JP6870007B2 - Test substance detection method and fluorescence detection method - Google Patents

Description

The present invention relates to a method for detecting a test substance and a method for detecting fluorescence, and more particularly to a technique for improving detection sensitivity.

Conventionally, as a method for detecting a test substance such as a gene or protein contained in a biological sample, a test substance detection method for detecting a test substance using a labeling substance that is activated when irradiated with light has been proposed. (See, for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2012-15007

As a method for detecting a test substance of this type, there is also a method for detecting a test substance by detecting the light emitted from the fluorescent substance when the fluorescent substance, which is a labeling substance bound to the test substance, is photoexcited. .. In this method for detecting a test substance, when the amount of the test substance is very small, the light emitted from the fluorescent substance bound to the test substance is also weak. Therefore, in order to detect the light emitted from the fluorescent substance with high sensitivity, it is important to improve the S / N ratio of the detection signal output from the detector.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a test substance detection method and a fluorescence detection method capable of improving the S / N ratio of a fluorescence detection signal.

The test substance detection method according to a certain viewpoint is a test substance detection method for detecting a test substance contained in a biological sample, and is 450 nm or more when irradiated with light having a first peak wavelength of 240 nm or more and 300 nm or less. A step of irradiating a complex containing a fluorescent substance and a test substance that emits light having a second peak wavelength of 900 nm or less with light having a first peak wavelength, and a light detector irradiating light with a first peak wavelength. A step of detecting the light of the second peak wavelength emitted from the fluorescent substance and obtaining an image of a fluorescence image, a step of irradiating the composite with light of a third peak wavelength different from the first peak wavelength, and a light detector. The light detector is provided with a step of obtaining a bright-field image of the test substance irradiated with light having a third peak wavelength, and the optical detector has a quantum efficiency of the first in the entire peak wavelength range of 450 nm or more and 900 nm or less. Ri der least twice the quantum efficiency at the peak wavelength, the light detector detects a plurality of colors of light having a peak wavelength in 900nm the range above 450nm.

The test substance detection method according to another viewpoint is a test substance detection method for detecting a test substance contained in a biological sample, and is a reaction between an enzyme and a substrate in a complex containing the test substance and the enzyme. The step of irradiating the fluorescent substance that emits the light of the second peak wavelength of 450 nm or more and 900 nm or less when the light of the first peak wavelength of 240 nm or more and 300 nm or less is irradiated with the light of the first peak wavelength and the light detection. A process of detecting the light of the second peak wavelength emitted from the fluorescent substance irradiated with the light of the first peak wavelength and obtaining an image of a fluorescence image, and the light of the third peak wavelength different from the first peak wavelength. The light detector is 450 nm or more and 900 nm or less. quantum efficiency at entire region of the peak wavelength of, der least twice the quantum efficiency at the first peak wavelength is, the light detector detects a plurality of colors of light having a peak wavelength in 900nm the range above 450nm.

In the fluorescence detection method according to a certain viewpoint, a fluorescent substance that emits light having a second peak wavelength of 450 nm or more and 900 nm or less when irradiated with light having a first peak wavelength of 240 nm or more and 300 nm or less is irradiated with light having a first peak wavelength. The step of detecting the light of the second peak wavelength emitted from the fluorescent substance irradiated with the light of the first peak wavelength by the light detector and obtaining the image of the fluorescence image are different from the step of obtaining the image of the fluorescence image. irradiating light of a third peak wavelength in a fluorescent substance, a photodetector, a step of light to obtain a bright field image of the fluorescent material irradiated in the third peak wavelength, comprising a photodetector, 450 nm 900nm or more following quantum efficiency at entire region of the peak wavelength, der least twice the quantum efficiency at the first peak wavelength is, to obtain an image of the fluorescence image by the light detector, a peak in 900nm the range above 450nm Detects light of multiple colors with wavelength .

According to the present invention, it is possible to provide a test substance detection method capable of improving the S / N ratio of a fluorescence detection signal and a fluorescence detection method.

It is a schematic block diagram which shows the test substance detection system which concerns on 1st Embodiment. It is a schematic block diagram when the test substance detection apparatus which concerns on 1st Embodiment is seen from the side. It is a figure which shows the sensitivity characteristic of the photodetector which concerns on 1st Embodiment. It is the schematic sectional drawing of the 2nd unit of the test substance detection apparatus which concerns on 1st Embodiment. It is a figure which schematically represented the processing procedure of the test substance detection method which concerns on 1st Embodiment. (A-1) to (a-3) are diagrams schematically showing the processing procedure of the test substance detection method according to the first embodiment, and (b-1) is a diagram schematically showing the processing procedure according to the first embodiment. It is a figure which shows the structure of the quantum dot, (b-2) is the band figure of the quantum dot which concerns on 1st Embodiment. It is a figure which shows the absorption spectrum and fluorescence spectrum of the quantum dot which concerns on 1st Embodiment. It is a figure which schematically represented the processing procedure of the test substance detection method which concerns on 2nd Embodiment. It is a figure which schematically represented the processing procedure of the test substance detection method which concerns on 2nd Embodiment. It is a schematic block diagram when the photodetector of the test substance detection apparatus which concerns on 3rd Embodiment is seen from the side. It is explanatory drawing which shows the pattern of the color filter assigned to 1 pixel of the photodetector in 3rd Embodiment. It is a figure which shows the sensitivity characteristic for each color of the photodetector in 3rd Embodiment. It is a figure which shows the sensitivity characteristic per pixel of the photodetector in 3rd Embodiment. It is a figure which shows an example of the optical spectrum of the deep ultraviolet light emitted from the LED which concerns on a modification. It is a figure which shows the fluorescence spectrum of the organic dye which concerns on a modification. (A-1) is an image obtained in Comparative Example 1, (b-1) is an image obtained in Comparative Example 2, and (c-1) is an image obtained in Example 1. (A-2) shows the intensity profile on the AA line of (a-1), (b-2) shows the intensity profile on the AA line of (b-1), and (c-2). ) Indicates the intensity profile on the AA line of (c-1). (A) shows the image obtained in Example 2, (b) is the intensity profile on the X (1) axis of the image of (a), and (c) is the intensity on the X (2) axis. Show profile. (A) shows the image obtained in the comparative example, (b) is the intensity profile on the X (1) axis of the image of (a), and (c) is the intensity profile on the X (2) axis. Is shown. (A) is a bright field image obtained in Example 3, and (b) is the same fluorescence image. It is a figure which shows the intensity profile of the fluorescence image shown in FIG. 19B. It is a figure which shows the formation procedure of the complex containing the test substance used in Example 4. FIG. It is an image obtained in Example 4. It is a figure which shows the intensity profile on the X-axis in FIG.

<First Embodiment>
<1> Configuration The configuration of the test substance detection system according to the first embodiment will be described. The test substance detection system includes a test substance detection device 1, a signal conversion device 2, and an image processing device 3.

The test substance detection device 1 includes a light source 11, a light diffusing member 12, a test substance holding unit 13, and a photodetector 6. The light source 11, the light diffusing member 12, the test substance holding portion 13, and the photodetector 6 are arranged in this order. A light diffusing member 12 is arranged on the light source 11 side of the test substance holding portion 13. The test substance detection device 1 includes a first unit 1a including a light source 11 and a light diffusing member 12, and a second unit 1b including a test substance holding unit 13 and a photodetector 6. The photodetector 6 includes a light receiving unit 16 described later.

The light source 11 emits light having a first peak wavelength. The first peak wavelength of the first embodiment is 270 nm. As the light source 11, a semiconductor light emitting element such as an LED (Light Emitting Diode) is used. Since the light source 11 is composed of a semiconductor light emitting element such as an LED whose power consumption is relatively low as compared with a light bulb or the like, power saving can be achieved as compared with a configuration using a light bulb or the like as the light source 11. When light is emitted from the light source 11, a complex containing the test substance and the fluorescent substance in the test substance holding unit 13 exists in the region S22. When the region S22 is irradiated with light from the light source 11, the fluorescent substance contained in the complex is photoexcited. The fluorescent substance is a fluorescent substance that emits light having a second peak wavelength whose peak wavelength is different from that of the first peak wavelength when excited by light having a first peak wavelength. In the first embodiment, the second peak wavelength is 705 nm. Quantum dots are used as the fluorescent substance. The quantum dots will be described in detail in <3> described later.

