JP2010210378A - Sensing method and sensing kit to be used of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable a high quantitative sensing operation to be carried out by improving an S/N ratio in a sensing method using a scattered light measurement. <P>SOLUTION: In the sensing method for detecting an amount of a substance to be detected A, a labeling complex whose amount corresponds to the amount of the substance to be detected A is formed at a sensor part 14 on the surface of a transparent carrier 10 on which a first capture substance B1 for forming the labeling complex containing a scatterer F is immobilized, and the refractive index of the surface of the transparent carrier 10 containing at least the sensor part 14 is adjusted to be 1.35 or more, and less than the refractive index of the transparent carrier itself, and then the scattered light measurement is carried out. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a sensing method for detecting scattered light of evanescent light by a scatterer and detecting the amount of a substance to be detected, and a sensing kit used therefor.

  Conventionally, a sensing method using total reflection illumination has attracted attention in bioassay for detecting proteins, DNA, and the like. This sensing method uses light that oozes out from the interface when the measurement light is totally reflected at the interface with different refractive indices, that is, evanescent light, and a detected substance contained in the sample or a label attached to the detected substance. In this method, the presence or amount of the substance to be detected is detected by analyzing optical interactions such as scattering, absorption, and light emission.

An example of such a sensing method is a fluorescence detection method using a fluorescent label.
The fluorescence detection method has become an indispensable tool for bio research, coupled with the enhancement of the performance of photodetectors such as cooled CCDs. Also in materials used for fluorescent labels, fluorescent dyes with high fluorescence quantum yield, particularly in the visible region, such as FITC (fluorescence: 525 nm, fluorescence quantum yield: 0.6) and Cy5 (fluorescence: 680 nm, fluorescence quantum yield). A fluorescent dye exceeding 0.2 which is a practical standard such as a rate of 0.3) has been developed and widely used. Furthermore, high-sensitivity detection that cuts 1 pM (picomolar) has been realized by increasing the fluorescence signal by using the enhancement of the electric field by surface plasmons.

  However, fluorescent dyes have a weak π bond in terms of chemical structure due to the property of absorbing and emitting light, so they are altered by irreversible destruction by strong light and chemical reactions with oxygen and ozone in the atmosphere. There is a problem. This causes a so-called discoloration in which the total amount of fluorescence emitted from the entire fluorescent dye decreases over time, so that the intensity of measurement light cannot be increased beyond a certain level, and there is a limit to increasing sensitivity.

  Therefore, another example of a sensing method using total reflection illumination is a scattered light detection method using a scattering label as shown in Patent Document 1 and Patent Document 2.

  The scattered light detection method uses scattering of evanescent light by a scattering label such as metal fine particles, and detects the amount of a substance to be detected based on the wavelength and intensity of the detected scattered light. Such a scattered light detection method does not have the above-mentioned problem of fading, and further, since the amount of detected light obtained is larger than that of a general fluorescence detection method using a fluorescent dye, higher detection sensitivity is required. It is used as a method used in such cases.

Japanese Patent No. 3608665 Special table 2007-525651 gazette

  However, in the scattered light detection method as described above, there is a problem of noise-like scattered light of evanescent light generated by impurities contained in the sample or present on the transparent carrier. This is because the noise-like scattered light increases the background against the scattered light by the scattering label that is originally a detection target, so that the S / N ratio is lowered and the quantification of scattered light detection is lost. is there.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a sensing method with improved S / N ratio and higher quantitativeness, and a sensing kit used therefor.

  In order to solve the above-mentioned problem, the inventor of the present application has a refractive index within a certain range when the refractive index on the surface of the transparent carrier is larger than air (refractive index 1.00) or water (refractive index 1.33). The inventors have found that it is possible to improve the S / N ratio in sensing, and have reached the present invention.

That is, the sensing method according to the present invention is:
A sensing method for detecting the amount of a substance to be detected,
Supplying a sample capable of containing a substance to be detected to a sensor unit on the surface of a transparent carrier, to which a first capture substance for forming a labeled complex containing a scatterer is fixed,
An amount of labeled complex corresponding to the amount of the substance to be detected is formed on the sensor part,
Adjusting the refractive index on the surface including at least the sensor part of the transparent carrier to be 1.35 or more and less than the refractive index of the transparent carrier;
Irradiate the transparent carrier with measurement light to generate evanescent light in the sensor unit,
It is characterized by detecting the scattered light of the evanescent light by the scatterer.

  Here, “detecting the amount of a substance to be detected” includes detection of the presence or absence of the substance to be detected, and not only a qualitative amount but also a quantitative amount or degree of activity can be detected. Shall mean.

  “Label complex” including a scatterer means an aggregate that includes a scatterer as a label and a first capture substance, and is formed on the sensor unit in an amount corresponding to the amount of the substance to be detected. And That is, for example, when an assay by the sandwich method is performed, the labeled complex means an assembly having a sandwich structure of a first capture substance-a substance to be detected-a second capture substance as described. On the other hand, in the case of performing an assay by a competitive method, the labeled capture substance means an aggregate having a structure of a first capture substance-third capture substance, as the label complex is described.

  The “capture substance” means a substance that specifically binds to a specific target substance. For example, when an antigen is considered as a specific target substance, an antibody that specifically binds to the antigen can be used as a capture substance. In the above case, another antibody that competes with the antigen and specifically binds to the antibody is also a capture substance.

  The “sensor part” means a region where the first capture substance on the surface of the transparent carrier is fixed.

  The label complex of “amount corresponding to the amount of the substance to be detected” means an amount of the label complex that correlates with the amount of the substance to be detected contained in the sample. Thereby, the amount of the substance to be detected is estimated from the amount of scattered light from the scatterer.

  Further, in the sensing method according to the present invention, the refractive index is preferably adjusted to be 1.38 or more and 1.51 or less, and more preferably adjusted to be 1.42 or more and 1.49 or less. preferable.

  And it is preferable to fill the surface of the transparent carrier with an adjustment liquid for adjusting the refractive index, and adjust the refractive index of this surface with the refractive index of the adjustment liquid. In this case, the adjustment liquid is preferably an organic solvent mixed with water or an aqueous solution of a solute having a high refractive index. Alternatively, it is preferable that an adhesive substance or a rubbery substance for adjusting the refractive index is brought into contact with the surface of the transparent carrier, and the refractive index of this surface is adjusted with the refractive index of the adhesive substance or the rubbery substance.

