Method for detecting a target particle

• US 9,354,176 B2
• Filed: 02/04/2014
• Issued: 05/31/2016
• Est. Priority Date: 08/11/2011
• Status: Active Grant

• Current Assignee: Olympus Corporation
• Original Assignee: Olympus Corporation
• Inventors: Nishikawa, Kazutaka
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/172,295
• Publication Number:
  US 20140147854A1
• Time in Patent Office: 847 Days
• Field of Search: 435/91.2, 435/6.1
• US Class Current: 1/1
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6493 by alternating fluorescence/transmission or fluorescence/reflection
• CPC Class Codes: G01N 21/278 Constitution of standards
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence

Abstract

This method for detecting a target particle has: (a) a step for preparing a sample solution containing target particles and one type or two or more types of a luminescent probe that binds to the target particles, and allowing two or more molecules of the luminescent probe to bind per one molecule of the target particles in the sample solution, and (b) a step for calculating the number of molecules of target particles bound to the luminescent probe present in the sample solution prepared in step (a) by a scanning molecule counting method by using as an indicator thereof the strength of light signals of the individually detected particles, and the luminescent probe is one type or two or more types of a luminescent probe to which the same type of luminescent substance is bound.

Claims

1. A method for detecting a target particle dispersed and moving randomly in a sample solution, comprising:

(a) preparing a sample solution containing the target particle and luminescent probes, and allowing two or more luminescent probes to bind per one target particle in the sample solution,

(b) moving a location of a photodetection region of an optical system of a confocal microscope or a multi-photon microscope in the sample solution,

(c) detecting light from the photodetection region during the moving of the location of the photodetection region in the sample solution over a plurality of units of time, and generating time series light intensity data of the light from the light detection region detected while moving the position of the light detection region in the sample solution,

(d) smoothing data values corresponding to light intensity variation over the plurality of units of time of a light signal from the luminescent probes bound to each target particle, said smoothing being conducted until a gap in the light intensity variation can be disregarded,

(e) individually detecting the target particle bound to the luminescent probes when the light intensity variation in the time series light intensity data over consecutive units among the plurality of units of time has a predetermined profile which is expected from a single light-emitting particle moving relatively inside the photodetection region, and

(f) counting the number of the target particles by using the light intensity of the target particle detected in the step (e) as an indicator thereof.

2. The method for detecting a target particle according to claim 1, wherein, in the moving of the location of the photodetection region, the location of the photodetection region is moved at a prescribed speed.

3. The method for detecting a target particle according to claim 1, wherein, in the moving of the location of the photodetection region, the location of the photodetection region is moved at a speed that is faster than the diffusion movement speed of the target particle bound to the luminescent probe.

4. The method for detecting a target particle according to claim 1,

wherein, in the individually detecting of the target particles by detecting a light signal from the individual target particle from the detected light, the entry of a single target particle bound to the luminescent probes into the photodetection region is detected based on the form of a chronologically detected light signal.

5. The method for detecting a target particle according to claim 1, wherein the luminescent probes comprise a labeling probe that binds with the target particle and a luminescent substance of which one or more substances bind per one labeling probe.