JP7356891B2 - Particle analyzer and particle analysis method - Google Patents

Description

The present invention relates to a particle analysis device and a particle analysis method for analyzing particles.

For example, exosomes secreted from abnormal cells such as cancer cells are thought to have the ability to act on other normal cells and cause them to become cancerous. In addition, exosomes are thought to reflect the characteristics of abnormal cells, and they contain nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins derived from abnormal cells. .

As shown in Patent Document 1, a diagnostic device is being considered that diagnoses the presence or absence of abnormal cells, that is, a disease, by analyzing microRNA present inside these exosomes.

JP2018-191636A

On the other hand, the inventor of the present application has discovered that tetraspanins (CD63, CD81, CD9, etc.), transmembrane proteins (CD13, etc.), membrane transport/binding proteins, etc., present on the surface of exosomes, rather than nucleic acids and proteins contained inside exosomes. We are considering detecting the presence or absence of abnormal cells using exosomes themselves as biomarkers, focusing on surface molecules such as (annexin, etc.), cell adhesion molecules (MFG-E8, etc.), sugar chains, or lipid rafts (cholesterol, etc.). ing.

Here, since the surface molecules of exosomes differ depending on the abnormal cell in which the exosomes are secreted, it is necessary to examine the state of the surface molecules depending on the disease to be diagnosed. However, some exosome surface molecules appear only in specific abnormal cells, while others are thought to be common to multiple abnormal cells. Therefore, testing only one surface molecule of exosomes may not necessarily be able to detect the presence or absence of abnormal cells characterized by that surface molecule, and may falsely detect the presence or absence of other abnormal cells. There is a fear.

Therefore, the main objective of the present invention is to enable efficient analysis of particles such as exosomes derived from a plurality of abnormal cells.

That is, the particle analyzer according to the present invention applies light having an excitation wavelength corresponding to each of the plurality of fluorescent markers to a sample in which a plurality of different surface molecules present on particles are modified with a plurality of different fluorescent markers. a light irradiation unit that irradiates light, a light detection unit that detects light emission from each of the plurality of fluorescent markers, and an analysis unit that analyzes particles modified with each of the fluorescent markers using a detection signal obtained by the light detection unit. The photodetection unit outputs moving image data as the detection signal, and the analysis unit tracks Brownian motion of particles modified with each fluorescent marker using the moving image data. The present invention is characterized in that the particle size distribution of the particles modified with each of the fluorescent markers is calculated from the diffusion rate.

With this particle analyzer, a sample whose particle surface molecules are modified with multiple fluorescent markers is irradiated with light of an excitation wavelength corresponding to each of the multiple fluorescent markers, so that particles modified with each fluorescent marker can be can be analyzed separately. For example, if the particles are exosomes, the abnormal cells that secreted each exosome can be analyzed, and diseases can be diagnosed from the analysis results. For example, if a fluorescent marker is attached to a surface molecule that appears only on a specific abnormal cell, the presence or absence of that specific abnormal cell can be detected. Furthermore, if a fluorescent marker is attached to a surface molecule that commonly appears on a plurality of abnormal cells, the presence or absence of a specific abnormal cell can be detected by combining the detection results of the plurality of fluorescent markers. In particular, the present invention uses particle tracking analysis (PTA or nano tracking analysis; NTA), and the analysis section uses moving image data to determine the brown color of exosomes modified with each fluorescent marker. Since the movement is tracked and the particle size distribution of the exosomes modified with each fluorescent marker is calculated from the diffusion rate, the particle size distribution of each exosome modified with each fluorescent marker can be measured individually. This makes it easier to analyze abnormal cells that secrete exosomes.

Here, if the plurality of fluorescent markers are modified with cancer-derived surface molecules in the exosome, they can be used for diagnosis and monitoring of cancer. In addition, by using a marker for a disease other than cancer or a marker for diagnosing a disease other than cancer as a fluorescent marker, diagnosis or monitoring of a disease other than cancer, or diagnosis for a disease other than cancer can be performed.

