Methods and devices for acoustophoretic operations in polymer chips

Abstract

The invention relates to a method of performing an acoustophoretic operation, comprising the steps of: a. providing an acoustophoretic chip comprising a polymer substrate in which a microfluidic flow channel is positioned, b. providing at least one ultrasound transducer in acoustic contact with one surface of the substrate, c. actuating the at least one ultrasound transducer at a frequency f that corresponds to an acoustic resonance peak of the substrate including the microfluidic flow channel filled with a liquid suspension, and d. providing the liquid suspension in the flow channel to perform the acoustophoretic operation on the liquid suspension. The invention further relates to an acoustophoretic device, a method of producing an acoustophoretic device, and a microfluidic system comprising the acoustophoretic device.

Claims

1. A method of performing an acoustophoretic operation, comprising the steps of: (a) providing an acoustophoretic chip comprising a polymer substrate defining a microfluidic flow channel; (b) providing at least two ultrasound transducers in acoustic contact with a first surface of the substrate; (c) actuating the at least two ultrasound transducers at a frequency f that corresponds to an acoustic resonance peak of the substrate including the microfluidic flow channel filled with a liquid suspension; and (d) providing the liquid suspension in the flow channel to perform the acoustophoretic operation on the liquid suspension.

2. The method according to claim 1, wherein the acoustic resonance peak corresponds to a three-dimensional volume resonance in the substrate including the microfluidic flow channel, which three-dimensional volume resonance cannot be described as a one- or two-dimensional resonance in the substrate.

3. The method according to claim 1, where the frequency f does not correspond to a resonance frequency of the channel alone.

4. The method according to claim 1, wherein in step (c), the at least two ultrasound transducers are actuated out of phase with respect to each other.

5. The method according to claim 4, wherein in step (c), the at least two ultrasound transducers are actuated in antiphase with respect to each other.

6. The method according to claim 4, wherein the at least two ultrasound transducers share a single common piezoelectric crystal.

7. The method according to claim 1, wherein the acoustophoretic operation comprises focusing particles, suspended in a suspension within the microfluidic flow channel, towards at least one discrete area of the microfluidic flow channel.

8. An acoustophoretic device for performing an acoustophoretic operation, comprising: an acoustophoretic chip, comprising a polymer substrate and a microfluidic flow channel in the substrate; at least two ultrasound transducers in acoustic contact with a first surface of the substrate; and a drive circuit connected to the at least two ultrasound transducers and configured to actuate the at least two ultrasound transducers at an acoustic resonance frequency f that corresponds to an acoustic resonance peak of the substrate including the microfluidic flow channel filled with a liquid suspension.

9. The acoustophoretic device according to claim 8, wherein the drive circuit is further configured to actuate the at least two ultrasound transducers out of phase relative to each other, at the acoustic resonance frequency f.

10. The acoustophoretic device according to claim 8, wherein the drive circuit is further configured to actuate the at least two ultrasound transducers in antiphase relative to each other.

11. The acoustophoretic device according to claim 8, wherein the substrate additionally comprises a further microfluidic flow channel, the further microfluidic flow channel being positioned so that an acoustic force arises, due to resonance in the substrate including the microfluidic flow channel and the further microfluidic flow channel, on a target particle in the further microfluidic channel.

12. A method of producing an acoustophoretic chip for performing an acoustophoretic operation, the acoustophoretic chip comprising a polymer substrate within which a microfluidic flow channel is provided, the method comprising the steps of: (a) determining an acoustic resonance of the substrate for each of a plurality of different combinations of parameter values, the parameters including polymeric substrate material, substrate dimensions, microfluidic flow channel dimensions, a microfluidic flow channel position within the substrate, properties of a liquid in the microfluidic flow channel, positions for at least two ultrasound transducers in acoustic contact with a first surface of the substrate, and an actuation frequency f of the at least two ultrasound transducers; (b) selecting, from among the plurality of different combinations of the parameter values of the parameters, a polymeric substrate material M, a set of substrate dimensions D.sub.S, a set of microfluidic flow channel dimensions D.sub.C, a microfluidic flow channel position P.sub.C within the substrate, properties of the liquid L in the microfluidic flow channel, a position P.sub.U for the at least two ultrasound transducers, and an actuation frequency f of the at least two ultrasound transducers, wherein the selected parameter values yield an acoustic resonance within the substrate including the microfluidic flow channel for performing the acoustophoretic operation; and (c) manufacturing the acoustophoretic chip made out of the substrate material M having the substrate dimensions D.sub.S and being provided with a microfluidic flow channel having the microfluidic flow channel dimensions D.sub.C and the microfluidic flow channel position P.sub.C within the substrate.

13. The method according to claim 12, wherein simulation is used in step (a), the simulation using as boundaries a polymer/air interface at an outer surface of the substrate, and a polymer/liquid interface at walls in the substrate defining the microfluidic flow channel.

14. The method according to claim 12, wherein step (a) further comprises determining an acoustic force on a target particle throughout the substrate for each of the plurality of different combinations of parameter values of substrate parameters, and step (b) further comprises determining a set of microfluidic flow channel dimensions D.sub.C and the microfluidic flow channel position P.sub.C within the substrate so that the microfluidic flow channel at least partly delimits a region of the substrate in which the acoustic force on the target particle is suitable for performing the acoustophoretic operation.

15. The method according to 12, wherein the acoustophoretic chip is configured for performing a further acoustophoretic operation, and wherein the parameters additionally comprise further microfluidic flow channel dimensions and a further microfluidic flow channel position within the substrate, for a further microfluidic flow channel.

