NL2008816C2

NL2008816C2 - Method for size-sorting microbubbles and apparatus for the same.

Info

Publication number: NL2008816C2
Application number: NL2008816A
Authority: NL
Inventors: Andreas Michael Versluis; Tim Joseph Segers
Original assignee: Univ Twente
Priority date: 2012-05-14
Filing date: 2012-05-14
Publication date: 2013-11-18
Also published as: WO2013172708A1; WO2013172708A8

Description

Title: Method for size-sorting microbubbles and apparatus for the same

Field of the invention

The present invention relates to microbubbles for use in biomedical applications, and more in particular to a method and an apparatus for sizesorting a polydisperse plurality of such microbubbles.

5

Background

Microbubbles may be used for various biomedical applications. One such application is their use as an ultrasound contrast agent (UCA) in ultrasound imaging, nowadays the most widely used medical imaging 10 technique. The mechanism by which microbubbles enhance the contrast in ultrasound echoes is twofold. Firstly, microbubbles reflect ultrasound more efficiently than tissue due to the larger difference in acoustic properties with their surroundings. Secondly, in response to an oscillating ultrasound pressure field, the microbubbles may undergo radial oscillations, which in turn may 15 generate secondary sound waves. The amplitude of the secondary sound wave generated by a microbubble depends on the relation between its resonance frequency and the frequency of the ultrasound pressure field in which it finds itself. A bubble resonant to the incident acoustic field will have the largest amplitude of oscillation, consequently generate the strongest secondary sound 20 wave, and thus enhance the contrast of the ultrasound echo optimally. Commercially available UCA are surfactant-coated microbubbles with radii typically ranging from 1 to 10 pm. The precise size distribution of microbubbles in an UCA sample is dependent on the manufacturer. However, since clinical ultrasound systems operate at a single frequency at a time, and 25 the resonance frequency of a microbubble is strongly size-dependent, resonance occurs only for a small portion of the microbubbles of an UCA having a wide microbubble size distribution.

In general, the sensitivity of contrast enhanced ultrasound imaging can be improved in two ways: by injecting more UCA into a patient’s blood 2 stream, or by narrowing down the microbubble size distribution of an UCA. Since clinical practice allows only a certain amount of gas volume to be injected into a human body, a method of narrowing the size distribution of a UCA is of interest. One way of realizing a monodisperse microbubble UCA is 5 enriching commercially available UCA by size-sorting the microbubbles therein, i.e. segregating microbubbles of different sizes and then collecting the microbubbles of the desired target size. This process is not trivial, however.

In the art, various methods of size-sorting microbubbles have been disclosed. Goertz et al., Attenuation and size distribution measurements of 10 Definity and manipulated Definity populations, Ultrasound in medicine & biology, 36(11):1919-32, November 2010, for example, discloses a method based on decantation. Although the method works in principle, it is insufficiently well-controlled for application in practice. United States patent application publication US 2011/0300078-Al (Borden et al.) discloses an alternative 15 method for isolating microbubbles of a selected size range from polydisperse microbubbles. The method is a batch process based on repeatedly centrifuging a suspension comprising the polydisperse microbubbles, and is rather laborious.

20 Summary of the invention

It is an object of the present invention to provide for a continuous method of size-sorting microbubbles, capable of being implemented by a lab-on-a-chip device, and suitable for isolating at least one monodisperse plurality of microbubbles from a polydisperse plurality of microbubbles, such as for 25 instance a commercially available microbubble UCA.

It is another object of the present invention to provide for an apparatus, in particular a microfluidic or lab-on-a-chip device, configured to continuously size-sort microbubbles, and suitable for isolating at least one monodisperse plurality of microbubbles from a polydisperse plurality of 30 microbubbles.

3

To this end, a first aspect of the present invention is directed to a continuous method of size-sorting microbubbles. The method may include providing a polydisperse plurality of microbubbles, and entraining these microbubbles in a fluid flow flowing in a flow direction towards a collection 5 location. The method may also include, at an insonification location upstream of the collecting location, subjecting the microbubbles in said fluid flow to an acoustic pressure wave propagating in a wave direction, which wave direction has with a non-zero directional component in a microbubble segregation dimension perpendicular to the flow direction, thereby segregating 10 microbubbles of different equilibrium radius in said microbubble segregation dimension as they pass the insonification location. The method may further include at said collection location, collecting the microbubbles present within at least two spatial ranges of said microbubble segregation dimension. In a particularly advantageous embodiment of the method, the acoustic pressure 15 wave is a travelling acoustic pressure wave, but a standing acoustic pressure wave may also be employed.

