WO2008007580A1 - Procédé d'analyse de particules fines - Google Patents

Procédé d'analyse de particules fines Download PDF

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Publication number
WO2008007580A1
WO2008007580A1 PCT/JP2007/063297 JP2007063297W WO2008007580A1 WO 2008007580 A1 WO2008007580 A1 WO 2008007580A1 JP 2007063297 W JP2007063297 W JP 2007063297W WO 2008007580 A1 WO2008007580 A1 WO 2008007580A1
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WIPO (PCT)
Prior art keywords
analysis
fine particles
scanning
region
beads
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PCT/JP2007/063297
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English (en)
Japanese (ja)
Inventor
Tamiyo Kobayashi
Masayoshi Kusano
Morinao Fukuoka
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Olympus Corporation
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Publication of WO2008007580A1 publication Critical patent/WO2008007580A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size

Definitions

  • the present invention relates to a method for analyzing fine particles such as biological materials with high sensitivity and high accuracy.
  • Patent Literature 1 JP 2001-502062
  • Patent Document 2 JP 2001-502066 Publication
  • the obtained data is known to have a large variation. [0004] Therefore, even when analyzing large biological materials such as cell membrane fragments, cells themselves, etc. having a diameter of 0.3 / zm or more, the obtained data is highly variable and highly accurate. Analysis cannot be performed. In particular, when preparing cell membrane fragments by cell membrane fractionation etc., it is difficult to homogenize the size of the cell membrane fragments, and these cell membrane fragments are often spherical with a diameter of 0.1 to 0.5 m. 0. Contains large molecules or particles of 3 m or more.
  • FCS single-molecule measurement methods
  • FIDA fluorescence intensity does not change more than 1.3 times before and after the interaction, the interaction cannot be detected.
  • a method may be used in which one of the interacting molecules is fixed to, for example, the surface of polystyrene particle beads and the interaction is detected.
  • Patent Document 1 In order to obtain highly accurate data, there is a limit to the size of a biological substance that can be measured. There was great variation, and it was only possible to scan the same area in a donut shape.
  • Non-Patent Document 1 Patent Documents 1 and 2 do not describe appropriate conditions such as scan speed and scan area when measuring beads with a size of 0.3 / zm or more. I got it. Means for solving the problem
  • the present invention has been made in view of the above circumstances, and even when the analysis target includes a size of 0.3 ⁇ m or more, the size of the analysis target microparticles or the binding of the microparticles Another problem is to provide a method for analyzing the presence or absence of deviation with high sensitivity and high accuracy. And
  • the present invention relates to a fine particle analysis method for scanning a minute region in a confocal region and measuring the signal strength of the minute region, and analyzing the size of the fine particle or the presence or absence of binding or detachment of the fine particle.
  • Fine particles whose volume of one micro area of a plurality of micro areas to be measured is 1 FL or less and whose total area area of the scanning area plane of the plurality of micro areas is 500 ⁇ m 2 to 40000 ⁇ m 2 This is an analysis method.
  • the scanning speed of one minute region for measuring the signal intensity may be 2.5 mmZsec to 25 mmZsec.
  • the analysis may be performed by at least one of fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and fluorescence polarization intensity distribution analysis.
  • the fine particles may be one or more selected from cells, cell membranes, cell membrane fragments, organic particles, inorganic particles, and one or more complex forces of these.
  • the size force of the fine particles may be 0.3 / ⁇ ⁇ to 10 / ⁇ ⁇ .
  • an analysis object includes a particle having a size of 0.3 m or more
  • the size of the particle to be analyzed or the presence or absence of the binding or detachment of the particle is highly sensitive and high. It can be analyzed with accuracy.
  • FIG. 1 is an explanatory diagram illustrating a comparison between the size of a fine particle that can be analyzed and analysis conditions.
  • FIG. 2A is a diagram illustrating a scanning method in the prior art.
  • FIG. 2B is a diagram illustrating a scanning method in the prior art.
  • FIG. 2C is a diagram illustrating a scanning method in the present invention.
  • FIG. 2B is a diagram illustrating a scanning method in the present invention.
