed in a mixed solution of hydrofluoric acid and ammonium (1: 6) for 5 minutes to remove the natural oxide film. Next, apply SU-8 (manufactured by MicroChem, NANO XP SU-850) to the surface of the silicon substrate with a spin coater, and use a hot plate to heat at 65 ° C: 10 minutes, 95 ° C: 30 minutes. Pre-bake was performed under the following conditions. In the spin coating, the spin coating conditions were changed so that the film thickness of SU-8 became 100 m, and coating was performed. Then, the pre-baked silicon substrate is irradiated with ultraviolet light by a mask alignment device through a mask pattern (manufactured by Yamada Photographic Engraving Co., Ltd.), and again, the temperature is 65 ° C .: 3 minutes, 95 ° C .: 10 on a hot plate. I did post-baking in minutes. This substrate The substrate was immersed in a SU-8 developer for about 10 minutes, and upon irradiation with ultraviolet light by a mask alignment device, SU-8 on a non-hardened substrate was removed and dried to obtain a target mold.

From the obtained mold, a chip using PDMS as a substrate was produced. For preparation, the PDMS prepolymer and the catalyst were mixed (10: 1) and thoroughly mixed, and then the pressure was reduced with a rotary vacuum pump in a bell jar to degas the bubbles present in the mixture. Next, PDMS was poured on a mold and heated and cured in an oven at 80 ° C. for 1 hour. In addition, when pouring PDMS onto the mold, the silicon rubber sheet provided a frame on the mold so that the PDMS would not spill. After curing by heating, PDMS was carefully removed from the mold, and a PDMS chip to which the mold was transferred was produced. The transferred chip was immersed overnight in a 1 N hydrochloric acid solution to make the chip surface hydrophilic, washed with ultrapure water, and then immersed in BT-PBS to perform blocking in the microchannel.

[0060] Next, disposable syringes-one dollar (1.2 x 38 mm) were used to form an inlet and an outlet for sample introduction and discharge to the chip. At the inlet and outlet, the FEP (fluonnated etnyiene propylene) system is included.

Before finally obtaining a microflow type antibody chip, while observing the microchannels using a stereomicroscope, ten antibody-fixed beads were placed in the microchannels of the produced PDMS chip with tweezers. At this time, the microchannel was not damaged by tweezers. After that, the micro channel side of the PDMS chip and the glass flat plate were pasted together to obtain a single channel type micro flow antibody type chip as shown in FIG. 3 to FIG. The microchannel formed in PDMS has a groove shape with a length of 30 mm, a width of 1000 μm, and a depth of 100 μm, and in the middle of the microchannel, the bottom force height of the microchannel is 50 A narrow portion opened by m is provided.

The operation procedure in the characterization of the microflow antibody chip basically conforms to Figure 1. First, using a syringe pump, a mixed solution of FK506 diluted with each concentration and horseradish peroxidase (POD) -labeled FK506 (FK506-POD) in the microchannels of the chip on which the antibody-immobilized beads were placed, using a syringe pump. The reaction was introduced for 5 minutes at a flow rate of 1 μl Z minutes, and the antigen-antibody reaction was performed by the competition method on the surface of the antibody immobilized beads. Next, in order to remove nonspecific adsorption of FK506 and FK506-POD on the surface of antibody-immobilized beads, BT-PBS was introduced for 5 minutes at a flow rate of 10 / z lZ minutes and washing operation was performed.

[0064] Next, a final concentration of peroxidase substrate 2. Hydrogen peroxide at a final concentration of 2OmM and a fluorescent substrate at a final concentration of 100 μl, 10, 3, 7-dihydroxydienoxazine (10 -acetyl-3, A mixed solution with 7-dihydroxyphenoxazine, Amplex (trademark) red) was introduced into the microchannel at a flow rate of 1 μl Z min. After 5 minutes of introduction, a fluorescence image of the resolvin generated by the enzyme reaction was taken using a CCD camera connected to a fluorescence microscope. Resolvin generated by Amplex (trademark) r ed was excited with excitation light at 546 nm, and a fluorescence image of fluorescence 590 nm was taken. The principle of detection using 10-asacetinole 3,7-dihydroxydienoxazine is shown in FIG. The obtained fluorescence image was analyzed by analysis software AQACOS MOS (manufactured by Hamamatsu Photonitas Co., Ltd.). At the time of analysis, ROI (Region of Interest) was taken around the bead, and fluorescence intensity was calculated as an average value per unit pixel in ROI.

The concentration of FK506 was measured in the same manner as in the above-described operation 1 for evaluating the calibration characteristics of the above-mentioned microfuge type 1 antibody chip, where the number of beads to be arranged on the microflow antibody chip is one. Also, blood samples were prepared assuming actual blood samples. Likewise, the concentration of FK506 was measured with a microflow antibody chip for blood samples.