A second aspect of the present invention is directed of an apparatus for continuously size-sorting microbubbles, configured to implement the method according to the first aspect of the present invention. The apparatus 20 may include an elongate segregation channel extending in a longitudinal flow direction between an inlet and a plurality of outlets, wherein said outlets are juxtaposed along a segregation dimension that is perpendicular to the flow direction. The apparatus may also include an acoustic pressure wave generator, disposed alongside the segregation channel and configured to 25 insonify at least a longitudinal portion thereof with a travelling acoustic pressure wave that propagates in a wave direction having a non-zero directional component in the microbubble segregation dimension.

These and other features and advantages of the invention will be more fully understood from the following detailed description of certain 4 embodiments of the invention, taken together with the accompanying drawings, which are meant to illustrate and not to limit the invention.

Brief descrintion of the drawings 5 Fig. 1A schematically illustrates how the primary acoustic radiation force experienced by a microbubble insonified by a travelling acoustic wave depends on the equilibrium radius of the microbubble;

Fig. IB schematically illustrates how the primary acoustic radiation force experienced by a microbubble insonified by a standing acoustic wave 10 depends on the equilibrium radius of the microbubble;

Fig. 2 schematically illustrates the construction and operation of an exemplary microfluidic apparatus for size-sorting microbubbles according to the present invention; and

Figs. 3A-B schematically illustrate how an apparatus according to 15 the present invention, similar to that shown in Fig. 2 but fitted with six instead of three microbubble collecting outlets, may acoustically sort a microbubble suspension having a size distribution as shown in Fig. 3B.

Detailed descrintion 20 The presently disclosed method and apparatus for size-sorting microbubbles are based on acoustic forcing. Without wishing to be bound by theory, a brief introduction to this topic is offered here in order to promote a better understanding of the invention.

A microbubble of volume V, disposed in a liquid that is subjected to 25 an oscillating acoustic pressure field p experiences a radiation force Fr that opposes the pressure gradient Vp of the pressure field, and changes periodically in both direction and magnitude. This radiation force Fr is given by

Fr=-m-vp(t), (1) 5 wherein V(t) denotes the time-varying volume of the microbubble, and Vp(t) denotes the time-varying pressure gradient of the pressure field at the position of the microbubble if it were absent. In case the microbubble couples with the oscillation of the pressure field, both V(t) and p(t) are oscillatory quantities, so 5 that the time average of the radiation force Fr need not be zero. This net, time-average of the radiation force may be referred to as the primary acoustic radiation force Fmd, given be given by

Frad=-(V(t)-Vp(t)). (2)

Even at low acoustic pressures the primary acoustic radiation force Frad may 10 be relatively large compared to other forces acting on the microbubble, such as viscous drag forces. It may therefore be purposefully used to displace or translate the microbubble in a direction parallel to the pressure gradient of the acoustic pressure field.

Eq. (2) reveals that the primary acoustic radiation force is dependent 15 on both the dynamics of an insonified microbubble and the characteristics of the acoustic pressure field p itself. The dynamics of an insonified microbubble may be described by one of the various forms of the Rayleigh-Plesset equation. A general treatment of microbubble dynamics may be found in T.G. Leighton, The Acoustic microbubble, Academic Press, 1994; a model applicable to in 20 particular surfactant-coated microbubbles is discussed by Philippe Marmottant et al., A model for large amplitude oscillations of coated microbubbles accounting for buckling and rupture, The Journal of the Acoustical Society of America, 118(6):3499, 2005, both of which publications are hereby incorporated by reference. A property of importance in the present 25 context is the resonance frequency of a microbubble, i.e. the frequency at which a pulsating microbubble exhibits its maximum amplitude of oscillation. The resonance frequency may typically be dependent on the size or equilibrium radius ro of a microbubble, in that a smaller microbubble may have a greater resonance frequency, and vice versa. As regards the characteristics of the 30 acoustic pressure field, the primary properties of importance in the present 6 context are its amplitude - which is directly linked to the magnitude of the primary acoustic radiation force Frad experienced by any microbubble, irrespective of its size - and its frequency. In addition, a distinction must be made between two regimes: a standing pressure wave, and a travelling 5 pressure wave.