  In addition, the second capture substance specifically binds to the substance to be detected, and a labeled capture substance composed of the second capture substance and a scatterer modified with the second capture substance is used as the first capture substance. It is preferable to form a labeled complex by specifically binding to a substance to be detected bound to a capture substance. Alternatively, the third capture substance competes with the substance to be detected and specifically binds to the first capture substance, and the third capture substance and the scatterer in which the third capture substance is modified are used. It is preferable to form a labeled complex by specifically binding a labeled capture substance to the first capture substance.

  Here, the “label capture substance” is bound on the sensor unit by an amount corresponding to the amount of the detected substance via the first capture substance that is fixed on the sensor unit and specifically binds to the detected substance. It is intended to mean a capture substance labeled with a scatterer. That is, for example, when an assay by the sandwich method is performed, the labeled capture substance is composed of a second capture substance that specifically binds to the substance to be detected and a scatterer in which the second capture substance is modified. The As a result, a sandwich structure of the first capture substance-the substance to be detected-the second capture substance is created, and the labeled capture substance is bound on the sensor unit. Here, the binding site for the first capture substance of the substance to be detected is different from the binding site for the second capture substance. On the other hand, in the case of performing the assay by the competitive method, the labeled capture substance competes with the substance to be detected and binds to the third capture substance that specifically binds to the first capture substance, and the third capture substance. And a modified scatterer. Thereby, the coupling | bonding of the 1st capture substance-third capture substance is made, and the label | marker capture substance couple | bonds on a sensor part.

  The scatterer is preferably fine particles of metal, metal oxide, or metal nitride. In this case, the particle size of the fine particles is preferably 30 nm to 300 nm.

  Here, the “particle size” of the fine particles refers to the diameter of a solid that moves independently in the dispersion liquid, usually in an aqueous solution. When stably dispersed in an aggregated state, the diameter of the aggregate is indicated. The particle size can be measured by, for example, a dynamic light scattering method. Specific methods for measuring particle size in water by dynamic light scattering include: Otsuka Electronics DLS-8000 series, DLS-6500 series, FPAR-1000, Malvern Zetasizer Nano series, Beckman Coulter Delsa Using a device such as Nano S, measurement can be performed according to the instruction manual of each device. When the fine particles are rod-shaped, the particle size indicates the length of the minor axis.

Furthermore, the sensing kit according to the present invention is:
In order to form an inlet for injecting a sample, a flow path for flowing the sample injected from the inlet, an air port for extracting air so that the sample flows, and a labeled complex including a scatterer And a transparent carrier chip having a sensor part on the surface of the flow path to which the first capture substance is fixed, and a transparent carrier having a refractive index of 1.35 or more for adjusting the refractive index on the surface of the flow path With adjustment liquid that is less than the refractive index of the chip,
A sample that can contain a substance to be detected is supplied to the sensor unit, a labeling complex of an amount corresponding to the amount of the substance to be detected is formed in the sensor unit, and the transparent carrier chip is irradiated with measurement light to emit evanescent light to the sensor unit. It is characterized in that it is used in a sensing method for detecting the amount of a substance to be detected by detecting scattered light of evanescent light caused by a scatterer.

Alternatively, the sensing kit according to the present invention is:
A transparent carrier chip having a sensor portion on which a first capture substance for forming a labeling complex including a scatterer is fixed, and a refractive index of 1. to adjust the refractive index on the surface of the transparent carrier chip. Comprising an adjustment liquid that is 35 or more and less than the refractive index of the transparent carrier chip;
A sample that can contain a substance to be detected is supplied to the sensor unit, a labeling complex of an amount corresponding to the amount of the substance to be detected is formed in the sensor unit, and the transparent carrier chip is irradiated with measurement light to emit evanescent light to the sensor unit. It is characterized in that it is used in a sensing method for detecting the amount of a substance to be detected by detecting the scattered light of evanescent light caused by a scatterer.

Alternatively, the sensing kit according to the present invention is:
A transparent carrier chip having a sensor portion on which a first capture substance for forming a labeling complex including a scatterer is fixed, and a refractive index of 1. to adjust the refractive index on the surface of the transparent carrier chip. Comprising an adhesive substance or a rubbery substance that is 35 or more and less than the refractive index of the transparent carrier chip,
A sample that can contain a substance to be detected is supplied to the sensor unit, a labeling complex of an amount corresponding to the amount of the substance to be detected is formed in the sensor unit, and the transparent carrier chip is irradiated with measurement light to emit evanescent light to the sensor unit. It is characterized in that it is used in a sensing method for detecting the amount of a substance to be detected by detecting scattered light of evanescent light caused by a scatterer.

And in the sensing kit according to the present invention,
It is preferable to provide a labeled capture substance comprising a second capture substance that specifically binds to the substance to be detected and a scatterer in which the second capture substance is modified. Alternatively, it is preferable to include a labeled capture substance composed of a third capture substance that specifically binds to the first capture substance in competition with the substance to be detected and a scatterer in which the third capture substance is modified.

  The sensing method according to the present invention and the sensing kit used therefor adjust the refractive index on the surface including at least the sensor part of the transparent carrier or flow path to be 1.35 or more and less than the refractive index of the transparent carrier. , Detecting scattered light. That is, compared with the conventional method in which the sensor unit is usually air (refractive index 1.00) or water (refractive index 1.33), the present invention increases the refractive index on the sensor unit to perform sensing. Yes. The inventor of the present application has found a phenomenon that when the refractive index on the surface of the transparent carrier is larger than that of air or water, the scattered light to be detected increases and the noise-like scattered light decreases at a certain range of refractive index. . Therefore, the S / N ratio can be improved as compared with the conventional method by making the refractive index on the surface of the transparent carrier larger than that of air or water. Thereby, it is possible to perform sensing with higher quantitativeness by the sensing method according to the present invention and the sensing kit used therein.