  • FIG. 2C is a diagram illustrating a scanning method in the present invention.
  • FIG. 3A is a graph showing the analysis results of Example 1.
  • FIG. 3B is a graph showing the analysis results of Example 1.
  • FIG. 3C is a graph showing the analysis results of Comparative Example 1.
  • FIG. 4A is a graph showing the results of five measurements of Example 2 and Comparative Example 2.
  • FIG. 4B is a graph showing five measurement results of Example 2 and Comparative Example 2.
  • FIG. 5A is a graph showing an average value of five measurement results of Example 2 and Comparative Example 2.
  • FIG. 5B is a graph showing an average value of five measurement results of Example 2 and Comparative Example 2.
  • FIG. 6 is a graph showing the analysis results of Example 3.
  • FIG. 7 is a graph showing the analysis results of Example 4.
  • FIG. 8 is a measurement image obtained in Example 5.
  • the volume of the minute region for measuring the signal intensity can be adjusted, for example, by changing the magnification of the objective lens to be used.
  • the volume of one minute region (each minute region in a plurality of minute regions) for measuring the signal intensity is set to 1 FL (humid torr) or less. If it is larger than 1FL, it may be difficult to detect the signal intensity with high sensitivity.
  • the total area value of the scanning region planes of the plurality of minute regions for measuring the signal intensity is set to 500 ⁇ m 2 to 40000 m 2 . Outside this range, the detection value of the signal intensity has a large variation, resulting in low reliability.
  • the scanning speed of one minute region may be adjusted as appropriate, but is preferably 2.5 mmZ seconds to 25 mmZ seconds. Within this range, the detected value of the signal intensity has a smaller variation, which is suitable for highly accurate analysis.
  • FCS fluorescence correlation spectroscopy
  • FIDA fluorescence intensity distribution analysis method
  • FIDA-polarization fluorescence polarization intensity distribution analysis method
  • the detected signal intensity changes depending on the size of the fine particles to be analyzed. Therefore, if the microparticles to be analyzed bind to other microparticles or dissociate into a plurality of microparticles, and the size changes, the binding or dissociation can be detected by the change in signal intensity.
  • the term “bond” as used herein refers to a bond based on an intermolecular attractive force such as a hydrogen bond or a hydrophobic bond that is not only a covalent bond.
  • the fine particles used as the analysis target are not particularly limited, and can be the analysis target by a conventional method, for example, a particle having a size of 0.1 m or less may be the analysis target.
  • a large size for example, a size of 0.3 m to 10 ⁇ m, which has been difficult to achieve, is suitable.
  • examples of such materials include biological substances, organic particles, inorganic particles, and one or more selected from complex forces that are one or more of these.
  • Preferred examples of the biological substance include cells, cell membranes and cell membrane fragments.
  • these fine particles those prepared by a conventionally known method or commercial products can be used. For example, as long as it is a bio-related substance, it is possible to use a sample extracted from the sample obtained by processing a sample obtained by collecting vital force by a conventionally known method.
  • Receptors present on the cell membrane such as GPCR, are thought to have many orphan receptors, but there are also important receptors that are targets for drug discovery. For this reason, recently, cell membrane fractions are often used to search for inhibitors or ligands. Thus, cells, cell membranes, cell membrane fragments and the like having important functions and large sizes are particularly suitable as the analysis target of the present invention.
  • the cell membrane usually exists as a ribosome in a solution.
  • membrane fractionation is performed by a conventionally known method, cell membrane fragments are obtained, and their sizes often vary by about 0.1 to 0.5 m in diameter.
  • Such items are conventionally labeled with radioisotopes (RI), spun down, and detected with a scintillation counter.
  • RI radioisotopes
  • analysis using RI is the mainstream.
  • the conventional single molecule measurement method is applied, only data with large variations can be obtained.
  • Fig. 1 shows an example of a comparison of the size of the fine particles that can be analyzed and the analysis conditions between the conventional method and the method of the present invention.
  • the scanning speed is approximated to a straight line because scanning is performed in a donut shape.
  • various biological substances fixed on the surface of beads having a diameter of 0.3 m to 10 m can be suitably used regardless of their sizes.