The light diffusing member 12 has a function of diffusing the light input from the light source 11. As the light diffusing member 12, for example, a transparent base material in which transparent particles having a refractive index different from that of the base material are dispersed, or a transparent base material whose surface is blasted may be used. As shown by the arrow L1 in FIG. 2, the light incident on one side of the light diffusing member 12 facing the light source 11 from the light source 11 is diffused in the direction orthogonal to the thickness direction of the light diffusing member 12, and FIG. As shown by the arrow L2 in the middle, the light is emitted from the other surface side of the light diffusing member 12 opposite to the light source 11 side.

As shown in FIG. 2, the test substance holding portion 13 is composed of a base material 13a and a trapping substance 21 immobilized on the surface side of the base material 13a facing the light diffusing member 12. The test substance holding unit 13 holds the test substance in a state where the trapping substance 21 has captured the test substance.

The base material 13a is formed of a transparent material in a plate shape, and is arranged so as to cover the photoelectric conversion element 161 of the photodetector 6 described later.

The trapping substance 21 is immobilized on the surface side of the base material 13a facing the light diffusing member 12. Immobilization of the trapping substance 21 on the base material 13a can be performed via a binding group or the like that binds to the base material 13a. Examples of the bonding group include a thiol group, a hydroxyl group, a phosphoric acid group, a carboxyl group, a carbonyl group, an aldehyde group, a sulfonic acid group, an amino group and the like. Immobilization of the trapping substance 21 on the base material 13a may be performed by a physical adsorption method, an ionic bonding method, or the like. The fixed amount of the trapping substance 21 on the base material 13a is not particularly limited, and can be set according to the application and purpose.

The trapping substance 21 can be appropriately selected according to the type of the test substance. For example, when the test substance is a nucleic acid, a nucleic acid probe that hybridizes to the nucleic acid, an antibody against the nucleic acid, a protein that binds to the nucleic acid, or the like can be used as the capture substance 21. When the test substance is a protein or peptide, an antibody against the protein or peptide can be used as the capture substance 21. In this way, the test substance holding unit 13 can selectively hold a specific organic substance corresponding to the trapping substance 21. Therefore, it is possible to take out only the test substance from the sample in which the test substance and other contaminants are mixed.

The capture substance 21 can capture the test substance, for example, under the condition that the capture substance 21 and the test substance are bound to each other. The conditions for binding the trapping substance 21 and the test substance can be appropriately selected according to the type of the test substance and the like. For example, when the test substance is a nucleic acid and the capture substance 21 is a nucleic acid probe that hybridizes to the nucleic acid, the capture of the test substance can be performed in the presence of a hybridization buffer. Also. When the test substance is a nucleic acid, protein or peptide and the capture substance 21 is an antibody against the nucleic acid or an antibody against the protein or an antibody against the peptide, the capture of the test substance is performed by phosphate buffered physiological saline, HEPES. It can be carried out in a solution suitable for carrying out an antigen-antibody reaction such as a buffer solution, a PIPES buffer solution, or a Tris buffer solution. Further, when the test substance is a ligand and the capture substance 21 is a receptor for the ligand, or when the test substance is a receptor and the capture substance 21 is a ligand for the receptor, the capture of the test substance is a ligand. It can be done in a solution suitable for binding to the receptor.

The photodetector 6 is mainly provided between the light receiving unit 16, the support substrate 162, the wire 164 for electrically connecting the light receiving unit 16 and the support substrate 162, and the support substrate 162 and the light receiving unit 16. The resin portion 162a is provided.

The light receiving unit 16 includes a first protective layer 14, a second protective layer 15, and a photoelectric conversion element 161. The first protective layer 14 is laminated on the second protective layer 15, and the second protective layer 15 is laminated on the photoelectric conversion element 161. Here, the first protective layer 14, the second protective layer 15, and the photoelectric conversion element 161 are integrally formed.

Such an optical detector 6 has the sensitivity characteristics shown in FIG. In the first embodiment, the photodetector 6 is a sensor in which a photoelectric conversion element using a silicon substrate is used for the light receiving unit 16, and more specifically, a CMOS in which a photodiode using a silicon substrate is used. It is an image sensor. This CMOS image sensor is a silicon substrate in which photodiodes, MOSFETs, wirings, etc. are built using well-known ion implantation technology, film formation technology, and the like.

The CMOS image sensor has a configuration in which a plurality of cells (not shown) including a photodiode and a MOSFET connected to the photodiode are arranged in a grid pattern. In this way, by using a solid-state image sensor such as a CMOS image sensor, it is possible to integrate a plurality of cells constituting the photodetector 6, so that the captured image obtained by the photodetector can be integrated accordingly. The resolution can be improved, and the detection sensitivity of the test substance can be improved. Since the power consumption of the CMOS image sensor is relatively low as compared with PMT (Photomultiplier Tube) or the like, power saving can be achieved as compared with a configuration using PMT or the like as a photodetector.

The sensitivity characteristics of the photodetector 6 according to the first embodiment are as shown in FIG. In FIG. 3, η indicates the quantum efficiency of the photodetector 6, ηmax indicates the maximum value of the quantum efficiency, and ηth indicates the quantum efficiency of 5%. The maximum quantum efficiency ηmax is included in the wavelength band of 450 nm or more and 900 nm or less. The photodetector 6 has a quantum efficiency of about 0% at the first peak wavelength, while the quantum efficiency at the second peak wavelength is about 40%, and the quantum efficiency at the second peak wavelength is the quantum efficiency at the first peak wavelength. It is far more than twice as much as.

The first protective layer 14 and the second protective layer 15 are layers that protect the photoelectric conversion element 161. The first protective layer 14 is composed of, for example, a silicon nitride (SiN) film, and the second protective layer 15 is composed of, for example, a silicon oxide (SiO2) film. The second protective layer 15 serves to reinforce the first protective layer 14. A microlens array composed of a plurality of microlenses is provided on the test substance holding portion 13 side of the first protective layer 14.

The photoelectric conversion element 161 is, for example, a photodiode using a silicon substrate. The light received by each cell of the photoelectric conversion element 161 is converted into a detection signal by a photodiode and sent to the support substrate 162 side. The first protective layer 14 is provided with one microlens at a position corresponding to each of the plurality of cells constituting the photoelectric conversion element 161. A terminal for extracting a signal from each cell to the outside is provided on the peripheral portion of the photoelectric conversion element 161.

The support substrate 162 supports the light receiving unit 16. The support substrate 162 is formed of a silicon substrate, has a recess formed in the center thereof, and is provided with a plurality of terminals (not shown) for taking out signals on the outer peripheral portion of the recess. A light receiving portion 16 is arranged inside the recess of the support substrate 162.

The wire 164 connects a terminal provided on the peripheral portion of the photoelectric conversion element 161 and a terminal provided on the support substrate 162. The wire 164 is embedded in the resin portion 162a.

By the way, the first unit 1a also includes, for example, a support member (not shown) that movably supports the light source 11 and a light source control unit (not shown) that controls the intensity, direction, position, etc. of the light emitted from the light source 11. (Not shown). Here, the support member has a light source moving mechanism for changing the direction and position of the light source 11.

FIG. 4 is a schematic cross-sectional view of the second unit 1b of the test substance detection device 1 according to the first embodiment. As shown in FIG. 4, the second unit 1b includes a housing 17 for accommodating the test substance holding portion 13 and the photodetector 6. The housing 17 has a window portion 17a for taking light into the light receiving portion 16 of the photodetector 6 and a signal line leading hole 17b for leading the signal line connected to the light receiving portion 16 to the outside of the housing 17. It is provided. The window portion 17a of the housing 17 is covered with the test substance holding portion 13. A bushing 18 is provided between the inner surface of the signal line lead-out hole 17b of the housing 17 and the outer surface of the signal line so as to maintain watertightness.

The signal conversion device 2 converts the signal acquired from the photoelectric conversion element 161 into image information and outputs the signal to the image processing device 3. The signal conversion device 2 includes, for example, an analog-digital converter that converts an analog signal acquired from the photoelectric conversion element 161 into a digital signal.

The image processing device 3 generates an image on the test substance holding unit 13 based on the image information input from the signal conversion device 2, and displays the generated image on the display unit 3a. Here, the display unit 3a is composed of, for example, a display or the like.

The image processing device 3 can calculate the amount of light detected by the photodetector 6 based on the image information input from the signal conversion device 2. The image processing device 3 holds calibration data showing the relationship between the amount of light that can be detected by the photodetector 6 and the amount of the fluorescent substance. The image processing device 3 has a function of calculating the amount of the fluorescent substance based on the calibration data and calculating the amount of the calibration substance from the calculated amount of the fluorescent substance.