Given knowledge of the dynamics of a microbubble and the characteristics of the pressure field, the primary acoustic radiation force Frad of eq.(2) may be calculated. The graph of Figs. 1A and IB schematically illustrate how the primary acoustic radiation force Frad may depend on the equilibrium 10 radius ro of a microbubble in a travelling pressure wave and in a standing pressure wave, respectively. In either case, the frequency of the pressure wave corresponds to the resonance frequency of a microbubble with equilibrium radius rres.

In the travelling pressure wave, the primary acoustic radiation force 15 Frad is positive for microbubbles of all sizes, and points into the direction of wave propagation. Its magnitude is microbubble-size dependent: microbubbles driven at their resonance frequency experience a maximum primary radiation force, while microbubbles driven below and above their respective resonance frequencies are subject to a smaller than maximum primary radiation force. As 20 a result, the primary acoustic radiation force in a travelling pressure wave may enable size-sorting of a polydisperse plurality of microbubbles. Two practical sorting modes are envisaged.

In a first sorting mode, a polydisperse plurality of microbubbles may be disposed in a travelling pressure wave whose frequency is selected to match 25 the resonance frequency of the microbubble-size that one wishes to separate from all the others. Thus, when a smallest microbubble in the polydisperse plurality of microbubbles has a resonance frequency fres,max, while a largest microbubble in the polydisperse plurality of microbubbles has a resonance frequency fres,min, with fres,min < fres,max, the frequency of the acoustic pressure 30 wave may be selected to have a frequency in the range of about [fres,min-2%, fres, 7 max+2%]. As may be inferred from Fig. 1A, the microbubbles driven at resonance will be displaced more than differently sized microbubbles. However, Fig. 1A also indicates that microbubbles having an equilibrium radius below the resonant radius, e.g. an equilibrium radius rres-An, may 5 experience a same primary radiation force Frad as microbubbles having a certain equilibrium radius, rres+Ar2, above the resonant radius. Microbubbles having respective equilibrium radii rres-An and rres+Ar2 may thus form ‘doubles’ , and may not be separable from each other by means of the first sorting mode as they will undergo approximately equal displacements.

10 A second sorting mode resolves this problem of non-segregated doubles by disposing a polydisperse plurality of microbubbles in a travelling pressure wave whose frequency is set either at or somewhat higher than the resonance frequency fees,max of the smallest microbubble in said plurality, or at or somewhat lower than the resonance frequency fres.mm of the largest 15 microbubble in said plurality. Practically, the frequency of the acoustic pressure wave may often be chosen around the minimum or the maximum resonance frequency, and be selected to be either in the range \fres,min - 20%, fres,min + 2%] or in the range \fres ,max -2%, fres, max + 20%]. By doing so, differently sized microbubbles may be subject to mutually different primary radiation 20 forces Frad, as a result of which they may be separated from each other.

In the context of a standing pressure wave, the primary acoustic radiation force Frad may be referred to as the primary Bjerknes force, or Fprim.Bj. It is positive for microbubbles driven above their resonance frequency and negative for microbubbles driven below their resonance frequency; see Fig. 25 IB. Accordingly, microbubbles having an equilibrium radius ro smaller than rres, i.e. the equilibrium radius of microbubbles resonant with the pressure field, may travel up the pressure gradient to aggregate at the pressure antinodes. Conversely, microbubbles having an equilibrium radius to greater than that of microbubbles resonant with the pressure field may travel down 30 the pressure gradient to aggregate at the pressure nodes. As with the primary 8 radiation force on a microbubble in a travelling wave pressure field, the force experienced on microbubbles near resonant size is the largest.

The presently disclosed invention applies the above described-theory 5 to provide for a continuous microbubble sorting method, and an apparatus implementing said method. Although the method and apparatus may in principle be based on both travelling and standing wave pressure fields, use of the latter type of field entails a number of drawbacks.