16. The method according to claim 15, wherein the step (b) further comprises determining a further set of microfluidic flow channel dimensions D.sub.C2 and a microfluidic flow channel position P.sub.C2 within the substrate so that the further microfluidic flow channel at least partly delimits a further region of the substrate in which the acoustic force on a target particle is suitable for performing the further acoustophoretic operation.

17. A microfluidic system, comprising: an acoustophoretic device, comprising an acoustophoretic chip, comprising a polymeric channel substrate and a microfluidic flow channel in the substrate; at least two ultrasound transducers in acoustic contact with a first surface of the substrate; and a drive circuit connected to the at least two ultrasound transducers and configured to actuate the at least two ultrasound transducers at a frequency f that corresponds to an acoustic resonance peak of the substrate including the microfluidic flow channel filled with a liquid suspension; a polymeric main substrate having a main substrate surface in which is formed a first set of surface features; and a polymeric lid substrate placed over the main substrate surface so as to define, together with the first set of surface features, at least one microfluidic flow channel; wherein a part of the microfluidic flow channel extends through an acoustophoretic region of the main substrate, in which acoustophoretic region an acoustophoretic operation is to be performed, the acoustophoretic region defining the acoustophoretic chip; wherein a second set of surface features is provided in the main substrate in or adjacent to the acoustophoretic region so as to at least partially separate the acoustophoretic region from the remainder of the main substrate; wherein the at least two ultrasound transducers are in acoustic contact with a side of the lid substrate facing away from the main substrate surface, the at least two ultrasound transducers being positioned on the lid substrate so as to cover at least part of the acoustophoretic region; and wherein the drive circuit is connected to the at least two ultrasound transducers and is configured to actuate the at least two ultrasound transducers at a frequency f corresponding to a resonance peak of the acoustophoretic region of the main substrate including the microfluidic flow channel filled with the liquid suspension and a part of the lid substrate facing the acoustophoretic region.

18. The system according to claim 17, wherein each of the first and second sets of surface features is selected from the group consisting of projections and depressions.

19. The system according to claim 17, wherein the drive circuit is further configured to actuate the at least two ultrasound transducers out of phase relative to each other, at the acoustic resonance frequency f.

20. The system according to claim 17, wherein the channel substrate additionally comprises a further microfluidic flow channel, the further microfluidic flow channel being positioned so that an acoustic force arises, due to resonance in the substrate including the microfluidic flow channel and the further microfluidic flow channel, on a target particle in the further microfluidic channel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS AND DETAILED DESCRIPTION

(1) A more complete understanding of the abovementioned and other features and advantages of the present invention will be apparent from the following detailed description of preferred embodiments in conjunction with the appended drawings, wherein:

(2) FIG. 1 shows the set up and results of 2D simulations of resonances in a PMMA chip substrate, FIG. 1A showing the setup, FIGS. 1B-D showing the resonance frequencies for chips having different widths, FIGS. 1E-1L showing the radiation force in the substrate and the channel at selected resonance frequencies, FIGS. 1M-1N showing the radiation force for symmetric actuation,

(3) FIG. 2 shows results of 3D simulations of resonances in a part of a PMMA chip substrate, FIG. 2A showing the resonance frequencies and FIGS. 2B-2C showing the radiation force on a 10 μm polystyrene bead in a water filled channel at the two main resonance frequencies,

(4) FIG. 3 shows results of further 3D simulations of resonances in two differently dimensioned PMMA chip substrate, FIGS. 3A and 3C showing the resonance frequencies and FIGS. 3B and 3D showing the radiation force on a 10 μm polystyrene bead in a water in a part of the flow channel,

(5) FIG. 4 shows microscope images of the flow channel during experimental validation of the resonance frequencies predicted by the simulations, FIGS. 4A-4C showing the results for the chip 1A (the channel (A), beads flowing through the channel at 50 μL/min without ultrasound (B) and beads at the same flow rate focused at a frequency of 1.3 MHz (C)), and FIGS. 4D-4G showing the results for chip 1B (the channel (D), beads flowing through the channel at 50 μL/min without ultrasound (E) and beads focused at a frequency of 1.55 MHz flowing at 100 μL/min (F) and beads focused at the same frequency flowing at 200 μL/min(G)),

(6) FIG. 5 schematically shows the construction of acoustophoretic chips having polymeric substrates, FIG. 1A showing the general construction including the split piezo-ceramic element, FIG. 5B showing an acoustophoretic device according to the third aspect of the present invention, and FIGS. 5C and 5D showing a top view and a cross sectional view, respectively, of a microfluidic system according to the fourth aspect of the present invention, and

(7) FIG. 6 showing flow sheets of embodiments of methods according to the first and third aspects of the present invention.

EXAMPLE 1A

Initial 2D Simulations of PMMA Chip

Materials and Methods

(8) For the 2D simulation experiments a PMMA ship was modeled using the geometry shown in FIG. 1A

(9) The parameter values were as follows:

(10) TABLE-US-00001 PMMA Chip dimension WB = 3.0 mm, HB = 1.0 ram, HL = 0.175 mm (variations: WB = 1.5 mm, 3.0 mm, 5.0 mm) PMMA Density (ρ) 1170 kg m.sup.−3 PMMA longitudinal speed of sound 2706 m s.sup.−1 (c.sub.L) PMMA transverse speed of sound 1105 m s.sup.−1 (c.sub.T) PMMA damping (α) 10 m.sup.−1 PMMA damping coefficient (Γ) 0.0043 Water (channel) dimensions w = 0.375 mm, h = 0.150 mm Water density (ρ.sub.0) 997 kg m.sup.−3 Water Speed of sound (c.sub.0) 1497 m s.sup.−1 Water damping coefficient (Γ) 0.004 Test particle spherical 10-μm-diameter polystyrene bead Test particle acoustophoretic 12 (μm s.sup.−1)/pN mobility (μ.sub.ac) Test particle buoyancy-corrected 0.26 pN gravity (F.sub.gr) Test particle time for 48 s sedimention h = 150 μm (t.sub.sed) Asymmetric Actuation at frequency f and amplitude 1 nm

(11) Simulations were run for a range of frequencies f from 0 to 2 MHz and the acoustic energy (E.sub.ac) was determined as shown in FIG. 1B (for W.sub.B=1.5 mm), FIG. 1C (for W.sub.B=3.0 mm), and FIG. 1D (for W.sub.B=5.0 mm)

(12) The simulations were based on the Finite element method (FEM) using the numerical FEM software COMSOL. FEM is a method where a discretized into a plurality of triangular mesh cells of finite sizes, i.e. into a plurality of finite element wherein a local approximation of the problem can be solved for each finite element and a global solution can be pieced together.