In order to accurately analyze particles modified with each fluorescent marker, it is desirable that the light irradiation unit sequentially irradiates light with an excitation wavelength corresponding to each fluorescent marker.

Preferably, the photodetector includes a filter that blocks light at the excitation wavelength.
With this configuration, scattered light of the excitation wavelength can be removed, so particles modified with each fluorescent marker can be analyzed with high accuracy.

Furthermore, the particle analysis method according to the present invention includes a modification step of modifying a plurality of mutually different surface molecules present on the particle with a plurality of mutually different fluorescent markers, and a modification step of modifying the plurality of surface molecules present on the particle with a plurality of different fluorescent markers. irradiating light with an excitation wavelength corresponding to each of the fluorescent markers, detecting the light emission of each of the plurality of fluorescent markers to obtain moving image data, and using the moving image data, each fluorescent marker is modified. The present invention is characterized by comprising an analysis step of tracking the Brownian motion of the particles and calculating the particle size distribution of the particles modified with each of the fluorescent markers from the diffusion rate thereof.

In order to generate a sample by removing impurities in a biological sample containing exosomes as the particles, a separation step of centrifuging the biological sample and a modification step are performed on the supernatant obtained by the separation step. It is desirable that this be done by adding the plurality of fluorescent markers.

In order to determine whether particles such as exosomes have been separated by the separation process and to efficiently analyze the particles, the particle analysis method requires particle size distribution measurement on the supernatant obtained from the separation process. It is desirable to further include a determination step of determining whether the supernatant contains particles of a predetermined particle size.

In order to improve the separation and recovery rate of exosomes, the separation step includes a first separation step of centrifuging the biological sample, and a supernatant obtained in the first separation step at a higher concentration than the first separation step. It is desirable to include a second separation step of rotating and centrifuging.

According to the present invention described above, particles such as exosomes derived from a plurality of abnormal cells can be efficiently analyzed.

1 is an overall schematic diagram of an exosome analysis device according to an embodiment of the present invention. It is a schematic diagram showing the particle size distribution of exosomes modified with each fluorescent marker of the same embodiment. It is a figure showing the procedure of the exosome analysis method of the same embodiment. FIG. 2 is a schematic diagram showing the relationship between the sizes of particles and cells in blood and fluorescent markers modified on each surface molecule.

Hereinafter, an exosome analyzer 100, which is one aspect of a particle analyzer according to an embodiment of the present invention, will be described with reference to the drawings.

The exosome analyzer 100 of this embodiment analyzes exosomes contained in body fluid samples such as blood and urine, and uses a sample in which a plurality of different surface molecules present in the exosomes are modified with a plurality of different fluorescent markers. Exosomes are analyzed by irradiating them with light and detecting the light emitted by each of multiple fluorescent markers.

Here, the surface molecules present in exosomes include tetraspanins (CD63, CD81, CD9, etc.), transmembrane proteins (CD13, etc.), membrane transport/binding proteins (Annexin, etc.), cell adhesion molecules (MFG-E8, etc.), and sugars. chains or lipid rafts (such as cholesterol). Furthermore, the fluorescent marker is one that binds to, for example, cancer-derived molecules on the surface of exosomes, and the surface molecules of exosomes are modified with fluorescent labels using antibodies.

Specifically, as shown in FIG. 1, the exosome analyzer 100 includes a light irradiation unit 2 that irradiates light with an excitation wavelength corresponding to each of a plurality of fluorescent markers, and a light detection unit that detects light emission from each of the plurality of fluorescent markers. 3, and an analysis section 4 that analyzes exosomes modified with each fluorescent marker using the detection signal obtained by the photodetection section 3.

Further, the exosome analyzer 100 is provided with a cell installation section (not shown) in which a cuvette cell 5 for batch measurement containing the sample is removably installed. In FIG. 1, the light irradiation section 2 and the light detection section 3 are provided such that the light irradiation direction of the light irradiation section 2 and the light detection direction of the light detection section 3 are orthogonal to each other, but the present invention is not limited to this.