The image processing device 3 includes a computer including a CPU and memories such as ROM and RAM. Various functions of the image processing device 3 are realized by the CPU executing a computer program stored in the memory.

<2> Test substance detection method Next, a test substance detection method using the test substance detection device 1 according to the first embodiment will be described.
5 and 6 (a-1) to 6 (a-3) are schematic views for explaining the processing procedure of the test substance detection method according to the first embodiment.

First, as shown in FIG. 5A, for example, using the dispenser D1, the sample S11 in which the test substance is dispersed is dropped onto the test substance holding portion 13. In sample S11, for example, the test substance is dispersed in a liquid containing a buffer solution for hybridization. By this step, the region S21 in which the test substance is captured by the capture substance is formed in the test substance holding portion 13.

At this time, as shown in FIGS. 6 (a-1) and 6 (a-2), the test substance 31 dispersed in the sample S11 is transferred by the trapping substance 21 provided on the test substance holding portion 13. Be captured. For example, when the test substance 31 is composed of a protein and an antibody against the test substance 31 is used as the capture substance 21, the capture substance 21 captures the test substance 31 composed of the protein. At this time, the capturing substance 21 selectively captures the test substance 31 in the liquid sample and does not capture any substance (contaminant substance) other than the test substance 31.

Next, as shown in FIG. 5B, sample S11 is removed from the test substance holding portion 13 using a cleaning solution W1 such as Tris Buffer Saline with Tween 20 or Phosphate buffered saline with Tween 20. At this time, the user may, for example, remove only the second unit 1b of the test substance detection device 1 from the test substance detection device 1 and clean it.

As a result, only the test substance 31 existing in the region S21 in the test substance holding portion 13 remains. In the following description, "Tris Buffer Saline with Tween 20" may be referred to as "TBS-T" or "TBS".

Subsequently, as shown in FIG. 5 (c), for example, a dispenser D2 is used to hold a quantum dot which is a fluorescent substance, and a binding substance which binds to the test substance 31 captured by the capturing substance 21 The dispersed reagent S12 is dropped onto the region S21 in the test substance holding portion 13. By this step, the region S22 in which the complex containing the capture substance 21, the test substance 31, and the quantum dots exists is formed in the test substance holding portion 13. As shown in FIG. 6A-3, a complex containing the trapping substance 21, the test substance 31, and the binding substance 41 holding the quantum dots is formed in the region S22. Reagent S12 is, for example, an antibody having quantum dots dispersed in a liquid containing a hybridization buffer. As the buffer solution for hybridization, for example, the same buffer solution contained in sample S11 may be adopted.

Then, as shown in FIG. 5D, the reagent S12 is removed from the test substance holding portion 13 using a cleaning solution W2 such as Tris Buffer Saline with Tween 20 or Phosphate buffered saline with Tween 20. At this time, the user may, for example, remove only the second unit 1b of the test substance detection device 1 from the test substance detection device 1 and clean it.

By this step, the contaminants are removed from the test substance holding section 13, and the capturing substance 21, the test substance 31, and the binding substance 41 holding the quantum dots, which are present in the region S22 of the test substance holding section 13, are included. Only the complex remains.

After that, as shown in FIG. 5E, by irradiating the region S22 in the test substance holding portion 13 with light L2 from the light source 11, the quantum dots contained in the complex existing in the region S22 fluoresce. .. The light K1 emitted from the quantum dots is received by the photoelectric conversion element 161 of the photodetector 6.

When the photodetector 6 receives the light K1 emitted from the quantum dots, the photodetector 6 inputs a detection signal corresponding to the light K1 to the signal conversion device 2. By this step, the signal conversion device 2 converts the detection signal acquired from the photodetector 6 into image information and outputs it to the image processing device 3. The image processing device 3 generates an image based on the image information and displays it on the display unit 3a.

<3> Quantum Dots Next, the quantum dots according to the first embodiment will be described in detail.

FIG. 6 (b-1) is a schematic configuration diagram of quantum dots used in the test substance detection method according to the first embodiment, and FIG. 6 (b-2) is a core in FIG. 6 (b-1). It is an energy band diagram of a part 411 and a shell 412.

As shown in FIG. 6 (b-1), the quantum dot 410 is a modifier bonded to the core 411, the shell 412 covering the core 411, the organic layer 413 covering the shell 412, and the organic layer 413. It is composed of 414 and.

The core 411 is composed of a first semiconductor. As shown in FIG. 6 (b-2), the shell 412 is composed of a second semiconductor having a wider bandgap than the first semiconductor. Examples of the combination of the first semiconductor and the second semiconductor include InP / ZnS, CuInS / ZnS, InP / ZnS, CdSe / ZnS and the like.

As the quantum dot 410, the core 411 and the shell 412 may be made of the same material. In this case, examples of the first and second semiconductors constituting the core 411 and the shell 412 include CdTe, CdSe, and CdS.

The organic layer 413 is composed of TOPO (trioctylphosphine oxide), HDA (hexadecylamine) and the like.

The modifier 414 is composed of a tripeptide such as glutathione. The modifying substance 414 is not limited to the tripeptide, and may be composed of, for example, synthetic compounds such as thiol and polyethyleneimine, peptides derived from natural products, sugar chains, and phospholipids.

FIG. 7 is a diagram showing an absorption spectrum and a fluorescence spectrum of the quantum dots 410. Here, the absorption spectrum and the fluorescence spectrum show the results of measurement for seven types of quantum dots having different average particle sizes. In the measured quantum dots 410, the first semiconductor constituting the core 411 is CdSe, and the second semiconductor constituting the shell 412 is ZnS. In FIG. 7, in the absorption spectra (j-2) (j = 1 to 7) and the fluorescence spectra (k-1) (k = 1 to 7), the larger the numbers j and k, the smaller the average particle size. Corresponds to things.

As shown in FIG. 7, the fluorescence spectra (k-1) (k = 1 to 7) are shifted to the long wavelength side with respect to the absorption spectra (j-2) (j = 1 to 7) of the quantum dots 410 ( Stokes shift). The fluorescence spectrum (k-1) (k = 1 to 7) of the quantum dot 410 shifts to the shorter wavelength side as the average particle size of the core 411 of the quantum dot 410 becomes smaller.

In this way, if the quantum dots 410 are adopted as the fluorescent material, the wavelength band of the light emitted from the quantum dots 410 can be changed by changing the average particle size of the quantum dots 410. Therefore, for example, by appropriately setting the average particle size of the quantum dots 410 according to the sensitivity characteristics of the photodetector 6, the detection sensitivity of the test substance can be improved. From this point of view, the average particle size of the quantum dots is preferably in the range of 10 nm to 50 nm.

The quantum dots 410 can be produced, for example, by using a chemical synthesis method performed in a liquid phase. Specifically, particles in which the surfaces of the core 411 and the shell 412 are coated with an organic layer such as TOPO or HDA (hereinafter referred to as "quantum dots before surface modification") are chemically synthesized in a coordinating organic solvent. It is produced by. The quantum dots can also be produced by a method (ligand substitution method) in which the organic layer on the surface of the quantum dots before surface modification is replaced with an amphipathic thiol compound or the like. Quantum dots can also be produced by coating the organic layer on the surface of the pre-surface modified quantum dots with an amphipathic polymer (encapsulation method).

As described above, in the test substance detection method according to the first embodiment, when the fluorescent substance is irradiated with light having a first peak wavelength of 190 nm or more and 350 nm or less, the fluorescent substance has a second peak wavelength of 450 nm or more and 900 nm or less. It emits light. The light of the second peak wavelength emitted from the fluorescent substance is detected by the photodetector 6 whose quantum efficiency at the second peak wavelength is at least twice the quantum efficiency at the first peak wavelength. As a result, the photodetector is more difficult to detect the light of the first peak wavelength than the light of the second peak wavelength, and the background level due to the detection of the light of the first peak wavelength is lowered, so that the light is weaker by that amount. It becomes possible to detect light having a second peak wavelength. That is, the S / N ratio of the fluorescence detection signal is improved. Therefore, the test substance can be detected with high detection sensitivity.

<Second Embodiment>
The test substance detection method according to the second embodiment uses the same test substance detection device as the test substance detection device according to the first embodiment. In the first embodiment, a binding substance holding a quantum dot is used as a binding substance that binds to the test substance 31 captured by the capturing substance 21, and light having a second peak wavelength emitted from the quantum dot is detected. , The test substance 31 was detected, but the test substance detection method according to the second embodiment is different from the first embodiment, and is enzyme-labeled as a binding substance that binds to the test substance 31 captured by the capture substance 21. The test substance 31 is detected by detecting the light having the second peak wavelength emitted from the fluorescent substance generated by the reaction between the enzyme and the substrate using the bound substance. Here, the description of the configuration of the test substance detection device will be omitted. Only the test substance detection method will be described.

