WO2008007580A1

WO2008007580A1 - Method for analyzing fine particles

Info

Publication number: WO2008007580A1
Application number: PCT/JP2007/063297
Authority: WO
Inventors: Tamiyo Kobayashi; Masayoshi Kusano; Morinao Fukuoka
Original assignee: Olympus Corporation
Priority date: 2006-07-13
Filing date: 2007-07-03
Publication date: 2008-01-17

Abstract

In a method for analyzing fine particles, signal strength in a fine region in a confocal region is measured by scanning the fine region and analyzing the sizes of the fine particles or existence of particle bonding or particle divergence. The volume of one fine region among a plurality of fine regions of which the signal strengths are to be measured is 1FL or less, and a total area of scanning region flat surfaces of the fine regions is 500μm2-40,000μm2.

Description

Specification

Analysis method of fine particles

Technical field

The present invention relates to a method for analyzing fine particles such as biological materials with high sensitivity and high accuracy. This application claims priority to Japanese Patent Application No. 2006-192742 filed on July 13, 2006, the contents of which are incorporated herein by reference.

Background art

[0002] As a method of detecting signal intensity and analyzing the magnitude of a biological substance or the presence or absence of binding or detachment of a biological substance with high sensitivity, such as fluorescence correlation spectroscopy analysis, for example, vibration centered on a standard position A method of using an apparatus in which a deflecting mirror array having objective lens side plane polarizing mirrors arranged in such a manner is arranged in a confocal microscope has been disclosed (see Non-Patent Document 1, Patent Documents 1 and 2). This mirror is designed to vibrate or rotate around a standard position and is usually called a beam scanner. This mirror technology used for fluorescence intensity distribution analysis prevents the analysis object from fading due to light such as a laser, and increases the number of analysis objects in the confocal region within a given measurement time. The data acquisition time is saved considerably.

Non-Patent Literature l: Pro. Natl. Acad. Sci USA, vol. 96, 1375-1378, 1999 Patent Literature 1: JP 2001-502062

Patent Document 2: JP 2001-502066 Publication

Disclosure of the invention

Problems to be solved by the invention

[0003] However, these documents only mention that the use of a beam scanner is effective for the analysis of a biological substance. For example, an appropriate scan according to the size of the biological substance to be measured is used. No details about scanning methods such as speed or area are given.

For example, when an object having a size of 0.3 m or more is analyzed, 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.

[0005] That is, in the method using the conventional beam scanner, there is an upper limit to the size of the biological substance that can be measured, and when analyzing a biological substance that exceeds the upper limit, the standard deviation is small. There was a problem that it was difficult to obtain stable data.

[0006] In addition, when detecting interactions between molecules by single-molecule measurement methods such as FCS and FIDA, in order to obtain highly accurate data, in FCS, the molecular force is more than 4 times 8 times before and after the interaction. Requires a change in quantity. On the other hand, in FIDA, if the fluorescence intensity does not change more than 1.3 times before and after the interaction, the interaction cannot be detected. In such a case, 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.

[0007] In this case, in order to obtain highly accurate data using a beam scanner as in the conventional method, even if the size of the molecule to be analyzed is 0.3 m or less, the particle size In other words, the upper limit of the diameter of the beads, which is 0.3 m, has a problem that the variation of data becomes larger at larger diameters.

[0008] As described above, in the conventional method, 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

[0009] The present invention has been made in view of the above circumstances, and even when the analysis target includes a size of 0.3 μm 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

[0010] That is, in order to solve the above problems,

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 μm ^{2 to} 40000 μm ² This is an analysis method.

In the present invention, the scanning speed of one minute region for measuring the signal intensity may be 2.5 mmZsec to 25 mmZsec.

In the present invention, the analysis may be performed by at least one of fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and fluorescence polarization intensity distribution analysis.

In the present invention, 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.

In the present invention, the size force of the fine particles may be 0.3 / ζ πι to 10 / ζ πι. The invention's effect

[0011] According to the present invention, even when 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.

Brief Description of Drawings

[0012] 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] 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.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

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. In the present invention, 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.

[0014] The total area value of the scanning region planes of the plurality of minute regions for measuring the signal intensity is set to 500 μm ^{2 to} 40000 m ² . Outside this range, the detection value of the signal intensity has a large variation, resulting in low reliability.

[0015] In the present invention, 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.

In the present invention, various methods can be applied to the measurement of signal intensity. A method of detecting fluorescence of fine particles labeled with a fluorescent dye is preferable. In this case, for example, fluorescence correlation spectroscopy (hereinafter abbreviated as FCS), fluorescence intensity distribution analysis method (hereinafter abbreviated as FIDA), fluorescence polarization intensity distribution analysis method (FIDA-polarization), etc. are preferred. Is The These may be performed singly or in combination of a plurality of methods. These measurements can be performed using, for example, a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) connected to a single molecule fluorescence analysis unit.

