Method and apparatus for contactless mixing of liquids

Abstract

The invention generally relates to an apparatus and a method for mixing of liquids (50) or of particles with a liquid (50). In a volume of liquid (50), a thermal convection flow is generated at at least one surface of the volume of liquid by irradiating IR radiation (30) into the volume of liquid. Thereby it is possible to avoid a depletion zone at the surface and to more accurately measure interactions of the particles with the surface by means of surface-based measurement methods.

Claims

1. Method for mixing liquids (50) or particles with a liquid (50), comprising the steps: a. providing a volume of liquid (50); b. generating a thermal convection flow at least one surface/boundary surface of the volume of liquid by irradiating infrared radiation up to 2000 nm (30) into the volume of liquid such that a depletion or concentration layer at said surface/boundary surface is avoided.

2. Method according to claim 1, wherein the volume of liquid (50) i) is provided in a sample chamber (45) having an inner diameter of from 0.05 mm to 0.8 mm, or ii) is provided as drop(s) on an object carrier.

3. Method according to claim 2, wherein the volume of liquid (50) is provided in a sample chamber (45) having an inner diameter of from 0.05 mm to 0.8 mm, and, wherein the surface of the volume of liquid is the boundary layer between the volume of liquid and a surface of the sample chamber.

4. Method according to claim 2, wherein the volume of liquid (50) is provided as drop(s) on an object carrier, wherein the surface of the volume of liquid is the boundary layer between the volume of liquid and a surface of the object carrier.

5. Method according to claim 1, wherein the liquid (50) is an aqueous solution.

6. Method according to claim 1, wherein the radiation (30) is directed parallel and/or antiparallel to gravitation and/or comprises a component that is oriented vertical to gravitation.

7. Method according to claim 1, wherein a temperature gradient of from 0.001 K/m to 2 K/m is generated with the irradiated radiation (30).

8. Method according to claim 7, wherein the generated temperature gradient is generated in an area of from 0.0001 mm.sup.2 to 12 mm.sup.2.

9. Method according to claim 8, wherein a detection region (80) for measuring properties of the liquid or of the particles in the liquid is spaced apart from the irradiation area of radiation (30).

10. Method according to claim 1, wherein flow rates of from 0.0005 mm/s to 2 mm/s are generated within the convection flow.

11. Method according to claim 1, wherein a sample chamber (45) is present in the form of a capillary or multi-well plate or microfluidic chip.

12. The method according to claim 1, wherein the infrared radiation is focused in the volume of liquid.

13. The method of claim 2, wherein the volume of liquid (50) is provided in a microcavity.

14. The method of claim 2, wherein the volume of liquid (50) is provided in a capillary.

15. The method of claim 14, wherein the capillary is made from glass.

16. The method of claim 1, wherein the radiation is produced by an LED or laser.

17. Method according to claim 9, wherein the detection region (80) for measuring properties of the liquid or of the particles in the liquid is spaced apart from the irradiation area of radiation (30) by at least 0.01 mm.

18. Method for analyzing molecular interactions of particles at and/or in a thin film in a volume of liquid, comprising the step of: providing, on an object carrier or a sample chamber (45), at least one volume of liquid (50) with particles present therein, and irradiating infrared radiation up to 2000 nm into the volume of liquid (50) for generating the thermal convection flow, measuring the interaction of the particles with a surface/boundary surface of a sample chamber or an object carrier, characterizing the interaction of the particles on the basis of the measurement.

19. Method according to claim 18, wherein the interaction is measured by reflectometric interference spectroscopy (RIfs).

20. Method according to claim 18, wherein the interaction is measured by surface plasmone resonance (SPR).

21. Method according to claim 18, wherein the interaction is measured by enzyme linked immunosorbent assay (ELISA).

22. Method according to claim 18, wherein the interaction is measured by a quartz crystal microbalance (QCM).

23. Method according to claim 18, wherein the interaction is measured by a surface acoustic wave (SAW).

24. Method according to claim 18, wherein the interaction is measured by at least one method from the group of: reflectrometric interference spectroscopy (RIfs), bio-layer interferometry (BLI), surface plasmone resonance (SPR), quartz crystal microbalance (QCM), surface acoustic wave (SAW), enzyme linked immunosorbent assay (ELISA), nanopores or transistors (next generation sequencing).

Description

SHORT DESCRIPTION OF THE FIGURES

(1) In the following, preferred embodiments of the present invention are described in detail with reference to the Figures.