The light irradiation unit 2 includes a plurality of light sources 21, 22, and 23 that irradiate light with excitation wavelengths corresponding to each of the plurality of fluorescent markers. The light sources 21, 22, and 23 are, for example, laser light sources. Since this embodiment uses three fluorescent markers, it has three light sources 21, 22, and 23. Note that one light source may irradiate light including three excitation wavelengths. Further, the number of light sources may be three or more.

The first light source 21 emits light with an excitation wavelength λ ₁ of the first fluorescent marker, and the second light source 22 emits light with an excitation wavelength λ ₂ of the second fluorescent marker. The third light source 23 emits light having an excitation wavelength λ ₃ of the third fluorescent marker. Here, the excitation wavelengths λ ₁ , λ ₂ , and λ ₃ are different from the fluorescence wavelengths λ ₁ ′, λ ₂ ′, and λ ₃ ′ generated by the three fluorescent markers emitting light. Further, the light emitted from each of these light sources 21, 22, 23 is guided to the cuvette cell 5 via the irradiation optical system 24, which includes reflective mirrors 241, 242, half mirrors 243, 244, condensing lens 245, etc. .

These light sources 21, 22, and 23 are controlled by a control unit (not shown) so as to sequentially irradiate light having excitation wavelengths λ ₁ , λ ₂ , and λ ₃ corresponding to each fluorescent marker. Note that these light sources 21, 22, and 23 can also be controlled so that they emit light at the same time.

The light detection unit 3 detects the light emitted from each of the plurality of fluorescent markers emitted from the cuvette cell 5, and in this embodiment, is an imaging camera 31 such as a CCD camera, and outputs moving image data as a detection signal. do. Further, the photodetector 3 includes a filter 33 that blocks light with excitation wavelengths λ ₁ , λ ₂ , and λ ₃ and transmits light with fluorescence wavelengths λ ₁ ′, λ ₂ ′, and λ _{3 ′} . This filter 33 is configured to selectively detect the light emitted from each fluorescent marker.

The analysis unit 4 tracks the Brownian motion of exosomes modified with each fluorescent marker using video image data, and determines the particle size and number of exosomes based on the Stokes-Einstein formula from the diffusion rate. Calculate. Then, the analysis unit 4 calculates the particle size distribution of exosomes modified with each fluorescent marker from the calculation results (see FIG. 2). For example, the analysis unit 4 calculates the diffusion rate of the exosome modified with the first fluorescent marker using the moving image data obtained by imaging the light emission from the first fluorescent marker, and determines the particle size of the exosome from the diffusion rate. Calculate the diameter distribution. The same applies to other fluorescent markers.

Next, the exosome analysis method of this embodiment will be explained with reference to FIG. 3.

(S1) Separation step First, a biological sample, which is a body fluid such as blood or urine, is centrifuged to separate exosomes. Specifically, a first separation step (S1-1) in which a biological sample is centrifuged, and a second separation step in which the supernatant obtained in the first separation step is centrifuged at a higher rotation speed than in the first separation step. (S1-2). Note that the second separation step may involve ultracentrifugation. Further, the separation step may be performed using a column, a filter, or a microchannel.

(S2) Determination step Particle size distribution measurement is performed on the supernatant obtained in the above separation step (second separation step), and it is determined whether the supernatant contains particles with a predetermined particle size. Here, the device used for particle size distribution measurement may be one using the nanotracking method described above, or may be of a laser diffraction/scattering type or a dynamic light scattering type.

As a result of this judgment step, if particles with a predetermined particle diameter are included, the process moves to the next detection step. On the other hand, if particles with a predetermined particle size are not included, the supernatant is either subjected to the second separation step again or discarded.

(S3) Modification step A plurality of fluorescent markers are added to the supernatant obtained in the separation step. Here, the plurality of fluorescent markers bind to cancer-derived surface molecules in the exosome. As a result, a plurality of different surface molecules present on the exosome are modified with a plurality of different fluorescent markers.

