JP5681077B2 - Measuring method and measuring device - Google Patents

Description

  The present invention relates to a measurement method and a measurement apparatus using a light detection method for analyzing a test substance by detecting light generated from a photoresponsive labeling substance bonded to the test substance.

  In bio-measurement and the like, the fluorescence method is widely used as a highly sensitive and easy measurement method. The fluorescence method is qualitative or qualitative by irradiating a sample considered to contain a substance to be detected that emits fluorescence when excited by light of a specific wavelength, by irradiating the excitation light of the specific wavelength and detecting the fluorescence emitted at this time. This is a method for quantitatively confirming the presence of a substance to be detected. In addition, when the substance to be detected is not a fluorescent material, the substance to be detected is labeled with a fluorescent label such as an organic fluorescent dye, and then the fluorescence is detected in the same manner. It is a method to confirm.

  In the above fluorescence method, for the reason that only a specific target substance can be efficiently detected while flowing a sample, the target substance is fixed on the surface of the sensor part of the analysis chip by the following two methods, and then fluorescence detection is performed. The technique to perform is common. One such technique is, for example, when the substance to be detected is an antigen, the antigen is specifically bound to the primary antibody immobilized on the surface of the sensor unit, and then a fluorescent label is attached. A secondary antibody that specifically binds to the antigen, and further binds to the antigen to form a primary antibody-antigen-secondary antibody binding state, and the fluorescence from the fluorescent label attached to the secondary antibody This is a so-called sandwich method for detection. The other is, for example, when the substance to be detected is an antigen, a secondary antibody in which an antigen and a fluorescent label are attached to the primary antibody immobilized on the surface of the sensor unit (unlike the above-described secondary antibody, Is a so-called competition method in which the primary antibody is bound to the primary antibody competitively and the fluorescence from the fluorescent label attached to the competitively bound secondary antibody is detected. .

  In addition, for the reason that the S / N ratio can be improved in fluorescence detection, an evanescent fluorescence method has been proposed in which a fluorescent label indirectly fixed to a sensor unit by the above-described method is excited by evanescent light. In the evanescent fluorescence method, excitation light is incident from the back surface of the sensor unit, and the fluorescent label is excited by evanescent light that oozes out to the surface of the sensor unit, and fluorescence generated from the fluorescent label is detected.

  On the other hand, in order to improve sensitivity in the evanescent fluorescence method, a method using the effect of electric field enhancement by plasmon resonance has been proposed in Patent Document 1, Non-Patent Document 1, and the like. In the surface plasmon enhanced fluorescence method, in order to generate plasmon resonance, a metal layer is provided in the sensor unit, surface plasmon is generated in the metal layer, and the S / N ratio is increased by increasing the fluorescence signal by the electric field enhancing action. It is to improve.

  Further, in the evanescent fluorescence method, as in the surface plasmon enhanced fluorescence method, as a method having the effect of enhancing the electric field of the sensor unit, a method using the electric field enhancement effect by the optical waveguide mode is proposed in Non-Patent Document 2. . In this optical waveguide mode enhanced fluorescence spectroscopy (OWF), a metal layer and an optical waveguide layer made of a dielectric or the like are sequentially formed on a sensor portion, and an optical waveguide mode is formed on the optical waveguide layer. The fluorescence signal is enhanced by the electric field enhancement effect.

  Further, in Patent Document 2 and Non-Patent Document 3, instead of detecting fluorescence from a fluorescent label as in the fluorescence method described above, the fluorescence is generated by newly inducing surface plasmon in the metal layer. A method for detecting synchrotron radiation (SPCE: Surface Plasmon-Coupled Emission) has been proposed.

  As described above, various methods have been proposed as measurement methods in biomeasurement and the like.