(13) The simulations made use of an Eigenmode analysis of Eigenfrequency for various widths of the simulated chip/substrate and introduced additional resonance modes beyond resonance in merely one dimension of the substrate. A frequency-response analysis established resonance frequencies of the substrate, and, taking into account and modeling the dissipative losses in the fluid (water filled channel) and the bulk material (the PMMA) the magnitude and direction of the displacement field in the substrate and the pressure field in the channel, could be determined. From this the acoustic radiation force on a potential particle in the

(14) $F^{\mathrm{rad}}=-\pi a^3\left[\frac{2{\kappa }_{\mathrm{wa}}}{3}\mathrm{Re}\left[f_1^{*}{p}^{*}\nabla p\right]-{\rho }_{\mathrm{wa}}\mathrm{Re}\left[f_2^{*}{v}^{*}.Math.\nabla v\right]\right]v=-i\frac{1}{{\rho }_0\omega }\nabla p$
channel could be determined using the formula:

Results

(15) As seen in FIGS. 1B-1D all three chip widths (W.sub.B) resulted in a number of actuation frequencies where the acoustic energy (E.sub.ac) peaked signifying a resonance in the whole chip.

(16) In FIG. 1B (W.sub.B=1.5 mm) the following resonance frequencies were found:

(17) TABLE-US-00002 Force Maximum Frequency Acoustic acting on pressure in f energy test particle channel pmax (MHz) E.sub.ac (J/m.sup.3 F.sub.rad (pN) (kPa) 0.18 0.5 0.2 10 0.993 68 107 552 1.206 57 139 598 1.644 10 36 253 1.815 2 10 153

(18) As is seen from the above table there are two frequencies, 0.993 MHz and 1.206 MHz, which give rise to strong forces on the test particle. These frequencies are far removed from the frequencies obtained in the prior art principle of actuating acoustophoretic chips as typically a frequency of 2 MHz would be used to actuate a channel having the width of 0.375 mm (the channel width corresponding to one half wavelength. However, in FIG. 1B there is no peak in acoustic energy at f=2.0 MHz, rather there is instead a walley here. A similar result is seen in FIG. 1D. For FIG. 1B there is no peak at 2.0 MHz—the peak at 2.025 MHz is lower than the peak at 1.860 MHz.

(19) Thus the conventional way of selecting actuation frequency based on the dimensions of the microfluidic flow channel results in non-optimal actuation of the PMMA chip.

(20) FIGS. 1E-F show the magnitude and direction of the displacement field in the substrate and the pressure field in the channel, and the magnitude and direction of the radiation force on a 10 μm diameter polystyrene bead in water in the channel, respectively, for f=0.993 MHz.

(21) FIGS. 1G-H the magnitude and direction of the displacement field in the substrate and the pressure field in the channel, and the magnitude and direction of the radiation force on a 10 μm diameter polystyrene bead in water in the channel, respectively, for f=1.206 MHz.

(22) As is shown in these figures the simulation results for both 0.993 MHz and 1.206 MHz give a strong, near-1D, focusing of the particles into a single band in the center of the channel

(23) In FIG. 1C (W.sub.B=3.0 mm) the following resonance frequencies were found:

(24) TABLE-US-00003 Force Maximum Frequency Acoustic acting on pressure in f energy test particle channel pmax (MHz) E.sub.ac (J/m.sup.3 F.sub.rad (pN) (kPa) 0.21 0.2 0.03 7 0.445 0.5 0.39 25 1.257 22 54 389 1.590 2 36 131 1.860 42 21 520

(25) FIG. 1I shows the magnitude and direction of the displacement field in the substrate and the pressure field in the channel (left/background) and the magnitude and direction of the radiation force on a 10 μm diameter polystyrene bead in water in the channel (right/foreground), respectively, for f=1.257 MHz.

(26) FIG. 1J shows the magnitude and direction of the displacement field in the substrate and the pressure field in the channel (left/background) and the magnitude and direction of the radiation force on a 10 μm diameter polystyrene bead in water in the channel (right/foreground), respectively, for f=1.590 MHz.

(27) As is shown in these figures also the wider chip (W.sub.B=3.0 mm) has a moderately strong focusing into one band in the center of the channel at 1.257 MHz. At 1.860 MHz the particles are focused into a central band and two lateral spots.

(28) In FIG. 1D (W.sub.B=5.0 mm) the following resonance frequencies were found:

(29) TABLE-US-00004 Force Maximum Frequency Acoustic acting on pressure in f energy test particle channel pmax (MHz) E.sub.ac (J/m.sup.3 F.sub.rad (pN) (kPa) 0.800 9 16 215 1.35 34 84 517 1.45 5 12 208 1.74 25 104 498 1.81 50 136 706

(30) FIG. 1K shows the magnitude and direction of the displacement field in the substrate and the pressure field in the channel (left/background) and the magnitude and direction of the radiation force on a 10 μm diameter polystyrene bead in water in the channel (right/foreground), respectively, for f=1.35 MHz.

