Method and a device for monitoring nucleic acid amplification reactions

• US 6,310,354 B1
• Filed: 07/13/1999
• Issued: 10/30/2001
• Est. Priority Date: 12/03/1996
• Status: Active Grant

• Current Assignee: Erkki Soini
• Original Assignee: Erkki Soini
• Inventors: Soini, Erkki, Hänninen, Pekka
• Primary Examiner(s): Epps, Georgia
• Assistant Examiner(s): HANIG, RICHARD E
• Application Number:
  US09/352,372
• Time in Patent Office: 840 Days
• Field of Search: 250/458.1, 250/459.1, 250/461.1, 250/462.2, 435/288.7, 422/82.08, 356/442
• US Class Current: 250/458.1
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/557 using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction

Abstract

A method for quantitatively measuring nucleic acid amplification reactions, especially the polymerase chain reaction, employing microparticles as hybridization solid phase, a probe sequence labeled with a fluorescent label and a fluorescence detection system which is based on two-photon fluorescence excitation, contacting all the amplification reaction components and the solid phase simultaneously in a closed cuvette, performing the amplification reactions in the same cuvette, focusing a two-photon exciting laser beam into the cuvette during the amplification cycles and measuring the fluorescence signal emitted by the microparticles from one particle at a time when they randomly float through the focal volume of the laser beam. The features of this invention allow a method and device for performing a fast quantitative nucleic acid amplification assay of single or multiple target sequences in a very small closed sample volume.

Claims

1. A method for quantitatively measuring a nucleic acid amplification reaction which employs microparticles to bind a nucleic acid sequence labeled with a fluorescent label, said method comprising

contacting all nucleic acid amplification reaction components in a single environmentally closed reaction volume;

performing a nucleic acid amplification reaction cycle;

scanning a two-photon excitation focal volume within said reaction volume using a mechanical or optical scanner and a two-photon exciting laser beam which is capable of two-dimensionally optically trapping the microparticles;

momentarily interrupting or reducing the scanning speed of said two-photon excitation volume when said two-photon excitation volume approaches a microparticle randomly located in the reaction volume;

optically trapping said microparticle such that it falls into a two-dimensional potential well crated by said laser beam; and

detecting fluorescence emission photon counts from said microparticle.

2. The method of claim 1, wherein movement of said scanner is controlled by a signal obtained from a detector measuring light scattering or reflection.

3. The method of claim 2, wherein an optical set-up for measuring said light scattering or reflection is confocal.

4. The method of claim 1, wherein said nucleic acid amplification reaction comprises a polymerase chain reaction.

5. A device for quantitatively measuring nucleic acid amplification reactions, comprising

a laser capable of generating a laser beam of sufficient power to two-photon excite and two-dimensionally optically trap a fluorescent label attached to a nucleic acid sequence bound to a microparticle in a reaction volume;

an optical system for focusing said laser beam through an objective lens to a focal volume within said reaction volume, said objective lens having a numerical aperture optimized for two-dimensionally optically trapping an individual microparticle, said system being capable of scanning said focal volume at a predetermined speed within said reaction volume;

a detector capable of detecting fluorescence emitted from said focal volume; and

a control system for momentarily interrupting or reducing said predetermined scanning speed to a second, slower scanning speed when a microparticle is present in said focal volume, thereby allowing said microparticle to be optically trapped for a period of fluorescence detection by said laser beam during said period of fluorescence detection.

6. The device of claim 5, further comprising a light scattering detector which is confocal with said laser beams.

7. The device of claim 6, wherein movement of said scanner is controlled by a trigger signal received from said light scattering detector.

8. The device of claim 6, wherein movement of said scanner is controlled by a trigger signal received from said light scattering detector.