8 and 9 are schematic views for explaining the processing procedure of the test substance detection method according to the second embodiment.
First, a region S21 (see FIG. 5B) in which the test substance is captured by the trapping substance is formed in the test substance holding portion 13 by the same method as the method described in the first embodiment. Here, as shown in FIG. 6A-2, the test substance 31 dispersed in the sample S11 is captured by the trapping substance 21 provided on the test substance holding portion 13.

Next, as shown in FIG. 8A, the reagent S212 in which the enzyme-labeled binding substance 241 is dispersed is dropped onto the region S21 in the test substance holding portion 13 using, for example, a dispenser D21. Then, the region S23 in which the complex containing the capture substance 21 and the test substance 31 and the enzyme-labeled binding substance 241 exists is formed in the test substance holding portion 13. Here, the reagent S212 is, for example, an enzyme-labeled antibody dispersed in a liquid containing a buffer solution for hybridization. As the enzyme (2411 in FIG. 9), for example, peroxidase or alkaline fosterase may be adopted. As the buffer solution for hybridization, for example, the same buffer solution as that contained in the sample S11 described in the first embodiment may be adopted.

As shown in FIGS. 9A and 9B, the enzyme-labeled binding substance 241 dispersed in the reagent S212 binds to the test substance 31 captured by the capturing substance 21 present in the region S23. .. As a result, a complex containing the trapping substance 21, the test substance 31, and the enzyme-labeled binding substance 241 is formed.

Next, as shown in FIG. 8B, the reagent S212 is removed from the test substance holding portion 13 using a cleaning solution W22 such as Tris Buffer Saline with Tween 20 or Phosphate buffered saline with Tween 20. As a result, only the complex existing in the region S23 in the test substance holding portion 13 remains. At this time, the second unit 1b can be removed from the test substance detection device 1 to clean the second unit 1b.

Subsequently, as shown in FIG. 8C, the reagent S213 in which the fluorescent substrate is dispersed is dropped onto the region S23 in the test substance holding portion 13 using, for example, a dispenser D22. This reagent S213 is, for example, a liquid in which a fluorescent substrate is dispersed in a liquid containing a buffer solution for hybridization.

As the fluorescent substrate, for example, a fluorescent substrate for peroxidase that reacts with peroxidase to produce resorphin, which is a fluorescent substance, and a fluorescent substrate for alkaline fostase, which reacts with alkaline fosterase to generate BBT-anion, which is a fluorescent substance, are adopted. do it. The resorphin produced from the fluorescent substrate for peroxidase is, for example, a fluorescent substance that emits stronger fluorescence than an organic dye. The BBT-anion produced from the fluorescent substrate for alkaline fosterase is, for example, a fluorescent substance having a large Stokes shift and a wide fluorescence spectrum as compared with organic dyes. As the buffer solution for hybridization, for example, the same buffer solution contained in the reagent S212 may be adopted.

By the above step, the fluorescent substance 243 produced by the reaction between the fluorescent substrate 242 and the enzyme of the complex existing on the region S23 is dispersed in the reagent S213. At this time, the region S24 in which the fluorescent substance 243 generated by the reaction between the fluorescent substrate 242 and the enzyme exists is formed in the test substance holding portion 13.

As shown in FIG. 9C, the fluorescent substrate 242 reacts with the enzyme of the complex present on the region S23 to produce the fluorescent substance 243. For example, when the enzyme is peroxidase, the enzyme reacts with the fluorescent substrate for peroxidase to produce resorphin, which is a fluorescent substance 243.

Then, as shown in FIG. 8D, the light L2 is dispersed in the reagent S213 by irradiating the region S24 in which the fluorescent substance 243 generated by the reaction between the fluorescent substrate 242 and the enzyme exists with light L2 from the light source 11. The fluorescent substance 243 is fluorescent. The light K2 emitted from the fluorescent substance 243 is received by the photoelectric conversion element 161 of the light receiving unit 16.

As described above, in the test substance detection method according to the second embodiment, when the fluorescent substance is irradiated with light having a first peak wavelength of 190 nm or more and 350 nm or less, the fluorescent substance has a second peak wavelength of 450 nm or more and 900 nm or less. It emits light. The light of the second peak wavelength emitted from the fluorescent substance is detected by the photodetector 6 whose quantum efficiency at the second peak wavelength is at least twice the quantum efficiency at the first peak wavelength. As a result, the photodetector 6 is more difficult to detect the light having the first peak wavelength than the light having the second peak wavelength, and the background level due to detecting the light having the first peak wavelength is lowered. It is possible to detect light having a second peak wavelength. That is, the S / N ratio of the fluorescence detection signal is improved. Therefore, the test substance can be detected with high detection sensitivity.

In the test substance detection method according to the first and second embodiments, the photodetector 6 has a fluorescent substance in the direction in which the light of the first peak wavelength is irradiated when the light of the first peak wavelength is irradiated. The photodetectors 6 are arranged in this order. In this way, both the light having the first peak wavelength emitted from the light source 11 and the light having the second peak wavelength emitted from the fluorescent substance of the complex existing in the test substance holding portion 13 are sent to the light detector 6. Even in a configuration in which light is easily incident, the light detector 6 has a quantum efficiency at the second peak wavelength that is more than twice the quantum efficiency at the first peak wavelength. It is difficult to detect light. Therefore, the background level due to the detection of the light having the first peak wavelength is lowered, and the light having the weak second peak wavelength can be detected accordingly.

Since the monochrome CMOS image sensor, which is the photodetector 106 of the first and second embodiments, does not absorb a part of the incident light by the color filter, the quantum efficiency is higher than that of the color CMOS image sensor using the same photoelectric conversion element. Will be higher.

<Third Embodiment>
In the third embodiment, as shown in FIG. 10, the configuration of the photodetector 106 is different from that in the first embodiment. The configuration of the test substance detection device other than the photodetector 106 and the test substance detection method can be the same as those in the first and second embodiments.

The photodetector 106 of the third embodiment is a color CMOS image sensor including a light receiving unit 116, a color filter 119, a microlens 120, and a cover member 121. The light receiving unit 116 includes a photoelectric conversion element 161a, a first protective layer 114 provided on the photoelectric conversion element 161a, and a second protective layer 115. The photoelectric conversion element 161a, the first protective layer 114, and the second protective layer 115 are laminated from the bottom in this order. As the photoelectric conversion element 161a, a photodiode can be used as in the first embodiment. The first protective layer 114 can also be formed of the silicon nitride film as in the first embodiment. The second protective layer 115 can also be formed of the silicon oxide film as in the first embodiment.

A plurality of color filters 119 are provided on the light receiving unit 116. Each of the plurality of color filters 119 is composed of an absorption filter that selectively transmits light in a specific wavelength range and absorbs light in other wavelength ranges. Specifically, the plurality of color filters 119 transmit light in the red, green, and blue wavelength ranges, respectively. Therefore, the photodetector 106 can identify and detect light in a wavelength range over a plurality of colors, that is, visible light of a plurality of colors by the color filter 119.

The light detector 106 uses a color filter 119 to obtain a first detector having a red spectral sensitivity with a peak wavelength in the range of 620 nm or more and less than 750 nm, and a green spectral sensitivity having a peak wavelength in the range of 495 nm or more and less than 570 nm. It functions as a second detection unit having a second detection unit and a third detection unit having a blue spectral sensitivity having a peak wavelength in the range of 450 nm or more and less than 495 nm.

The red filter R, which is the color filter 119 of the first detection unit, constitutes a first absorption unit that absorbs light other than the red wavelength region. The green filter G, which is the color filter 119 of the second detection unit, constitutes a second absorption unit that absorbs light other than the green wavelength region. The blue filter B, which is the color filter 119 of the third detection unit, constitutes a third absorption unit that absorbs light other than the blue wavelength region.

A plurality of microlenses 120 are provided so as to correspond to each color filter 119. Each microlens 120 collects the light incident from above on the photoelectric conversion element 161a of the light receiving unit 116 via the color filter 119.

The cover member 121 is made of a transparent plate material such as a quartz glass plate, and covers the microlens 120 from above. The complex containing the test substance is provided on the cover member 121, and the excitation light emitted by the light source 11 is irradiated from above. The cover member 121 functions as the base material 13a of the first and second embodiments when the complex containing the test substance described in the first and second embodiments is used.