Schematic diagram showing one process of sensing method (1) Schematic diagram showing one process of sensing method (Part 2) Schematic diagram showing one step of sensing method related to design change (1) Schematic diagram showing one step of sensing method related to design change (Part 2) Schematic showing scattered light measurement in the sensing method according to the first embodiment Schematic showing scattered light measurement in the sensing method according to the second embodiment Schematic which shows the sensing kit which concerns on 1st Embodiment Schematic which shows the sensing method using the kit which concerns on 1st Embodiment (the 1) Schematic (2) which shows the sensing method using the kit which concerns on 1st Embodiment Schematic showing scattered light measurement using the kit according to the first embodiment Schematic which shows the sensing kit which concerns on 2nd Embodiment. Schematic which shows the sensing kit which concerns on 3rd Embodiment Schematic showing the relationship between refractive index and signal or S / N ratio Schematic showing the relationship between antigen concentration and signal Schematic showing the measurement image in the example

  Hereinafter, although an embodiment of the present invention is described using a drawing, the present invention is not limited to this.

"Sensing method"
<First Embodiment of Sensing Method>
First, the sensing method according to the present embodiment will be described. This sensing method is a sandwich type sensing method. FIG. 1 is a schematic diagram illustrating a part of the steps of the sensing method according to the present embodiment.

  As shown in FIGS. 1A and 1B, the sensing method fixes a first capture substance that specifically binds to a target substance to a predetermined region on a transparent carrier to form a sensor unit (Step 1a), and detects the target. A sample that can contain a substance is supplied to the sensor unit (Step 1b). After the substance to be detected is bound to the first capture substance, the sensor part is washed (Step 1c), and then a label that specifically binds to the substance to be detected. The capture substance is supplied onto the sensor part (Step 1d), the label capture substance is bound to the detection substance bound to the first capture substance, and then the sensor part is washed (Step 1e), and a gap is formed on the sensor part. A cover glass 12 is placed and an adjustment liquid for adjusting the refractive index is injected into the gap (Step 1f), and then the scattered light is measured. Here, the label capture substance is composed of a second capture substance that specifically binds to the substance to be detected and a scatterer in which the second capture substance is modified. Another method is to perform Step 1b and Step 1d at the same time without performing Step 1c, that is, a method in which a sample that can contain a substance to be detected and a label capture substance are mixed in advance and then supplied onto the sensor unit.

  The transparent carrier 10 has a sensor portion formed in a predetermined region on one surface thereof. The transparent carrier 10 is made of a transparent material such as transparent resin or glass. When a resin substrate is used, there is an advantage that the substrate is inexpensive, and when a glass substrate is used, scattering due to impurities and surface irregularities (this scattering becomes noise for the detection signal) is small. Since there is an advantage, it is desirable to select appropriately according to the application and detection conditions. In such a transparent carrier, the refractive index of the transparent carrier is approximately 1.5 to 1.6. As the resin material forming the transparent carrier 10, polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO) containing cycloolefin, and the like are preferable. The transparent carrier becomes a waveguide for total reflection illumination in the scattered light measurement. Here, the “waveguide” means a two-dimensional total internal reflection element in which light is totally internally reflected at multiple points and forms substantially uniform evanescent light on the entire surface or almost the entire surface. . The structure of the two-dimensional waveguide may be planar or curved.

  The sensor unit is a region where the first capture substance on the transparent carrier surface is fixed. There may be one sensor unit or a plurality of sensor units. When there are a plurality of sensor units, a plurality of substances to be detected can be detected, and so-called multi-item array detection is possible. When there are a plurality of sensor units, it is preferable that the sensor units are separated by a non-sensor unit (that is, a region where the first capture substance is not fixed). Further, the two-dimensional shape of the sensor unit is not particularly limited, such as a rod shape or a circular shape.

  The first capture substance is a capture substance that specifically binds to the substance to be detected. Such a capture substance is not particularly limited, and can be appropriately selected according to detection conditions (particularly the substance to be detected 2). For example, when the substance to be detected is an antigen, examples of such a capture substance include an antibody against it. For example, when the antigen is an hCG antigen (molecular weight 38000 Da), a monoclonal antibody that specifically binds to the antigen can be used. As the immobilization method, a method of physically adsorbing to a transparent carrier, a functional group such as a carboxyl group, an amino group, a thiol group, etc. is introduced by applying surface modification to the transparent carrier, and electrostatically there Alternatively, a method of immobilizing via a chemical bond, a method of preliminarily introducing a structure that binds to a capture molecule and the like can be mentioned. As a chemical bonding method, for example, a so-called amine coupling method in which the transparent carrier is carboxylated and further activated to bond with the amino group of the first capture substance can be used. In addition, a method in which a layer such as a linker or a gel is interposed between the first capture substance and the surface carrier is also exemplified.

  The labeled capture substance is a capture substance labeled with a scatterer that binds on the sensor unit 14 by an amount corresponding to the amount of the substance A to be detected. As shown in FIG. 1, the labeled capture substance comprises a capture substance that specifically binds to the substance A to be detected and a scatterer when assaying by the sandwich method. Further, the labeled capture substance is composed of a capture substance and a scatterer that compete with the substance to be detected when assaying by the competition method described later is performed. More specifically, when the transparent carrier 10 is used in which the first capture substance B1 that specifically binds to the substance A to be detected is fixed to the sensor unit 14, the label capture substance BF in the sandwich method is: It consists of a second capture substance B2 that specifically binds to the substance A to be detected and a scatterer in which the second capture substance B2 is modified. On the other hand, in the same case, the labeled capture substance in the competition method includes a third capture substance that specifically binds to the first capture substance in competition with the substance to be detected, and a scatterer in which the third capture substance is modified. It consists of When the substance to be detected A is an antigen, an antibody may be used as the first and second capture substances.

  The scatterer F is appropriately selected according to detection conditions without particular limitation, but is preferably metal fine particles, metal oxide fine particles, or metal nitride fine particles from the viewpoint of scattering properties and diameter control. Further, the metal fine particles are metal fine particles composed of at least one metal selected from the group consisting of Au, Ag, Cu, Pt, Pd, Rh, Ru, Co, Fe, and Ni. preferable. Examples of the metal oxide fine particles or metal nitride fine particles include silica, alumina, titania, zirconia, zeolite, zinc oxide, yttrium oxide, cerium oxide, tin oxide, iron oxide, silicon nitride, aluminum nitride, and nitride. Examples thereof include titanium, chromium nitride, and iron nitride. Here, the particle diameter of the fine particles is preferably 30 nm or more, more preferably 50 nm from the viewpoint of scattering intensity, and from the viewpoint of the maximum amount of binding of the scatterer per unit area, in turn from the viewpoint of dynamic range, 300 nm or less, more Preferably it is 200 nm or less.