(31) FIG. 1L shows the magnitude and direction of the displacement field in the substrate and the pressure field in the channel (left/background) and the magnitude and direction of the radiation force on a 10 μm diameter polystyrene bead in water in the channel (right/foreground), respectively, for f=1.810 MHz.

(32) As is shown in these figures also the widest chip (W.sub.B=5.0 mm) has a strong focusing of the particles into one band in the center of the channel.

(33) In a further simulation the geometry of the PMMA chip shown in FIG. 1A with W.sub.B=3 mm was inverted with the transducers attached to the lid. This variant also resulted in a number of strong resonances:

(34) TABLE-US-00005 Frequency Force acting on test f (MHz) particle F.sub.rad (pN) 1.040 81 1.130 53 1.380 38 1.455 174 1.785 411 1.908 107

(35) In summary Example 1 shows that chips having substrates made from PMMA and other similar polymeric materials can be actuated to provide strong useful resonances, but that the actuation frequencies cannot be determined as for the conventional silicon or glass chips based on the dimensions of the microfluidic flow channel alone, but rather requires considering the resonances in the whole substrate including the microfluidic flow channel.

(36) Further it should be noted that in the FIGS. 1E-1L the force at the side walls of the channel is non-zero.

(37) Further simulations show that the radiation force F.sub.rad and the acoustic energy density E.sub.ac is only weakly affected by a gap between the ultrasound transducers or a lateral shift of the flow channel.

EXAMPLE 1B

Simulation Comparing Asymmetric and Symmetric Actuation

Materials and Method

(38) As in example 1 for W.sub.S=3.0 mm and a total height of the chip=1.18 mm. The frequency f=1.745 MHz was selected and simulations were performed for an asymmetric actuation and symmetric actuation.

Results

(39) FIG. 1M shows F.sub.rad for symmetric actuation at 1.380 MHz. The force vectors are directed towards the side walls of the channel.

(40) FIG. 1N shows F.sub.rad for symmetric actuation at 1.745 MHz. The force vectors are directed towards the ceiling of the channel and also towards the side walls of the channel.

EXAMPLE 2

3D Simulation of Part of PMMA Chip

Materials and Method

(41) A PMMA chip 1B, see example 4 for dimensions was simulated using the parameters of example 2. The simulation was made using the ¼-symmetry:
0<x<L.sub.s/2L.sub.s=40 mm
0<y<W.sub.s/2W.sub.s=3 mm
0<z<H.sub.s H.sub.s=1.18 mm

(42) The asymmetric actuation, defined as (0.1 nm)*tan h(50*y/Ws) was applied in the xy plane at z=0.

(43) The xy plane at y=0 has antisymmetric boundary conditions due to actuation and the yz-plane at x=0 has symmetrical boundary conditions due to the symmetry away from the center plane in the chip along the x-axis.

Results

(44) The two largest resonances, as measured using the acoustic energy E.sub.ac was found for 1.29 MHz, which corresponds exactly to the experimental value, see example 4, and 1.63 MHz which is about 105% of the experimental value of 1.55 MHz, see FIG. 2A.

(45) For 1.29 MHz the maximum F.sub.rad was 4.0 pN (note here that the amplitude of the actuation is 1/10 of the amplitude used in example 1, hence the lower F.sub.rad. FIG. 2B shows the quarter of the flow channel along the section 0<z<0.8 mm, as seen the force vectors point towards the center of the channel (y=0)—this would yield a qualitatively good focusing of particles into a vertical band at the center of the flow channel.

(46) For 1.63 MHz the maximum F.sub.rad was 2.7 pN. FIG. 2C shows the quarter of the flow channel along the section 0<z<1.3 mm, as seen the force vectors point towards the center of the channel (y=0)—this would yield a qualitatively good focusing of particles into a vertical band at the center of the flow channel.

EXAMPLE 3

3D Simulation of Full Chip

Materials and Method

(47) Chips 1B (W.sub.S=3.0 mm) and 1F (W.sub.S=5.0 mm), both having the height (H.sub.S=1.18 mm) and length (L.sub.S=50 mm) over the full height using the quarter vertical transverse symmetry plane and the vertical axial anti-symmetry to reduce the geometry to a quarter (0<x<L.sub.S/2=25 mm and 0<y<W.sub.S/2=1.5 mm or 2.5 mm) as in example 2.

Results

(48) The table below compares the resonance frequencies predicted by the simulation with those identified in the experiments, see example 4.

(49) Chip 1B

(50) TABLE-US-00006 f (MHz)/ f (MHz)/ f (MHz)/ f (MHz)/ Resonance F.sub.rad (pN) F.sub.rad (pN) F.sub.rad (pN) F.sub.rad (pN) Simulation 1.132/0.38 1.277/0.05 1.381/0.20 1.455/0.88 Experiment 1.29 — — 1.550

(51) FIG. 3A shows the acoustic energy E.sub.ac for chip 1B, and FIG. 3B shows F.sub.rad in the center of the channel for f=1.456 MHz.

(52) Chip 1F

(53) TABLE-US-00007 f (MHz)/ f (MHz)/ f (MHz)/ f (MHz)/ f (MHz)/ Resonance F.sub.rad (pN) F.sub.rad (pN) F.sub.rad (pN) F.sub.rad (pN) F.sub.rad (pN) Simulation 1.027/ 1.330/0 1.415/ 1.731/ 1.790/ 0.07 1.05 0.54 0.31 Experiment 1.120 1.330 1.460 1.770 —

(54) FIG. 3C shows the acoustic energy E.sub.ac for chip 1F, and FIG. 3D shows F.sub.rad in the center of the channel for f=1.415 MHz.