The plurality of color filters 119 are arranged in the arrangement pattern shown in FIG. Specifically, one pixel α of the CMOS image sensor, which is the photodetector 106, is divided into 2 × 2 four compartments α1 to α4, one of which compartments α1 is provided with a red filter R, and the other one compartment. A blue filter B is provided in α4, and a green filter G is provided in the remaining two compartments α2 and α3. Then, the arrangement pattern shown in FIG. 11 is regularly arranged over the entire light receiving unit 116.

FIG. 12 shows the quantum efficiency per pixel of a color CMOS image sensor for each color of the color filter. η _red , η _green , and η _blue are the quantum efficiencies of the compartments α1 to α4 provided with the red filter R, the green filter G, and the blue filter B, respectively.

_{On the other hand, the quantum efficiency η pixel} per pixel α of the color CMOS image sensor shown in FIG. 11 is expressed as the average of the quantum efficiencies of all the compartments α1 to α4 in the one pixel α. That is, the quantum efficiency η _pixel can be obtained by the following equation (1).
η _pixel = (η _blue + η _green + η _green + η _red ) / 4
= 1/4η _blue + 1 / 2η _green + 1/4η _red・ ・ ・ (1)

FIG. 13 shows the quantum efficiency η _pixel , its components 1/4η _blue , 1 / 2η _green , and 1/4η _red , respectively, per pixel α. Since the color CMOS image sensor is configured by integrating a plurality of pixels α, the quantum efficiency η _pixel per pixel α and the quantum efficiency η _{all of the} entire color CMOS image sensor are the same. That is, the following equation (2) holds.
η _all = η _pixel ... (2)

In FIG. 13, ηmax _{indicates the maximum value of the quantum efficiency η pixel} per pixel α, that is, the quantum efficiency η _all of the color CMOS image sensor, and ηth indicates the quantum efficiency of 5%. The wavelength at which the quantum efficiency ηmax is obtained is included in the wavelength range of 450 nm or more and 900 nm or less. The photodetector 106 of the present embodiment, which is composed of a color CMOS image sensor, has a quantum efficiency of about 0% with respect to the light having the first peak wavelength of 190 nm or more and 350 nm or less emitted by the light source 11. On the other hand, the light of the second peak wavelength emitted by the fluorescent substance is about 22% as shown in FIG. Therefore, even when the photodetector 106 of the third embodiment is used, the quantum efficiency at the second peak wavelength is more than twice the quantum efficiency at the first peak wavelength.

As a result, the light detector 106 is more difficult to detect the light of the first peak wavelength than the light of the second peak wavelength, and the background level due to detecting the light of the first peak wavelength is lowered by that amount. It is possible to detect weak light having a second peak wavelength. That is, the S / N ratio of the fluorescence detection signal is improved. Therefore, the test substance can be detected with high detection sensitivity. Further, the detector 106 can identify and detect the color of the light having the second peak wavelength.

<Modification example>
The present invention is not limited to the above-described embodiments, but includes all aspects within the scope of the claims. For example, the following modification is included.

(1) The test substance detection method according to the first to third embodiments has described an example in which the present invention is used for detecting a test substance, but the present invention is not limited thereto. For example, it may be used not only in the test substance but also in a fluorescence detection method for detecting fluorescence from a fluorescent substance. In this case, the trapping substance used in the first to third embodiments is not essential. Therefore, as the device used in this fluorescence detection method, for example, a device in which the test substance holding portion 13 of the test substance detection device 1 in the first embodiment is replaced with the base material 13a can be used.

(2) In the first to third embodiments, an example in which the first peak wavelength is 270 nm has been described, but the present invention is not limited thereto. In the present invention, the first peak wavelength may be 190 nm or more and 350 nm or less.

In the present invention, the first peak wavelength may be between 242 nm and 243 nm, for example, as shown in FIG. FIG. 14 is a diagram showing an example of an optical spectrum of deep ultraviolet light emitted from the LED according to the modified example. Note that FIG. 14 shows the optical spectra when the magnitude of the forward bias to the LED is set to seven different magnitudes. In FIG. 14, the larger the number among (1) to (7), the larger the forward bias.

Since the light output of the light source decreases as the wavelength becomes shorter, the first peak wavelength is preferably 240 nm or more in the present invention from the viewpoint of improving the intensity of the fluorescence detection signal. In the present invention, the first peak wavelength is preferably 300 nm or less from the viewpoint of suppressing the background level of the detection signal caused by the light incident on the photodetector. As described above, from the viewpoint of improving the S / N ratio of the fluorescence detection signal, the first peak wavelength is preferably 240 nm or more and 300 nm or less in the present invention. As a general-purpose light source, it is easy to obtain a light source having a wavelength longer than 190 nm. On the other hand, if the wavelength of the light emitted from the light source is longer than 350 nm, a general-purpose photodetector may detect the light from the light source and cause background noise.

(3) In the first to third embodiments, an example in which the second peak wavelength is 705 nm has been described, but the present invention is not limited thereto. In the present invention, the second peak wavelength may be 450 nm or more and 900 nm or less. Since the Illuminating Engineering Institute of Japan uses a spectral sensitivity of 360 nm to 830 nm to define brightness, in the present invention, if the second peak wavelength is 830 nm or less, it is easy to use a general-purpose photodetector and a fluorescent substance. , More suitable. Therefore, the second peak wavelength is preferably 450 nm or more and 830 nm or less.

(4) In the test substance detection method according to the first to third embodiments, the photodetectors 6 and 106 have a quantum efficiency of about 0% with respect to light at the first peak wavelength, and the quantum efficiency at the second peak wavelength. Although the example in which is about 40% or about 22% has been described, the present invention is not limited thereto. In the present invention, the photodetector may have a quantum efficiency at the second peak wavelength that is at least twice the quantum efficiency at the first peak wavelength.

In the present invention, from the viewpoint of suppressing the background level of the detection signal caused by the light incident on the photodetector, the photodetector has a quantum efficiency of less than 10% with respect to the light having the first peak wavelength. preferable. In other words, the first peak wavelength is preferably a wavelength that the photodetector does not detect with a quantum efficiency of 10% or more. With such a configuration, the S / N ratio of the fluorescence detection signal can be further improved.

In the present invention, from the viewpoint of further suppressing the background level of the detection signal caused by the light incident on the photodetector, the photodetector has a quantum efficiency of less than 5% with respect to the light having the first peak wavelength. preferable. In other words, the first peak wavelength is more preferably a wavelength that the photodetector does not detect with a quantum efficiency of 5% or more. With such a configuration, the S / N ratio of the fluorescence detection signal can be further improved.

(5) In the test substance detection method according to the first to third embodiments, an example in which a CMOS image sensor including a photoelectric conversion element using a silicon substrate is used as the photodetectors 6 and 106 has been described. The invention is not limited to this.

If the photodetector includes a photoelectric conversion element using a silicon substrate, the silicon blocks the light having the first peak wavelength of 190 nm or more and 350 nm or less, so that the quantum efficiency of the photodetector with respect to the light of the first peak wavelength is low. Become. Therefore, from the viewpoint of suppressing the background level of the detection signal caused by the light incident on the photodetector, the photodetector preferably includes a photoelectric conversion element using a silicon substrate. Specifically, as a photodetector including a photoelectric conversion element using a silicon substrate, a CMOS image sensor, a microPMT (Photomultiplier Tube), a PiN (Positive-Intrinsic-Negative) photodiode, an APD (Avacanche PhotoMix), and an MP -Pixel Photodiode), EMCCD (Electron Multiplier Tube Coupled Device), CCD (Charge Coupled Device) image sensor, or NMOS (Negative Digital) Image Sensor, etc. Among them, the CMOS image sensor and the microPMT have particularly low quantum efficiency at the first peak wavelength, so that the S / N ratio in the fluorescence detection signal can be improved. Therefore, in the present invention, the photodetector is a CMOS image. Sensors or microPMTs are preferred.

(6) In the test substance detection method according to the first to third embodiments, an example in which quantum dots are used as the fluorescent substance has been described, but the present invention is not limited thereto. For example, an organic dye may be used as the fluorescent substance. As the organic dye, for example, an organic dye synthesized from coumarin, rhodamine, xanthene or cyanine can be adopted.