  The second capture substance is a capture substance that specifically binds to the substance to be detected and modifies the scatterer. Here, the first and second capture substances bind to different binding sites (epitope in the case of antibodies) of the substance to be detected. Thus, the sensing method according to the present embodiment is a sandwich type that forms a sandwich structure of the first capture substance-the substance to be detected-the second capture substance. When a plurality of substances to be detected are detected by a plurality of sensor units in a sandwich format, a substance that specifically binds to a common structure of the plurality of substances to be detected can be used as the second capture substance. Specifically, when an antibody against a specific antigen is detected as a substance to be detected, a substance that binds to a common structure such as an Fc part of an antibody can be used as the second capture substance.

The adjustment liquid is for adjusting the refractive index on the surface of the transparent carrier. The adjustment liquid preferably has a refractive index of 1.35 or more and less than the refractive index of the transparent carrier chip. By disposing the adjustment liquid on the sensor unit, the refractive index on the surface of the transparent carrier is adjusted with the refractive index of the adjustment liquid. Further, the adjustment liquid preferably has a refractive index of 1.38 or more and 1.51 or less, and more preferably 1.42 or more and 1.49 or less. For example, substances having various refractive indexes can be appropriately selected from those shown in Table 1 below. However, the adjustment liquid for adjusting the refractive index is not limited to the following substances.

  Further, it is preferable that the adjustment liquid is not disposed over a wide range more than necessary on the surface of the transparent carrier, and is disposed only in a range in the vicinity of the sensor unit including at least the sensor unit. This is because when the total reflection illumination type measurement is performed, the refractive index of the transparent carrier and the refractive index on the surface thereof are close to each other, so that light energy easily leaks as evanescent light, and the utilization efficiency of the light energy decreases.

  Next, the scattered light measurement in the sensing method will be described with reference to FIG. FIG. 3 is a schematic cross-sectional view showing a detection device used for scattered light measurement. The detection apparatus shown in FIG. 3 includes a transparent carrier having a sensor part, a gap cover glass covering the sensor part, an adjustment liquid injected into the gap cover glass so as to adjust the refractive index of the sensor part, and a transparent carrier. Due to the light source that irradiates the measurement light toward the end face, the light shielding plate arranged at the end of the transparent carrier so that the measurement light is transmitted only through the end face and guided in the transparent carrier, and the measurement light is guided And a photodetector for detecting the scattered light of the evanescent light generated as described above. FIG. 3 also shows a first capture substance fixed to the sensor unit, a detection substance bound to the first capture substance, and a labeled capture substance bound to the detection substance.

  The measurement light may be, for example, single wavelength light obtained from a laser light source or the like, or broad light obtained from a white light source or the like, and is not particularly limited, but can be appropriately selected according to detection conditions. In the detection apparatus according to this embodiment, since the measurement light is irradiated from the end face of the transparent carrier and guided through the transparent carrier, it is possible to easily generate an evanescent wave. The wavelength of the measurement light Lo can be a value in the visible, ultraviolet and near infrared regions, and is not particularly limited.

  The light source 21 may be a laser light source, for example, and is not particularly limited, but can be appropriately selected according to detection conditions. If necessary, the light source 21 guides the measurement light Lo so that the measurement light Lo is incident on the surface of the transparent carrier 10 through the dielectric transparent carrier 10 so as to satisfy the total reflection condition on the surface. Therefore, a light guide system such as a mirror or a lens can be appropriately combined. The light shielding plate is for minimizing the influence of stray light generated from the light source. The light shielding plate is more effective when a light absorbing material is used.

  As the photodetector 30, a CCD, PD (photodiode), photomultiplier, c-MOS, or the like can be used as appropriate. In particular, from the viewpoint of detection sensitivity, it is preferable to use a cooled CCD. Further, the photodetector 30 can be used in combination with a spectroscopic means such as an optical filter or a spectroscope depending on detection conditions. As a preferable photodetector 30, for example, LAS-1000 plus (trade name) manufactured by FUJIFILM Corporation can be exemplified.

  The inventor of the present application has found a phenomenon that when the refractive index on the surface of the transparent carrier is larger than that of air or water, the scattered light to be detected increases and the noise-like scattered light decreases at a certain range of refractive index. . Usually, it is known that the intensity of evanescent light increases as the refractive index of a transparent carrier approaches the refractive index on the surface thereof. Therefore, technically, the intensity of scattered light (that is, scattered light from a scatterer) to be detected increases, and noise-like scattered light (that is, scattered light from contaminants on the surface of a transparent carrier) increases. The strength is also expected to increase. However, the present inventor has found a particularly surprising phenomenon that “noisy scattered light decreases”. Thereby, as described later, the S / N ratio can be remarkably improved by the sensing method according to the present invention.

  In the sensing method according to the present invention, the refractive index on the surface including at least the sensor portion of the transparent carrier is adjusted to be 1.35 or more and less than the refractive index of the transparent carrier to detect scattered light. That is, compared with the conventional method in which the sensor unit is usually air (refractive index 1.00) or water (refractive index 1.33), the present invention increases the refractive index on the sensor unit to perform sensing. Yes. Therefore, the S / N ratio can be improved as compared with the conventional method by making the refractive index on the surface of the transparent carrier larger than that of air or water. Thereby, it is possible to perform sensing with higher quantitativeness by the sensing method according to the present invention and the sensing kit used therein.

<Design changes>
In the above description, the sandwich type sensing method has been described. However, the present invention is not limited to the above aspect. That is, as shown in FIG. 2A and FIG. 2B, a competitive sensing method may be used.

