Method for detecting a target particle
First Claim
1. A method for indirectly detecting a particle dispersed and moving randomly in a solution using a luminescent probe, comprising:
- (a) preparing a solution containing a target particle to be detected and a first luminescent probe that directly or indirectly binds to the target particle;
(b) forming a complex containing the target particle and the first luminescent probe in the solution;
(c) removing first luminescent probes not bound to the target particles from the solution containing the complex, thereby recovering the complex;
(d) dissociating the first luminescent probe from the recovered complex and separately separating the free first luminescent probe and the target particle, thereby separately recovering the free first luminescent probe and the target particle;
(e) repeating one or more times a cycle consisting of the following operations (a′
) to (d′
);
(a′
) preparing a solution by adding a second luminescent probe to the target particle recovered by separating from the free first luminescent probe in the (d);
(b′
) forming a complex containing the target particles and the second luminescent probe in the solution following the (a′
);
(c′
) separating second luminescent probes not bound to the target particle, thereby recovering the complex from the solution following the (b′
); and
,(d′
) dissociating the second luminescent probe from the complex recovered in the (c′
) followed by mutually separating and recovering free second luminescent probes and the target particle;
followed by preparing a single measurement sample solution containing the total amount of the free first and second luminescent probes recovered in the (d) and the (d′
); and
,(f) calculating the number of molecules of the first and second luminescent probes in the measurement sample solution by a method consisting of detecting light emitted from the first and second luminescent probes in a photodetection region of an optical system of a confocal microscope or multi-photon microscope while moving the location of the photodetection region in the measurement sample solution using the optical system.
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Accused Products
Abstract
The present invention provides a method for indirectly and with high sensitivity detecting a particle dispersed and moving randomly in a solution using a luminescent probe. In the present invention, (a) a solution is prepared that contains a target particle and one or more types of a luminescent probe that directly or indirectly binds to the target particle, (b) a complex is formed that contains the target particle and the luminescent probe in the solution, (c) the complex is recovered by separating luminescent probe not bound to the target particle from the solution containing the complex, followed by (d) dissociating the luminescent probe from the recovered complex and mutually separating and separately recovering the free luminescent probe and target particle, (e) again binding a luminescent probe to the recovered target particle followed by dissociating the luminescent probe and mutually separating and recovering free luminescent probe and the target particle are repeated, followed by preparing a single measurement sample solution containing the total amount of the recovered free luminescent probe, and (f) the number of molecules of the luminescent probes in the measurement sample solution is calculated.
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Citations
11 Claims
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1. A method for indirectly detecting a particle dispersed and moving randomly in a solution using a luminescent probe, comprising:
-
(a) preparing a solution containing a target particle to be detected and a first luminescent probe that directly or indirectly binds to the target particle; (b) forming a complex containing the target particle and the first luminescent probe in the solution; (c) removing first luminescent probes not bound to the target particles from the solution containing the complex, thereby recovering the complex; (d) dissociating the first luminescent probe from the recovered complex and separately separating the free first luminescent probe and the target particle, thereby separately recovering the free first luminescent probe and the target particle; (e) repeating one or more times a cycle consisting of the following operations (a′
) to (d′
);(a′
) preparing a solution by adding a second luminescent probe to the target particle recovered by separating from the free first luminescent probe in the (d);(b′
) forming a complex containing the target particles and the second luminescent probe in the solution following the (a′
);(c′
) separating second luminescent probes not bound to the target particle, thereby recovering the complex from the solution following the (b′
); and
,(d′
) dissociating the second luminescent probe from the complex recovered in the (c′
) followed by mutually separating and recovering free second luminescent probes and the target particle;followed by preparing a single measurement sample solution containing the total amount of the free first and second luminescent probes recovered in the (d) and the (d′
); and
,(f) calculating the number of molecules of the first and second luminescent probes in the measurement sample solution by a method consisting of detecting light emitted from the first and second luminescent probes in a photodetection region of an optical system of a confocal microscope or multi-photon microscope while moving the location of the photodetection region in the measurement sample solution using the optical system. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification
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- Resources
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Current AssigneeOlympus Corporation
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Original AssigneeOlympus Corporation
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InventorsNakata, Hidetaka
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Primary Examiner(s)SALMON, KATHERINE D
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Application NumberUS14/322,010Publication NumberTime in Patent Office790 DaysField of SearchUS Class Current1/1CPC Class CodesC12Q 1/682 Signal amplificationG01N 21/6428 Measuring fluorescence of f...G01N 21/6458 Fluorescence microscopy flu...