JP-A-10-307141 US Patent Application Publication No. 2005/0053974

W. Knoll et al., Analytical Chemistry 77 (2005), p.2426-2431 2007 Spring Japan Society of Applied Physics Proceedings No.3, P.1378 Thorsten Liebermann Wolfgang Knoll, "Surface-plasmon field-enhanced fluorescence spectroscopy" Colloids and Surfaces A 171 (2000) 115-130

  In the analysis chip used in the measurement apparatus using the fluorescence method as described above, in order to reduce the amount of the sample solution collected from the living body and increase the detection speed, μTAS (Micro Total Analysis Systems) technology is often used. However, since the microchannel formed in the μTAS type analysis chip is generally very narrow, the higher the viscosity of the sample solution, the lower the sample solution feeding speed. In addition to this, the liquid feeding speed of the sample liquid also changes depending on the wet state in the microchannel.

  Since the amount of the substance to be detected supplied on the sensor unit per unit time changes when the liquid feed rate of the sample liquid supplied on the sensor unit in the microchannel changes, the reaction in the sensor unit in particular When the amount of a substance to be detected is specified by measuring the rate of change of the sample (rate measurement), the measurement results vary due to differences in the viscosity of the sample liquid, the wet state in the microchannel, and so on. Can not do.

  The present invention has been made in view of the above problems, and in a measuring apparatus that analyzes light to be detected by detecting light generated from a photoresponsive labeling substance that is bound to a substance to be detected, An object of the present invention is to provide a measurement method and a measurement apparatus in which the measurement accuracy is improved by eliminating the influence.

  The measurement method of the present invention uses an analysis chip in which a microchannel for allowing a sample liquid to flow in a channel member is provided, and a sensor part is disposed in a part of the microchannel, The sample liquid containing the substance to be detected is brought into contact with the sensor section by feeding the sample liquid to the sensor section, and an amount of the photolabel binding substance according to the amount of the substance to be detected contained in the sample liquid is placed on the sensor section. In a measurement method for measuring the amount of a substance to be detected by detecting light generated due to excitation and detecting light generated due to excitation, a sample liquid is predetermined from a predetermined measurement start position in a microchannel. Measure the time required to reach the measurement end position and correct the measurement result so that the amount of the detected substance increases as the required time increases with respect to the measurement result of the quantity of the detected substance. Features.

  The measuring device of the present invention is a measuring device used in the above-described measuring method, and a photolabel binding substance is placed at a position of a housing portion for housing an analysis chip and a sensor portion of the analysis chip housed in the housing portion. An excitation light irradiation optical system for irradiating excitation light for excitation, a light detection means for detecting light caused by excitation, and an operation for calculating the amount of a substance to be detected based on a detection result by the light detection means Means, a required time measuring means for measuring a time required for the sample liquid to reach a predetermined measurement end position from a predetermined measurement start position in the microchannel, and a calculation result of the amount of the substance to be detected, And a correction unit that corrects the calculation result so that the amount of the substance to be detected increases as the required time increases.

  The measuring apparatus of the present invention further includes a pressure measuring means for measuring the pressure in the microchannel, and the required time measuring means starts measurement based on the pressure change in the microchannel when the sample liquid is fed. It may be detected that the sample liquid has passed at the position and / or the measurement end position.

  Further, the required time measuring means may include a sensor that optically detects that the sample liquid has passed at the measurement start position and / or the measurement end position.

  In addition, the sensor part of the analysis chip may be formed by stacking a metal film on a dielectric plate that generates an evanescent wave when irradiated with excitation light.

  Note that “light generated due to excitation” is not limited to light such as fluorescence, phosphorescence, delayed fluorescence, and Raman scattered light generated from a photolabel-binding substance by excitation, but between a photolabel-binding substance and another substance. Any light may be used as long as it is triggered by irradiation with excitation light, such as light generated by fluorescence resonance energy transfer (FRET).

  As for the excitation of the photolabel binding substance, the photolabel binding substance may be directly excited by excitation light, or evanescent light is generated in the sensor part by irradiating the sensor part with the excitation light. The photolabel binding substance may be excited.

