Fast thermo-optical particle characterisation

• US 8,431,903 B2
• Filed: 11/20/2007
• Issued: 04/30/2013
• Est. Priority Date: 11/20/2006
• Status: Active Grant

• Current Assignee: NanoTemper Technologies GmbH
• Original Assignee: Ludwig-Maximilians-Universitat Munchen
• Inventors: Duhr, Stefan, Baaske, Philipp
• Primary Examiner(s): Porta, David
• Assistant Examiner(s): GREEN, YARA B
• Application Number:
  US12/515,641
• Publication Number:
  US 20100044586A1
• Time in Patent Office: 1,988 Days
• Field of Search: 250/458.1, 250/459.1, 250/251, 250/362, 250/361.R, 250/458.9, 435/6.12, 435/6.11
• US Class Current: 250/362
• CPC Class Codes: B01F 33/3033 using heat to mix or move the fluids
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0451 Thermophoresis; Thermodiffusion; Soret-effect
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01K 11/12 using changes in colour, translucency or reflectance
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/0205 by optical means
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 2015/0092 Monitoring flocculation or agglomeration
• CPC Class Codes: G01N 2015/025 Methods for single or grouped particles
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/171 with calorimetric detection, e.g. with thermal lens detection
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• 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 21/6458 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 2201/02 : Mechanical
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 2201/0638 : Refractive parts
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a method and an apparatus for a fast thermo-optical characterization of particles. In particular, the present invention relates to a method and a device to measure the stability of (bio)molecules, the interaction of molecules, in particular biomolecules, with, e.g. further (bio)molecules, particularly modified (bio)molecules, particles, beads, and/or the determination of the length/size (e.g. hydrodynamic radius) of individual (bio)molecules, particles, beads and/or the determination of length/size (e.g. hydrodynamic radius).

Claims

1. A device for measuring inter- and/or intra-molecular interactions of particles in a solution, comprising:

a receiver for receiving a solution containing marked particles;

a light source for fluorescently exciting the marked particles;

a fluorescence detector for detecting the excited fluorescence in said solution; and

a laser for irradiating a laser light beam into the solution to obtain a spatial temperature distribution in the solution around the irradiated laser light beam

wherein the laser and the detector are arranged on the same side with respect to the receiver,

wherein the laser is a high power laser within the range of from 4 W to 6 W.

2. The device according to claim 1, wherein the laser and the detector are arranged such that fluorescence detection and laser focussing are performed through the same optical unit.

3. The device according to claim 1, wherein the light source is a LED.

4. The device according to claim 1, wherein the device further comprises an optic for magnifying the detected region.

5. The device according to claim 1, wherein the device further comprises an optic for focusing or defocusing the laser beam.

6. The device according claim 5, wherein the optic is a single lens.

7. The device according to claim 1, wherein the detector is a CCD camera.

8. The device according to claim 1, wherein the detector is a photo diode.

9. The device according of claim 1, wherein said particles are selected from the group consisting of:

(i) (a) (bio)molecule(s), (ii) (nano)particles, (iii) (micro)beads, (iv) (an) organic substance(s), (v) (an) inorganic substance(s) and (vi) combinations of any of (i)-(v).

10. A method of thermo-optically measuring the characteristics of particles in a solution, comprising:

(i) providing a sample solution comprising marked particles;

(ii) exciting fluorescently the marked particles and detecting a first fluorescence of the excited particles;

(iii) irradiating a laser light beam into the solution to obtain a spatial temperature distribution in the solution around the irradiated laser light beam;

(iv) detecting a second fluorescence of the excited particles in the solution at a predetermined time of 1 ms to 250 ms after initiation of said irradiating a laser into the solution to obtain a spatial temperature distribution, and

(v) characterizing the particles based on said two detections.

11. A method of thermo-optically measuring the characteristics of particles in a solution, comprising:

(i) providing a sample solution comprising marked particles;

(ii) exciting fluorescently the marked particles and detecting a first fluorescence of the excited particles;

(iii) irradiating a laser light beam into the solution to obtain a spatial temperature distribution in the solution around the irradiated laser light beam;

(iv) detecting a second fluorescence of the excited particles in the solution, wherein the detection time is in the range of 1ms to 50ms; and

(v) characterizing the particles based on said two detections.

