JP2010008351A - Scattered light measuring method and scattered light measuring device used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily measuring scattered light with high sensitivity and a scattered light measuring device used for the same. <P>SOLUTION: A sample is irradiated with light from the light source and the light that irradiates the sample is transmitted through the sample. The arrival of the direct light, which is transmitted, to a light-receiver is intercepted to get scattered light received by the light-receiver. The interception of the direct light in this manner allows the scattered light to be measured easily with high sensitivity. This device includes the light source to emit light, the light receiver to receive light, a sample arranging section to arrange the sample, and an interception section to intercept light, in which the light receiver is arranged in the light-path direction of the irradiating light from the light source, while the sample arranging section is arranged between the light source and the light receiver, and the interception section is arranged between the sample arranging section and the light receiver and also on the way of the light-path of the direct light that transmits through the sample. Thus, the device allows the method to be easily conducted. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a scattered light measurement method, a scattered light measurement apparatus used for the method, and a sample holding tool.

  Conventionally, measurement of scattered light has been adopted in measurement of particle diameter and detection of a target substance. Precision is important for the measurement of scattered light. For example, a very strict structural design is required for a scattered light measurement apparatus. Specifically, for example, use of a light source capable of emitting coherent light, strict arrangement of optical lenses, strict position determination of a light receiving unit for receiving scattered light in a specific direction (angle), and the like are performed. Yes. In order to efficiently receive scattered light, for example, a method of installing a plurality of light receiving units at a plurality of angles or moving the light receiving unit to an area where scattered light of a required angle can be received in the apparatus. It has been taken.

  However, strict structural design is costly, and there is a problem that measurement accuracy is affected if strictness is lost. In addition, by strictly arranging the optical lens, the light receiving unit, and the like, there is a problem that, for example, the apparatus becomes large or the assembly is difficult. Furthermore, there is a possibility that the measurement accuracy is lowered by appropriately moving the light receiving unit.

In recent years, disposable small analysis tools such as a microchip and a micro total analysis system (μTAS) have attracted attention. Since these tools have fine channels and reaction parts, for example, reduction of samples, reduction of measurement time, reduction of waste, and the like are expected. However, on the other hand, since the flow path and the reaction part are fine, the scattered light generated is extremely small, and there is a problem that reliable measurement sensitivity cannot be obtained. For this reason, when these small analytical tools are used, detection is performed with direct light, particularly in place of scattered light whose signal is too small.
JP 2006-292410 A JP 2003-215139 A

  Therefore, an object of the present invention is to provide a method for measuring scattered light easily and with high sensitivity, and a scattered light measuring apparatus and a sample holding tool used therefor.

The scattered light measurement method of the present invention is a scattered light measurement method for measuring scattered light,
A light irradiation step of irradiating the sample with light from a light source;
A light receiving step of receiving light irradiated to the sample by a light receiving unit,
In the light receiving step, direct light transmitted through the sample in the irradiation light is blocked from reaching the light receiving unit, and the scattered light is received by the light receiving unit.

The scattered light measurement device of the present invention is a scattered light measurement device used in the scattered light measurement method of the present invention,
A light source that emits light, a light receiving unit that receives light, a sample placement unit that places a sample, and a light blocking unit that blocks light,
The light receiving unit is disposed in the optical path direction of the irradiation light from the light source,
The sample placement portion is placed between the light source and the light receiving portion;
The light-shielding portion is disposed between the sample placement portion and the light-receiving portion, and is disposed in the middle of the optical path of the direct light that passes through the sample.

The sample holding tool of the present invention is a sample holding tool used in the scattered light measurement method of the present invention,
A sample holding part and a light shielding part for blocking light;
The light-shielding part is arranged in the middle of an optical path of direct light that passes through the sample.

  According to the present invention, it is possible to easily and efficiently receive scattered light by blocking direct light transmitted through the sample. In particular, when a sample is placed in a fine area, such as microchips and microtuses that have attracted attention in recent years, the conventional method must use precise measurement and special equipment as described above. However, there was a problem that the scattered light could hardly be received and the required measurement sensitivity could not be obtained. However, according to the present invention, for example, even when a microchip or the like is used, it is possible to measure necessary scattered light only by blocking direct light transmitted through the sample. For this reason, it can be said that the method of the present invention, and the scattered light measuring device and the sample holding tool used therein are extremely useful in all fields where scattered light measurement is required.

<Method for measuring scattered light>
As described above, the scattered light measurement method of the present invention is a scattered light measurement method for measuring scattered light, and includes a light irradiation step of irradiating light on a sample from a light source, and irradiation light irradiated on the sample. A light receiving step for receiving light at the light receiving portion, wherein in the light receiving step, direct light transmitted through the sample in the irradiation light is blocked from reaching the light receiving portion, and the scattered light is received by the light receiving portion. It is characterized by.

  Scattered light generally means light whose path has been changed in a medium. However, in the present invention, the meaning of reflected light, refracted light, and the like is included in a broad sense.

  The present invention is characterized in that effective direct reception of scattered light is easily realized by blocking the direct light transmitted through the sample, and there is no limitation on how to block the direct light. Not. Examples of the blocking method include a method of reflecting or absorbing the direct light transmitted through the sample before the direct light reaches the light receiving unit.

  The kind of the sample to which the scattered light measurement method of the present invention is applied is not limited at all, and any sample can be applied as long as the measurement of scattered light is required. Further, the form of the sample is not limited at all, and examples thereof include liquids such as solutions, dispersions, and suspensions, gels, solids, gases, and aerosols. Specific examples are applicable to, for example, analysis and quantification of target components, measurement of particle diameter, and the like. The present invention is not limited to these.