  In this case, in the sensing method, a first capture substance that specifically binds to the substance to be detected is fixed to a predetermined region on the transparent carrier to form a sensor unit (Step 2a), and a sample that can contain the substance to be detected is detected by the sensor. (Step 2b), after binding the substance to be detected to the first capture substance, the sensor part is washed (Step 2c), and then competes with the substance to be detected and specifically binds to the first capture substance. The label capture substance to be supplied is supplied onto the sensor part (Step 2d), the label capture substance is bonded to the first capture substance, and then the sensor part is washed (Step 2e), and a gap cover glass is placed on the sensor part. Then, an adjustment liquid for adjusting the refractive index is injected into the gap (Step 2f), and then the scattered light is measured.

<Second Embodiment of Sensing Method>
First, the sensing method according to the present embodiment will be described. This sensing method is a sandwich type sensing method. The sensing method according to the present embodiment has the same configuration as the sensing method according to the first embodiment, but has a refractive index of 1.35 or more and less than the refractive index of the transparent carrier chip, or a rubbery material Is used to adjust the refractive index on the surface of the transparent carrier. Therefore, the description of the same components as the sensing method according to the first embodiment is omitted unless particularly necessary.

  The adhesive substance or rubbery substance is for adjusting the refractive index on the surface of the transparent carrier. Since it is easy to use, a tape-like thing (adjustment tape) which consists of a support body and the adhesive substance layer or rubber-like substance layer formed on the support body is preferable. Here, from the viewpoint of light detection, the support is preferably transparent. However, it is not necessarily transparent. In this case, the side of the adjustment tape on which the adhesive substance layer or rubber substance layer is formed is brought into contact with the sensor portion, so that the refractive index of the adhesive substance or rubber substance can be refracted on the surface of the transparent carrier. Adjust the rate. The adhesive substance or rubbery substance preferably has a refractive index of 1.35 or more and less than the refractive index of the transparent carrier chip. Furthermore, the adhesive substance or the rubbery substance preferably has a refractive index of 1.38 or more and 1.51 or less, and more preferably 1.42 or more and 1.49 or less.

  The sensing method according to the present embodiment also detects scattered light by adjusting the refractive index on the surface including at least the sensor part of the transparent carrier to be 1.35 or more and less than the refractive index of the transparent carrier. That is, compared with the conventional method in which the sensor unit is usually air (refractive index 1.00) or water (refractive index 1.33), the present invention increases the refractive index on the sensor unit to perform sensing. Yes. Therefore, it is possible to obtain the same effect as in the first embodiment.

Sensing kit
<First Embodiment of Sensing Kit>
The sensing kit 60a of this embodiment is demonstrated. FIG. 5 is a schematic diagram showing the configuration of the sensing kit 60a. The sensing kit 60a includes a transparent carrier chip having a flow path, a sensor section, and a label capture substance dried and fixed on the upstream side of the sensor section, and an adjustment liquid for adjusting the refractive index on the surface of the flow path. ing. Here, the adjustment liquid is the same as that in the first embodiment of the sensing method. In FIG. 5, the adjustment liquid 63 is put in an ampoule 62 and sealed.

  The transparent carrier chip 50 includes a base 51 made of a dielectric plate, a spacer 53 that holds the liquid sample S on the base 51 and forms a flow path 52 for the liquid sample S, and an inlet 54a for injecting the sample S. And an upper plate 54 made of a glass plate provided with an air hole 54b serving as a discharge port for discharging the sample flowing down the channel 52, and the sample contact between the inlet 54a and the air hole 54b of the channel 52 Sensor portions 58 and 59 are provided on a predetermined region of the base 51 serving as a surface. Further, a membrane filter 55 is provided at a location from the inlet 54 a to the flow path 52, and a waste liquid reservoir 56 is formed at a portion connected to the air hole 54 b downstream of the flow path 52. A label capture substance is dried and fixed on the upstream side of the sensor unit.

In the sensing method of the present invention, FIG. 6A and FIG. 6B show the procedure for performing an assay by the sandwich method as to whether or not an antigen as a substance to be detected is contained in blood (whole blood) using the sensing kit 60a of the present embodiment. The description will be given with reference.
Step 6a: Blood (whole blood) So to be examined is injected from the injection port 54a. Here, the case where the antigen A which is a to-be-detected substance is contained in this blood So is demonstrated. In FIG. 6A, blood So is indicated by a shaded area.
step 6b: The blood So is filtered by the membrane filter 55, and large molecules such as red blood cells and white blood cells become residues.
Step 6c: Plasma S (blood separated by the membrane filter 55) leaks into the flow path 52 by capillary action. Alternatively, in order to accelerate the reaction and shorten the detection time, a pump may be connected to the air hole 54b, and the plasma S may be caused to flow down by pump suction and push-out operations. In FIG. 6A, plasma S is indicated by a hatched area.
Step6d: scatterers plasma S and antibody B ₂ exuded into the flow path 52 is attached (labeled antibody BF, namely labeling capture substance) and are mixed, the antigen A in plasma S bind with the labeled antibody BF.
Step 6e: The plasma S gradually flows along the flow path 52 toward the air hole 54b, and the antigen A bound to the labeled antibody BF binds to the immobilized antibody B ₁ immobilized on the first measurement area 58. and so-called sandwich antigen A is sandwiched with immobilized antibody B ₁ and the labeled antibody BF is formed.
step 6f: A part of the labeled antibody BF that has not bound to the antigen A binds to the immobilized antibody B ₀ immobilized on the second measurement area 59. Even if further labeled antibody BF that did not bind with the antigens A or immobilized antibody B ₀ remains on the measurement area, a subsequent plasma S is responsible for washing, floating and non-specific adsorption on the plate Wash away the labeled antibody BF.
Step 6g: An adjustment liquid for adjusting the refractive index on the channel surface is injected from the injection port 54a.
Step 6h: The adjustment liquid oozes out to the flow path 52 by capillary action. Similarly to the above, in order to shorten the detection time, a pump may be connected to the air hole 54b, and the adjustment liquid may be caused to flow down by pump suction and push-out operations.
Step 6i: When the adjustment liquid almost covers the sensor unit, the replacement of the refractive index of the flow path surface is completed.

  Thereafter, the scattered light measurement is performed using a detection apparatus as shown in FIG. This detection apparatus includes an accommodation unit 19 that accommodates a transparent carrier chip, an excitation light irradiation optical system 20 that irradiates the sensor unit 14 with the measurement light Lo, and an amount corresponding to the detected substance A that is generated by the irradiation of the measurement light Lo. And a light detection means 30 for detecting the light.