[0017] According to the signal intensity measurement method as described above, 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.

[0018] In the present invention, 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 μm, 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. As 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.

[0019] 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.

[0020] For example, the cell membrane usually exists as a ribosome in a solution. When 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. However, analysis using RI is the mainstream. In addition, when the conventional single molecule measurement method is applied, only data with large variations can be obtained. However, according to the present invention, even if a thing with a size exceeding 0.3 m is included as an analysis object, analysis can be performed with high accuracy without using a dangerous RI. 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. In the conventional method, the scanning speed is approximated to a straight line because scanning is performed in a donut shape.

[0021] In addition, 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. For example, particle assembly of biological materials bound to such beads can be performed with high accuracy. When 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. It is possible to increase the number of biological substances by one bead. That is, the larger the bead used, the more the signal intensity of the biological substance can be amplified during measurement. The present invention is particularly suitable when such large beads, particularly beads having a diameter of 0.3 μm to 10 μm are used.

[0022] The use of beads having a large particle diameter is also preferable in terms of easy preparation and handling of beads having a biological substance immobilized thereon. When beads with a small particle size, for example, those with a diameter of 0.3 / zm or less, are used for the fixation force reaction of biologically related substances to the beads and the centrifugation process associated with each measurement procedure. 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.

[0023] When using beads in the present invention, any material can be used as long as it does not affect the measurement of signal intensity such as fluorescence intensity. For example, various materials such as polystyrene are used. Conventionally known ones such as plastic beads or metal beads may be used. In addition, 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.

In the present invention, conventionally known fluorescent dyes can be used, and examples thereof include fluorescein, rhodamine, alexa, ATTO dye and the like.

[0025] 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, and FIGS. 2C to 2E are main scans. Each preferred scan of the invention is shown. In the present invention, as described in FIG. 2C, 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. In addition, as another scanning method in the present invention, as shown in FIG. 2D, 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. For example, when 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 μm 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.

[0026] When 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. In contrast, in the present invention, 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 μm ^{2 to} 40000 μm 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. Furthermore, by enlarging the scan area, 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.

[0027] Normally, when scanning is performed, the scan area is preliminarily set before the measurement is started. When the acquisition of the signals of the number of fine particles that have been preliminarily set is completed, the scan is terminated. You can also. In this way, the measurement time can be shortened.

[0028] In the present invention, 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.

Example

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.

[Example 1] Receptor monoligand binding inhibition experiment using cell membrane fractionation (PAF inhibitor: PAF receptor antagonist WEB2086 study) (cell membrane fractionation treatment)

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 μm in diameter.

(1) Human PAF-expressing CHO cells in the tube were homogenized on ice using approximately 10 times (wZw) of HEPES buffer.

(2) Centrifugation was performed at 4 ° C and 800 X G for 20 minutes, and then the supernatant was carefully removed and transferred to another tube.

(3) Further, the mixture was centrifuged at 4 ° C, 100000 X G for 60 minutes, and then the supernatant was removed.

(4) The precipitate was pipetted with HEPES buffer.

(5) Using a homogenizer, the suspension was suspended 10 times on ice.

(6) Centrifugation was performed at 4 ° C and 100000 XG for 60 minutes, and then the supernatant was removed. (7) The precipitate was loosened with the same amount of HEPES buffer as the cells.

(8) Using a homogenizer, the suspension was suspended 10 times on ice.

(9) The amount of protein was measured and adjusted to 0.5 mgZml.

[0030] (Reaction)

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.

[0031] (FIDA analysis)

Using 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 μW, measurement time: 30 seconds X 5 times, scanning speed: 25 mm / decrease, total scanning area: 500 μm ² , 25000 μ rn

[0032] [Comparative Example 1] Receptor-one-ligand binding inhibition experiment by conventional method

Analysis was performed in the FIDA measurement mode in the same manner as in Example 1 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 Fig. 3C.

Measurement conditions: Laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds x 5 times, beam scanner: 2. 85 mm / second, total scan area: 100 μ rn

[0033] 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 μm 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.

[0034] [Example 2] Confirmation of detection sensitivity when beads of different sizes are used (production of fluorescent beads with a diameter of 10 μm)

Polybead Carboxylate 10. Omicron microspheres, trade name, catalog number 18133; manufactured by Polyscienses) 250 μL 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 μg), 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.

[0035] (Preparation of fluorescent beads with a diameter of 0.5 μm)

To prevent bead aggregation, 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 μL 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 μL of Biotin—ΑΤΤ065 5 ( After adding 10 m), the total liquid volume was 1000 μL, and the mixture was reacted at room temperature for 1 hour. After completion of the reaction, centrifuge at 20000 XG for 5 seconds, remove the supernatant, add 1 mL of PBS—0.05% tween20, and centrifuge again at 20000 XG for 5 seconds to remove the supernatant. It was. Then, PBS-0.05% tween20 was added in 1 mL and well suspended to obtain a fluorescent bead solution having a diameter of 0.5 μm, which was subjected to the following analysis.