  • particle assembly of biological materials bound to such beads can be performed with high accuracy.
  • an antibody or antigen is immobilized on the bead surface and a biological substance is detected by forming a specific bond between the antigen and antibody
  • the larger the bead particle size the greater the number of antibodies or antigens that can be immobilized on the bead surface.
  • the present invention is particularly suitable when such large beads, particularly beads having a diameter of 0.3 ⁇ m to 10 ⁇ m are used.
  • beads having a large particle diameter is also preferable in terms of easy preparation and handling of beads having a biological substance immobilized thereon.
  • beads with a small particle size for example, those with a diameter of 0.3 / zm or less
  • the beads to be analyzed that have settled in the lower layer may spread immediately to the upper layer after centrifuge processing. For this reason, the beads to be analyzed are removed together with the upper layer during recovery and washing, and this causes the disadvantage that the concentration of biological substances cannot be measured accurately. This is done using beads with a diameter of 0.3 / zm or more. If the analysis method of the invention is applied, such inconvenience does not occur.
  • any material can be used as long as it does not affect the measurement of signal intensity such as fluorescence intensity.
  • various materials such as polystyrene are used.
  • Conventionally known ones such as plastic beads or metal beads may be used.
  • a conventionally well-known method may be applied also when immobilizing a biological substance or the like to be analyzed on beads. It should be noted that here, mainly bio-related substances are immobilized on the bead surface.
  • the force fixed to the bead surface is not limited to a biological substance as long as it is an analysis object of the present invention, and any material may be used.
  • conventionally known fluorescent dyes can be used, and examples thereof include fluorescein, rhodamine, alexa, ATTO dye and the like.
  • FIGS. 2A to 2E are diagrams illustrating scanning methods.
  • FIG. 2A is a point scan
  • FIG. 2B is a scan of the center of the prior art
  • a donut scan
  • FIGS. 2C to 2E are main scans.
  • Each preferred scan of the invention is shown.
  • a plurality of microscopic regions arranged in a straight line (a plurality of egg shapes in FIG. 2C) are sequentially scanned, and then, adjacent to the plurality of microscopic regions arranged in a straight line.
  • the confocal region is scanned by repeating the operation of sequentially scanning a plurality of minute regions arranged in a straight line.
  • the central force of the scan region is also directed toward the outer peripheral portion, and a plurality of minute regions arranged in a spiral shape are applied to the central portion.
  • a method of scanning forcefully as shown in Fig. 2E, a method of scanning a plurality of minute regions arranged in a spiral shape with the outer peripheral force of the scan area directed toward the central portion, and the outer peripheral force also moving toward the central portion.
  • These spiral scan methods can scan a desired area without changing the scan speed to zero or slowing down in order to change the scan direction. Therefore, it is more preferable because the scanning time can be shortened than when scanning in a straight line.
  • the beam scanner of the prior art uses MF20 (trade name; manufactured by Olympus Corporation), the confocal area is scanned in a donut shape, so the area where data can be acquired is as small as about 100 m 2. ( Figure 2B). Therefore, for example, cells with a size of 0.3 ⁇ m or more, such as cells, cell membranes, cell membrane fragments, or nanoparticles with a diameter of 0.3 / zm or more, have a very low probability of passing through the measurement area. Even if it is possible to measure, there is a large variation and data with high reliability cannot be obtained.
  • the measurement area is simply enlarged, for example, when the confocal area is enlarged by lowering the magnification of the objective lens, it is necessary to label the fine particles! Because the excitation energy is constant for any reason, the signal intensity per particle to be analyzed becomes small, making it impossible to detect with high sensitivity.
  • one confocal region for measuring the signal intensity is set to 1 FL or less, and the scan region is kept as a minimal region. The region is expanded from 500 ⁇ m 2 to 40000 ⁇ m 2 . Therefore, by using FID A, it is possible to detect fine particles having sufficient signal intensity. For example, a change in fluorescence intensity of 1.3 times or more due to the interaction of fine particles can be captured.