  According to the measurement method and the measurement apparatus of the present invention, an analysis chip is provided in which a microchannel for allowing a sample liquid to flow is provided in a channel member, and a sensor unit is disposed in a part of the microchannel. The sample liquid containing the substance to be detected is brought into contact with the sensor unit by sending the sample liquid into the microchannel, and the amount of the photolabel binding substance according to the quantity of the substance to be detected contained in the sample liquid In a measurement method and a measurement apparatus for measuring the amount of a substance to be detected by exciting a photolabel binding substance, detecting light generated due to excitation, and measuring the amount of the substance to be detected. The time required to reach the predetermined measurement end position from the predetermined measurement start position is measured, and as the required time is longer than the measurement result of the amount of the detected substance, the amount of the detected substance increases. The measurement results are corrected for the Even if the liquid feeding speed of the sample liquid supplied onto the sensor unit in the micro flow path changes due to the difference in the wet state, it is possible to correct the influence of this and obtain an accurate measurement result. .

Schematic diagram of a fluorescence detection apparatus which is an embodiment of the measurement apparatus of the present invention Block diagram of the fluorescence detection device Schematic diagram showing an example of an analysis chip used in the fluorescence detection device FIG. 2 is a schematic diagram showing how a sample is extracted from a sample container using a nozzle tip by the sample processing means of FIG. FIG. 2 is a schematic diagram showing how the sample in the nozzle tip is injected and stirred into the reagent cell by the sample processing means of FIG. Sectional view taken along line VI-VI in FIG. The graph which shows an example of the pressure change in the microchannel at the time of liquid feeding in the said fluorescence detection apparatus Schematic diagram showing an example of the analysis chip, light irradiation means and fluorescence detection means The graph which shows an example of the measurement result by the said fluorescence detection apparatus

  Hereinafter, a fluorescence detection apparatus according to an embodiment of the measurement apparatus of the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic diagram of a fluorescence detection apparatus according to an embodiment of the measurement apparatus of the present invention, FIG. 2 is a block diagram of the fluorescence detection apparatus, and FIG. 3 is a schematic diagram illustrating an example of an analysis chip used in the fluorescence detection apparatus. 4 is a schematic diagram showing a state in which the sample is extracted from the sample container using the nozzle tip by the sample processing means in FIG. 2, and FIG. 5 is a diagram in which the sample in the nozzle chip is injected into the reagent cell by the sample processing means in FIG. FIG. 6 is a cross-sectional view taken along the line VI-VI in FIG. 3, FIG. 7 is a graph showing an example of a pressure change in the microchannel during liquid feeding in the fluorescence detection device, and FIG. 8 is a schematic diagram showing an example of the analysis chip, light irradiation means, and fluorescence detection means, and FIG. 9 is a graph showing an example of measurement results obtained by the fluorescence detection apparatus.

  This fluorescence detection device 1 is an immunological analysis device using surface plasmon resonance, and when performing analysis by the fluorescence detection device 1, a sample container CB containing the sample shown in FIG. 1 and a sample and a reagent are extracted. The nozzle chip NC used at the time, and the analysis chip 10 in which the reagent cell and the microchannel are formed are loaded. Note that the sample container CB, the nozzle tip NC, and the analysis chip 10 are all disposable items that are discarded once they are used. Then, the fluorescence detection apparatus 1 performs a quantitative or qualitative analysis on the test substance in the sample while flowing the sample through the microchannel 15 of the analysis chip 10.

  The fluorescence detection apparatus 1 includes a sample processing unit 20, a light irradiation unit 30, a fluorescence detection unit 40, a light irradiation unit 50, a light detection unit 60, a data analysis unit 70, and the like. The sample processing means 20 extracts a sample from the sample container CB containing the sample using the nozzle chip NC, and generates a sample solution obtained by mixing and stirring the extracted sample with a reagent. Further, the data analysis means 70 functions as a calculation means for calculating the amount of the substance to be detected, the time t required for the sample liquid to reach the predetermined measurement end position from the predetermined measurement start position in the flow path 15. And a function as a required time measuring means for measuring the amount of the substance to be detected and a function as a correcting means for correcting the calculation result of the amount of the substance to be detected. The operation of the data analysis means 70 will be described later in detail.