(2) FIG. 1 shows an exemplary IR absorption spectrum of water or an aqueous solution with shown absorption maxima;

(3) FIGS. 2A & 2B schematically show the influence of the orientation of an irradiated IR laser beam relative to gravitation on the generated thermal convections;

(4) FIG. 3 shows a schematic representation of the preferred detection region for measuring the specific and unspecific interaction of particles within a sample chamber;

(5) FIG. 4 shows a further embodiment of an arrangement for the method of the present invention wherein in particular the beam path of the respective light beams is schematically shown;

(6) FIG. 5 shows a schematic representation of a test arrangement according to the present invention for measuring specific and/or unspecific interactions of particles with a surface;

(7) FIG. 6 shows a schematic representation similar to FIG. 5, but with a means for coupling a laser beam for generating convection in the measuring cell, which is arranged further above;

(8) FIGS. 7A & 7B show schematic representations of the irradiation of IR radiation into multi-well plates; and

(9) FIG. 8 shows a schematic representation of an exemplary test arrangement according to the invention for measuring multi-colored (multiplexing) fluorescence in a multi-well plate in which fluorescence excitation and fluorescence detection as well as IR radiation focussing is effected by the same optical system.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(10) FIG. 3 shows, by way of example, the detection region 80 for measuring the specific and unspecific interactions of particles. The shown sample chamber 45 is, by way of example, a capillary.

(11) The detection region 80 is preferably located at the surface/boundary surface of the measurement volume within the capillary 45, i.e. on the inside of sample chamber 45. The detection region 80 may, for example, be selected around the region of an irradiated IR radiation 30 such that it is smaller, larger or has the same size as the surface swept by the thermal convection 90.

(12) The detection region 80 may, for example, be a thin film and contain, for example, antibodies for the specific detection of antigens. In other words, the detection region is located at the surface of the liquid or at the surface of the capillary 45. The detection region 80 may, for example, also be composed of a plurality of differently thin films, which differ, for example, in their refractive index, polarizability or their fluorescence. The convection is preferably adjusted such that it transports particles from far outside the detection region, for example, from some millimeters away, into the detection region.

(13) It is possible to vary the order; the decisive factor is that there is a specific distance between IR laser focus (determining the convection rolls) and the place where the interaction at the surface is detected. The distance between both is important since, depending on output and chamber thickness and chamber geometry, the IR laser will generate different thermal convection flows. The whole setup must be controlled such that the correct thermal convection flows are present at the point where the interaction is observed (for preventing a depletion layer of the molecules).

(14) FIG. 4 shows an exemplary arrangement in which the method of the invention is used. Here, infrared laser radiation 30 is irraditated from below into a sample chamber 45 with a volume of liquid, here an aqueous solution with particles 50, and generates a thermal convection 90 in the sample chamber 45. The flow rates in the liquid are represented by corresponding vectors. Moreover, a symmetrical convection around the irradiated laser beam 30 is discernible in this example. The reflectometric interference spectroscopy (RIfs) method is used as detection method for measuring the interaction of the particles 105 dissolved in the liquid with a thin film (hatched) of functionally immobilized molecules/particles 103.

(15) In summary, the RIfS is a physical method based on the interference of white light at thin films. This method is used in practice, e.g., for analyzing molecular interactions. The underlying measuring principle corresponds to the Fabry-Prot interferometer. RIfs is primarily used as detection method in chemosensors and biosensors. As sensitive layers, mostly non-selectively measuring polymers are used which sort analytes either according to their size (the so-called molecular sieve effect with microporous polymers) or on the basis of different polarities (e.g., functionalized polydimethyl siloxanes). In the field of biosensors, for example, polymers, such as polyethylene glycols or dextranes, are applied to the layer system, and identification structures for biomolecules are immobilized thereon. Generally, all substance classes can be used as identification structures (proteins, such as antibodies; DNA/RNA, such as aptamers; small organic molecules, such as Estron; but also lipids, such as phospholipid membranes).

(16) Additionally, FIG. 4 shows a carrier 46 which may be component of the sample chamber 45. However, it is also possible that the sample to be analyzed is provided on the carrier as drop or liquid film. The carrier 46 may be made, for example, from glass or plastics. On this carrier 46, the thin film to be analyzed, comprising a layer 103 of functionally immobilized molecules and a further layer 102 of molecules which is arranged between the layer 103 and the carrier 46, is schematically represented. The layer 102 serves, in particular, for better adhesion of the thin film on the carrier 46. The film/layer 102 of molecules (e.g., PEG or dextrane, etc.) which is arranged between the layer 103 and the carrier 46 (schemically represented) may, for example, serve as spacer and/or as immobilization means for the functional molecules of film 103. In particular, the layer 102 is also used for a better adhesion of the layer/thin film 103 on the carrier.