FIG. 15 shows an example of the fluorescence spectrum of the organic dye used in the test substance detection method according to the modified example. Here, the organic dyes shown as an example are all organic dyes manufactured by Molecular Probe Co., Ltd. In FIG. 15, (1-3) is Alexa Fluor 790, (2-3) is Alexa Fluor 750, (3-3) is Alexa Fluor 680, and (4-3) is Alexa Fluor 647, (5). -3) is Alexa Fluor 633, (6-3) is Alexa Fluor 594, (7-3) is Alexa Fluor 555, (8-3) is Alexa Fluor 488, (9-3) is Alexa Fluor 405, (10-3) corresponds to Alexa Fluor 355.

In addition, as the organic dye, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 660, Alexa Fluor 680 and Alexa Fluor 700 (all manufactured by Molecular Probes) may be used.

Furthermore, as organic dyes, 9-phenylxanthene dyes, cyanine dyes, methalocyanine dyes, xanthene dyes, triphenylmethane dyes, acrydin dyes, oxazine dyes, coumarin dyes, and merocyanine dyes. Dyes, rodacyanine dyes, polymethine dyes, porphyrin dyes, phthalocyanine dyes, rhodamine dyes, xanthene dyes, chlorophyll dyes, eosin dyes, Mercurochrome dyes, Indigo dyes, BODIPY dyes, CALFluor dyes Oregon green dye, Rhodol green, Texas red, Cascade blue, Nucleic acid (DNA, RNA, etc.), Cyaninated cadmium, Tellurized cadmium, Ln2O3: Re, Ln2O2S: Re, ZnO, CaWO4, MO ・ xAl2O3: Eu , Zn2SiO4: Mn, LaPO4: Ce, Tb, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5 and Cy9 (all manufactured by Amasham Bioscience); DY-610, DY-615, DY -630, DY-631, DY-633, DY-635, DY-636, EVOblue10, EVOblue30, DY-647, DY-650, DY-651, DY-800, DYQ-660 and DYQ-661 (all of which). (Manufactured by Dyomics); Atto425, Atto465, Atto488, Atto495, Atto520, Atto532, Atto550, Atto565, Atto590, Atto594, Atto610, Atto611X, Atto610, Atto611X, Atto620, Atto611X, Atto620, Atto635 (Manufactured by Atto-TEC GmbH); VivoTagS680, VivoTag680, VivoTagS750 (all manufactured by VisEnMedical) and the like may be adopted. In addition, the said Ln represents La, Gd, Lu or Y, Re represents a lanthanide group element, M represents an alkaline earth metal element, and x represents a number of 0.5 to 1.5.

It should be noted that the quantum dots have higher brightness than, for example, organic dyes and fluorescent proteins, and are less likely to be discolored by the light emitted from the light source. Therefore, fluorescent substances such as organic dyes and fluorescent proteins are more likely to fade due to short wavelength light than long wavelength light, but even when the fluorescent substance is irradiated with such short wavelength light, it is fluorescent. By using quantum dots as a substance, fading due to light of a short wavelength is unlikely to occur, which is advantageous for detecting a trace amount of the test substance and observing the test substance for a long time. Therefore, in the present invention, the fluorescent substance is preferably quantum dots.

(7) In the first to third embodiments, an example in which the light source 11 is composed of a semiconductor light emitting element such as an LED has been described, but the type of the light source 11 is not limited to this. For example, the light source 11 may be composed of a discharge lamp (for example, a HID lamp or the like) or the like.

(8) Further, in the first to third embodiments, an example in which the modifying substance 414 of the quantum dot 410 is composed of one kind of peptide such as glutathione has been described, but the modifying substance 414 is only one kind of organic substance. It is not limited to those consisting of. For example, the modifier 414 may be composed of a peptide and a ligand for an antibody or receptor bound to the peptide.

(9) Although the photodetector 106 of the third embodiment includes an absorption filter as a filter that transmits a part of the light emitted from the fluorescent substance, it may be an interference filter instead of the absorption filter. The photodetector 106 of the third embodiment includes a red, green, and blue color filter as the color filter 119, but may include a cyan, magenta, and yellow color filter. The photodetector 106 may not include the color filter 119. For example, as a photodetector 106, spectroscopy of a laminated image sensor that identifies and detects a plurality of colors by utilizing the difference in absorption depth depending on the color, an organic film image sensor in which a photoelectric conversion film is laminated, a deflector, or the like. A spectroscopic image sensor or the like provided with a portion can be used.

Hereinafter, the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited thereto.

<Example 1>
(Fixing of fluorescent material on CMOS image sensor)
A sample containing 1 μM quantum dots as a fluorescent substance on a microlens array arranged in a two-dimensional array by removing the cover glass provided in advance in the monochrome CMOS image sensor constituting the detector 6 of the first embodiment. TBS-T was added dropwise as a reference sample. Then, the sample was dried by leaving the CMOS image sensor in an environment of 50 ° C. for 10 minutes. The sensitivity characteristics of the CMOS image sensor are as shown in FIG. As the monochrome CMOS image sensor, MT9M001 manufactured by Aptina Imaging Co., Ltd. was used. For the quantum dots, Qdot 705 Streptavidin Conjugate Q10163MP manufactured by Life Technologies Co., Ltd. was used.

(Irradiation of excitation light)
Then, the excitation light was irradiated from the upper part of the CMOS image sensor via the light diffusing member (Diffuser). Each excitation light intensity was set to the maximum intensity within a range in which the pixel value of the CMOS image sensor was not saturated.
In Comparative Example 1, a light source having a blue LED was used to irradiate light having a peak wavelength of 470 nm.
In Comparative Example 2, a light source having an ultraviolet LED was used to irradiate light having a peak wavelength of 365 nm.
In this embodiment, a light source having a deep ultraviolet LED was used to irradiate light having a peak wavelength of 270 nm.

(Experimental result)
16 (a-1) is an image obtained in Comparative Example 1, FIG. 16 (b-1) is an image obtained in Comparative Example 2, and FIG. 16 (c-1) is an image obtained in Example. is there. 16 (a-2) shows the intensity profile in line AA of FIG. 16 (a-1), and FIG. 16 (b-2) shows the intensity in line AA of FIG. 16 (b-1). The profile is shown, and FIG. 16 (c-2) shows the intensity profile at line AA of FIG. 16 (c-1). In the graphs of FIGS. 16 (a-2), (b-2), and (c-2), the position on the line AA is shown as the X coordinate.

As shown in FIGS. 16 (a-1) and 16 (a-2), in the case of Comparative Example 1, the detection intensity and the detection intensity of the region to which TBS-T was added as a reference (hereinafter referred to as region Ref) in the image. The detection intensity of the region to which the quantum dots are added (hereinafter, region S25) is lower than the detection intensity of other regions in the image. This is because the peak wavelength (470 nm) of the light emitted from the light source using the blue LED exists in the wavelength band where the sensitivity of the CMOS image sensor is relatively high, so that the detection signal output from the CMOS image sensor is backed up. This is probably because the ground level is high.

As shown in FIGS. 16 (b-1) and 16 (b-2), in the case of Comparative Example 2, the detection sensitivity in the region S25 is higher than the detection sensitivity in the region in the image excluding the region S25 and the region Ref. The detection sensitivity in the region Ref is slightly lower than the detection sensitivity in the regions in the image excluding the region S25 and the region Ref. This is because the CMOS image sensor is less sensitive to the light of Comparative Example 2 having a peak wavelength of 365 nm than the light of Comparative Example 1 having a peak wavelength of 470 nm, and therefore is output from the CMOS image sensor as compared with Comparative Example 1. It is considered that the background level of the detected signal is lowered. Also in the case of Comparative Example 2, the background level of the detection signal is about 80% of the detection intensity in the region S25, and it cannot be said that the S / N ratio in the detection signal is sufficiently high.

On the other hand, as shown in FIGS. 16 (c-1) and 16 (c-2), in the case of Example 1, the detection sensitivity in the region S25 is higher than the detection sensitivity in the region in the image excluding the region S25 and the region Ref. It is extremely high. The detection sensitivity in the region Ref is about the same as the background level, which is extremely lower than the detection sensitivity in the region S25. It is considered that this is because the CMOS image sensor has substantially zero sensitivity to the wavelength of 270 nm, so that the increase in the background level is suppressed. In the case of the first embodiment, the background level of the detection signal is about 4% of the detection intensity in the region S25, and a sufficiently high S / N ratio can be obtained.

From these results, the peak wavelength of the light irradiating the fluorescent substance is set to 270 nm, which is substantially insensitive to the CMOS image sensor, and the wavelength of fluorescence emitted from the fluorescent substance is set to 705 nm, which is sufficiently sensitive to the CMOS image sensor. It can be seen that by setting the wavelength to, it is possible to suppress an increase in the background level of the detection signal output from the CMOS image sensor and improve the S / N ratio in the fluorescence detection signal.