  FIG. 3 is a schematic diagram illustrating an example of the analysis chip 10. The analysis chip 10 has a structure in which an inlet 12, an outlet 13, sample cells 14 a and 14 b, and a flow path 15 are formed in a main body 11 made of a dielectric plate such as a light transmissive resin. The injection port 12 communicates with the discharge port 13 via the flow channel 15, and by applying a negative pressure from the discharge port 13, the specimen is injected from the injection port 12, flows into the flow channel 15, and is discharged from the discharge port 13. The The sample cells 14a and 14b are containers for storing a fluorescent reagent (second antibody) to be mixed with the specimen in the specimen container CB. Note that the openings of the sample cells 14a and 14b are sealed with a sealing member, and the sealing member is pierced when the specimen and the fluorescent reagent are mixed.

  Further, a test region TR as a sensor unit for detecting a test substance in the specimen and a control region CR provided on the downstream side of the test region TR are formed in the flow channel 15. A first antibody is immobilized on the test region TR, and the labeled antibody is captured by a so-called sandwich method. In addition, a reference antibody is fixed to the control region CR, and the reference antibody captures the fluorescent substance when the sample solution flows on the control region CR. Two control regions CR are formed, a so-called negative control region CR for detecting non-specific adsorption, and a so-called positive control region CR for detecting a difference in reactivity due to a difference in specimen. Is formed.

  The sample processing means 20 includes a pump and a pressure sensor (not shown), and when the start of analysis is instructed, a nozzle tip NC is attached to the tip of the nozzle of the sample processing means 20 as shown in FIG. A specimen is aspirated from the container CB. Thereafter, the sample processing means 20 punctures the seal member of the sample cell 14a as shown in FIG. 5, mixes and stirs the sample with the reagent in the sample cell 14a, and then sucks the sample solution again using the nozzle tip NC. . This operation is similarly performed for the sample cell 14b. Then, a sample solution is generated in which the second antibody B2, which is a second binding substance specifically binding in the reagent, to the test substance (antigen) A present in the sample is modified on the surface. The sample processing means 20 then installs the nozzle tip NC containing the sample solution on the injection port 12, inserts the nozzle tip into the discharge port 13, sucks it from the discharge port 13, and creates a negative pressure in the flow path 15. By setting the pressure, the sample solution in the nozzle tip NC is caused to flow into the flow path 15.

  In order to stabilize the liquid feeding speed of the sample solution at the time of the main measurement, in the fluorescence detection apparatus 1 of the present embodiment, liquid feeding (primary liquid feeding) for wetting the flow path 15 is performed prior to the main measurement.

  In addition, if the liquid feeding speed of the sample liquid supplied onto the sensor unit in the microchannel changes due to the difference in the viscosity of the sample solution or the wet state in the channel 15, the measurement result varies. In order to correct the influence, in the fluorescence detection device 1 of the present embodiment, the sample solution is measured from the predetermined measurement start position in the flow path 15 to the predetermined measurement end position in the primary liquid feeding. The required time t is measured simultaneously. Here, the entrance of the sensor area where the sensor unit (test area TR and control area CR) is provided is the measurement start position, and the exit is the measurement end position.

  As shown in FIG. 6, since the depth of the flow path in the sensor region is shallower than the depth of the flow path in the passage portion communicating with the sensor region, the pressure in the flow path 15 during the primary liquid feeding is measured. As shown in the graph of FIG. 7, when the sample solution reaches the sensor region from the nozzle tip NC, the pressure in the flow path 15 increases.