[0036] (FIDA analysis)

Using a confocal laser microscope FV1000 (trade name; manufactured by Olympus Corporation) with a single-molecule fluorescence analysis unit connected, analysis of the two types of fluorescent bead solution is performed under the following measurement conditions, using the FIDA measurement mode. Went.

The measurement was performed 5 times, and the detected fluorescence intensity (Hz), the average value, and the standard deviation were calculated. The results are shown in Tables 1 and 2. Table 1 shows 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, and Figures 4B and 5B show the results when using fluorescent beads with a diameter of 10 m.

Measurement conditions; laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds, scanning speed: 25 mmZ seconds, total scanning area: 25000 μm ² [0037] [Comparative Example 2] Confirmation of detection sensitivity by conventional method

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.

Measurement conditions; laser wavelength: 543 nm, laser power: 250 μW, measurement time: 30 seconds, scanning speed: 2. 85 mmZ seconds, total scanning area: 100 m ²

[0038] In Example 2, it was possible to detect fluorescent beads with a diameter of 0.5 μm and a diameter of 10 μm! 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.

[0039] [Table 1]

¾ diameter of light peas 0.5 rn

[0040] [Table 2] Fluorescent bead diameter 10 am

[0041] [Example 3] Confirmation of influence of scanning speed on detection accuracy

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.

The results are shown in Table 3 and FIG. Table 3 shows the detected values of the first to fifth fluorescence intensities, and 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 μW), 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

[0042] 2. The detected values were more stable for 5, 10, 20, and 25 mmZ seconds, whereas data variations (error bars) were observed for lm mZ seconds and 50 mmZ seconds. From this, it was confirmed that the scanning speed in the present invention is preferably 2.5 to 25 mmZ seconds.

[0043] [Table 3] mm / sec SO 25 20 10 2.5 1

1st 1256.1 1796 2149.β 2123 2489.6 554.7

2nd 1724.1 2436.2 2741.7 190t.9 3279.1 2879.1

3rd 2452.8 2140.1 1858.1 2834.6 2639.9

4th 2480.3 Z1S7.1 17C4.1 2083.5 3I9B.4 1749.4

5th 2466.6 2197.4 2161.6 E534.3 3315.2 321.6 Average 2071.66 2207.9 2179.46 2100.16 3023.18 1628.94 Standard deviation 556.3787 266.669 369- 0626 j 267.9444 354.1296 1168.608 [0044] [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 μm 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.

The measurement was performed five times for each objective lens magnification. The results are shown in Table 4 and FIG. Table 4 shows the detected values of the 1st to 5th fluorescence intensities, and 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 μW), 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

[0045] It was confirmed that 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.

[0046] [Table 4]

[0047] [Example 5] Confirmation of the number of particles to be detected by fluorescence observation

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 μW), scanning speed: 20 mm / sec, total scanning area: 25 μηιΧ 25 μm to 200 ^ m X 200 μm, used Objective lens magnification: X 60

[0048] About 10 to 20 fluorescent beads of the same size as the cells were observed to move slowly. In other words, it is possible to perform FIDA analysis sufficiently with about 10 to 20 fluorescent beads as detection targets, and the scan area at this time is 25 m X 25 μm to 200 m X 200 m The range was confirmed to be appropriate.

[0049] In the present example, force using beads as an analysis target. This is a technique usually used as a standardized measurement method for cells in flow cytometry. The same result can be obtained even if a cell or the like is used as the measurement object.

Industrial applicability

[0050] 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.

Claims

The scope of the claims

[1] A fine particle analysis method that scans a small region within a confocal region and measures the signal intensity in the small region to analyze the size of the fine particle, whether or not there is a binding or detachment of the fine particle,

Fine particles in which the volume of one micro area of the plurality of micro areas for measuring signal intensity is 1 FL or less, and the total area value of the scanning area planes of the plurality of micro areas is 500 / ζ πι ^{2 to} 40000 m ² Analysis method.

[2] The method for analyzing fine particles according to [1], wherein the scanning speed of one minute region for measuring the signal intensity is 2.5 mmZ second to 25 mmZ second.

[3] The method for analyzing fine particles according to claim 1 or 2, wherein the analysis is performed by at least one of fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, and fluorescence polarization intensity distribution analysis.

[4] The microparticle according to any one of claims 1 to 3, wherein the microparticle is a cell, a cell membrane, a cell membrane fragment, an organic particle, an inorganic particle, or one or more selected from these complex forces. The microparticle analysis method described.

[5] The method for analyzing fine particles according to any one of [1] to [4], wherein a size force of the fine particles is 0.3 m to 10 μm.