  Such a scattered light measurement method of the present invention can be realized, for example, by using the scattered light measurement device of the present invention described below and the reagent holding tool of the present invention. The present invention is not limited to these.

<Scattered light measuring device>
As described above, the scattered light measurement device of the present invention is a scattered light measurement device used in the scattered light measurement method of the present invention, and includes a light source for irradiating light, a light receiving unit for receiving light, and a sample. A sample placement unit and a light blocking unit that blocks light, the light receiving unit is arranged in an optical path direction of the irradiation light from the light source, and the sample placement unit is disposed between the light source and the light receiving unit. The light-shielding portion is disposed between the sample placement portion and the light-receiving portion, and is disposed in the middle of an optical path of direct light that passes through the sample.

  According to the scattered light measurement device of the present invention, for example, as in a conventional scattered light measurement device, the light receiving unit is moved to a necessary angle to receive scattered light, or a plurality of light receiving units are set to a plurality of angles. There is no need to install. Moreover, according to the scattered light measuring apparatus of this invention, the position of a light source, a light-receiving part, and a sample arrangement | positioning part can be fixed, for example. Since the positional relationship can be stabilized in this way, for example, the reliability of measurement accuracy can be maintained.

  The light shielding part preferably absorbs light or reflects light, for example. Specifically, the light shielding part preferably includes a light absorbing material or a light reflecting material, and particularly preferably includes a light reflecting material. The light shielding part may include both a light absorbing material and a light reflecting material. The light absorbing material is not particularly limited, and examples thereof include black paint, black powder (inorganic powder) such as cupric oxide and cobalt oxide, carbon, black plastic, and black alumite. The light reflecting material is not particularly limited, and examples thereof include a mirror surface material, a mirror surface processing material, a white paint, and white powder such as titanium oxide and aluminum oxide. Examples of the mirror surface material and the mirror surface processing material include ceramics and metals. For example, the light shielding part as a whole may include the light absorbing material or the light reflecting material as described above, or the surface of the base material may be coated with the material as described above or may include the material as described above. It can also be formed by attaching (sealing) a film. The material of the base material into which the various materials are mixed in the former or the material of the base material to be coated with the various materials in the latter is not particularly limited, and examples thereof include permeable polymers such as acrylic. Further, in the cover member that covers the light receiving unit, the region that needs to be shielded from direct light may include the above-described material. In this case, the cover member is preferably made of a material that transmits light as described later, except for the light shielding portion. In addition, for example, on the surface of the cover member that covers the light receiving portion, a region as described above may be coated with a material as described above, or a film including the material as described above may be attached. The cover member is preferably made of, for example, a material that transmits the light.

  In particular, the light shielding part preferably reflects light. Thus, when light is reflected by the light-shielding part, the sample is irradiated with the light multiple times (twice or more) because the sample is irradiated with the reflected light. Thereby, the scattered light received by the light receiving unit can be further increased.

  The size and shape of the light-shielding part are not particularly limited as long as the direct light transmitted through the sample can be blocked. For example, the light-blocking part may have a size and shape that can similarly block scattered light at an unnecessary angle. .

  Moreover, the scattered light measurement apparatus of the present invention may further include, for example, a light blocking unit that blocks scattered light at an unnecessary angle that has passed through the sample, in addition to the light blocking unit that blocks direct light. As a result, for example, it becomes possible to select and receive only scattered light at a necessary angle.

  In the scattered light measurement apparatus of the present invention, for example, it is preferable that a sample holding tool having a sample holding portion is arranged in the sample arrangement portion at the time of measurement. In this case, for example, prior to the light irradiation step described above, a sample is supplied (arranged) to the sample holding part of the sample holding tool, and then placed in the sample arrangement part of the scattered light measurement device, so that the sample The sample in the holding tool is irradiated with light (emitted light) from the light source. Prior to the light irradiation step, the sample holding tool may be placed in the sample placement portion of the scattered light measurement apparatus, and then the sample may be supplied (placed) in the sample holding portion of the sample holding tool.

  As the sample holding tool, for example, a structure including a sample holding portion whose sectional shape is a concave portion can be exemplified. Further, for example, a structure including a flow path may be used. As a specific example, a structure having a sample introduction port, a flow path, and a sample holding section, and the introduction port and the sample holding section are connected by the flow path can be exemplified. Further, for example, an inlet and a channel may be provided, the inlet and the channel may be connected, and a part of the channel may also serve as a sample holding unit. Such a sample holding tool is not particularly limited, and conventionally known tools such as a cell, a chip, a microchip, a microtube, and a bioreactor can be used.

  In the sample holding tool, the length of the sample holding portion in the optical path direction of the irradiation light to the sample is not particularly limited, but is, for example, in the range of 10 to 1000 μm, preferably 50 to 500 μm, and more preferably. Is 100 to 400 μm. This length can also be referred to as, for example, a cell length or an optical path length.

  In the scattered light measurement apparatus of the present invention, for example, a sample may be directly supplied (arranged) to the sample arrangement unit. In this case, examples of the sample placement portion include a channel and a recess for holding the sample formed in the scattered light measurement device. The shape and size of the sample placement portion are not particularly limited, but when a sample is placed in the sample placement portion, the length of the region occupied by the sample in the optical path direction of the irradiation light from the light source, that is, inside the apparatus The thickness of the filled sample in the optical path direction is, for example, in the range of 10 to 1000 μm, preferably 50 to 500 μm, and more preferably 100 to 400 μm.