  The excitation light irradiation optical system 20 includes a light source 21 made of a semiconductor laser (LD) or the like that outputs measurement light Lo, and a prism 22 that is arranged so that one surface thereof is in contact with the transparent carrier chip 10. The prism 22 guides the measurement light Lo into the transparent carrier chip 10 so that the measurement light Lo is totally reflected at the interface of the transparent carrier 10. The prism 22 and the transparent carrier chip 10 are in contact with each other through a refractive index matching oil. The light source 21 is arranged so that the measurement light Lo is incident on the sample contact surface of the sensor chip 10 from the other surface of the prism 22 at a total reflection angle or more. Furthermore, you may arrange | position a light guide member between the light source 21 and the prism 22 as needed.

  The accommodating portion 19 is configured such that when the transparent carrier 10 is accommodated, the sensor portion 14 of the transparent carrier is disposed on the prism 22 and the scattered light can be detected by the photodetector 30. The transparent carrier 10 can be taken in and out in the direction of the arrow X in the figure with respect to the housing part 19.

  Then, it is possible to detect the amount of the substance to be detected by irradiating the predetermined area with the measurement light to generate the evanescent light D and detecting the scattered light of the evanescent light by the scatterer in the labeled antibody. It becomes possible.

Thus, by injecting the blood So from the inlet 54a, after Step6i from step6a to sandwich antigen A on the first measurement area 58 is sandwiched by the immobilized antibody _{B 1} and the labeled antibody BF is formed By detecting a scattered light signal (hereinafter referred to as a detection signal) from the first measurement area 58, it is possible to detect the presence and / or concentration of the antigen. Thereafter, the sample cell 50 is moved in the X direction so that the detection signal from the second measurement area 59 can be detected, and the detection signal from the second measurement area 59 is detected. Detection signal from the second measurement area 59 that the immobilized antibody B ₀ which binds the labeled antibody BF fixed is believed falling amounts of labeled antibodies BF, the activity is a signal that reflects the reaction conditions, such as By using this detection signal as a reference and correcting the detection signal from the first measurement area 58, a more accurate detection result can be obtained. Further, even when a known amount of a labeling substance (light scattering substance, metal fine particles) is fixed in advance in the second measurement area 59, similarly, the first optical signal from the second measurement area 59 is used as a reference. The detection signal from the measurement area 58 can be corrected.

  The sensing kit according to the present invention detects scattered light by adjusting the refractive index on the surface including at least the sensor part of the flow path to be 1.35 or more and less than the refractive index of the transparent carrier. That is, compared with the conventional method in which the sensor unit is usually air (refractive index 1.00) or water (refractive index 1.33), the present invention increases the refractive index on the sensor unit to perform sensing. Yes. Therefore, the S / N ratio can be improved as compared with the conventional method by making the refractive index on the surface of the transparent carrier larger than that of air or water. Thereby, it is possible to perform sensing with higher quantitativeness by the sensing method according to the present invention and the sensing kit used therein.

<Second Embodiment of Sensing Kit>
The sensing kit 60b of this embodiment will be described. FIG. 8A is a schematic diagram showing the configuration of the sensing kit 60b. The sensing kit 60b includes a transparent carrier having a sensor part, an adjustment liquid for adjusting the refractive index on the surface of the transparent carrier, and a labeling liquid containing a label capture substance. Here, the adjustment liquid is the same as that in the first embodiment of the sensing method. In addition, the label capture substance is composed of a second capture substance and a scatterer in which the second capture substance is modified. Further, in FIG. 8A, the adjustment liquid 63 and the labeling liquid 65 are sealed in the ampoules 62 and 64, respectively.

  Sensing similar to the sensing method described in <First embodiment of sensing method> can be performed by the sensing kit according to the present embodiment.

  The sensing kit according to the present invention also detects scattered light by adjusting the refractive index on the surface including at least the sensor portion of the transparent carrier to be 1.35 or more and less than the refractive index of the transparent carrier. That is, compared with the conventional method in which the sensor unit is usually air (refractive index 1.00) or water (refractive index 1.33), the present invention increases the refractive index on the sensor unit to perform sensing. Yes. Therefore, the same effects as those of the first embodiment of the sensing kit can be obtained.

<Third embodiment of sensing kit>
The sensing kit 60c of this embodiment will be described. FIG. 8B is a schematic diagram showing the configuration of the sensing kit 60c. The sensing kit 60c includes a transparent carrier having a sensor part, an adjustment tape 66 for adjusting the refractive index on the surface of the transparent carrier, and a labeling liquid 65 containing a label capturing substance. Here, the adjustment tape 66 is the same as that of the second embodiment of the sensing method. In addition, the label capture substance is composed of a second capture substance and a scatterer in which the second capture substance is modified. Further, in FIG. 8B, the labeling liquid 65 is put in an ampoule 64 and sealed.

  Sensing similar to the sensing method described in <Second Embodiment of Sensing Method> can be performed by the sensing kit according to the present embodiment.

  The sensing kit according to the present invention also detects scattered light by adjusting the refractive index on the surface including at least the sensor portion of the transparent carrier to be 1.35 or more and less than the refractive index of the transparent carrier. That is, compared with the conventional method in which the sensor unit is usually air (refractive index 1.00) or water (refractive index 1.33), the present invention increases the refractive index on the sensor unit to perform sensing. Yes. Therefore, the same effects as those of the first embodiment of the sensing kit can be obtained.

  An embodiment of the sensing method according to the present invention is shown below.