[0066] Blood samples were prepared as follows. Approximately 1 ml of blood is collected from the vein of the blood using a syringe treated with mercury, and the FK 506 standard solution (5 ng Z ml diluted with methanol) 100 1 is concentrated in a nitrogen stream in a dry container. Eh. A solution of 0.2 M phosphate buffer (PH 7.0) and ethyl acetate: hexane = 1: 1 was added thereto and extracted three times. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The above operation was performed in 2 trains.

[0067] Results and Discussion

Confirmation of antibody fixation and evaluation of the optimal concentration of FK506-POD (96-well plate)> Confirm the immobilization of antibody on polystyrene beads, and the optimum concentration of FK506-POD For calculation, the change in absorbance for each concentration of FK506-POD was measured. The results are shown in Figure 10. From this result, it was suggested that it is necessary to use a 15000-fold dilution or less as an FK506-POD solution that fills all the binding sites of the beads. Therefore, in all the subsequent experiments, we used 15,000-fold diluted FK506-POD.

[0068] Examination of preparation of calibration curve by OPD (approximately 100 beads)>

Once the optimal FK506-POD concentration was confirmed, the assay was performed using FK506 dilution. At this time, since it is very time-consuming to accurately count 100 beads, in order to simplify the method, approximately 100 beads are included by dispensing a suspension of beads of a predetermined concentration. I was supposed to be The results are shown in FIG. As shown in Fig. 11, although the variation is large, the correlation was obtained up to the concentration of 0. IngZml. The cause of this variation was considered to be the variation in the number of beads. When the bead solution was dispensed in suspension, the number of beads was actually counted, and an error of about 10% occurred. From this, it is judged that it is difficult to obtain the accuracy of the number of beads by this method and the number of beads. Therefore, in the following, the number of beads was reduced, and by actually counting the number of beads, the number of beads was made accurate and the same experiment was performed.

Taking into account the fact that concentration evaluation is performed with a short flow path microchip, experiments were performed with one, three and ten beads, respectively. However, in the case of 1 bead and 3 beads, the correlation was obtained in IngZm, and the correlation could not be recognized in the point where the concentration was weak. In addition, when the number of beads was small, it was considered that it was not suitable for quantification because the rate of change in absorbance was insufficient. On the other hand, in the case of 10 beads, the correlation was obtained for 0. lpgZm (Fig. 12). Therefore, in order to ensure sufficient sensitivity, it was considered appropriate to set the number of beads to ten.

The calibration characteristics at the time of using ten beads were examined at 0. lpg Zml to 1000 ng Zm. As a result, lpgZml to 1000ngZm showed a good correlation (r ² = 0. 947) (FIG. 13). From these results, it is possible to sense enough concentration of FK506 by using 10 beads. It turned out that a degree could be obtained. Therefore, it was decided to use 10 beads for which sufficient sensitivity can be obtained for the microflow antibody chip.

After the antigen-antibody reaction and subsequent washing with PBS, when the substrate solution was flowed in the micro channel of the chip, fluorescence emission started when reaching around the substrate solution force beads. The steady state was reached in about 30 seconds, and at least 15 minutes thereafter, no difference in fluorescence intensity was observed. A photograph of the periphery of the produced single-channel microflow antibody chip is shown in FIG. 14 and a calibration characteristic is shown in FIG. From this result, it was possible to quantify FK506 at a concentration of 0.01 ng / ml to: L000 ng / ml (r ² = 0. 966) by using this microflow antibody chip. Also, although this experiment was not an experiment conducted on the same day, it still showed a good correlation, suggesting that the experiment was highly reproducible. This calibration characteristic was about 10 times more sensitive than the assay on a 96-well plate using beads. From the above results, it was found that by using a fluorescent substrate using 10 beads, it has about 1000 times the sensitivity compared to the ELISA method reported so far.

By the way, in the actual medical field, for example, the sensitivity (0. IngZml) of the conventional ELISA may be sufficient in the actual medical field, for example. Further simplification of the examination and improvement of the examination time in terms of high speed can be said to be crucial. So, in the next experiment, the number of beads was reduced and the same experiment was performed (with one bead). In the case of one bead, the sensitivity is expected to decrease according to the result of absorbance measurement using a 96-well plate. Ten beads have a sensitivity 1000 times that of the conventional ELISA method. Also in the case of individual, sensitivity equal to or higher than ELISA can be expected.