  In the scattered light measurement apparatus of the present invention, the type of the light receiving unit is not particularly limited, and examples thereof include light receiving elements such as photodiodes, phototransistors, and photomultiplier tubes. The number of light receiving elements in the light receiving unit is not particularly limited, but is preferably a small number, particularly preferably one.

  In the scattered light measurement apparatus of the present invention, the type of light source is not particularly limited, and examples thereof include LED, LD, laser, mercury lamp, halogen lamp, xenon lamp, tungsten lamp and the like. The wavelength is not particularly limited, but is, for example, in the range of 260 to 1100 nm, preferably 310 to 650 nm, and more preferably 365 to 415 nm. The diameter of the light emitted from the light source can be adjusted by, for example, the stop of the emission unit. The said output part and a light source are connected by the optical fiber etc., for example.

  In the scattering measurement apparatus of the present invention, examples of the light source include a small-diameter light source (also referred to as “point light source”) and a linear light source. In the case of a light source with a small diameter, for example, since the scattered light in the circumferential direction can be received, the sensitivity can be improved. In the case of a line light source, for example, the sensitivity can be improved by using a slit structure.

  The scattered light measuring apparatus of the present invention further includes, for example, an amplifier that amplifies a voltage generated when the light receiving unit receives scattered light, an I / V circuit that changes a current change into a voltage change, and converts the voltage into a digital value. An AD converter or the like may be provided. In addition, the scattered light measurement device further converts a current value into light intensity (scattered light intensity), adjusts light emission of a light source, converts a digital value of current into light intensity (scattered light intensity), and the like. A CPU or the like may be provided.

  Hereinafter, specific examples of the scattered light measurement apparatus of the present invention will be described with reference to FIGS. These embodiments are examples of a scattered light measurement apparatus that uses a sample holding tool arranged in a sample arrangement portion. Note that the present invention is not limited to these embodiments.

(Embodiment 1)
Embodiment 1 is an example of a scattered light measurement apparatus including a small-diameter light source as a light source.

  FIG. 1 is a perspective view of a scattered light measurement device in which a sample holding tool is arranged, and FIG. 2 is a cross-sectional view in the AA direction of FIG. In addition, FIG. 1 is the schematic which separated and showed each structural member for convenience. As shown in both drawings, the scattered light measurement apparatus includes a light source 11, a light receiving unit 16, a first light shielding unit 14, and a second light shielding unit 13. In the present embodiment, the first light shield 14 is a member for blocking direct light that has passed through the sample, and the second light shield 13 is for blocking scattered light at an unnecessary angle that has passed through the sample. It is a member. The light receiving unit 16 is arranged at a position facing the light source 11, specifically, arranged in the optical path direction of the irradiation light X from the light source 11. A first light shielding part 14 and a second light shielding part 13 are disposed on the light source 11 side surface of the light receiving part 16. In use of the scatterometer, the sample holding tool 12 in which the sample 15 is held by the sample holding part is arranged on the light receiving part 16 via the first light shielding part 14 and the second light shielding part 13 in use. Is done. In the scattered light measurement device, a portion where the sample holding tool 12 is arranged is a so-called sample arrangement portion. The sample holding tool 12 includes a cover substrate 12a and a support substrate 12b. A concave groove is formed as the sample holding portion 15 on the surface of the support substrate 12b, and the cover substrate 12a is covered on the surface where the groove is formed. In this scattered light measurement apparatus, the first light shielding unit 14 is disposed in the optical path of the direct light X ′ transmitted through the sample 15 in the irradiation light X from the light source 11, and the second light shielding unit 13 is Of the irradiation light X from the light source 11, the light is transmitted through the sample 15 and is disposed in the middle of the optical path of the scattered light whose path is changed to an unnecessary angle.

  In the scattered light measurement device, the shape of the first light shielding unit 14 is not particularly limited, and can be determined as appropriate depending on, for example, the type of the light source. When the light source is a small-diameter light source as in the present embodiment, the planar shape of the light shielding portion 14 is preferably circular. In addition, the said plane means the surface where the direct light which permeate | transmitted the sample is irradiated. Moreover, in the scattered light measurement apparatus, it is preferable that the center of the first light shielding unit 14 is opposed to the center of the irradiation light from the small-diameter light source.

  The size of each part in the scattered light measurement device is not particularly limited, and can be appropriately determined depending on the size of the sample holding tool 12, the size of the sample holding part 15 in the sample holding tool 12, and the like. Further, when the scattered light measuring device of the present invention is manufactured using an existing light intensity measuring device such as a spectrophotometer, for example, the size of a light shielding portion or the like is adjusted in accordance with the size of each part of the spectrophotometer. Can be set. Hereinafter, the size of each part of the scattered light measurement device will be described on the assumption that the sample holding tool is a general microchip, but these descriptions are merely examples and do not limit the present invention.

  The size of the first light-shielding part 14 is not particularly limited. For example, the diameter of the irradiation light from the small-diameter light source 11, the position where the irradiation light is emitted from the small-diameter light source 11, and the sample surface on the light source side It can be appropriately determined in consideration of the distance, the thickness of the sample holding tool, and the like. As a specific example, for example, the ratio of the diameter of the irradiation light from the light source having a small diameter and the diameter of the flat surface of the light shielding part is preferably 1: 2 to 100, more preferably 1:20 to 100. Yes, particularly preferably 1: 40-60. In addition, the diameter of irradiation light here means the diameter of irradiation light at the time of emission, for example.