<Example 1>
Example 1 was performed by the following steps. A well was prepared by placing a PDMS plate with a hole of φ5 mm on a MAS glass slide (manufactured by Matsunami Glass Industry), and washed twice with PBS. Then, 8 ul of 100 nM anti-hCG antibody in PBS was dispensed, allowed to stand at room temperature for 1 hour, and then washed 3 times with PBS-T. Next, 20 ul of a 1% casein TBS solution was dispensed, allowed to stand at room temperature for 1 hour, and then washed three times with PBS-T. 20 ul of 1 nM or 10 nM hCG antigen PBS solution or PBS alone was dispensed and allowed to stand at room temperature for 1 hour, followed by washing 4 times with PBS-T. Then, 10 ul of anti-hCG antibody-immobilized gold colloid suspension (suspension with a particle size of 50 nm in water, 520 nm absorbance of 3.46 diluted 10-fold with PBS 2% PBS solution) was dispensed at room temperature. Let stand for 1 hour. The plate was washed 4 times with PBS-T and once with pure water to completely remove the solution. Then, the PDMS plate forming the well was peeled off, and a gap cover glass was placed thereon, and isopropyl alcohol (IPA) was injected into the gap. Finally, measurement light was irradiated from the side surface of the glass slide so as to propagate through the slide with total reflection, and the scattered light intensity in the sensor part on which the antibody was immobilized was detected with a cooled CCD. In this embodiment, the refractive index on the transparent carrier surface is adjusted to 1.377.

<Example 2>
The same process as in Example 1 was performed except that DMF was used instead of IPA. In this embodiment, the refractive index on the transparent carrier surface is adjusted to 1.427.

<Example 3>
The same process as in Example 1 was performed except that DMSO was used instead of IPA. In this embodiment, the refractive index on the transparent carrier surface is adjusted to 1.478.

<Example 4>
The same process as in Example 1 was performed except that matching oil was used instead of IPA. In this embodiment, the refractive index on the transparent carrier surface is adjusted to 1.510.

<Comparative Example 1>
The same process as in Example 1 was performed except that the sensor part was dried without applying IPA. In this example, the refractive index on the transparent carrier surface is 1.000.

<Comparative example 2>
It implemented by the process similar to the process of the said Example 1 except using a pure water instead of IPA. In this example, the refractive index on the transparent carrier surface is 1.333.

  FIG. 9 shows implementation data for <Example 1> to <Example 4> and <Comparative Example 1> and <Comparative Example 2>.

  FIG. 9A is a graph showing the intensity of scattered light from the sensor unit in each of the examples and the comparative examples. Here, the vertical axis is normalized and displayed based on the case where the surface of the transparent carrier is air (refractive index: 1.000). From this graph, it can be seen that as the refractive index on the surface increases, the intensity of scattered light from the sensor section also increases.

  FIG. 9B is a graph showing (noise-like) scattered light intensity from the non-sensor part in each of the examples and the comparative examples. Here, the vertical axis is normalized and displayed based on the case where the surface of the transparent carrier is air (refractive index: 1.000). From this graph, it can be seen that as the refractive index on the surface increases, the noisy scattered light intensity first decreases and then increases. As described above, this phenomenon is a surprising phenomenon because the noise-like scattered light intensity is considered to increase as the refractive index of the transparent carrier and the refractive index on the surface approach each other. This is considered to be due to the fact that impurities on the surface are not recognized by the evanescent light due to an increase in the refractive index on the surface of the transparent carrier. That is, it is considered that a trade-off relationship is caused between the increase in noise accompanying the increase in the intensity of the evanescent light and the non-recognition of impurities and the like accompanying the increase in the refractive index.

  FIG. 9C is a graph showing the S / N ratio in each example and comparative example. Here, the vertical axis is normalized and displayed based on the case where the surface of the transparent carrier is air (refractive index: 1.000). From this graph, it can be seen that as the refractive index on the surface increases, the S / N ratio first increases and then decreases. In particular, when DMF (refractive index: 1.427) is used as the adjustment liquid, the S / N ratio is improved by 12 times or more. That is, it can be said that measurement can be performed with a very high S / N ratio within a certain range of the refractive index on the transparent carrier surface.

  Next, an example in which an adjustment tape for adjusting the refractive index is used and an adjustment liquid is applied over a wide area will be described.

<Example 5>
It implemented by the process similar to the process of the said Example 1 except sticking an adhesive tape (acrylic resin adhesive tape) on a sensor part instead of inject | pouring IPA. In this embodiment, the refractive index on the transparent carrier surface is adjusted to about 1.4 to 1.5. Also in this embodiment, the result that the S / N ratio was improved by 9 times was obtained. From this, it was shown that the refractive index can be adjusted with an adhesive substance or a rubber substance in addition to the adjustment liquid.

<Example 6>
The same process as in Example 2 was performed, except that the gap cover glass was placed on a transparent carrier and the region where DMF was injected was expanded to the non-sensor part on the light irradiation side. In this embodiment, the refractive index on the transparent carrier surface is adjusted to 1.427, and the range of the adjustment is wide. In this embodiment, the result that the scattered light from the sensor part which is a detection object decreases a little compared with the said Example 2 was obtained. As described above, this is presumably because light energy is lost while the measurement light is guided to the sensor unit. However, the S / N ratio was improved by nearly 12 times. Therefore, it was shown that the present invention has a remarkable effect that the S / N ratio can be sufficiently improved even if the utilization efficiency of light energy is slightly inferior.

  Furthermore, the following Examples and Comparative Examples using CRP antigen and its antibody are shown.

<Example 7>
Example 7 was performed by the following steps. A well was prepared by placing a PDMS plate with a hole of φ5 mm on a MAS glass slide (manufactured by Matsunami Glass Industry), and washed twice with PBS. Then, 8 ul of 100 nM anti-CRP antibody in PBS was dispensed, allowed to stand at room temperature for 1 hour, and then washed 3 times with PBS-T. Next, 20 ul of a 1% casein TBS solution was dispensed, allowed to stand at room temperature for 1 hour, and then washed three times with PBS-T. 20 ul of 0, 9, 300, 9000 pM CRP antigen PBS solution or PBS alone was dispensed and allowed to stand at room temperature for 1 hour, followed by washing 4 times with PBS-T. Next, 10 ul of anti-CRP antibody-immobilized gold colloid suspension (suspension with absorbance at 520 nm of 3.46 diluted 10-fold with PBS 2% PBS solution) was dispensed and allowed to stand at room temperature for 1 hour. . The plate was washed 4 times with PBS-T and once with pure water to completely remove the solution. Then, the PDMS plate forming the well was peeled off, and a gap cover glass was placed thereon, and DMF was injected into the gap. Finally, measurement light was irradiated from the side surface of the glass slide so as to propagate through the slide with total reflection, and the scattered light intensity in the sensor part on which the antibody was immobilized was detected with a cooled CCD. In this embodiment, the refractive index on the transparent carrier surface is adjusted to 1.427. That is, the same process as in Example 2 was performed except that the CRP antigen and its antibody were used and the concentration was set to 0, 9, 300, 9000 pM.