12. The method of thermo-optically measuring the characteristics of particles in a solution, according to claim 11, comprising:

(i) providing a sample solution comprising marked particles;

(ii) exciting fluorescently the marked particles and detecting a first fluorescence of the excited particles;

(iii) irradiating a laser light beam into the solution to obtain a spatial temperature distribution in the solution around the irradiated laser light beam;

(iv) detecting a second fluorescence of the excited particles in the solution at a predetermined time of 1 ms to 250 ms after initiation of said irradiating a laser into the solution to obtain a spatial temperature distribution, wherein the detection time is in the range of 1 ms to 50 ms, and (v) characterizing the particles based on said two detections.

13. The method according to claim 12, further comprising the step of measuring the temperature distribution in said solution around the irradiated beam with a temperature sensitive dye.

14. The method according to claim 13, wherein the temperature distribution is determined based on detected. fluorescence of the temperature sensitive dye, wherein the solution comprising said temperature sensitive dye is heated by the irradiated laser beam and the fluorescence spatial fluorescence intensity is measured substantially perpendicular around the laser beam.

15. The method according to claim 14, wherein in said predetermined time concentration change(s) within the spatial temperature distribution in the solution due to thermophoretic effects and such (an) concentration change(s) is(are) detected by a change of the distribution of fluorescence.

16. The method according to claim 14, wherein the laser beam is focused such that a temperature gradient within the temperature distribution is achieved in the range of from 0.001 to 10 K/μ

m.

17. The method according to claim 14, wherein said fluorescence is detected with a CCD camera.

18. The method according to claim 14, wherein the brightness of said fluorescence is detected with a photodiode or a single pixel with the CCD in the centre of the laser beam.

19. The method according to claim 12, wherein the particles are biomolecules and/or nanoparticles and/or microbeads and/or combinations thereof.

20. The method according to claim 12, wherein the laser light has a frequency within the range of from 1200 nm to 2000 nm.

21. The method according to claim 12, wherein the laser has a power of 0.1 W to 10 W.

22. The method according to claim 21, wherein the laser has a power of 4 W to 6 W.

23. The method according to claim 12, wherein the solution is an aqueous solution with a particle concentration within the range of from 1 atto Molar to 1 M.

24. The method according to claim 23, wherein the aqueous solution has a particle concentration within the range of from 1 atto Molar to 100 μ

Molar.

25. The method according to claim 12, wherein the solution is a saline solution with concentrations in the range of from 0 to 1 M.

26. The method according to claim 12, wherein the spatial temperature distribution is between 0.1°

C. and 100°

C.

27. The method according to claim 12, wherein the irradiation of the laser and the detection of the fluorescence is conducted from the same side with respect to the sample probe.

28. The method according to claim 12, wherein the solution has a thickness in direction of the laser light beam from 1 μ

m to 500 μ

m.

29. The method according to claim 12, wherein the fluorescence is detected within a range of from 1 nm to 500μ

m in direction of the laser beam.

30. The method according to claim 12, wherein the fluorescence is detected substantially perpendicular with respect to the laser light beam with a CCD camera.

31. The method according to claim 30, wherein the second fluorescence detection is spatial measurement of the fluorescence in dependence of the temperature distribution substantially perpendicular with respect to the laser light beam.

32. The method of claim 12, wherein said particles are selected from the group consisting of:

(i) (a) (bio)molecule(s), (ii) (nano)particles, (iii) (micro)beads, (iv) (an) organic substance(s), (v) (an) inorganic substance(s) and (vi) combinations of any of (i)-(v).

33. The method of claim 31, wherein said (bio)molecule is selected from the group consisting of:

(i) (a) protein(s), (ii) (a) peptide(s), (iii) (a) nucleic acid(s), (iv) (a) proteinnucleic acid fusion molecule(s), (v) (a)PNA(s) and (vi) (a) locked DNA(s) (LNAs).

34. The method according to claim 12 wherein the laser beam is defocused such that a temperature gradient within the temperature distribution is in the range of from 0.0 to 5K/μ

m.

35. The method according to claim 34 wherein the laser beam is defocused such that a temperature gradient within the temperature distribution is in the range of from 0.0 to 2K/μ

m.

36. The method according to claim 34, wherein the laser beam is irradiated through an optical element into the solution.

37. The method according to claim 34, wherein the optical element is a single lens.

38. The method according to claim 34, wherein said temperature gradient is created within 0.1μ

m to 500μ

m in diameter around the laser beam.