(55) Where not explicitly discussed in example 4 the experimental resonance frequencies in the above tables were determined as in example 4.

EXAMPLE 4

Evaluation of Prototype PMMA Chips

Materials and Method

(56) A number of 20 PMMA chips were ordered from Microfluidic ChipShop, Germany.

(57) The basic common properties for all chips are given in the table below:

(58) TABLE-US-00008 Chip material PMMA Chip length 50 mm Lid thickness H.sub.lid 175 μm Channel length (1) 40 mm Channel width (w) 375 ± 15 μm Channel depth (h) 150 ± 15 μm

(59) A number of parameters were varied as detailed in the table below:

(60) TABLE-US-00009 Chip Substrate Width of Thickness of width thickness Piezoceramic Piezoceramic Chip (W) (H.sub.base) transducer transducer Transducer name (mm) (mm) (mm) (mm) position 1A 3 1.18 5 1 Opposite* 1B 3 1.18 5 1 1C 5 1.8 5 1 1D 5 1.75 7 1 1E 5 1.18 7 1 Misaligned 1F 5 1.18 7 1 2A 3 1.18 5 2.2 2B 3 1.68 5 2.2 Opposite* 2C 5 1.69 7 2.2 2D 5 1.18 7 2.2 *Here the transducer was attached to the base substrate, and not onto the lid substrate. Thus the transducers in these chips were further away from the flow channel than in the other chips.

(61) The microfluidic flow channel was provided on one surface of the substrate to which the lid was bonded so as to seal the channel. A planar piezoceramic crystal was provided with a common grounded single bottom electrode attached to its bottom surface. First and second top electrode were formed on the top surface by deposition on an electrode material after which the electrode material was divided into the first and second top electrodes by sawing through the electrode material and approximately 400 μm into the top surface of the piezoceramic crystal. The gap between the first and second top electrodes was approximately 100 μm.

(62) For the evaluation, a solution of 8 μm diameter polystyrene beads in water with Tween (detergent) was used. The piezoceramic crystal was actuated in an asymmetric manner, i.e. with the part of the piezoceramic crystal defined between the first top electrode and the single bottom electrode being actuated out of phase, by 180°, to the part of the piezoceramic crystal defined between the second top electrode and the single bottom electrode. The frequency was manually scanned in 10 kHz steps from 0.6 to 2 MHz. The function generator was set to 10 Vpp with a 180° phase difference between the transducers.

Results

(63) The table below shows the different resonance frequencies f.sub.1, f.sub.2, f.sub.3, f.sub.4 found for each chip.

(64) TABLE-US-00010 Chip f.sub.1 f.sub.2 f.sub.3 f.sub.4 name (MHz) (MHz) (MHz) (MHz) 1A 1.3 1.82 1.98 1B 1.29 1.55 1C 0.96 1.3 1.56 1.8 1D 1.03 1.27 1.7 1E 1.34 1.45 1.69 1F 1.12 1.33 1.46 1.7 2A 1.16 Testing was discontinued for these chips 2B 1.2 after finding the first resonance frequency 2C 1.25 f.sub.1 2D 1.11

(65) FIG. 4A shows a microscope bright field image of chip 1A, showing the channel.

(66) FIG. 4B is a fluorescence image showing beads in channel without ultrasound at 50 μl/min. As seen from the image there is no focusing of the beads in the channel.

(67) FIG. 4C shows how beads are focused to the center of the channel when the chip is actuated asymmetrically at a frequency of 1.3 MHz, with an amplitude of 10 V.sub.pp, and at a flowrate of 50 μl/min.

(68) Further resonances, i.e. acoustophoretic focusing effects, were obtained at 1.82 and 1.98 MHz also at 50 μL/min.

(69) These results should firstly be compared to the simulations, see example 1, of resonance frequencies in a chip with a width W.sub.B of 3.0 mm, se FIG. 1C. Here the simulation predicts a resonance at 1.225 MHz (1.3 MHz) 1.590 MHz, and 1.860 MHz (1.82 MHz, 1.98 MHz). Accordingly the qualitative results of the simulations, i.e. that there are effective actuation frequencies that are not determined by the dimensions of the microfluidic flow channel, are confirmed in the experiments.

(70) Secondly, these results may also be compared to previous attempts were significantly higher ultrasound energies, such as 70 Vpp, has been used in order to be able to focus particles at the same flow rate.

(71) FIG. 4D shows a microscope bright field image of chip 1B, which is of the same type as chip 1A.

(72) FIG. 4E is a Fluorescence image showing beads in channel without ultrasound at 50 μL/min. As seen from the image there is no focusing of the beads in the channel.

(73) FIG. 4F shows how beads are focused to the center of the channel when the chip is actuated asymmetrically at a frequency of 1.55 MHz, with an amplitude of 10 V.sub.pp, and at a flowrate of 100 μl/min.

(74) FIG. 4G shows how beads are focused to the center of the channel when the chip is actuated asymmetrically at a frequency of 1.55 MHz, with an amplitude of 10 V.sub.pp, and at a flowrate of 200 μl/min.

(75) Further resonance, i.e. acoustophoretic focusing effects, were obtained at 1.29 MHz at 150 μL/min.

(76) Here the simulations predicts a resonance at 1.225 MHz (1.3 MHz) 1.590 (1.55 MHz) MHz, and 1.860 MHz. Thus also here the simulation results are confirmed at least quantitatively.

(77) At the higher flowrates shown in FIGS. 4F and 4G the separation efficiency is decreased as some particles are not focused into the center of the channel but instead occupies positions along the walls. However, it should be noted that these results are obtained at low ultrasound energies (10 V.sub.pp) and at very high flow rates (100-200 μL/min).