<Comparative Example 3>
The same process as in Example 7 was performed except that the sensor part was dried without applying DMF. In this example, the refractive index on the transparent carrier surface is 1.000.

  Implementation data for the above <Example 7> and <Comparative Example 3> are shown in FIGS.

  10A and 10B show the change in scattering intensity with respect to the change in CRP antigen concentration for Example 7 and Comparative Example 3, respectively. On the other hand, FIGS. 11A to 11C show measurement image images of Example 7, measurement image images of Comparative Example 3, and density information of each sensor unit in the image images, respectively. From these, it was shown that the sensing method according to the present invention is a method having a very remarkable effect of significantly improving the S / N ratio by reducing noise and increasing the detection target signal.

2 Target substance 6 Dielectric prism substrate 9 Excitation light 10 Transparent carrier 11 Well 12 Gap cover glass 14 Sensor part 19 Storage part 20 Excitation light irradiation optical system 21 Light source 22 Prism 30 Photodetector 60a Sensing kit 60b Sensing kit 60c Sensing kit 63 Adjustment liquid 65 Label liquid 66 Adjustment tape A Target substance BF Label capture substance D Evanescent light F Scattering body Lo Measurement light

Claims (15)

A sensing method for detecting the amount of a substance to be detected,
Supplying a sample that can contain the substance to be detected to a sensor unit on the surface of the transparent carrier, to which a first capture substance for forming a labeled complex containing a scatterer is fixed,
Forming the labeled complex in an amount corresponding to the amount of the substance to be detected in the sensor unit;
Adjusting the refractive index on the surface including at least the sensor part of the transparent carrier to be 1.35 or more and less than the refractive index of the transparent carrier;
Irradiating the transparent carrier with measuring light to generate evanescent light in the sensor unit,
A sensing method comprising detecting scattered light of the evanescent light by the scatterer.   The sensing method according to claim 1, wherein the refractive index is adjusted to be 1.38 or more and 1.51 or less.   The sensing method according to claim 2, wherein the refractive index is adjusted to be 1.42 or more and 1.49 or less.   The sensing according to any one of claims 1 to 3, wherein the surface of the transparent carrier is filled with an adjustment liquid for adjusting the refractive index, and the refractive index of the surface is adjusted by the refractive index of the adjustment liquid. Method.   The sensing method according to claim 4, wherein the adjustment liquid is an organic solvent mixed with water or an aqueous solution of a solute having a high refractive index.   An adhesive substance or a rubbery substance for adjusting the refractive index is brought into contact with the surface of the transparent carrier, and the refractive index of the surface is adjusted by the refractive index of the adhesive substance or the rubbery substance. The sensing method according to claim 1. A second capture substance specifically binds to the substance to be detected;
By specifically binding a labeled capture substance composed of the second capture substance and the scatterer modified with the second capture substance to the detected substance bound to the first capture substance, The sensing method according to claim 1, wherein the label complex is formed. A third capture substance that specifically binds to the first capture substance in competition with the substance to be detected;
A labeled capture substance composed of the third capture substance and the scatterer modified with the third capture substance is specifically bound to the first capture substance to form the labeled complex. The sensing method according to claim 1, wherein:   The sensing method according to any one of claims 1 to 8, wherein the scatterer is a fine particle of metal, metal oxide, or metal nitride.   The sensing method according to claim 9, wherein a particle diameter of the fine particles is 30 nm or more and 300 nm or less. An inlet for injecting a sample, a channel for flowing the sample injected from the inlet, an air inlet for extracting air so that the sample flows, and a labeling complex including a scatterer A transparent carrier chip having a sensor part on the surface of the flow path to which a first capture substance to be formed is fixed, and a refractive index of 1. to adjust the refractive index on the surface of the flow path. Comprising an adjustment liquid that is 35 or more and less than the refractive index of the transparent carrier chip;
The sample that can contain the substance to be detected is supplied to the sensor unit, the labeled complex corresponding to the amount of the substance to be detected is formed in the sensor unit, and the transparent carrier chip is irradiated with measurement light. A sensing kit, which is used in a sensing method for generating evanescent light in the sensor unit, detecting the scattered light of the evanescent light by the scatterer, and detecting the amount of the substance to be detected. A transparent carrier chip having a sensor part to which a first capture substance for forming a labeling complex including a scatterer is fixed, and a refractive index of 1 for adjusting the refractive index on the surface of the transparent carrier chip An adjustment liquid that is 35 or more and less than the refractive index of the transparent carrier chip,
The sample that can contain the substance to be detected is supplied to the sensor unit, the labeled complex corresponding to the amount of the substance to be detected is formed in the sensor unit, and the transparent carrier chip is irradiated with measurement light. A sensing kit, which is used in a sensing method for generating evanescent light in the sensor unit, detecting the scattered light of the evanescent light by the scatterer, and detecting the amount of the substance to be detected. A transparent carrier chip having a sensor part to which a first capture substance for forming a labeling complex including a scatterer is fixed, and a refractive index of 1 for adjusting the refractive index on the surface of the transparent carrier chip An adhesive substance or a rubbery substance that is 35 or more and less than the refractive index of the transparent carrier chip,
The sample that can contain the substance to be detected is supplied to the sensor unit, the labeled complex corresponding to the amount of the substance to be detected is formed in the sensor unit, and the transparent carrier chip is irradiated with measurement light. A sensing kit, which is used in a sensing method for generating evanescent light in the sensor unit, detecting the scattered light of the evanescent light by the scatterer, and detecting the amount of the substance to be detected.   14. A label capture substance comprising a second capture substance that specifically binds to the substance to be detected and the scatterer modified with the second capture substance. Sensing kit according to.   A labeled capture substance comprising: a third capture substance that specifically binds to the first capture substance in competition with the substance to be detected; and the scatterer modified with the third capture substance. The sensing kit according to claim 11, wherein the sensing kit is characterized in that