One drawback of a standing wave pressure field is that it may only 10 serve to perform binary sorting. I.e., microbubbles of smaller than resonant size may be separated from microbubbles of greater than resonant size, but to achieve a finer segregation multiple consecutive binary segregation operations may be necessary. Such extra operations may complicate the design of an apparatus implementing the size-sorting method, and thus increase its 15 manufacturing costs. Another drawback is that an apparatus based on a standing wave pressure field may require a segregation space - i.e. a space in which the actual size-segregation of the microbubbles is to take place by insonification - whose dimensions are tailored to a certain acoustic wavelength in order to be able to sustain the standing wave. Once the dimensions of the 20 segregation space have been set, the usable frequencies of the standing wave are fixed as well. Accordingly, the frequency of the standing wave may not be free tunable to the resonance frequencies of the plurality of microbubbles at hand, which limits the versatility of the apparatus. Furthermore, to size-sort UCA-microbubbles with equilibrium radii in the range of about 1-10 pm and 25 resonance frequencies in the range of about 0.05-5 MHz, the segregation space may require dimensions at least two orders of magnitude larger than the size of the bubbles. For instance, assuming that the fluid in which the microbubbles are entrained during segregation is water, half a wavelength of an acoustic wave with a frequency of 1 MHz is still 0.75 mm. This is rather 9 large compared to the microbubble diameters, and may inhibit both the miniaturization and the efficient operation of the apparatus.

Travelling wave pressure fields, on the other hand, may perform full and fine size-segregation of a plurality of microbubbles in one pass. In 5 addition, the usable frequencies of travelling pressure waves may not be rigidly tied to the dimensions of a segregation space, so the frequency of the travelling pressure wave can be varied for one and the same device. This allows the of segregation process induced by the primary acoustic radiation force Frad to be controlled, and, as will become clear below, the output 10 microbubble size(s) of the presently disclosed method and apparatus to be tuned by tuning the frequency of the travelling pressure wave. In addition, an apparatus based on travelling wave pressure fields may advantageously be miniaturized. Preferred embodiments of the present invention may therefore be based on travelling wave pressure fields.

15

Fig. 2 schematically illustrates both the construction and operation of an exemplary microfluidic embodiment of the apparatus 1 according to the present invention.

The apparatus 1 may include at least one elongate segregation 20 channel 40; some embodiments of the apparatus 1 may include multiple segregation channels 40, which may operate in parallel to boost the production of the apparatus 1. The at least one segregation channel 40 may preferably be straight, and extend between an inlet 42 and a plurality of outlets 44a-f. The outlets 44a-f may be juxtaposed in a width or segregation dimension y that is 25 perpendicular to the longitudinal dimension x of the segregation channel 40. During operation, microbubbles 70 to be segregated may be entrained in a laminar fluid flow and so sequentially pass through the segregation channel 40, from the inlet 42 to one of the outlets 44a-f.

Both the width (measured in the y-dimension) and the height 30 (measured in the 2-dimension) of the segregation channel 40 may preferably be 10 significantly larger than the diameters of the microbubbles 70 passing therethrough in order to avoid microbubble-wall interactions (due to secondary Bjerknes forces). In addition, a width:height ratio of the segregation channel 40 may preferably be at least 4:1 so as to ensure an at least approximately 5 constant fluid velocity across the largest part of the width of the channel, to be used for segregation of the microbubbles 70. The constantness of the fluid velocity implies the absence of a lateral fluid velocity gradient across the diameter of a microbubble 70 entrained in the fluid flow, and hence the absence of a lift force that might otherwise counteract the action of the 10 primary radiation force Fmd and effective push microbubbles 70 back towards the middle of the segregation channel 40, i.e. towards the region in the flow with the largest/maximum velocity. Microbubbles 70 may thus preferably be entrained in a region of the fluid flow with a maximum or near-maximum fluid flow velocity; here, the phrase ‘near-maximum’ may be construed to mean the 15 maximum fluid flow velocity ± 15%.