(17) Sample particles 105 may adhere/bond to the functionally immobilized molecules/particles of layer 103. As a consequence, the thickness of the thin film increases, the distance of its upper boundary surface 104 from the phase boundary between thin film and carrier (or from the lower boundary surface of the thin film) increases. After adhesion/bonding of the sample particles 105, light 31 irradiated from below, which is used for measuring, is now also reflected at the boundary surface 104. The boundary surface 104 is formed with respect to the solution with particles 50, for example, when the particles from the aqueous solution bind to the functionally immobilized molecules in the thin film 103. The reflected beam 113 at this boundary surface 104 is schematically shown. Further shown are the reflected beams 112, 111a, 111b and 110 which are reflected at boundary surfaces lying under the boundary surface 104. Since the irradiated light has to travel a longer path length to the boundary surface 104, this causes a displacement of the interferogram which is generated by superposition of the reflected electromagnetic radiation 110, 111a, 111b, 112 and 113. It is possible to measure this displacement time-resolved, which allows exact conclusions as to the change in film thickness and thus to the interaction between the disassociated/dissolved particles 105 and the functionally immobilized particles 103.

(18) For example, the particles 105 in the aqueous solution are biomolecules, such as DNA, RNA, proteins, antibodies, antigenes, etc., small molecules, nanoparticles, polymers, peptides, PNA, etc., or even cells, viruses, bacteria, vesicles, liposomes, microbeads, nanobeads, nanodiscs, etc.

(19) FIG. 5 shows, by way of example, the use of the method of the invention in a specific test arrangement, without, however, being limited thereto. The reference numeral 1 designates a light source that is used for the measuring. For example, the light source 1 can be one or more LED(s), one or more laser(s) and/or one or more SLED(s) (superluminescent LED). The light of the light source 1 is primarily used for irradiating a sample 50 to be analyzed, preferably for vertically irradiating it. The light emitted from the light source 1 can be changed by known optical means, for example by a diffusor 4 and/or a lens system (not shown). A diffusor 4 may, for example, be used for evenly distributing the light, and a lens system may be used for concentrating the light as desired. In this embodiment, the light subsequently passes a polarizer 5, e.g. for generating linearly polarized light. In addition, the light may also cross a filter 14, so that a light beam 31 with defined properties is irradiated on the sample 50. The filter 14 may be, for example, a wavelength filter, such as a band-pass filter, or a long pass filter or a short pass filter.

(20) The beam splitter 7 in the embodiment shown is used for splitting the beam of light into a measuring beam or measuring beam path 9 and a reference beam or reference beam path 11, with the measuring beam path being shown towards the bottom and the reference beam path 11 being shown left towards the reference detector array 19. The beam splitter 7 has preferably a polarizing property. However, it is also possible to omit the beam splitter 7 in specific embodiments. In that case, there will be no reference beam path 11 either, nor the whole reference object comprising reference beam path 11, reference lens system 17, reference detector filter 23 and reference detector 19.

(21) In the embodiment shown with a reference object, the reference detector array 19 may be, e.g., a photodiode, a photomultiplier (photomultiplier tube, PMT), a CCD camera (Charge-Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), a diode array or an avalanche photodiode. Upstream of the reference detector array 19, the reference object may also have a reference lens system 17 for the depiction/focusing on the reference detector array 19, and/or a reference detector filter 23, such as a band pass filter, or a long pass filter or a short pass filter.

(22) Prior to striking the sample 50, the measuring beam path 9 can be changed by additional optical means which are arranged after/downstream of the beam splitter 7. By way of example, a spot filter 21 is shown and a (first) optical correcting element 35, so as to compensate/correct, for example, the phase shift, the change in polarization and/or the beam path change, possibly generated by the second beam splitter 34 for coupling in the infrared laser radiation. In addition, a second optical correcting element 36 is shown, which may optionally be supplemented with a lens or a lens/lens system, for compensating/correcting, for example, the phase shift, the change in polarisation and/or the beam path change, which may be associated with the (second) beam splitter 34 for coupling in the infrared laser radiation. The second optical correcting element 36 and/or the optional lens or the optional lens system may also be used for focusing the beam paths on the sample 50.