  • an analysis object having a diffusion speed of 0. or more with a slow diffusion rate has a sufficiently high probability of passing through the measurement area. Therefore, it is possible to measure with high sensitivity even those with a size exceeding 0.3 m, which was difficult to measure in the past.
  • the scan area is preliminarily set before the measurement is started.
  • the scan is terminated. You can also. In this way, the measurement time can be shortened.
  • one micro area for measuring signal intensity, the total area value of the scanning area plane, and the scanning speed of the micro area are as described above. Therefore, you can set the optimal conditions according to the situation.
  • Cell membrane fractionation was performed according to the following procedure, and cell membrane fragments were obtained as ribosomes having a variation of about 0.1 to 0.5 ⁇ m in diameter.
  • the cell membrane fraction solution 14 obtained above was mixed with 7 L of unlabeled PAF solution 7 adjusted to a concentration of 2 nM to 160 nM and reacted with TAMRA-labeled PAF solution 7 L at 25 ° C for 1 hour.
  • FIGS. 3A to 3C show a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) with a single molecule fluorescence analysis unit. Analysis was performed in the FIDA measurement mode under the following measurement conditions. The results are shown in FIGS. 3A to 3C.
  • Figure 3A shows a total scan area of 500 m 2 and
  • Figure 3B shows a total scan area of 25000 m 2 .
  • Measurement conditions Laser wavelength: 543 nm, laser power: 250 ⁇ W, measurement time: 30 seconds X 5 times, scanning speed: 25 mm / decrease, total scanning area: 500 ⁇ m 2 , 25000 ⁇ rn
  • Measurement conditions Laser wavelength: 543 nm, laser power: 250 ⁇ W, measurement time: 30 seconds x 5 times, beam scanner: 2. 85 mm / second, total scan area: 100 ⁇ rn
  • Example 1 From the results of Example 1 and Comparative Example 1, according to the present invention, if the total value of the scanned area is 500 ⁇ m 2 or more, stable data with small variations and high reproducibility can be obtained. It was. In other words, the data obtained in Example 1 was more reliable than the conventional method, which has no practical problem.
  • Polybead Carboxylate 10. Omicron microspheres, trade name, catalog number 18133; manufactured by Polyscienses) 250 ⁇ L was taken and centrifuged at 500 XG for 5 minutes to obtain purified beads. Next, PolyLink Protein Coupling Kit for COOH Microp Using a kit of articles (trade name, catalog number PL01N; manufactured by Bangs Laboratories), the purified beads were reacted with Alexa647—Albumin (1 ⁇ g), and 200 L of fluorescent bead solution with a diameter of 10 m to which Alexa647 was bound was added. Obtained. A 10-fold diluted solution was used for the following analysis.
  • Streptavidin Coated Microspheres (trade name, catalog number CP01N; manufactured by Bangs Laboratories) 0.49 m was diluted with PBS—0.05% t Ween20 and sonicated for 5 seconds. The washing operation for removing the supernatant was repeated three times. Then, 998 ⁇ L of PBS—0.05% tween20 was added to 10 / z L of PBS—0.05% tween20 solution of the obtained washed beads, followed by 2 ⁇ L of Biotin— ⁇ 065 5 ( After adding 10 m), the total liquid volume was 1000 ⁇ L, and the mixture was reacted at room temperature for 1 hour.
  • Tables 1 and 2 show the results when 0.5 m diameter fluorescent beads are used, and Table 2 shows the results when 10 m diameter fluorescent beads are used.
  • 4A and 4B are graphs showing the fluorescence intensity detection values for the first to fifth times, and FIG. 5A and FIG. 5B are graphs showing the average values of these detection values.
  • Figures 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 m
  • Figures 4B and 5B show the results when using fluorescent beads with a diameter of 10 m.
  • Example 2 Analysis was performed in the FIDA measurement mode in the same manner as in Example 2 except that the single molecule fluorescence analysis system MF20 (trade name; manufactured by Olympus Corporation) was used and the measurement conditions were as follows. The results are shown in Tables 1 and 2, FIGS. 4A, 4B, 5A, and 5B. Table 1 shows the results when fluorescent beads with a diameter of 0.5 m were used, and Table 2 shows the results when fluorescent beads with a diameter of 10 m were used. 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 / m, and FIGS. 4B and 5B show the results when using fluorescent beads with a diameter of 10 / z m.