  Further, at the measurement end position, a light irradiation means 50 such as an LED and a light detection means 60 are arranged so as to sandwich the analysis chip 10. This is to see whether the tip of the sample solution has reached by light transmission. For example, when light is irradiated from the light irradiation means 50 from below to above the analysis chip 10 and the amount of light is detected by the upper light detection means 60, if the sample solution has not reached the measurement end position, A part of light (about 4%) is reflected at the boundary between the lower member 11 and the flow path and the boundary between the flow path and the upper member 12 constituting the road member, and finally the light quantity is about 8%. When the sample solution has reached the measurement end position, the refractive index of the sample solution and the transparent resin is so close that no light is reflected, so that the amount of light hardly decreases. Therefore, as shown in the graph of FIG. 7, when the sample solution has reached the measurement end position, the detected light amount is about 8% higher than when the sample solution has not reached. In this way, it is possible to know whether the tip of the sample solution has reached the measurement end position (that is, whether all of the detection area portion has been passed) by the change in the detected light amount.

  As described above, since the timing at which the sample solution has passed the measurement start position in the flow channel 15 and the timing at which the sample solution has reached the measurement end position can be measured, the data analysis means 70 determines the measurement start position based on these pieces of information. The time required t until the measurement end position is reached is measured.

  Next, the operation during this measurement will be described. FIG. 8 is a schematic diagram showing an example of an analysis chip, light irradiation means, and fluorescence detection means. In FIG. 6, the description will be given focusing on the test region TR, but the excitation light L is similarly applied to the control region CR.

  Specifically, the main body 11 of the analysis chip 10 includes a base 11a and an upper lid 11b formed of a dielectric material such as a light transmissive resin. The substrate (dielectric plate) 11a is provided with a groove for forming a flow path 15 (sample liquid holding portion), and an upper lid 11b is attached on the groove so that the groove portion becomes the flow path 15 (sample liquid). Functions as a holding unit). A metal film 16 is laminated on the bottom surface of the test region TR (same for the control region CR) of the flow path 15.

  The light irradiating means 30 irradiates the dielectric plate 17 and the metal film 16 in the test region TR through the prism with the excitation light L from the back side of the analysis chip 10 at an incident angle that is a total reflection condition. The fluorescence detection means 40 is composed of, for example, a CCD, a CMOS, etc., and obtains an image signal FS by photographing the test area TR.

  A specimen solution is supplied into the flow path 15, and the excitation light L is incident on the interface between the dielectric plate 17 and the metal film 16 at a specific incident angle greater than the total reflection angle by the light irradiation means 30. An evanescent wave Ew oozes out from the sample S on the metal film 16, and surface plasmons are excited in the metal film 16 by the evanescent wave Ew. This surface plasmon causes an electric field distribution on the surface of the metal film 16 to form an electric field enhancement region. Then, the fluorescent labeling substance F bound to the first antibody B1 fixed on the metal film 16 is excited by the evanescent wave Ew to generate enhanced fluorescence.

  In detection using plasmon enhancement, metal quenching may occur and the sensitivity may be lowered. For example, if a quenching prevention layer made of a silica layer, a polystyrene layer, or the like is provided on the metal layer 16, Such a problem can be solved. In addition, as for the fluorescent labeling substance F, for example, as a quenching preventive substance such as a fluorescent dye encapsulated in polystyrene particles or silica particles, or a gold colloid surface coated with polystyrene, the problem of metal quenching is solved. be able to.

  The data analysis means 70 in FIG. 2 analyzes the test substance based on the temporal change of the fluorescence signal FS detected by the fluorescence detection means 40. Specifically, since the fluorescence intensity changes depending on the amount of the fluorescent labeling substance F bound thereto, the fluorescence intensity changes with time as shown by the solid line in the graph of FIG. The data analysis means 70 acquires a plurality of fluorescent signals FS in a predetermined sampling period (for example, 5 seconds) in a predetermined period (for example, 5 minutes), and analyzes the change rate of the fluorescence intensity over time, thereby analyzing the test in the sample. Perform quantitative analysis of substances (rate method).