  The first light-shielding part 14 only needs to be able to shield light, and its thickness is not limited at all. As shown in the figure, when a member that blocks light is disposed as the light shielding portion, the upper limit of the thickness is preferably, for example, 1 mm or less, particularly preferably 0.1 mm, and the lower limit of the thickness is, for example, , 0.05 mm (50 μm) or less is preferable. In the case of coating, sealing, etc., the upper limit of the thickness is preferably 1 mm or less, particularly preferably 0.1 mm, and the lower limit of the thickness is not particularly limited, but is 0.05 mm (50 μm, for example). )

  Although the diameter of the irradiation light from the light source having a small diameter (for example, the irradiation light at the time of emission) is not particularly limited, it is generally preferably 20 to 800 μm, more preferably 20 to 400 μm, and particularly preferably. 40-100 μm.

  The distance between the position where the irradiation light is emitted from the small-diameter light source 11 and the surface of the sample 15 on the light source side or the light source side surface of the sample holding tool 12 is not particularly limited, but is generally 0 to 10 mm. More preferably, it is 1-5 mm, Most preferably, it is 1-3 mm.

  The distance between the surface of the sample 15 on the light source side and the surface of the light receiving unit 16 is not particularly limited, but is generally preferably 1 to 50 mm, more preferably 1 to 20 mm, and particularly preferably 2 to 5 mm. is there.

  The size, shape, material and the like of the sample holding tool 12 are not particularly limited, but a general microchip can be exemplified. In the case of a general microchip, the size is, for example, an overall length of 20 to 200 mm, an overall width of 20 to 150 mm, and an overall thickness of 0.1 to 10 mm. The thickness of the sample holder 15, that is, the length (cell length) of the irradiation light from the light source 11 in the optical path direction is, for example, in the range of 10 to 1000 μm, and preferably 100 μm.

  The shape of the sample holding part 15 in the sample holding tool 12 is not particularly limited. For example, in the case of a small-diameter light source, the planar shape is preferably circular. The plane means, for example, a surface of the sample holder 15 that is irradiated with the irradiation light X from the light source 11. The plane diameter of the sample holding unit 15 is preferably, for example, 0.1 to 10 mm, more preferably 0.5 to 1.2 mm, and particularly preferably 0.7 to 0.8 mm. Moreover, it is preferable that the volume of the sample hold | maintained at the sample holding part 15 is 1-1000 microliters, for example.

  The material of the sample holding tool 12 is not particularly limited, but generally, a light transmissive material is preferable in order to reduce the background. Examples of the material include polymers such as polymethyl methacrylate (PMMA), polydimethylpolyoxane (PDMS), polyethylene terephthalate (PET), and PS (polystyrene), and glass. The specific shape of the sample holding tool is the same as, for example, the sample holding tool of the present invention described later. However, the sample holding tool used in the scattered light measurement apparatus of the present invention does not have to include a light shielding portion as described later.

  Using this scattered light measurement apparatus, for example, the scattered light measurement method of the present invention can be implemented as follows. First, as described above, after the sample 15 is supplied to the sample holding portion of the sample holding tool 12, it is arranged at a predetermined position. Then, the irradiation light X is emitted from the light source 11 to irradiate the sample 15 of the sample holding tool 12. Then, the direct light X ′ transmitted through the sample 15 among the irradiation light X irradiated to the sample 15 is blocked by the first light shielding unit 14, and is prevented from reaching the light receiving unit 16 positioned below. Of the irradiation light X irradiated to the sample 15, scattered light that has passed through the sample 15 and changed its path to an unnecessary angle is blocked by the second light-shielding unit 13, and the light-receiving unit located below Access to 16 is prevented (not shown). On the other hand, among the scattered light generated by contact with the sample 15, the scattered light that has passed through the sample 15 and reached between the first light-shielding part 14 and the second light-shielding part 13 is positioned below. Light is received by the light receiving unit 16. In this way, the necessary scattered light can be received more efficiently by blocking the direct light and further blocking unnecessary scattered light. In addition, the 2nd light-shielding part 13 is arbitrary and does not limit this invention.

  Moreover, it is preferable that especially the 1st light-shielding part 14 reflects light among light-shielding parts. In this case, in FIG. 2, when the direct light X ′ transmitted through the sample 15 reaches the first light-shielding portion 14, the path is changed in the direction of the sample 15 by reflection. By irradiating the sample 15 with the reflected light, further scattered light is generated, and therefore the amount of generated scattered light can be further increased. For this reason, the measurement sensitivity can be further improved. Moreover, when the 1st light-shielding part 14 reflects light, you may provide a light-receiving part further in the side which irradiates irradiation light. By providing the light receiving portion in this manner, for example, direct light that has passed through the sample 15 again can be received out of the direct light X ′ that has passed through the sample 15. Thereby, only the target scattered light is received by the light receiving unit 16, and the direct light (so-called transmitted light) transmitted through the sample by the further light receiving unit can also be received.

  In addition, although this embodiment is a form which arrange | positions the members 13 and 14 which consist only of a light-shielding part on the light-receiving part 16, it is not restrict | limited to this. For example, a cover having a light shielding portion as described above may be disposed on the light receiving portion 14. This can be produced, for example, by preparing a cover member that transmits light and sealing a film that blocks light in an area that needs to be blocked or coating a material that blocks light.

(Embodiment 2)
The second embodiment is an example of a scattered light measurement device including a line light source as a light source.

  FIG. 3 is a perspective view of the scattered light measurement device in which the sample holding tool is arranged, and is a schematic view showing the constituent members separated for convenience. Unless otherwise indicated, it is the same as the first embodiment.