(78) Symmetric actuation of the chips resulted in the particles being pushed towards the walls of the channel, the reverse to focusing, as detailed in the below table. This unexpected feature is not possible for particles with positive acoustic contrast in silicon/glass chips and not predicted by the one-dimensional channel resonance model that is normally used for channel design. It was, however, now predicted by whole substrate resonance simulation, see example 1.

(79) TABLE-US-00011 Chip f.sub.1 (MHz) name (symmetric) observation 1A 0.67 Reverse focusing 1B 2.02 Reverse focusing 1C 2 Reverse focusing 1D 2.02 Reverse focusing 1E 1.95 Reverse focusing 1F 2.39 Reverse focusing 2A 3.01 Reverse focusing 2B 2.95 Reverse focusing 2C 3 Reverse focusing 2D 2.96 Reverse focusing

(80) The general construction of an acoustophoretic chip of an acoustophoretic device according to the second aspect of the present invention is shown schematically and in cross section in FIG. 5A. The acoustophoretic chip or device 10 thus comprises a polymeric substrate 12 made up of a base substrate 14 into which lower surface 16 (or upper surface depending on the orientation) a microfluidic flow channel 18 is provided, either during a moulding step when the base substrate is moulded, such as by injection moulding, or in a subsequent step of precision machining, such as by milling. The microfluidic flow channel 18 thus initially resembles a groove or trough on one of the surfaces of the base substrate 14, a floor, or roof depending on the orientation, to the channel 18 is provided by bonding, such as by solvent bonding (where a solvent partially dissolves the surfaces of two objects to be joined) or using an adhesive, a lid substrate 20 to the lower surface 16 of the base substrate 14. A fluid may then be led through the flow channel 18 so as to introduce and/or pass a liquid or fluid sample through the chip 10.

(81) Actuation of the polymeric substrate is provided by first and second ultrasound transducers 22A and 22B which are constructed so as to share a single common piezoelectric crystal 24. An electrode material is provided on the upper surface 26 of the piezoelectric crystal 24, whereafter a cut is made through this layer of electrode material and also preferably, as shown, partially down into the upper surface 26 of the piezoelectric crystal 24 to form a cut-out or groove 28 in the electrode material and the upper surface 26, thus leading to the formation of first and second 30A, 30B spaced apart electrodes. On the bottom surface 32 of the piezoelectric crystal 24 a layer of electrode material is similarly applied, however no cut is needed as this layer is to form a common ground electrode 34 for the first and second electrodes 30A; 30B. The thus formed two ultrasound transducers 22A and 22B are then attached to the lid substrate 20 by a bonding layer of for instance adhesive 36. In operation a liquid or suspension 2 is provided in the flow channel 18. Acoustic forces then affect particles in the liquid, such as particle 4 in the further microfluidic flow channel 18′, and thereby perform an acoustophoretic operation in the liquid and the particles.

(82) The generally non-homogenous pressure fields arising in the substrate when in resonance, see in particular the simulation results outside the microfluidic channel in inter alia FIG. 1E of example 1, can be used by placing a further, or a plurality of further, microfluidic flow channel(s) 18′ in the substrate 12. If the forces arising on the particle 4 in the further microfluidic flow channel are the same as would affect the same particle in the microfluidic flow channel 18, then both microfluidic flow channel 19 and 18′ may be used to perform the same acoustophoretic operation. If that is not the case different acoustophoretic operations may be performed in the different flow channels.

(83) It should also be noted that, the ultrasound transducers 22A, 22B here are attached to the lid substrate 20, thus providing a shorter distance between the ultrasound actuators and the microfluidic flow channel 18.

(84) FIG. 5B shows an acoustophoretic device according to the second aspect of the present invention including, in addition to the substrate with the ultrasound transducers shown in FIG. 5A also the drive circuit. Thus a drive circuit 38 includes two function generators 40A and 40B capable of sending out signals at or near a resonance frequency of the substrate 12 including the base substrate 14 and the lid substrate 20 (see FIG. 5A) by first and second signal leads 42A and 42B connected to the first and second electrodes 30A and 30B on the piezoelectric crystal 24. The ground electrode 34 is then connected to ground 44 via a ground lead 46. In operation drive circuit 38 outputs, using function generators 40A and 40B, signals, which preferably are in antiphase, which are led to the first and second electrodes 30A and 30B, so as to actuate the polymeric substrate 12 asymmetrically at the resonance frequency of the polymeric substrate 12 in order to perform an acoustophoretic operation in the channel 18. Preferably, as described earlier, the resonance frequency is the resonance frequency of the combination of the polymeric substrate 12, preferably including the microfluidic flow channel 18, and the ultrasound transducer 22A, 22B (including the piezoelectric crystal 24 with the electrodes 30, 30B and 34).

(85) FIGS. 5C and 5D shows a top view and a cross sectional view, respectively, of a microfluidic system 100 according to the fourth aspect of the present invention.

(86) The microfluidic system 100 includes a main substrate 102, which is made from a polymeric material, and which includes at least one microfluidic channel 104 having an at least one inlet 106 and one or more outlets 108, 110, 112, the channel being formed by milling or moulding grooves or troughs in the surface of the main substrate 102. Microfluidic systems typically comprise modules for performing various functions such as mixing, reacting, collecting a fluidic sample, such modules being exemplified by a collection cavity 114 for collecting a fluid sample, and also by holding and/or mixing section 116 in which the channel 104 is convoluted.

(87) As microfluidic systems typically are made from polymeric materials, the inclusion of an acoustophoretic region or module where acoustophoretic operations are to be carried out would be difficult or complicated if silicon or glass were to be used for these functions, as these materials differ from the material of the main substrate 102 of the microfluidic system 100, thus requiring separate manufacturing followed by assembling the silicon/glass parts with the main substrate.