The calibration characteristics of FK506 by the microflow antibody chip using one bead showed a good correlation (r ² = 0. 987) in the range of 0.01 ng Zml to 1000 ng Zml, as shown in FIG. The sensitivity of this experiment was found to be about 100 times smaller than that obtained using 10 beads. Even with this method, the sensitivity was about 10 times that of the conventional ELISA method. Also, in the case of 10 beads, the arrangement of the beads changes (changes with each flow) every measurement, but there is a disadvantage that an analytical error is likely to occur, but in the case of one bead, R OI There is an analysis advantage in that there is less operational error (Fig. 17). Furthermore, it is very easy because it takes less time to count 10 beads.

Also, from the evaluation results of whole blood samples (FIG. 16), it was confirmed that no biological components were mixed in the data and no abnormality was found in the quantification of FK506. As described above, in this experiment, monkey blood was covered with FK506 standard solution to obtain whole blood samples. The blood concentration was adjusted to 5 ng Z ml, and the evaluation was performed using a microflow antibody chip. The results are shown by the white circles in FIG. From this experiment, the result of 3. IngZml was obtained in an average of 2 cases.

As described above, by using the microflow-type antibody chip in which the polystyrene beads to which the antibody has been adsorbed is disposed, the achievement can not be achieved by the conventional ELISA or MEIA (microparticle enzyme-mediated immunoassay). The high sensitivity and short time measurement of FK506 concentration was realized.

Claims

The scope of the claims

[1] A method for quantifying a substance to be measured in a sample solution by enzyme immunoassay, which comprises one or more bead carriers on which an antibody or antigen specifically binding to the substance to be measured is immobilized in a microchannel A substrate solution containing a substrate is prepared by transferring a mixed solution in which the sample solution and a solution containing the substance to be measured which has been enzyme-labeled are mixed so as to be arranged in approximately one row in the width direction of the microchannel. A method for quantifying a substance, comprising detecting luminescence or coloring in the vicinity of the bead carrier while feeding the solution.

[2] The method for quantifying a substance according to claim 1, wherein the luminescence is fluorescence.

[3] The method for quantifying a substance according to claim 1, wherein the washing solution is fed after feeding the mixed solution, and then the substrate solution is fed next.

[4] The method for quantifying a substance according to claim 3, wherein the mixed solution, the washing solution and the substrate solution are continuously fed to the microchannel.

[5] The method for quantifying a substance according to claim 1, wherein the bead carrier is disposed so as not to overlap in the height direction in the microchannel.

[6] The method for quantifying a substance according to claim 1, wherein the flow rate of the substrate solution during the liquid transfer is set to 1 μ 1 Z minutes or more and 10 μ 1 Z minutes or less.

[7] The method for quantifying a substance according to claim 1, wherein the flow rate of the mixed solution at the time of feeding is 1 μ 1 Z minutes or more and 10 μ 1 Z minutes or less.

[8] When a plurality of microchannels are used, in which a sample introduction unit for introducing a liquid is shared and a sample discharge unit for discharging a liquid after liquid transfer is individually formed.

After closing all the sample discharge parts, release any one force of the sample discharge part and also introduce the mixed solution from the sample introduction part and feed it to any one of the microchannels. The method for quantifying a substance according to claim 1, wherein the process is sequentially performed on each of the microchannels.

[9] A quantitative device used for quantifying a substance to be measured in a sample solution by enzyme immunoassay,

A microchannel provided with a bead carrier to which an antibody or an antigen specifically binding to a substance to be measured is immobilized, and a microchannel provided in the middle of the microchannel for flowing a liquid and carrying the bead Have narrow areas that prevent the body from moving downstream,

In the narrowing portion, one bead carrier or approximately one line in the width direction of the microchannel is arranged in the narrow channel, and the sample solution and a solution containing an enzyme-labeled substance to be measured are mixed in the microchannel. After the mixed solution is fed, luminescence or coloring in the vicinity of the bead carrier is detected while a substrate solution containing a substrate is fed.

[10] The quantitative device according to claim 9, wherein the microchannel is a groove formed on a substrate.

[11] A lid disposed on the substrate and having a convex portion on the surface opposite to the surface on which the groove of the substrate is formed,

The quantitative device according to claim 10, wherein the narrowing portion is formed by the convex portion and the groove.

[12] The quantification device according to claim 10, wherein the bead carriers are arranged so as not to overlap in the height direction.

[13] The depth of the micro channel exceeds the diameter of the bead carrier, and the diameter of the bead carrier is

The quantitative device according to claim 10, characterized in that it is not more than 2 times.

[14] A plurality of the micro flow channels are provided, and each micro flow channel shares a sample introduction portion into which the liquid is introduced, and a sample discharge portion into which the liquid after liquid delivery is discharged is individually formed. The measuring device according to claim 9, characterized in that the sample discharger can be opened and closed independently.