  In this case, the slower the liquid feeding speed of the sample solution, the smaller the amount of the fluorescent labeling substance F that passes through the sensor area per unit time, and the lower the detection signal value. About the influence on the detection signal value due to the difference in the liquid feeding speed, measurement using a test liquid having a known concentration is performed in advance, and the correction coefficient is obtained based on the detection signal value when the liquid feeding speed is changed. Thereby, in the data analysis means 70, based on the required time t measured at the time of the primary liquid feeding, the measurement result is corrected so that the binding amount of the fluorescent labeling substance F increases as the required time t increases. Specifically, as shown in the graph of FIG. 9, the difference in liquid feeding speed is obtained by multiplying the result obtained during the actual measurement (indicated by a solid line in the graph) by a larger coefficient as the required time t is longer. As a result of correcting the influence of (a dotted line in the graph), the result can be obtained. Here, the degree of the coefficient to be multiplied can be appropriately selected in consideration of the measurement results acquired in advance and various conditions.

  Moreover, you may make it correct | amend further using the measurement result in control area | region CR with respect to the result corrected as mentioned above.

  The finally obtained analysis result is output from the information output means 4 comprising a monitor, a printer or the like.

  As mentioned above, although preferable embodiment of this invention was described, this invention is not limited to the said embodiment.

  For example, as for the method for detecting the timing at which the sample solution passes the measurement start position in the flow channel 15 and the timing at which the sample solution reaches the measurement end position, both may be detected based on the pressure change in the flow channel 15. Both may be detected optically using sensors such as the light irradiation means 50 and the light detection means 60. Moreover, you may make it detect by methods other than the above.

  Further, the method for correcting the measurement result is not limited to the above, and any method may be used such as adding a larger numerical value as the required time t is longer.

  In addition to the surface plasmon enhanced fluorescence method and evanescent fluorescence method, the fluorescence detection apparatus of the present invention can be adapted to various systems such as an optical waveguide mode enhanced fluorescence spectroscopy.

  In addition to the above, it goes without saying that various improvements and modifications may be made without departing from the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Fluorescence detection apparatus 10 Analytical chip 20 Sample processing means 30 Light irradiation means 40 Fluorescence detection means 50 Light irradiation means 60 Light detection means 70 Data analysis means CR Control area FS Fluorescence signal L Excitation light TR Test area

Claims (3)

A microchannel is provided in the channel member for circulating the sample liquid, and the cross-sectional area in the microchannel changes at a predetermined measurement start position in the microchannel. Using an analysis chip in which the sensor part is arranged in the part,
An amount of light corresponding to the amount of the substance to be detected contained in the sample liquid is obtained by bringing the sample liquid into the microchannel to contact the sample liquid containing the substance to be detected on the sensor unit. Binding a label binding substance onto the sensor unit;
Exciting the photolabel binding substance;
In a measurement method for detecting the light caused by the excitation and measuring the amount of the substance to be detected,
Detecting that the sample liquid has passed through a predetermined measurement start position in the microchannel based on a pressure change in the microchannel, and the sample liquid determines a predetermined measurement end position in the microchannel. that has passed by detecting optically measures the time required for the said sample solution reaches the measurement end position from the measurement start position,
A measurement method, wherein the measurement result is corrected so that the amount of the substance to be detected increases as the required time increases with respect to the measurement result of the quantity of the substance to be detected. A measurement device used in the measurement method according to claim 1,
An accommodating portion for accommodating the analysis chip;
An excitation light irradiation optical system for irradiating excitation light for exciting the photolabel binding substance to the position of the sensor part of the analysis chip accommodated in the accommodation part;
A light detection means for detecting light caused by the excitation;
An arithmetic means for calculating the amount of the substance to be detected based on a detection result by the light detection means;
Pressure measuring means for measuring the pressure in the microchannel;
A sensor that optically detects that the sample liquid has passed at the measurement end position;
A required time measuring means for measuring the required time,
A measuring apparatus comprising: a correction unit that corrects the calculation result so that the amount of the substance to be detected increases as the required time increases with respect to the calculation result of the quantity of the substance to be detected. 3. The measuring apparatus according to claim 2 , wherein the sensor portion of the analysis chip is formed by laminating a metal film on a dielectric plate that generates an evanescent wave when irradiated with the excitation light.

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