(88) However, as the present invention now provides the possibility of efficiently performing acoustophoretic operations in polymeric materials, the acoustophoretic operations may be performed using a module or chip integrated with the main substrate 102 of the microfluidic system 100. As shown in FIGS. 5C and 5D a section 118 of the microfluidic channel 104 may thus be arranged to pass through a region 120 of the base substrate 102 in which region 120 an acoustophoretic operation is to be carried out. Turning briefly to FIG. 5D, which is a cross section of FIG. 5C through the line AA′, it can be seen that the main substrate 102 comprises a main base substrate 122 joined to is joined with a lid substrate 124, which similar to the device in FIG. 5A serves to define the floor or ceiling of the channel 118. Similarly to FIGS. 5A and 5B the ultrasound transducers 22A and 22B are attached to the lid substrate 124 opposite the region 120. To further isolate the region 120 from the remainder of the main substrate material cutouts or groove 126A and 126B are provided around the region 120, these grooves may even pass right through the main base substrate 122 all the way to the other surface 128 so as to define a chip 130 that is integrated in the base substrate 102 and which only connects to the remainder of the base substrate 102 where the channel 118 enters and exits the region 120.

(89) Thus in use the ultrasound transducers 22A and 22B are actuated. A sample flowing through the region 120 is exposed to acoustic forces in the channel 118, such as for example forces that focus particles towards the center of the channel 118. Where the channel 118 branches into the first and second side channels 132 and 134, the concentrated and focused particles thus flow, due to the laminar nature of the flow, into the central channel 136 and outlet 110, whereas other parts of the sample are led to outlet 108 and 112.

(90) FIG. 6A shows the method according to the first aspect of the present invention, including the steps of:

(91) providing, designated the reference numeral 1, an acoustophoretic chip comprising a polymer substrate in which a microfluidic flow channel is positioned,

(92) providing, designated the reference numeral 3, at least one ultrasound transducer, in acoustic contact with one surface of the substrate,

(93) actuating, designated the reference numeral 5, the at least one ultrasound transducer at a frequency f that corresponds to an acoustic resonance peak of the substrate including the microfluidic flow channel filled with a liquid suspension (2), and

(94) providing, designated the reference numeral 7, the liquid suspension in the flow channel to perform the acoustophoretic operation on the liquid suspension.

(95) FIG. 6B shows the method according to the third aspect of present invention, including the steps of:

(96) determining, designated the reference numeral 9, by calculation or simulation, the acoustic resonances of the substrate for each of a plurality of different combinations of parameter values of substrate parameters, the substrate parameters including polymeric substrate material, substrate dimensions, microfluidic flow channel dimensions, microfluidic flow channel positions within the substrate, properties of a liquid in the microfluidic flow channel, positions for at least one ultrasound transducer, and actuation frequency f, and
Selecting, designated the reference numeral 11, among the plurality of different combinations of the parameter values of the substrate parameters, a polymeric substrate material M, a set of substrate dimensions D.sub.S, a set of microfluidic flow channel dimensions D.sub.C, a microfluidic flow channel position P.sub.C within the substrate, properties of the liquid L in the microfluidic flow channel, a position P.sub.U for at least one ultrasound transducer, and an actuation frequency f, which yield acoustic resonance within the substrate including the microfluidic flow channel, and manufacturing, designated the reference numeral 13, the acoustophoretic chip made out of the substrate material M having the substrate dimensions D.sub.S and being provided with a microfluidic flow channel having the microfluidic flow channel dimensions D.sub.C and the microfluidic flow channel position P.sub.C within the substrate.

Feasible Modifications of the Invention

(97) The invention is not limited only to the embodiments described above and shown in the drawings, which primarily have an illustrative and exemplifying purpose. This patent application is intended to cover all adjustments and variants of the preferred embodiments described herein, thus the present invention is defined by the wording of the appended claims and the equivalents thereof. Thus, the equipment may be modified in all kinds of ways within the scope of the appended claims.

(98) For instance, it shall be pointed out that structural aspects of embodiments of the method according to the first aspect of the present invention shall be considered to be applicable to embodiments of the system according to the second aspect of the present invention, and conversely, methodical aspects of embodiments of the system according to the second aspect of the present invention shall be considered to be applicable to embodiments of the method according to the first aspect of the present invention.

(99) It shall also be pointed out that all information about/concerning terms such as above, under, upper, lower, etc., shall be interpreted/read having the equipment oriented according to the figures, having the drawings oriented such that the references can be properly read. Thus, such terms only indicates mutual relations in the shown embodiments, which relations may be changed if the inventive equipment is provided with another structure/design.

(100) It shall also be pointed out that even thus it is not explicitly stated that features from a specific embodiment may be combined with features from another embodiment, the combination shall be considered obvious, if the combination is possible.

(101) Throughout this specification and the claims which follows, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or steps or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

(102) Further embodiments of aspects of the invention are set out in the following points:

(103) 1. A method of performing an acoustophoretic operation, comprising the steps of:

(104) a. providing an acoustophoretic chip (10) comprising a polymer substrate (12) in which a microfluidic flow channel (18) is positioned, b. providing at least one ultrasound transducer (22A, 22B) in acoustic contact with the substrate, c. actuating the at least one ultrasound transducer at a frequency f that corresponds to an acoustic resonance peak of at least the substrate, preferably including the microfluidic flow channel filled with a liquid suspension (2), and d. providing the liquid suspension in the flow channel to perform the acoustophoretic operation on the liquid suspension.
2. The method according to point 1, wherein the acoustic resonance peak corresponds to three-dimensional volume resonance in the substrate, preferably including the microchannel, which three-dimensional volume resonance cannot be described as a one- or two-dimensional resonance in the substrate.
3. The method according to point 1 or 2 where the frequency f does not correspond to a resonance frequency of the channel alone.
4. The method according to any of the point 1-3, wherein in step b at least two ultrasound transducers (22A, 22B) are provided in acoustic contact with the substrate, and wherein in step c the at least two ultrasound transducers are actuated out of phase, preferably in antiphase, with respect to each other.
5. The method according to point 4, wherein the at least two ultrasound transducers share a single common piezoelectric crystal (24).
6. The method according to any of the points 1-5, wherein the acoustophoretic operation comprises focusing particles, suspended in a suspension within the microfluidic flow channel, towards one or more discrete areas of the microfluidic flow channel.
7. A device for performing an acoustophoretic operation, comprising: an acoustophoretic chip (10) comprising a polymer substrate (12) and a microfluidic flow channel (18) positioned within the substrate, at least one ultrasound transducer (22A, 22B) in acoustic contact with the substrate, and a drive circuit (38) connected to the at least one ultrasound transducer and being configured to actuate the at least one ultrasound transducer at a frequency f that corresponds to an acoustic resonance peak of at least the substrate, preferably including the microfluidic flow channel filled with a liquid suspension.
8. The acoustophoretic device according to point 7, comprising at least two ultrasound transducers (22A, 22B) in acoustic contact with the substrate, wherein the drive circuit is further configured to actuate the at least two ultrasound transducers, out of phase relative to each other, at the acoustic resonance frequency f.
9. The acoustophoretic device according to any of the points 7-8, wherein the substrate additionally comprises a further microfluidic flow channel (18′), the further microfluidic flow channel being positioned so that that an acoustic force arises, due to resonance in the substrate preferably including the microfluidic flow channel and the further microfluidic flow channel, on a target particle (4) in the further microfluidic channel, the acoustic force being the same, or different, from an acoustic force arising on a target particle in the microfluidic channel.
10. A method of producing an acoustophoretic chip (12) for performing an acoustophoretic operation, the acoustophoretic chip comprising a polymer substrate (12) in which a microfluidic flow channel (18) is provided, comprising the steps of: a. determining, by calculation or simulation, the acoustic resonances of the substrate for each of a plurality of different combinations of parameter values of substrate parameters, the substrate parameters including polymeric substrate material, substrate dimensions, microfluidic flow channel dimensions, microfluidic flow channel positions within the substrate, properties of a liquid in the microfluidic flow channel, positions for at least one ultrasound transducer, and actuation frequency f, and b. selecting, among the plurality of different combinations of the parameter values of the substrate parameters, a polymeric substrate material M, a set of substrate dimensions D.sub.S, a set of microfluidic flow channel dimensions D.sub.C, a microfluidic flow channel position P.sub.C within the substrate, properties of the liquid L in the microfluidic flow channel, a position P.sub.U for at least one ultrasound transducer, and an actuation frequency f, which yield acoustic resonance within the substrate including the microfluidic flow channel, and c. manufacturing the acoustophoretic chip made out of the substrate material M having the substrate dimensions D.sub.S and being provided with a microfluidic flow channel having the microfluidic flow channel dimensions D.sub.C and the microfluidic flow channel position P.sub.C within the substrate.
11. The method according to point 10, wherein simulation is used in step a, the simulation using as boundaries the polymer/air interface at the outer surfaces of the substrate and the polymer/liquid interface at walls of the microfluidic flow channel.
12. The method according to any of the points 10-11, wherein step a further comprises determining the acoustic force on a target particle (4) throughout the substrate for each of the plurality of different combinations of parameter values of substrate parameters, and step b further comprises determining the set of microfluidic flow channel dimensions D.sub.C and the microfluidic flow channel position P.sub.C within the substrate so that the microfluidic flow channel at least partly delimits a region of the substrate in which the acoustic force on the target particle is suitable for performing the acoustophoretic operation.
13. The method according to any of the points 10-12, wherein the acoustophoretic chip is suitable for performing a further acoustophoretic operation, and wherein the substrate parameters additionally comprises further microfluidic flow channel dimensions and further microfluidic flow channel positions within the substrate, for a further microfluidic flow channel (18′).
14. The method according to points 13, wherein the acoustophoretic operation and the further acoustophoretic operation are different, and wherein step b further comprises determining a further set of microfluidic flow channel dimensions D.sub.C2 and microfluidic flow channel positions P.sub.C2 within the substrate so that the further microfluidic flow channel at least partly delimits a further region of the substrate in which the acoustic force on a target particle is suitable for performing the further acoustophoretic operation.
15. A microfluidic system comprising a polymeric main substrate (122) having a substrate surface in which is formed a first set of projections, such as walls, or depressions, such as grooves, a polymeric lid substrate (124) placed over the substrate surface so as to define, together with the first set of projections or depressions, at least one microfluidic channel (104),  wherein a part (118) of the microfluidic flow channel extends through an acoustophoretic region (120) of the main substrate, in which region an acoustophoretic operation is to be performed,  wherein a second set of projections or depressions (126A, 126B) are provided in the polymeric main substrate in or adjacent the acoustophoretic region so as to at least partially separate the acoustophoretic region from the remainder of the polymeric main substrate, and at least two ultrasound transducers (22A, 22B) in acoustic contact with the polymeric lid substrate on the side of the polymeric lid substrate facing away from the substrate surface, the at least two ultrasound transducers being positioned on the polymeric lid substrate so as to cover at least part of the acoustophoretic region, and a drive circuit (38) connected to the at least two ultrasound transducers and being configured to actuate, preferably out of phase or in antiphase, the at least two ultrasound transducers at a frequency f corresponding to a resonance peak of the acoustophoretic region of the polymeric main substrate, preferably including the microfluidic flow channel and/or a part of the polymeric lid substrate facing the acoustophoretic region.