  In the scattered light measurement apparatus according to the present embodiment, as shown in FIG. 3, the light source 11 is a linear light source, and the planar shape of the first light shielding part 34 and the shape of the inner frame of the second light shielding part 33 are each square. There is the same as the first embodiment except that the planar shape of the sample holding part 35 in the sample holding tool 12 is a quadrangle. In FIG. 3, the arrow Y is referred to as a longitudinal direction, and the arrow Z is referred to as a direction perpendicular to the longitudinal direction.

  In the scattered light measurement apparatus, the shape of the first light shielding part 34 is not particularly limited, but when the light source is a linear light source as in the present embodiment, the planar shape of the light shielding part is preferably a quadrangle. In addition, the said plane means the surface where the direct light which permeate | transmitted the sample is irradiated. In the scattered light measurement apparatus, it is preferable that the first light shielding unit 34 has a longitudinal direction (Y direction) facing a longitudinal direction (Y direction) of irradiation light from the line light source.

  The size of the first light shielding part 34 is not particularly limited. For example, the length of the irradiation light from the linear light source in the longitudinal direction (Y direction) and the length of the light shielding part in the longitudinal direction (Y direction) The ratio is preferably 1: 1 to 100, more preferably 1: 1 to 10, and particularly preferably 1: 1 to 3. The ratio of the length in the vertical direction (Z direction) to the longitudinal direction of the irradiation light and the length in the vertical direction (Z direction) of the light shielding part is preferably 1: 1 to 100, for example. More preferably, it is 1: 1-10, Most preferably, it is 1: 2-5. In the present invention, the length in the longitudinal direction (Y direction) of the irradiation light from the line light source means, for example, the length at the time of emission.

  The length of light irradiated from the line light source at the time of emission (length in the Y direction) is not particularly limited, but is generally preferably 10 to 3000 μm, more preferably 100 to 2000 μm, and particularly preferably. Is 800-1500 μm.

  The shape of the sample holding part 35 in the sample holding tool 12 is not particularly limited. For example, in the case of a line light source, the planar shape is preferably a square. The plane means, for example, a surface on the side of the sample holder 35 where the irradiation light X from the light source 11 is irradiated. In the sample holder 35, the length in the longitudinal direction (Y direction) is preferably, for example, 10 to 150 mm, more preferably 20 to 100 mm, and particularly preferably 50 to 80 mm. In the sample holder 35, the length in the direction perpendicular to the longitudinal direction (Z direction) is, for example, preferably 10 to 150 mm, more preferably 20 to 100 mm, and particularly preferably 50 to 80 mm. It is.

<Sample holding tool>
As described above, the sample holding tool of the present invention is a sample holding tool used in the scattered light measurement method of the present invention, and includes a sample holding part and a light shielding part that blocks light, and directly transmits the sample. It arrange | positions in the optical path of light, It is characterized by the above-mentioned.

  According to the sample holding tool of the present invention, for example, as in a conventional scattered light measurement device, in order to receive scattered light, the light receiving unit is moved to a necessary angle, or a plurality of light receiving units are installed at a plurality of angles. There is no need to do. Further, according to the sample holding tool of the present invention, for example, the positions of the light source, the light receiving unit, and the sample placement unit in the scattered light measurement device can be fixed. Since the positional relationship can be stabilized in this way, for example, the reliability of measurement accuracy can be further improved. Moreover, since it is sufficient to provide the sample holding tool with a light-shielding portion, it can be applied to, for example, an existing light intensity measuring device.

  The light shielding part in the sample holding tool of the present invention is not particularly limited as long as it is disposed in the middle of the optical path of the direct light passing through the sample. Moreover, the said light-shielding part is the same as the light-shielding part in the scattered light measuring apparatus of this invention mentioned above except having arrange | positioned at the sample holding tool. Moreover, the sample holding tool of this invention is the same as that of the sample holding tool used for the scattered light measuring apparatus of this invention mentioned above, for example except having a light-shielding part.

  The sample holding tool of the present invention only needs to include the light shielding portion as described above, and can be used by being disposed in a light intensity measuring device such as an existing spectrophotometer or scattered light measuring device. Further, as the apparatus, for example, the scattered light measurement apparatus of the present invention can be used, and in this case, the scattered light measurement apparatus does not need to include the above-described light shielding unit.

  In the sample holding device of the present invention, for example, a desired reagent may be further arranged in the sample holding portion or a flow path connecting the sample inlet and the sample holding portion as will be described later. When detecting or quantifying target components in a sample by receiving scattered light, for example, various reagents such as antigens, antibodies, enzymes, and chromogenic substrates bound with antigens, antibodies, latex, enzymes, etc., and target components in a sample And the reaction product is irradiated with light to measure scattered light. Therefore, for example, depending on the type of the target component, a necessary sample is appropriately arranged to perform a reaction necessary for detection in the sample holding tool, and the same sample holding tool can be used to detect scattered light. Detection can also be performed.

  Hereinafter, specific examples of the sample holding tool of the present invention will be described with reference to FIGS. Note that the present invention is not limited to these embodiments.

(Embodiment 3)
The third embodiment is an example of a sample holding tool in which the planar shape of the sample holding unit is a circle. In addition, the sample holding tool of this embodiment is the same as the sample holding tool used for the scattered light measuring apparatus of this invention mentioned above except having a light-shielding part.