(23) The sample 50 can be provided on a carrier 46 in the form of a drop or in a sample chamber 45, as shown in FIGS. 2 to 4. In particular, the sample chamber may be a capillary, a microcavity, a reaction vessel (Eppi), a microfluidic system, or a pipette tip, without being limited thereto. The sample 50 to be analyzed is preferably a liquid, preferably an aqueous solution, with particles 105 present therein (see FIG. 4) which may be in dissolved or undissolved form.

(24) The carrier 46 is preferably at least partly transparent. The shown carrier 46 is an object carrier glass, formed from glass, on which a thin film 103 is formed. The thin film 103 to be analyzed comprises, for example, a layer of functionally immobilized molecules.

(25) The thin film 103 is affected by the sample 50 to be analyzed. For example, an interaction of the molecules on the thin film 103 with the corresponding particles 105 in the sample leads to a change in film thickness (see FIG. 4). This change in film thickness also affects the light passed via the measuring beam path 9 to the carrier 46 and reflected at the surface of the thin film, which light is diverted by the beam splitter 7 and depicted on a detector array 19. The measuring branch (right of the beam splitter 7) is preferably designed similar to the detector array 19 or even identical to the reference branch (right of the beam splitter 7). In the shown embodiment, the detector array 19 may be, for example, a photodiode, a photomultiplier (photomultiplier tube, PMT), a CCD camera (Charge-Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), a diode array or an avalanche photodiode. In front of/upstream of the detector array 19, the measuring branch may comprise a lens system 17 for depiction/focusing on the detector array 19 and/or a detector filter 23, such as a band pass filter, or a long pass filter or a short pass filter.

(26) As already described with reference to FIG. 4, preferably multiple reflections at the boundary surfaces of the thin film are used for measurement, with the reflected beams being detected with the two detector arrays 19, 19. The two detector arrays 19, 19 are connected with an evaluation unit which is not further described herein.

(27) To ensure good mixing in the thin film 103 and to avoid a depletion layer, according to the invention light of a laser 32 is irradiated into the sample 50. For irradiation of the laser light, in FIG. 5, the second beam splitter 34 already mentioned above is arranged below the first beam splitter 7. In a further embodiment of the invention according to FIG. 6, in which identical reference signs refer to identical components, the second beam splitter 34 is shown, by way of example, above the first beam splitter 7. By means of the second beam splitter 34, the infrared laser radiation 30, which is emitted by the laser 32 and optionally changed by optical means 33, e.g. by lenses or a lens system, such as a collimator for parallelizing and/or focusing the infraread laser radiation, is coupled into the measuring beam path 9. The beam splitter 34 may be similar or identical to the beam splitter 7, or have other properties. For example, the beam splitter 34 may be a dichroic reflector or a hot mirror. Here, it is again emphasized that the irradiated electromagnetic radiation 31 is used for measuring, whereas the irradiated electromagnetic radiation 30 is used for generating convection.

(28) Here again it is explicitly emphasized that the above described test arrangement is only one of many examples of the invention and that the invention is by no means limited to a specific arrangement of the above-described optical means. In particular, the test arrangement is not limited to the shown orientation. Thus, instead from above, the light may also come from the bottom left or from the right, and the corresponding optical means can be shifted or rotated accordingly. Furthermore, the order of the optical means is not limited to the embodiment shown and can be changed depending on the desired properties for irradiation and measurement. According to the invention, a transmission may be measured instead of the reflection shown. However, a person skilled in the art will readily see that the method of the invention for generating convections can easily be implemented also in such a transmission test arrangement. In this connection, too, reference is made to FIG. 6, which shows a test arrangement very similar to the one of FIG. 5, where the light of the IR laser irradiates at another site.

(29) FIGS. 2A and 2B show, by way of example, the influence of the orientation of an irradiated IR laser beam 30 relative to gravitation on the thermal convection within a sample chamber 45 containing an aqueous solution 50 with particles dissolved therein (not shown). Drawn are also the velocity vectors (arrows) and the flow lines (lines) of the thermal convection 90.

(30) When the laser radiation, as shown in FIG. 2A, is oriented antiparallel to gravitation, the radiation pressure/light pressure increases the thermal convection, i.e. the flow rate is higher than when the laser radiation is directed parallel or vertical to gravitation.

(31) When the laser radiation is directed parallel to gravitation, as shown in FIG. 2B, the radiation pressure/light pressure mitigates the thermal convection; the flow rate is lower than when the laser radiation is directed antiparallel or vertical to gravitation.