  • Table 1 shows the results when fluorescent beads with a diameter of 0.5 m were used
  • Table 2 shows the results when fluorescent beads with a diameter of 10 m were used.
  • 4A and 5A show the results when using fluorescent beads with a diameter of 0.5 / m
  • FIGS. 4B and 5B show the results
  • Example 2 it was possible to detect fluorescent beads with a diameter of 0.5 ⁇ m and a diameter of 10 ⁇ m! On the other hand, in Comparative Example 2, fluorescent beads with a diameter of 0.5 / zm may be detected in some cases, but fluorescent beads with a diameter of 10 m that have no reproducibility are completely undetectable. That is, it was confirmed that the present invention has higher sensitivity and higher accuracy than the conventional method.
  • the fluorescent bead solution having a diameter of 10 m prepared in Example 2 was added to a 384-well microphone opening plate, and a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) was connected to a 1-molecule fluorescence analysis unit. Using this, we examined whether the difference in scanning speed would affect the detection value under the following measurement conditions. The measurement was performed 5 times per scanning speed.
  • FIG. Table 3 shows the detected values of the first to fifth fluorescence intensities
  • FIG. 6 shows a graph of the average of the detected values of the first to fifth fluorescence intensities.
  • Measurement conditions Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 ⁇ W), measurement time: 30 seconds x 5 times, measurement mode: FIDA, scan speed: 1, 2.5, 10, 20, 25 , 50m mZ second, total scanning area: 160 mX 160 / zm, objective lens magnification: X 60
  • Example 4 Confirmation of influence of difference in signal intensity acquisition region (magnification of objective lens) on detection accuracy Fluorescent bead solution with a diameter of 10 ⁇ m prepared in Example 2 was applied to a 384-well microphone mouthplate. Add a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) and connect a 1-molecule fluorescence analysis tube. We examined whether the volume of the sample affects the detection value.
  • FIG. Table 4 shows the detected values of the 1st to 5th fluorescence intensities
  • Fig. 7 shows a graph of the average value of the detected 1st to 5th fluorescence intensities.
  • Measurement conditions Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 ⁇ W), measurement time: 30 seconds x 5 times, measurement mode: FIDA, scanning speed: 20 mmZ seconds, total scan area: 160 m x 160 m, Objective lens magnification: X 10, X 20, X 40, X 60
  • the signal intensity can be detected with high sensitivity when the volume of the confocal region is 1FL or less, that is, when the magnification of the objective lens used is X20 to X60.
  • the fluorescent bead solution with a diameter of 10 m prepared in Example 2 was analyzed under the following measurement conditions using a confocal laser microscope FV 1000 (trade name; manufactured by Olympus Corporation) with a single molecule fluorescence analysis unit connected. Then, the number of fluorescent beads was confirmed. The results are shown in Fig. 8. Measurement conditions: Laser wavelength: 633 nm, laser power: 41% (equivalent to about 300 ⁇ W), scanning speed: 20 mm / sec, total scanning area: 25 ⁇ 25 ⁇ m to 200 ⁇ m X 200 ⁇ m, used Objective lens magnification: X 60
  • the present invention can be used in the fields of clinical testing, hygiene testing, biochemical research, and the like, and is useful for analysis of biological samples.

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Abstract

L'invention concerne un procédé d'analyse de particules fines, dans lequel une intensité de signal dans une région fine d'une région à foyer commun est mesurée par balayage de la région fine et analyse des dimensions des particules fines ou de l'existence de liaison de particules ou de divergence de particules. Le volume d'une région fine parmi une pluralité de régions fines dont les intensités de signal doivent être mesurées est 1FL ou moins, et une aire totale des surfaces planes de régions de balayage des régions fines est de 500 μm2-40.000 μm2.
PCT/JP2007/063297 2006-07-13 2007-07-03 Procédé d'analyse de particules fines WO2008007580A1 (fr)

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