<Example 2>
0.5 μL of each of the 1 μM quantum dots and the reference shown below as fluorescent substances was dropped onto the cover member 121 of the color CMOS image sensor, which is the photodetector 106 shown in FIG. 10, and heated at 50 ° C. for 10 minutes. It was dried. As Example 2, a fluorescence image was obtained by irradiating deep ultraviolet light having a peak wavelength of 270 nm from an LED light source. As a comparative example, a bright field image was obtained by irradiating ultraviolet light having a peak wavelength of 405 nm from a light source.

MT9M001C12STC manufactured by Aptina Imaging Co., Ltd. was used as the color CMOS image sensor.

The following (1) was used as a reference, and the following (2) to (6) were used as quantum dots.
(1) TBS
(2) Qdot 525 Streptavidin Conjugate Ex. 425nm / Em. 525nm (Q10141MP, manufactured by Life Technologies)
(3) Qdot 585 Streptavidin Conjugate Ex. 425nm / Em. 585nm (Q10111MP, manufactured by Life Technologies)
(4) Qdot 625 Streptavidin Conjugate Ex. 425nm / Em. 625nm (A10196, manufactured by Life Technologies)
(5) Qdot 705 Streptavidin Conjugate Ex. 425nm / Em. 705nm (Q10161MP, manufactured by Life Technologies)
(6) Qdot 800 Streptavidin Conjugate Ex. 425nm / Em. 800nm (Q10173MP, manufactured by Life Technologies)

FIG. 17 shows the result of Example 2, and FIG. 18 shows the result of Comparative Example. 17 (a) and 18 (a) show images of a fluorescence image and a bright-field image, respectively. The circled numbers 1 to 6 and the wavelength values on the image correspond to the reference of (1) and the quantum dots of (2) to (6), respectively. 17 (b) and 18 (b) show intensity profiles on the X (1) axis shown by dotted lines on the images of FIGS. 17 (a) and 18 (a), respectively. 17 (c) and 18 (c) show the intensity profile on the X (2) axis shown by the dotted line on the images of FIGS. 17 (a) and 18 (a).

In the comparative example shown in FIG. 18, the blue detection intensity is high in both the reference (1) and the quantum dots (2) to (6) above, and the blue detection sensitivity is also high in the surrounding area. became. This is because, as shown in FIG. 12, the peak wavelength of ultraviolet light of 405 nm overlaps with the blue spectral sensitivity of the photodetector 106, so that the background level of the detection signal output from the photodetector 106 becomes high. it is conceivable that. On the other hand, in the reference and quantum dot regions, the detection intensities of red, green, and blue were lower than the detection intensities around them. As a result, the bright field image of the reference and the quantum dot could be confirmed.

In Example 2 shown in FIG. 17, the fluorescence emitted from the quantum dots (2) to (6) was detected in a color corresponding to the wavelength, and the detection intensity around the quantum dots became extremely low. In addition, the reference (1) that does not emit fluorescence was hardly detected. Therefore, the S / N ratio of the fluorescence detection signal was improved, and the fluorescent substance could be detected with high detection sensitivity. Further, by using the photodetector 106 of the third embodiment, it was possible to acquire both a bright field image and a fluorescence image of a plurality of colors by changing the wavelength of the light source.

<Example 3>
The fluorescent beads as the fluorescent substance shown in (2) to (5) below and the non-fluorescent beads shown in (1) below are each mixed by 10 μL to prepare a 50 μL solution, which is shown in FIG. It was dropped onto the cover member 121 of the color CMOS image sensor, which is the photodetector 106, heated at 50 ° C. for 30 minutes, and dried and fixed. As Example 3, a fluorescence image was obtained by irradiating a light source with deep ultraviolet light having a peak wavelength of 270 nm. As a comparative example, a bright field image was obtained by irradiating ultraviolet light having a peak wavelength of 405 nm from a light source.

The following (1) was used for the non-fluorescent beads, and the following (2) to (5) were used for the fluorescent beads.
(1) 15 μm polystyrene non-fluorescent beads (18328, manufactured by Polyscience)
(2) 15 μm polystyrene fluorescent beads Ex. 365 nm / Em. 415 nm (F-8837, manufactured by Life Technologies, Inc.)
(3) 15 μm polystyrene fluorescent beads Ex. 505 nm / Em. 515 nm (F-8844, manufactured by Life Technologies, Inc.)
(4) 15 μm polystyrene fluorescent beads Ex. 540 nm / Em. 560 nm (F-8841, manufactured by Life Technologies, Inc.)
(5) 15 μm polystyrene fluorescent beads Ex. 580 nm / Em. 605 nm (F-8842, manufactured by Life Technologies, Inc.)

FIG. 19 (a) shows the results of the comparative example, and FIG. 19 (b) shows the results of the third embodiment. 19 (a-1) and (b-1) show the whole image of the bright field image and the fluorescent image, respectively, and FIGS. 19 (a-2) and 19 (b-2) are shown in FIG. 19 (a-1). And (b-1) shows a partially enlarged image. The numbers 1 to 5 on the image of FIG. 19 (b-2) correspond to the non-fluorescent beads of (1) and the fluorescent beads of (2) to (5). In addition, the intensity profile on the dotted line crossing the non-fluorescent beads (1) and the fluorescent beads (2) to (5) on the image of FIG. 19 (b-2) is shown in FIG. In FIG. 20, the position on the dotted line in FIG. 19 (b-2) is set as the X coordinate.

As shown in FIG. 19A, by irradiating ultraviolet light having a peak wavelength of λpeak of 405 nm, the photodetector 106 obtains bright-field images of the non-fluorescent beads (1) and the fluorescent beads (2) to (5). I was able to get it. As shown in FIG. 19B, the fluorescent images of the fluorescent beads (2) to (5) could be obtained by the photodetector 106 by irradiating with deep ultraviolet light having a peak wavelength of λpeak of 270 nm. As shown in FIG. 20, in the photodetector 106, the fluorescence emitted from the fluorescent beads (2) to (5) was detected in a color corresponding to the wavelength, and the detection intensity around the fluorescent beads became extremely low. .. Therefore, the S / N ratio of the fluorescence detection signal was improved, and the fluorescent substance could be detected with high detection sensitivity. Further, by using the photodetector 106 of the third embodiment, it was possible to acquire both a bright field image and a fluorescence image of a plurality of colors by changing the wavelength of the light source.

<Example 4>
As shown in FIG. 21, a composite in which the biotin-binding primary antibody 321 captured in the streptavidin-bound fluorescent magnetic particles 320 and the antigen 331 are bound, and the secondary antibody 341 labeled with the enzyme 3411 is further bound to the antigen 331. A body was generated, and a solution in which this complex was suspended in a fluorescent substrate solution was prepared. A fluorescent substance is produced by the reaction between the fluorescent substrate and the enzyme 3411. Specifically, using an automatic pretreatment device, 50 μL of an antibody solution for antigen capture (HISCL®-2000i R1 reagent, manufactured by Sysmex Corporation) and 0 IU / mL or 2500 IU / mL recombinant HBs antigens ( 20 μL of HISCL HBsAg (manufactured by Sysmex Corporation) was reacted at 42 ° C. for 3 minutes. 30 μL of a magnetic bead suspension (R2 reagent for HISCL-2000i, manufactured by Sysmex Corporation) was added, reacted at 42 ° C. for 3 minutes, and then magnetically separated. By this reaction, as shown in FIG. 20 (I), the antigen-capturing antibody was captured by the magnetic beads.

Then, a combination cleaning of 300 μL of the cleaning liquid and magnetic separation was performed twice in total. 100 μL of an ALP-labeled antibody solution (R3 reagent for HISCL-2000i, manufactured by Sysmex Corporation) was added, reacted at 42 ° C. for 3 minutes, and then magnetically separated. As a result, as shown in (II) of FIG. 20, the enzyme-labeled secondary antibody bound to the antigen. Then, a combination cleaning of 300 μL of the cleaning liquid and magnetic separation was performed a total of three times. Further, a combination cleaning of 150 μL of the cleaning liquid and magnetic separation was performed. Then, 50 μL of a dispersion liquid (R4 reagent for HISCL-2000i, manufactured by Sysmex Corporation) was added, and the mixture was mixed and stirred. Further, 20 μL of AttoPhos (registered trademark) solution (S1000, manufactured by Promega Co., Ltd.), which is a fluorescent substrate for ALP, was added, and the mixture was mixed and stirred.

