Fast thermo-optical particle characterisation
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).
1. A method for measuring inter- and/or intra-molecular interactions of particles in a solution comprising:
a) providing a sample with marked particles in a solution;
b) exciting fluorescently the marked particles and detecting a first fluorescence of the excited particles;
c) irradiating a laser light beam into the solution to obtain a spatial temperature distribution in the solution around the irradiated laser light beam;
d) detecting a second fluorescence of the particles in the solution at a predetermined time after irradiation of the laser into the solution has been started, and
e) characterizing the inter- and/or intra-molecular interactions of the particles based on the first and second detections,
wherein the brightness of said fluorescence is detected with a photomultiplier tube, a photodiode or a single pixel with a CCD in the centre of the laser beam.
2. The method of claim 1 , wherein both fluorescence detection and infrared laser focussing are performed through a single optical unit.
3. The method of claim 2 , wherein both fluorescence detection and infrared laser focussing are performed through a single objective.
4. The method of claim 1 , wherein the predetermined time is within the range of 1 ms to 250 ms.
5. The method of claim 1 , wherein the detection time is in the range of 1 ms to 50 ms.
6. The method according to claim 1 , wherein the spatial temperature distribution in the solution around the irradiated laser light beam is 0.0 to 5 K/μm.
7. The method of claim 6 , wherein the spatial temperature distribution in the solution around the irradiated laser is 0.0 to 2 K/μm.
8. The method of claim 1 , wherein the laser beam is irradiated through an optical element into the solution.
9. The method of claim 8 , wherein the optical element is a single lens.
10. The method of claim 1 , wherein the solution further comprises measuring the temperature distribution in the solution around the irradiated beam by detecting the color intensity of a temperature sensitive dye.
11. The method of claim 10 , 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.
12. The method of claim 11 , wherein the spatial temperature distribution is 0.001 to 10K/μm.
13. The method of claim 11 , wherein said fluorescence is detected with a CCD camera.
14. The method of claim 1 , wherein the predetermined time is within the range of 0.5 s to 250 s.
15. The method of claim 1 , further comprising detecting a distribution of fluorescence, wherein a change in the distribution of fluorescence within the spatial temperature distribution in the solution during the predetermined time measures a change in concentration of the particles.
16. The method of claim 1 , wherein the particles are selected from (i) a biomolecule; (ii) a nanoparticle; (iii) a microbead; and (iv) combinations thereof.
17. The method of claim 16 , wherein the biomolecules are selected from the group consisting of proteins, peptides, nucleic acids, protein-nucleic acid fusion molecules, PNAs, and locked DNAs (LNAs).
18. The method of claim 1 , wherein the laser light has a wavelength of 1200 nm to 2000 nm.
19. The method of claim 1 , wherein the laser is a high power laser of 0.1 W to 10 W.
20. The method of claim 19 , wherein the laser is 4 W to 6 W.
21. The method of claim 1 , wherein the solution is an aqueous solution with a particle concentration of 1 atto Molar to 1 M.
22. The method of claim 21 , wherein the solution is an aqueous solution with a particle concentration of 1 atto Molar to 100 μM.
23. The method of claim 1 , wherein the solution is a saline solution with a concentration in the range of from 0 to 1 M.
24. The method of claim 1 , wherein the spatial temperature distribution is between 0.1° C. and 100° C.
25. The method of claim 24 , wherein the temperature gradient is created within 0.1 μm to 500 μm in diameter around the laser beam.
26. The method of claim 1 , wherein the solution is provided with a thickness in direction of the laser light beam from 1 μm to 500 μm.
27. The method of claim 1 , wherein the detection of the fluorescence is detected within a range of from 1 nm to 500 μm in the direction of the laser beam.
28. The method of claim 1 wherein the particles to be measured are selected from the group consisting of biomolecules, nanoparticles, microbeads, organic substances, inorganic substance and/or combinations of these.
29. A device for measuring inter- and/or intra-molecular interactions of particles in a solution according to claim 1 , wherein the device comprises:
a receiving means for receiving marked particles within a solution;
means for fluorescently exciting the marked particles;
means for detecting the excited fluorescence in said solution wherein selected from a photomultiplier tube, a photodiode or a single pixel with a CCD in the centre of the laser beam; 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.
30. The device of claim 29 , wherein the laser and the means for detecting the excited fluorescence are arranged such that fluorescence imaging and infrared laser focussing are performed through a single optical unit (1).
31. The device of claim 30 , wherein the fluorescence imaging and infrared laser focussing are performed through a single objective.
32. The device of claim 29 , wherein the means for fluorescently exciting the marked particles is an LED.
33. The device of claim 29 wherein the laser is a high power laser of 0.1 W to 10 W.
34. The device of claim 33 , wherein the laser is 4 W to 6 W.
35. The device of claim 29 , wherein the device further comprises an optic for magnifying the detected region.
36. The device of claim 29 , wherein the device further comprises an optic for focusing or defocusing the laser beam.
37. The device of claim 36 , wherein the optic is a single lens.
38. The device of claim 29 , wherein the detecting means (31) is a CCD camera.
39. The device of claim 29 , wherein the detecting means (31) is a photo diode.