  FIG. 4 is a perspective view of the sample holding tool 40, (a) is a schematic view showing the components separated for convenience, and (b) is a perspective view from the upper surface side of the sample holding tool 40. (C) is a perspective view from the back side of the sample holding tool 40. As shown in FIG. 4, the sample holding tool 40 includes a support substrate 42 and a cover substrate 41. On the surface of the support substrate 42, a channel 47 and a sample holder 45 connected to the channel 47 are formed as concave grooves. The cover substrate 41 is coated on the surface of the support substrate 42 on which the concave grooves are formed, whereby the end (the right end in the figure) of the flow path 47 becomes the sample inlet 46. Then, as shown in FIG. 3C, a first light shielding portion 44 and a second light shielding portion 43 are formed on the back surface of the sample holding tool 40. The first light-shielding portion 44 is formed in the middle of the optical path of direct light that passes through the sample when the sample held on the sample holding portion 45 is irradiated with irradiation light from the surface of the cover substrate 41 in the sample holding tool 40. . Further, the second light-shielding portion 43 transmits the sample when irradiated from the surface of the cover substrate 41 of the sample holding tool 40 to the sample held by the sample holding portion 45, and travels to an unnecessary angle. Is formed in the middle of the optical path of the scattered light. In addition, the 1st light-shielding part 44 in the sample holding tool of this embodiment is the same as that of the 1st light-shielding part in the scattering measuring apparatus of Embodiment 1 unless otherwise indicated. Moreover, the 2nd light-shielding part 43 is arbitrary and does not limit this invention.

  The type of irradiation light with which the sample holding tool 40 of the present embodiment is irradiated is not particularly limited. However, since the planar shape of the sample holding portion 45 that holds the sample is circular, for example, light emitted from a small-diameter light source Is preferably irradiated. The size of the sample holder, the condition of the irradiation light from the light source having a small diameter, etc. are not particularly limited and are the same as described above.

  Using the sample holding tool 40, for example, the scattered light measurement method of the present invention can be implemented as follows. First, a sample is supplied to the sample holding part 45 of the sample holding tool 40. Then, the sample holding tool 40 holding the sample is disposed in a device capable of receiving light and measuring its intensity, such as an existing spectrophotometer or scattered light measuring device. The configuration of such an apparatus is not particularly limited. For example, the apparatus includes a light source that emits light, a light receiving unit that receives light, and a sample placement unit that places a sample. And an apparatus in which the sample placement portion is placed between the light source and the light receiving portion. A specific configuration is, for example, the same as that of the scattered light measurement device of the present invention. However, in this embodiment, the light shielding unit may not be arranged in the scattered light measurement device. The sample holding tool 40 is placed in the sample placement portion of such an apparatus, and the sample of the sample holding portion 45 is irradiated from the surface of the cover substrate 41 of the sample holding tool 40 with irradiation light from the light source. Then, the direct light which permeate | transmits a sample among the irradiation lights to a sample is interrupted | blocked by the 1st light-shielding part 44. FIG. In addition, of the light irradiated to the sample, scattered light that has passed through the sample and has changed its course to an unnecessary angle is blocked by the second light shielding unit 43. On the other hand, of the scattered light generated by the contact with the sample, the scattered light that has passed through the sample and reached the region between the first light shielding part 44 and the second light shielding part 43 is the sample holding tool 40. Is received by a light receiving portion located below the sample holding tool 40. In this way, necessary scattered light can be received efficiently by blocking direct light and further blocking unnecessary scattered light. In addition, the 2nd light-shielding part 43 is arbitrary and does not limit this invention.

  Note that the method of supplying the sample to the inside of the sample holding tool 40 is not limited at all, and the sample holding tool 40 may be provided with a further part, for example, in order to introduce the sample. For example, the cover substrate 41 may have an air hole above the sample holding unit 45. According to such a form, the sample can be introduced into the inside from the sample inlet 46 by, for example, capillary action. Further, the sample can be introduced into the sample holding tool 40 by pressurization using a syringe or the like. Further, the sample holding unit is further connected to another channel, and the sample is introduced into the inside from the inlet 46 by reducing the pressure inside the sample holding tool 40 from the end opening of the channel. You can also Further, the sample can be introduced into the interior from the introduction port 46 by sucking from the end opening of the flow path using, for example, a pump or a syringe.

(Embodiment 4)
The fourth embodiment is an example of a sample holding tool in which the flow path also serves as a sample holding unit. In addition, the sample holding tool of this embodiment is the same as the sample holding tool used for the scattered light measuring apparatus of this invention mentioned above except having a light-shielding part.

  5A and 5B are perspective views of the sample holding tool 50. FIG. 5A is a schematic view showing the components separated from each other for convenience. FIG. 5B is a perspective view of the sample holding tool 50 from the upper surface side. (C) is a perspective view from the back side of the sample holding tool 50. As shown in FIG. 5, in the sample holding tool 50 of the present embodiment, a part of the channel 57 also serves as the sample holding unit 55 that receives light irradiation. The inner frame of the light shielding portion 53 is the same as that of the third embodiment except that the shape of the inner frame is a quadrangle. Specifically, on the surface of the support substrate 42, the channel 57 and the waste liquid part 51 connected to the channel 57 are formed as concave grooves. Further, as shown in FIG. 5C, a first light shielding part 54 and a second light shielding part 53 are formed on the back surface of the sample holding tool 50. As in the third embodiment, the first light shielding part 54 is formed in the middle of the optical path of the direct light that passes through the sample, and the second light shielding part 53 passes through the sample and travels to an unnecessary angle. Is formed in the middle of the optical path of the scattered light. In addition, the 1st light-shielding part 54 in the sample holding tool 50 of this embodiment is the same as that of the 1st light-shielding part in the scattering measurement apparatus of Embodiment 2, for example unless otherwise indicated. Moreover, the 2nd light-shielding part 53 is arbitrary and does not limit this invention.