(32) By way of example, FIGS. 7A and 7B show the irradiation of radiation, preferably of IR radiation, e.g. of laser radiation 30 into the well 45 of a multi-well plate, e.g. a 96-, 384- or 1536-multi-well plate, that is filled with an aqueous solution 50. The irradiated IR radiation 30 generates a thermal convection 90 in the irradiated sample chamber well 45. In FIG. 7A, the IR radiation 30 is irradiated through a transparent floor 47 of a multi-well plate.

(33) In FIG. 7B, the IR radiation 30 is irradiated through the floor but directly into the aqueous solution 50 in the sample chamber well 45, here from above. For example, this multi-well plate may have a non-transparent floor 48, but it may, e.g., also have a transparent floor or a partly transparent floor.

(34) FIG. 8 shows, by way of example, the application of the method of the invention in a concrete test arrangement, without, however, being restricted thereto. Again, identical reference numerals refer to identical or similar parts. Reference numeral 1a designates a light source which is used for measurement. For example, light source 1a may be one or more LED(s), one or more laser(s) and/or one or more SLED(s) (superluminescent LED). Reference numeral 1b designates a light source used for measurement. For example, light source 1b may be one or more LED(s), one or more laser(s) and/or one or more SLED(s) (superluminescent LED). Light source 1b preferable has a wave length or a wave length range differing from that of light source 1a. The light of light source 1a and/or 1b is preferably used for irradiating a sample 50 to be analyzed. The light emitted from light sources 1a and/or 1b can be modified by known optical means, e.g. by a lens 26 and/or by a lens system (not shown) or an aperture (not shown) or a polarization filter. Subsequently, the light from the light source 1a preferably passes an excitation filter 25, preferably a band pass filter, and the light from the light source 1b preferably passes an excitation filter 24, preferably a band pass filter. The excitation filter 24 preferably has another transmission range than the excitation filter 25. Reference numeral 23 refers to an optional detector filter, such as a band pass filter or a long pass filter or a short pass filter or a dual pass filter or a multi pass filter. In the case of fluorescence, the filter 23 may also be designated as an emission filter.

(35) The light from the two excitation light sources is preferably combined, for example by means of the dichroic mirror 28 and is subsequently preferably reflected by a further dichroic mirror 29 into the direction of the lens system 38. The dichroic mirror 29 is preferably also used for separating the excitation light from the detection light. After reflection at the dichroic mirror 29, the excitation light preferably passes a further dichroic mirror 34 (Hot Mirror) and is preferably subsequently focussed by the lens system 38 through the transparent floor 47 of the multi-well plate into the aqueous solution 50, in the sample chamber 45, which is preferably a well of a multi-well plate. There the excitation light activates the fluorescence of fluorescent particles 105, such as proteins with intrinsic fluorescence and/or fluorescently labelled biomolecules or other fluorescent substances. The fluorescent light is collected by the lens system 38, preferably a lens, a combination of lenses or a microscope objective; it subsequently passes the dichroic mirrors 34 and 29, then the detection filter 23, which is preferably an emission filter, such as a band pass filter, dual pass filter or multi-pass filter, and is then focussed by a lens 17, for example, an asphere, onto the detector 19, such as a photodiode, a PMT, a CCD camera, a CMOS camera, a diode array, an avalanche photodiode.

(36) With this detector it is possible to measure, and then to electronically process and save the intensity and/or phase and/or the temporal sequence of the fluorescence intensity. The infrared radiation for generating thermal convection is preferably produced with a fiber-coupled infrared laser 32. The fiber of the laser is coupled, for example, by means of a fiber coupling 27, into the optics or the optic system. The infrared radiation can be modified by means of known optical means, for example, by means of a lens 26 and/or of a lens system (not shown) or by means of an aperture (not shown) or a polarization filter. For example, it can be parallelized or focussed by the lens 26, such as an asphere. Subsequently, the infrared radiation is reflected through the dichroic mirror 34 (Hot Mirror) into the lens system 38. The lens system 38 then focusses the infrared radiation 30 through the transparent floor 47 of the multi-well plate into the aqueous solution 50 of the sample chamber 45, preferably a well of a multi-well plate. The multi-well plate is preferably a 96-well plate or a 384-well plate or a 1536-well plate. Depending on actual focus, the infrared radiation 30 generates therein a defined thermal convection 90 for mixing the particles 105 in the aqueous solution 50.

(37) The particles are, for example, biomolecules such as DNA, RNA, PNA, proteins, antibodies, antigens, or small molecules, cells, viruses, bacteria, microbeads, nanobeads, nanoparticles, polymers, peptides. The apparatus may also be used, for example, for detecting and quantifying biomolecule aggregation, e.g. the aggregation of proteins, or therapeutic antibodies.