In case the apparatus 1 is configured to operate based on a travelling wave pressure field, the channel 40 may preferably be provided in, and thus be bounded by walls of, a material having an acoustic impedance as close as possible to that of the fluid in which the microbubbles 70 are to be transported 20 therethrough, so as to minimize reflections of the incident acoustic wave by the channel walls. Practically, where the acoustic impedances of the fluid and the wall material are denoted as Zfiuid and Zwaii, respectively, Zwaii may preferably be in the range Zfiuid ± 30%. For instance, in case water is to be used as the transport fluid, such that Zfiuid - 1.4 M Rayl [1 Rayl = 1 Pa s/m], 25 polydimethylsiloxane (PDMS), having an acoustic impedance of about 1.04 M Rayls, may be suitable as the wall material for bounding the segregation channel 40.

The plurality of outlets 44a-f provided at the downstream extremity of the segregation channel 40 may be juxtaposed in the width or segregation 30 dimension y. Together, the outlets 44a-f may cover the entire width of the 11 segregation channel 40. The outlets 44a-f may, but need not, be of equal size; i.e. some outlets may be larger than others, and thus cover a greater spatial range than others.

The apparatus 1 may also include at least one acoustic pressure 5 wave generator 50, which may be disposed alongside the segregation channel 40 at an insonification location 60, and be configured to insonify a longitudinal portion of the segregation channel 40 with an acoustic pressure wave 52. The wave direction in which the acoustic pressure wave 52 propagates may be at least partially, and preferably substantially, perpendicular to the flow 10 direction x in which microbubbles will pass the segregation channel 40 during operation. In the depicted embodiment, the acoustic wave generator 50 is set up to generate an acoustic pressure wave 52 that travels along the width dimension y of the segregation channel 40, and thus in a direction perpendicular to the flow direction x.

15 The acoustic pressure wave generator 50 may be configured to effect a travelling wave pressure field or a standing wave pressure field within the insonified longitudinal portion of the segregation channel 40. The precise implementation of the acoustic pressure wave generator 50 may differ for these cases. Typically, however, it may include at least one electroacoustic 20 transducer. In embodiments of the apparatus 1 based on a travelling wave, no more may be required. In embodiments of the apparatus 1 based on a standing wave, the acoustic pressure wave generator 50 may further include an acoustic reflector, disposed opposite of the electroacoustic transducer, on an opposite side of the segregation channel 40. The function of an acoustic reflector may be 25 fulfilled by a segregation channel wall having an acoustic impedance that is large relative to that of the fluid that is used to transport the microbubbles through the segregation channel 40 during operation.

In an embodiment of the apparatus configured to size-sort microbubbles having an equilibrium radius ro in the range of about 1-10 pm, 30 the acoustic pressure field may have a frequency in the range of 0.05-5 MHz.

12

It is noted that this frequency need not be the fundamental frequency of the acoustic wave generator 50; it may, for instance, be a subharmonic frequency thereof. Accordingly, a Surface Acoustic Wave (SAW) transducer having for example a fundamental frequency of 40 Mhz may generate an acoustic 5 pressure field having a frequency in the mentioned range through a subharmonic frequency of ((1/8) -40 MHz=) 5 MHz. In a preferred embodiment, the acoustic pressure wave generator 50 may be configured to generate acoustic pressure waves at various alternative frequencies and/or amplitudes, and to enable adjustment or user-selection of the frequency and/or amplitude 10 at which it provides an acoustic pressure wave 52, so as to enable a user to control the size-sorting process.

The apparatus 1 may also include a sample fluid flow focuser 64, which may be disposed at the inlet 42 of the segregation channel 40. The sample fluid flow focuser 64 may include a central sample fluid supply channel 15 20, having an outlet 24 that discharges into a flow focusing junction 30 at the inlet 42 of the segregation channel 40, and at least two sheath fluid supply channels 10, 10’, each of which may have an outlet 14, 14’ that discharges into the flow focusing junction 30. The sample fluid flow focuser 64 may be configured such that, in operation, a flow of sample fluid 26 discharging from 20 the outlet 24 of the sample fluid supply channel 20 is engaged in co-flow by focusing flows of sheath fluid 16, 16’ discharging from the outlets 14, 14’ of the sheath fluid supply channels 10, 10’ under the formation of a composite fluid flow 46 that extends into the segregation channel 40. It is understood that the sample fluid 26 may be a microbubble suspension with a relatively wide 25 microbubble size distribution that is to be sorted. The microbubbles 70 in the suspension may be coated with a surfactant, which may, for example, include a film-forming (mixture of) phospholipid(s), e.g. a mixture of DPPC, DPPA, and DPPE-PEG5000. The sample fluid flow focuser 64 may serve to dilute this microbubble suspension, and to simultaneously focus the flow of sample fluid 30 26, so as to effect a laminar, composite flow 46 in which the microbubbles 70 13 are arranged in a bubble train. To avoid or at least minimize bubble-bubble interactions, in particular during insonification at the insonification location 60, each two microbubbles 70 in the composite flow 46 may preferably be spaced apart in the flow direction x by a distance d of at least ten times their 5 average equilibrium radius ro. The distance d between consecutive microbubbles 70 may be controlled through adjustment of the fluid flow rates of the sheath and sample fluid flows 16, 16’, 26.