  The type of light irradiated to the sample holding tool 50 of the present embodiment is not particularly limited. However, since the planar shape of the flow path 57 (sample holder 55 irradiated with light) is a quadrangle, for example, It is preferable to irradiate light emitted from a linear light source. Note that the size of the sample holder 55, the condition of the irradiation light from the line light source, and the like are not particularly limited and are the same as described above.

  The sample holding tool of this embodiment can also be used in the same manner as the sample holding tool of Embodiment 3, for example. Further, for example, the surplus of the sample introduced into the sample holding tool 50 may be sent to the waste liquid unit 51.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

  The scattered light intensity was measured for the antigen-antibody reaction solution of the antigen CRP and the antibody using the scattered light measuring apparatus of the present invention provided with a light-shielding part for directly blocking light.

(Preparation of avidin-biotinylated anti-CRP antibody)
First, biotin was bound to the anti-CRP antibody. 494.2 μl of a 10 mM biotin aqueous solution (solvent: water) was added to 3 ml of 12.2 mg / ml anti-CRP antibody (Oriental Yeast), and the mixture was allowed to stand at room temperature. NHS-LC-LC-biotin (PIERCE) was used as the biotin. The mixture was applied to a centrifugal desalting column Zeba (trademark) Desalt Spin Columns (PIERCE), and centrifuged at 1,000 × g for 2 minutes at 20 ° C. to remove unreacted biotin. Then, the solution containing the biotinylated anti-CRP antibody remaining in the column is collected, subjected to an ultrafiltration filter (fractional molecular weight 100 kDa), and centrifuged at 3,600 × rpm, 20 minutes, 20 ° C. The biotinylated anti-CRP antibody was concentrated. Here, the biotin labeling rate of the anti-CRP antibody was determined by the HABA substitution method. As a result, it was confirmed that one or more biotin molecules were bound per antibody molecule. Prior to the subsequent avidinization, the amount of protein was measured for the concentrated solution containing the biotinylated anti-CRP antibody. The amount of protein was measured by measuring the absorbance at 280 nm. Subsequently, avidin was further bound to the biotinylated anti-CRP antibody. 1.35 ml of a PBS solution containing 2 mg / ml avidin (Calbiochem) was added to the concentrated solution containing the biotinylated anti-CRP antibody, and the mixture was allowed to stand at room temperature for 5 minutes. Subsequently, the mixed solution is subjected to an ultrafiltration filter (fractional molecular weight: 100 kDa) and centrifuged at 3,600 × rpm for 20 minutes at 20 ° C. to remove unreacted avidin and avidin-biotinylated CRP antibody concentration was performed. Thus, an avidin-biotinylated anti-CRP antibody was obtained.

(Placement of dry reagent in microcell)
A liquid reagent having the following composition was prepared using the avidin-biotinylated anti-CRP antibody. 29 μl of this liquid reagent was supplied to the sample holder 65 of the sample holding tool 60 shown in FIG. 6 and dried by lyophilization. In this way, the dry reagent was placed on the sample holding tool 60. FIG. 6 is a perspective view showing an example of a sample holding tool, and is a schematic diagram showing the constituent members separated for convenience. As shown in the figure, the sample holding tool 60 includes a support substrate 63 and a cover substrate 61. On the surface of the support substrate 63, a sample introduction part 64, a sample holding part 65 in which a dry reagent is disposed, a waste liquid part 66, and a channel 67 connecting them are formed as concave grooves. The cover substrate 61 includes a sample introduction port 62 at a location corresponding to the sample introduction portion 64 of the support substrate 63, and is covered on the surface of the support substrate 63 where the groove is formed. In the sample holding tool 60, the planar shape of the sample holding portion 65, that is, the shape of the surface irradiated with light was circular. The details of the sample holding tool 60 are as follows.
Cover substrate Material: Silicone polyethylene terephthalate (PET)
Thickness: 150 μm
Support substrate Material: Acrylic resin Thickness: 5 mm (length from the bottom of the sample holder to the bottom of the support substrate)
Sample holder diameter: 1.3 mm
Depth: about 300μm

(Scattered light measuring device)
The scattered light measurement apparatus shown in FIG. 1 was assembled. The details of each part of the scattered light measurement device are as follows. In this embodiment, the sample holding tool 12 in FIG. 1 is the sample holding tool 60 in FIG.
Light source: LED (Nitride)
Wavelength: 365nm
Irradiation light diameter: 0.4 mm
Receiver: Photodiode (trade name S2386-44K, manufactured by Hamamatsu Photonics)
1st light shielding part Material: Acrylic containing 50% (w / v) light shielding agent carbon black Diameter: 0.8mm
Thickness: 10 μm
Second light-shielding part Material: acrylic containing 50% (w / v) of light-shielding agent carbon black Internal diameter: 25 mm
Thickness: 10 μm

(CRP concentration measurement method)
CRP (Capricon Products Inc.) is added to CRP-free serum collected from human whole blood (Oriental Yeast Co., Ltd.) to a predetermined concentration (0, 0.5, 1 mg / 100 ml) to prepare a sample did. Then, 20 μl of each sample is added from the introduction port 62 of the sample holding tool 60 described above, the sample is moved from the sample introduction unit 64 to the sample holding unit 65, incubated at 25 ° C. for 4 minutes, and then the scattered light measurement device. , The sample holder 65 was irradiated with light, and the scattered light intensity (365 nm) was measured. In the scattered light measurement apparatus shown in FIG. 1, the sample holding tool 60 is arranged such that light is irradiated to the sample holding unit 65 and the direct light transmitted through the sample is blocked by the first light blocking unit. On the other hand, as a comparative example, the scattered light intensity was measured in the same manner using a scattered light measurement apparatus having the same configuration except that no light shielding part was disposed on the light receiving part. Then, the optical density (OD) was calculated from the measured scattering intensity.