(S4) Analysis step The sample obtained in the modification step is accommodated in the cuvette cell 5, and the cuvette cell 5 is installed in the cell installation section of the exosome analyzer 100. Thereafter, in the exosome analyzer 100, the cuvette cell 5 is irradiated with light having an excitation wavelength corresponding to each of the plurality of fluorescent markers, and the luminescence of each of the plurality of fluorescent markers is detected. Thereby, the analysis unit 4 calculates the particle size distribution of the exosomes modified with each fluorescent marker based on the detection signal (moving image data) obtained by the photodetection unit 3. The calculated particle size distribution of each exosome (see FIG. 2) is displayed on the display of the exosome analyzer 100 or the like.

Although exosomes can be separated to some extent by centrifugation, particles with the same physical properties (e.g., viruses, destroyed cell debris, etc.) are also included in the supernatant (separation layer) (see Figure 4). However, in this embodiment, since a fluorescent marker is attached to an antibody against a surface molecule of a specific exosome, only a specific exosome can be specifically detected by antigen-antibody reaction and receptor binding.

<Effects of this embodiment>
According to the exosome analyzer 100 of this embodiment configured in this way, a sample in which surface molecules of exosomes are modified with a plurality of fluorescent markers is irradiated with light having an excitation wavelength corresponding to each of the plurality of fluorescent markers. , each fluorescent marker-modified exosome can be analyzed separately. Therefore, abnormal cells that secrete each exosome can be analyzed, and diseases can be diagnosed from the analysis results. For example, if a fluorescent marker is attached to a surface molecule that appears only on a specific abnormal cell, the presence or absence of that specific abnormal cell can be detected. Furthermore, if a fluorescent marker is attached to a surface molecule that commonly appears on a plurality of abnormal cells, the presence or absence of a specific abnormal cell can be detected by combining the detection results of the plurality of fluorescent markers.

<Other modified embodiments>
Note that the present invention is not limited to the above embodiments.

For example, the exosome analyzer 100 of the embodiment uses the nanotracking method (NTA), but it uses a dynamic scattering method that calculates the particle size distribution based on fluctuations in the emission intensity accompanying Brownian motion of exosomes. may be used. Moreover, other optical analysis methods can also be applied.

In addition, the exosome analyzer has a diagnosis section that diagnoses a disease based on particle information such as particle size distribution, number of particles, and concentration of exosomes modified with each fluorescent marker obtained by the analysis section 4. It's okay. This diagnostic unit can diagnose a disease using, for example, relational data indicating a correspondence between particle information and a disease stored in the memory.

Although the photodetector 3 in the embodiment described above is an imaging camera such as a CCD camera, it may also be one using a photomultiplier tube (PMT). When PMT is used, sensitivity can be improved compared to when a CCD camera is used, and even a small amount of light emission can be detected. As a result, it becomes possible to diagnose cancer in its early stages, for example.

In the embodiment described above, exosomes are separated by centrifugation, but magnetic beads (magnetic particles) may also be used to separate exosomes. As a method for isolating exosomes using magnetic beads, exosomes are isolated by modifying magnetic beads bound with biotin-labeled anti-exosome antibodies to exosomes. Exosomes are then separated by separating the magnetic beads from the isolated exosomes.

Furthermore, the analysis unit 4 may be configured to measure the zeta potential in addition to the particle diameter and number (concentration) of exosomes. In this case, the average zeta potential of the entire exosome modified with multiple fluorescent markers is measured. Further, it is desirable that the particle size, number (concentration), and zeta potential of exosomes be displayed on one screen, for example, on a three-dimensional graph, on a display device such as a display of the exosome analyzer 100. This allows more accurate analysis of abnormal cells that secreted each exosome. Furthermore, by measuring the zeta potential, the degree of reaction between exosomes and antibodies can be investigated.