The working of the apparatus 1 shown in Fig. 2 is as follows. During operation, a sample fluid 26 in the form of a microbubble suspension is fed to 10 the inlet 22 of the sample fluid supply channel 20, and the sample fluid flow focuser 64 effectively outputs a laminar composite flow 46, comprising a bubble train of longitudinally spaced apart microbubbles 70, into the segregation channel 40. In the depicted embodiment, the bubble train is centered on the width of the segregation channel 40, where the fluid flow 46 has a maximum 15 fluid flow velocity. As the microbubbles 70 pass the insonification location 60, they are insonified by the acoustic pressure wave 52 generated by the acoustic pressure wave generator 50. The acoustic pressure wave 52 propagates along the microbubble segregation direction y, which is perpendicular to the flow direction x of the microbubbles. Consequently, the insonified microbubbles 70 20 experience a net radiation force Frad that translates each of them over a certain distance in the microbubble segregation dimension y.

The exact distance over which an individual microbubble 70 is displaced depends on the primary radiation force Frad experienced. The magnitude of the primary radiation force Frad, and hence the magnitude of the 25 displacement of a microbubble 70, depends on the amplitude of the acoustic pressure wave 52, and, as explained above with reference to Figs. 1A-B, on the relation between the microbubble’s resonance frequency/equilibrium diameter ro and the frequency of the acoustic pressure wave 52. In addition, the direction in which an individual microbubble 70 is displaced depends on 30 whether the acoustic pressure wave 52 in the insonified portion of the 14 segregation channel 40 traversed by the bubble forms a travelling pressure wave or a standing pressure wave. In a travelling pressure wave, all microbubbles 70 are displaced in the wave direction; in the embodiment of Fig. 2, this is the positive y-direction. In a standing pressure wave, microbubbles 70 5 of a smaller and a larger than resonant size may travel in opposite directions, i.e. in the positive and the negative y-directions respectively. - In the situation depicted in Fig. 2, the apparatus 1 operates on the basis of a travelling wave pressure field 52. Accordingly, all insonified microbubbles 70 are displaced in the positive y-direction.

10 The lateral displacement of the microbubbles 70 effects a size segregation. In addition, it gets the microbubbles in lane for one of the outlets 44a-c at the downstream end of the segregation channel 40. - Indeed, in the case of a travelling wave pressure field as shown, the outlets 44d-f are effectively dummy-outlets that merely serve to discharge microbubbleless fluid 15 46. In case the operation of the apparatus 1 would be based on a standing wave pressure field, fluid 46 discharged into any of the outlets 44a-f might contain microbubbles 70. - Each of the outlets 44a-c covers a certain spatial range of the microbubble segregation dimension y, and will thus collect microbubbles 70 within one or two selected size ranges. It will be clear that the 20 size range(s) that end up in a certain outlet 44a-f may be controlled through selection of the amplitude and frequency of the pressure wave 52, and the spatial ranges across which the outlets 44a-f extend.

The method of size-sorting microbubbles in a travelling wave 25 pressure field according to the present invention is, somewhat more precisely, illustrated in Figs.3A-B. The concrete sorting process illustrated may be carried out by means of an apparatus 1 similar to that illustrated in Fig.2, but with six instead of three (i.e. 44a-c) microbubble collecting outlets.