A color CMOS image of the photodetector 106 shown in FIG. 10 showing a mixed solution in which 0 IU / mL recombinant HBs antigen was reacted as a negative control and a mixed solution in which 2500 IU / mL recombinant HBs antigen was reacted as a positive control. 2 μL of each was dropped onto the sensor. Then, a fluorescence image was acquired by irradiating deep ultraviolet light with a peak wavelength of 270 nm with an LED light source. The fluorescence image is shown in FIG. Further, the intensity profile (pixel value) on the X axis in the fluorescence image of FIG. 22 is shown in FIG.

As shown in FIGS. 22 and 23, in the fluorescent image of the negative control, since the amount of the fluorescent substance is small, the image has a low detection intensity as a whole. On the other hand, since the positive control contains a large amount of fluorescent substance produced by the reaction between the enzyme label and the fluorescent substrate, a color image having high detection intensity and different colors according to the wavelength was obtained.

1 Test substance detection device 1a 1st unit 1b 2nd unit 2 Signal conversion device 3 Image processing device 3a Display unit 6 Photodetector 11 Light source 12 Light diffusion member 13 Test substance holding unit 13a Base material 14 1st protective layer 15 Second protective layer 16 Light receiving part 17 Housing 17a Window part 17b Signal line lead-out hole 18 Bushing 21 Capture substance 31 Test substance 41,241 Coupled substance 106 Photodetector 116 Light receiving part 119 Color filter 161 Photoelectric conversion element 162 Support substrate 162a Resin Part 164 Wire 242 Fluorescent substrate 243 Fluorescent substance 331 Quantum dot 410 Quantum dot 411 Core 412 Shell 413 Organic layer 414 Modulator 2411 Enzyme 3411 Enzyme

Claims (19)

This is a test substance detection method for detecting a test substance contained in a biological sample.
A first complex containing a first fluorescent substance and a test substance that emits light having a second peak wavelength of 450 nm or more and 900 nm or less when irradiated with light having a first peak wavelength of 240 nm or more and 300 nm or less, and the first peak. A second complex containing a second fluorescent substance and a test substance that emit light having a fourth peak wavelength of 450 nm or more and 900 nm or less, which is a wavelength different from the second peak wavelength when irradiated with light of a wavelength. And the step of irradiating the sample containing the above with light of the first peak wavelength.
From the second peak wavelength light emitted from the first fluorescent substance irradiated with the light of the first peak wavelength and the second fluorescent substance irradiated with the light of the first peak wavelength by the light detector. A step of detecting the emitted light having the fourth peak wavelength and obtaining an image of a fluorescence image, and
A step of irradiating the complex with light having a third peak wavelength different from the first peak wavelength,
A step of obtaining a bright-field image of the test substance irradiated with light having the third peak wavelength by the photodetector is provided.
The photodetector has a quantum efficiency in the entire peak wavelength range of 450 nm or more and 900 nm or less, which is more than twice the quantum efficiency in the first peak wavelength.
The photodetector detects the light having the second peak wavelength and the light having the fourth peak wavelength as light having a peak wavelength in the range of 450 nm or more and 900 nm or less.
Test substance detection method. The third peak wavelength has a longer wavelength than the first peak wavelength.
The method for detecting a test substance according to claim 1. The photodetector identifies and detects light of a plurality of colors having a peak wavelength in the range of 450 nm or more and 900 nm or less.
The test substance detection method according to claim 1 or 2. The photodetector is arranged between a light receiving portion that receives light, the light receiving portion, the first fluorescent substance, and the second fluorescent substance, and the light emitted from the first fluorescent substance and the second fluorescent substance. Equipped with a filter that transmits a part of
The method for detecting a test substance according to claim 3. The filter includes a first filter that transmits light having a wavelength in the first range, a second filter that transmits light having a wavelength in a second range different from the first range, and the first and second filters. Includes a third filter that transmits light in a third range of wavelengths that is different from the range of
The method for detecting a test substance according to claim 4. The first filter transmits light having a blue wavelength, the second filter transmits light having a green wavelength, and the third filter transmits light having a red wavelength.
The method for detecting a test substance according to claim 5. The filter is an absorption filter.
The test substance detection method according to any one of claims 4 to 6. The photodetector has a quantum efficiency of less than 10% at the first peak wavelength.
The test substance detection method according to any one of claims 1 to 7. The photodetector has a quantum efficiency of less than 5% at the first peak wavelength.
The method for detecting a test substance according to claim 8. The photodetector includes a photoelectric conversion element using a silicon substrate.
The test substance detection method according to any one of claims 1 to 9. The photodetector is a microPMT, a PiN photodiode, an APD, an MPCC, an EMCCD, a CCD image sensor, a CMOS image sensor, or an NMOS image sensor.
The method for detecting a test substance according to claim 10. The photodetector is a microPMT or CMOS image sensor.
The method for detecting a test substance according to claim 11. The fluorescent substance is a quantum dot or an organic dye.
The test substance detection method according to any one of claims 1 to 12. When the light of the first peak wavelength is irradiated, the photodetector of the first fluorescent substance, the second fluorescent substance , and the photodetector in the direction of being irradiated with the light of the first peak wavelength. Arranged in order,
The test substance detection method according to any one of claims 1 to 13. The test substance is a nucleic acid, protein, or peptide.
The test substance detection method according to any one of claims 1 to 14. The step of forming the complex by binding the fluorescent substance and the test substance is further included.
The test substance detection method according to any one of claims 1 to 15. This is a test substance detection method for detecting a test substance contained in a biological sample.
When the light of the first peak wavelength of 240 nm or more and 300 nm or less is irradiated, which is generated by the reaction of the enzyme and the substrate in the first complex containing the test substance and the enzyme, the second peak wavelength of 450 nm or more and 900 nm or less It is generated by the reaction between the first fluorescent substance that emits light and the enzyme in the second complex containing the test substance and the enzyme, and is irradiated with light having a first peak wavelength of 240 nm or more and 300 nm or less. A step of irradiating a sample containing a second fluorescent substance having a wavelength different from the second peak wavelength and emitting light having a fourth peak wavelength of 450 nm or more and 900 nm or less with light having the first peak wavelength. ,
From the second peak wavelength light emitted from the first fluorescent substance irradiated with the light of the first peak wavelength and the second fluorescent substance irradiated with the light of the first peak wavelength by the light detector. A step of detecting the emitted light having the fourth peak wavelength and obtaining an image of a fluorescence image, and
A step of irradiating the complex with light having a third peak wavelength different from the first peak wavelength,
A step of obtaining a bright-field image of the test substance irradiated with light having the third peak wavelength by the photodetector is provided.
The photodetector has a quantum efficiency in the entire peak wavelength range of 450 nm or more and 900 nm or less, which is more than twice the quantum efficiency in the first peak wavelength.
The photodetector detects the light having the second peak wavelength and the light having the fourth peak wavelength as light having a peak wavelength in the range of 450 nm or more and 900 nm or less.
Test substance detection method. The enzyme is peroxidase or alkaline phosphatase,
The method for detecting a test substance according to claim 17. When the light of the first peak wavelength of 240 nm or more and 300 nm or less is irradiated, the first fluorescent substance which emits the light of the second peak wavelength of 450 nm or more and 900 nm or less, and the second when the light of the first peak wavelength is irradiated. A step of irradiating a sample containing a second fluorescent substance having a wavelength different from the peak wavelength and emitting light having a fourth peak wavelength of 450 nm or more and 900 nm or less with light having the first peak wavelength.
From the second peak wavelength light emitted from the first fluorescent substance irradiated with the light of the first peak wavelength and the second fluorescent substance irradiated with the light of the first peak wavelength by the light detector. A step of detecting the emitted light having the fourth peak wavelength and obtaining an image of a fluorescence image, and
A step of irradiating the fluorescent substance with light having a third peak wavelength different from the first peak wavelength,
A step of obtaining a bright-field image of the fluorescent substance irradiated with light having the third peak wavelength by the photodetector is provided.
The photodetector has a quantum efficiency in the entire peak wavelength range of 450 nm or more and 900 nm or less, which is more than twice the quantum efficiency in the first peak wavelength.
In the step of obtaining the image of the fluorescence image, the light of the second peak wavelength and the light of the fourth peak wavelength are used as light of a plurality of colors having a peak wavelength in the range of 450 nm or more and 900 nm or less by the photodetector. , Detect,
Fluorescence detection method.

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