Here, as a configuration for measuring the zeta potential, a configuration in which the cuvette cell 5 is provided with an electrode for measuring the zeta potential can be considered. Further, in addition to the cuvette cell 5, a flow cell for continuous measurement may be used. The flow cell is provided with an electrode for measuring zeta potential. When using a flow cell, the flow of the sample into and out of the cell is stopped when measuring the zeta potential, and the sample is allowed to flow after the measurement of the zeta potential is completed.

Although the above embodiment shows an example in which exosomes are analyzed as particles, the present invention can analyze particles other than exosomes, such as microorganisms, cells, intracellular organelles (microendoplasmic reticulum, nucleus, Golgi apparatus, mitochondria, leaflets, etc.). green bodies, etc.), apoptotic bodies, extracellular endoplasmic reticulum (EV), receptors, ligands, antagonists, agonists, nucleic acids, antibodies, proteins, peptides, amines, other biological substances, viruses, inorganic particles, polystyrene beads, etc. It can also be applied to organic particles.

In addition, various modifications and combinations of the embodiments may be made as long as they do not go against the spirit of the present invention.

100...Exosome analyzer (particle analyzer)
2...Light irradiation unit 21...First light source 22...Second light source 23...Third light source 3...Light detection unit 31...Imaging camera 32...Filter 4 ...Analysis Department

Claims (8)

a determination unit that determines whether or not the supernatant contains particles with a predetermined particle size based on the result of particle size distribution measurement of a supernatant obtained by centrifuging a biological sample containing particles;
When the determination section determines that the particles contain particles of a predetermined particle size, a plurality of fluorescent markers are added to the supernatant, and the plurality of different fluorescent markers are added to the plurality of mutually different surface molecules present on the particles. a light irradiation unit that irradiates the modified sample with light having an excitation wavelength corresponding to each of the plurality of fluorescent markers;
a light detection unit that detects light emission from each of the plurality of fluorescent markers;
an analysis section that analyzes particles modified with each of the fluorescent markers using the detection signal obtained by the photodetection section;
The photodetector outputs moving image data as the detection signal,
The analysis section uses the moving image data to track Brownian motion of the particles modified with each fluorescent marker, and calculates the particle size distribution of the particles modified with each fluorescent marker from the diffusion rate. can be,
A particle analysis device that detects the presence or absence of specific abnormal cells based on analysis results obtained by the analysis section. The particle analysis device according to claim 1, wherein the particles are exosomes. The particle analysis device according to claim 2, wherein the plurality of fluorescent markers are modified with cancer-derived surface molecules in the exosome. The particle analysis device according to any one of claims 1 to 3, wherein the light irradiation unit sequentially irradiates light having an excitation wavelength corresponding to each fluorescent marker. The particle analyzer according to any one of claims 1 to 4, wherein the photodetector includes a filter that blocks light at the excitation wavelength. a separation step of centrifuging a biological sample containing particles;
A determination step of performing particle size distribution measurement on the supernatant obtained in the separation step and determining whether or not the supernatant contains particles with a predetermined particle size;
When it is determined that the supernatant contains particles of a predetermined particle size in the determination step, a plurality of fluorescent markers are added to the supernatant, and a plurality of different surface molecules present on the particles are labeled with a plurality of different fluorescent markers. a modification step of modifying the fluorescent marker;
The sample obtained in the modification step is irradiated with light having an excitation wavelength corresponding to each of the plurality of fluorescent markers, and the luminescence of each of the plurality of fluorescent markers is detected to obtain moving image data; an analysis step of tracking the Brownian motion of particles modified with each fluorescent marker using moving image data, and calculating the particle size distribution of the particles modified with each fluorescent marker from the diffusion rate;
A particle analysis method that detects the presence or absence of specific abnormal cells based on the analysis results obtained in the analysis step. 7. The particle analysis method according to claim 6, wherein the separation step involves centrifuging a biological sample containing exosomes as the particles. The separation step includes a first separation step of centrifuging the biological sample, and a second separation step of centrifuging the supernatant obtained in the first separation step at a higher rotation speed than the first separation step. , The particle analysis method according to claim 6 or 7.