Fig. 3B depicts the size distribution of microbubbles 70 in the sample 30 fluid 26. Various microbubble size ranges A-F have been hatched differently in 15 the depicted distribution; size-ranges indicated by a same hatch type, and referred to by the same capital A-F, are centered on the same resonance frequency. It is noted that size ranges A-D are split or double ranges, while size range F is singular. Fig.3A schematically illustrates what happens when 5 the microbubbles 70 from the various size ranges are sequentially passed through the segregation channel 40, a longitudinal portion of which is insonified with a travelling pressure wave 52 having a relative strength as indicated by the curve 52 in Fig. 3A, and a frequency equal to the resonance frequency on which size range F is centered.

10 The applied sorting method corresponds to the first sorting mode discussed above with, and aims to isolate microbubbles from size range F. Microbubbles falling in size range F are driven at or close to resonance, and will therefore be displaced the most; they will end up in a dedicated outlet (the one with the greatest y-coordinate). Microbubbles falling in the split size range 15 E, which encloses size range F in the size distribution of Fig.3B, will experience a net radiation force Frad that is smaller than that experienced by the microbubbles from size range F, but greater than that experienced by the microbubbles from split size range D, which in turn encloses both size ranges E and F in the size distribution of Fig. 3B. Accordingly, the microbubbles from 20 size range E will be displaced less than microbubbles from size range F, but more than microbubbles from size range D, and thus end up in an outlet spanning a spatial range of the y-dimension between those spanned by the outlets in which the microbubbles from size ranges F and E are collected, respectively.

25

With regard to the terminology used in this text and not elaborated on above, the following may be noted. The term ‘bubble’ may be construed to refer to a typically globular body of a gas (the dispersed phase), disposed within a liquid (the continuous phase). The term ‘microbubble’ may be 30 construed to refer to a microbubble having an equilibrium radius ro < 500 pm.

16

The term 'poly disperse’, where used in this text to characterize a plurality of microbubbles, may be construed to mean that the polydispersity index (PDI) of the plurality, mathematically defined as PDI = s/n, wherein n denotes the average equilibrium microbubble radius and s the standard deviation of the 5 microbubble radii, is greater than 5 10 2. That is, a plurality of microbubbles having a PDI > 5% may be considered to be polydisperse. In contrast, term ‘monodisperse’ may be used to characterize a plurality of microbubbles having a PDI < 5%.

Although illustrative embodiments of the present invention have 10 been described above, in part with reference to the accompanying drawings, it is to be understood that the invention is not limited to these embodiments. Variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Reference throughout this 15 specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not 20 necessarily all referring to the same embodiment. Furthermore, it is noted that particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner to form new, not explicitly described embodiments.

17

List, of elements 1 microfluidic microbubble sorter 10 sheath fluid supply channel 12 sheath fluid supply channel inlet 5 14 sheath fluid supply channel outlet 16 sheath fluid flow 20 sample fluid supply channel 22 sample fluid supply channel inlet 24 sample fluid supply channel outlet 10 26 sample flow 30 flow focusing junction 40 segregation channel 42 segregation channel inlet 44a,b,c,... segregation channel outlets 15 46 fluid flow through segregation channel 50 acoustic pressure wave generator 52 acoustic pressure wave 60 insonification location 62 collection location 20 64 sample fluid flow focuser 70 microbubble d longitudinal distance between successive microbubbles in segregation channel, upstream of insonification location Frad primary acoustic radiation force 25 ro equilibrium radius of a microbubble rres radius of microbubble resonant with a certain acoustic pressure field x longitudinal dimension of segregation channel, flow direction (+) y width dimension of segregation channel, microbubble segregation dimension, wave direction (+) 30 z height dimension of segregation channel

Claims

A continuous method for sorting microbubbles by size, comprising: • providing a polydisperse plurality of microbubbles (70); Entraining said microbubbles (70) in a fluid stream (46) that flows in a flow direction (x +) to a collection location (62); • at an insonification location (60) upstream of the collection location (62), subjecting the microbubbles (70) in said fluid stream (46) to an acoustic pressure wave (52) propagating in a wave direction with a zero-unequal directional component in a microbubble separation dimension (y) extending perpendicular to the flow direction (r +), so that microbubbles with a different equilibrium radius (r6) are separated from each other in said microbubble separation dimension (y) as they pass through the insonification location; and • at said collection location (62), collecting microbubbles (70) located within at least two spatial ranges (44a-c) of the microbubble separation dimension (y).

