Method for detecting a target particle

• US 9,575,060 B2
• Filed: 08/21/2014
• Issued: 02/21/2017
• Est. Priority Date: 04/18/2012
• Status: Active Grant

• Current Assignee: Olympus Corporation
• Original Assignee: Olympus Corporation
• Inventors: Nishikawa, Kazutaka, Hanashi, Takuya
• Primary Examiner(s): Wallenhorst, Maureen
• Application Number:
  US14/465,208
• Publication Number:
  US 20140356969A1
• Time in Patent Office: 915 Days
• Field of Search: 436/501, 436/94, 436/164, 436/165, 436/172, 422/82.05, 422/82.08, 422/82.09, 435/6.1, 435/7.1
• US Class Current: 1/1
• CPC Class Codes: G01N 15/1434 Optical arrangements
• 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
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G02B 21/00 Microscopes
• CPC Class Codes: G02B 21/0024 Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Provided is a method for detecting a target particle that is a method for detecting a non-luminescent target particle dispersed and randomly moving in a sample solution using an optical system of a confocal microscope or multi-photon microscope, having: (a) preparing a sample solution containing target particles, and labeling particles of which the average outer diameter is less than 15% of the diameter of a photodetection region of the optical system, binding two or more molecules of the labeling particles per molecule of the target particles in the sample solution, and forming a non-luminescent complex of which the outer diameter is 15% or more of the diameter of the photodetection region; and, (b) calculating the number of molecules of the complex in the sample solution prepared in the (a) using an inverse scanning molecule counting method.

Claims

1. A method for detecting a target particle dispersed and randomly moving in a sample solution using an optical system of a confocal microscope or multi-photon microscope, comprising:

(a) preparing a sample solution containing target particles, and labeling particles of which an average outer diameter is less than 15% of a diameter of a photodetection region of the optical system, and forming a non-luminescent complex of which an outer diameter is 15% or more of the diameter of the photodetection region by binding two or more molecules of the labeling particles to one molecule of the target particles in the sample solution; and

,

(b) calculating number of molecules of the complex in the sample solution prepared in step (a), comprising;

moving a location of the photodetection region of the optical system in the sample solution,

generating chronological light intensity data by detecting light containing substantially constant background light from the photodetection region while moving the location of the photodetection region of the optical system in the sample solution,

individually detecting a decrease in light intensity in the chronological light intensity data that occurs when the complex has entered the photodetection region as a signal representing the presence of an individual complex, and

counting number of the complexes detected during movement of the location of the photodetection region by counting the number of signals representing the presence of the individually detected complexes.

2. The method for detecting a target particle according to claim 1, wherein the outer diameter of the complex is 35% or more of the diameter of the photodetection region.

3. The method for detecting a target particle according to claim 1, wherein the target particle and the labeling particle bind specifically.

4. The method for detecting a target particle according to claim 1, wherein the target particle is a nucleic acid.

5. The method for detecting a target particle according to claim 1, wherein the background light is fluorescent light, phosphorescent light, chemiluminescent light, bioluminescent light, or scattered light generated by a substance dispersed in the sample solution.

6. The method for detecting a target particle according to claim 1, wherein the background light is illumination light.

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

8. The method for detecting a target particle according to claim 1, wherein the location of the photodetection region in the sample solution is moved by altering a light path of the optical system in the moving of the location of the photodetection region.

9. The method for detecting a target particle according to claim 1, wherein a single complex is judged to have entered the photodetection region when a signal has been detected having light intensity lower than a prescribed threshold value as determined from the intensity of the background light in the individually detecting of a signal representing the presence of the complex.

10. The method for detecting a target particle according to claim 1, wherein the chronological light intensity data is smoothed;

and a downwardly convex, bell-shaped pulsed signal, having intensity below that of a threshold value determined from the intensity of the background light in the smoothed chronological light intensity data, is detected as a signal representing the presence of the complex in the individually detecting of a signal representing the presence of the complex.

11. The method for detecting a target particle according to claim 1, further comprising:

determining number density or concentration of the target particles in the sample solution based on the number of the complexes detected and counted in step (b).

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

the moving of the location of the photodetection region of the optical system in the sample solution,

the detecting of light containing substantially constant background light from the photodetection region while moving the location of the photodetection region of the optical system in the sample solution, and

the detecting of the signal representing the presence of the individual complex,

are repeated until number of signals representing the presence of the complex reaches a predetermined number, and

concentration of the target particles in the sample solution is determined based on an amount of time required for the number of signals representing the presence of the complex to reach the predetermined number.