  These results are shown in FIG. The figure is a graph showing optical density at each CRP density. In the figure, ■ is the result of Example 1 measured with a scattered light measurement device provided with a light shielding part, and ◆ is the result of Comparative Example 1 measured with a scattered light measurement device without a light shielding part. .

  As shown in the figure, Comparative Example 1 (♦) without a light shielding part also received direct light, and thus hardly scattered light could be detected. On the other hand, Example 1 (■) provided with a light shielding part could detect scattered light with high sensitivity by direct light shielding.

  The scattered light intensity was measured for the diluted milk using a scattered light measuring device provided with a light-shielding part for blocking direct light.

  The scattered light was measured in the same manner using the same apparatus as in Example 1 except that the diluted milk was used as the specimen. In addition, the ratio (dilution density | concentration) of the milk in a dilution liquid was set to 0.0005, 0.001, 0.0015, 0.002, 0.0025 by setting undiluted milk as 1.

  These results are shown in FIG. The figure is a graph showing the light intensity at each milk dilution concentration. In the figure, ■ is the result of Example 2 measured with a scattered light measurement device provided with a light shielding part, and ◆ is the result of Comparative Example 2 measured with a scattered light measurement device without a light shielding part. .

  As shown in the figure, Comparative Example 2 (♦) without a light shielding part also received direct light, and thus hardly scattered light could be detected. On the other hand, Example 2 (■) provided with a light shielding part was able to detect scattered light with higher sensitivity than the comparative example by direct light shielding.

  From the above results, it was found that scattered light can be detected effectively by providing a blocking portion and blocking direct light that passes through the sample.

  As described above, according to the present invention, it is possible to easily receive scattered light with high sensitivity by blocking direct light transmitted through the sample. In particular, when a sample is placed in a fine area, such as microchips and microtuses that have attracted attention in recent years, the conventional method must use precise measurement and special equipment as described above. However, there was a problem that the scattered light could hardly be received and the required measurement sensitivity could not be obtained. However, according to the present invention, for example, even when a microchip or the like is used, it is possible to measure necessary scattered light only by blocking direct light transmitted through the sample. For this reason, it can be said that the method of the present invention, and the scattered light measuring apparatus and sample holding tool used therein are extremely useful in all fields where scattered light measurement is required.

FIG. 1 is a perspective view showing a scattered light measurement apparatus according to an embodiment of the present invention. FIG. 2 is a cross-sectional view of the scattered light measuring apparatus shown in FIG. FIG. 3 is a perspective view showing a scattered light measurement device according to another embodiment of the present invention. 4A and 4B are schematic views showing a sample holding tool according to another embodiment of the present invention, in which FIG. 4A is a perspective view with components separated, FIG. 4B is a perspective view from the upper surface side, and FIG. FIG. 3 is a perspective view from the back side. 5A and 5B are schematic views showing a sample holding tool according to another embodiment of the present invention, in which FIG. 5A is a perspective view showing components separated, FIG. 5B is a perspective view from the upper surface side, and FIG. FIG. 3 is a perspective view from the back side. FIG. 6 is a perspective view showing an outline of a sample holding tool in Embodiment 1 of the present invention. FIG. 7 is a graph showing the relationship between the scattered light intensity and the CRP concentration measured using a scattered light measurement device including a light shielding unit in Example 1 of the present invention. FIG. 8 is a graph showing the relationship between the scattered light intensity measured using a scattered light measurement device including a light shielding unit and the milk dilution concentration in Example 2 of the present invention.

Explanation of symbols

11: Light source 12, 40, 50, 60: Sample holding tool 13, 14, 33, 34, 43, 44, 53, 54: Light shielding unit 15, 35, 45, 55, 65: Sample, Sample holding unit 16: Light reception Parts 12a, 41, 61: cover substrates 12b, 42, 63: support substrate 46, 62: inlets 47, 57, 67: flow path 64: sample introduction part 51, 66: waste liquid part

Claims (3)

A scattered light measurement method for measuring scattered light,
A light irradiation step of irradiating the sample with light from a light source;
A light receiving step of receiving light irradiated to the sample by a light receiving unit,
In the light receiving step, the scattered light measurement method is characterized in that direct light transmitted through the sample in the irradiation light is blocked from reaching the light receiving unit, and the scattered light is received by the light receiving unit.   The scattered light measurement method according to claim 1, wherein direct light is blocked by at least one of absorption and reflection of the direct light. A scattered light measurement device used in the scattered light measurement method according to claim 1 or 2,
A light source that emits light, a light receiving unit that receives light, a sample placement unit that places a sample, and a light blocking unit that blocks light,
The light receiving unit is disposed in the optical path direction of the irradiation light from the light source,
The sample placement portion is placed between the light source and the light receiving portion;
The scattered light measurement apparatus, wherein the light-shielding portion is disposed between a sample placement portion and the light-receiving portion and in the middle of an optical path of direct light that passes through the sample.

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