The method of claim 1, wherein the wave direction (y +) is substantially perpendicular to the flow direction (x +). 20

The method of any one of claims 1-2, wherein the fluid flow (46) is laminar.

The method of claim 3, wherein the microbubbles (70) are entrained in a region of the fluid flow (46) with a maximum or near-maximum fluid flow rate.

The method of any one of claims 1-4, wherein each two microbubbles (70) in the fluid stream (46) are separated from each other in the flow direction (x +) by a distance (d) of at least ten times their average equilibrium radius (ro).

The method of any one of claims 1-5, wherein the microbubbles 5 (70) are coated with a surfactant.

The method of any one of claims 1-6, wherein the fluid flow (46) extends through an elongated separation channel (40) which, at the collection location (62), flows into a plurality of outlets (44a-f) that 10 are placed side by side in the microbubble separation dimension (y), and wherein collecting the microbubbles (70) within at least two spatial ranges of the microbubble separation dimension (y) distributing the fluid stream (46) between the plurality of outlets (44a) f).

The method of any one of claims 1-7, wherein the acoustic pressure wave (52) is a standing wave.

The method of any one of claims 1-7, wherein the acoustic pressure wave (52) is a running wave, 20

The method of claim 9, wherein a smallest microbubble (70) in the polydisperse plurality of microbubbles has a resonance frequency fres, max, while a largest microbubble in the polydisperse plurality of microbubbles has a resonance frequency fres, min, where fres, min fres , max, and wherein the acoustic pressure wave (52) has a frequency in the range \ fres, min-2%, fres, max + 2% \.

The method of claim 9, wherein a smallest microbubble (70) in the polydisperse plurality of microbubbles has a resonance frequency fres, max 30, while a largest microbubble in the polydisperse plurality of microbubbles has a resonance frequency fres, mm, where fres, min ^ fres, max, 611 where the acoustic pressure wave (52) has a frequency that falls in at least one of the ranges \ fns, min - 20%, fns.mm + 2%] and | fres, max-2%, fres , max + 20%].

A device (1) for continuously sorting microbubbles 5 (70) by size, comprising: • an elongated separation channel (40) extending in a longitudinal flow direction (x +) between an inlet (42) and a plurality of outlets (44a-c), wherein said outlets are placed side by side in a microbubble separation dimension (y) that is perpendicular to the flow direction 10 (x *); and • an acoustic pressure wave generator (50) provided along the separation channel (40) and adapted to sonicate at least one longitudinal part thereof with a running acoustic pressure wave (50) that propagates in a wave direction with a zero-directional directional component in the direction microbial separation dimension (y).

The device of claim 12, wherein said longitudinal portion of the separation channel (40) is at least partially bounded by a wall with an acoustic impedance Zwaii in the range 1.4 ± 30% MPa • s / m.

The device of claim 12 or 13, wherein said acoustic pressure wave (52) has a frequency in the range of 0.05-5 MHz.

15. The device according to any of claims 12-14, wherein the acoustic pressure wave generator (50) is arranged such that it can produce acoustic pressure waves (52) with various alternative frequencies, and that adjustment of the frequency at which it receives the acoustic pressure wave (50). ) produces is possible.

The device of any one of claims 12-15, wherein the separation channel (40) has a width in the microbubble separation dimension (y), and a height in a dimension perpendicular to both the longitudinal flow direction (x +) and the microbubble separation dimension (y) and wherein a width: height ratio of the separation channel (40) is at least 4: 1. 5

The device according to any of claims 12-16, comprising a sample fluid flow focuser (64), comprising: • a sample fluid supply channel (20) with an outlet (24) that flows into a flow focusing node (30) at the inlet (42) of the separation channel (40); • at least two sheath fluid supply channels (10, 1 (1), each with an outlet (14, 14 ') that opens into the flow focusing node (30) and arranged such that, during operation, a sample fluid stream (26) emerges from the outlet (24) of the sample fluid supply channel (20) flows co-current 15 can be engaged by focusing skull fluid flows (16, 16 ') flowing out of the outlets (14, 14') of the skull fluid supply channels (10, 10 '), forming a fluid flow ( 46) extending into the separation channel (40).