WO2014178782A1

WO2014178782A1 - Microsystem and method to focus cells or particles using acoustophoresis

Info

Publication number: WO2014178782A1
Application number: PCT/SE2014/050520
Authority: WO
Inventors: Maria Nordin; Thomas Laurell; Per Augustsson
Original assignee: Acousort Ab
Priority date: 2013-04-30
Filing date: 2014-04-29
Publication date: 2014-11-06

Abstract

The invention relates to a method and system to focus cellsor particles having a size less than 2 µm. This is achieved by using acoustophoresis and a specific design of the channel, which enables the possibility to focus cells or particles having a size less than 2 µmfor the first time.

Description

MICROSYSTEM AND METHOD TO FOCUS CELLS OR PARTICLES

USING ACOUSTOPHORESIS

FIELD OF THE INVENTION

The invention relates to a method and system to focus cells or particles having a size less than 2 μιη. This is achieved by using acoustophoresis and a specific design of the channel, which enables the possibility to focus cells or particles having a size less than 2 μπι for the first time.

BACKGROUND OF THE INVENTION

The ability to handle and process sub-micrometer bioparticles, such as bacteria or sub-cellular organelles, is becoming increasingly important in biomedical, environmental and food analysis. Handling methods such as blood culture of bacteria or sub-cellular fractionation are, however, laboursome, complicated and time consuming. Microfluidics offers a means of automatisation of the handling and analysis processes of sub-micrometer bioparticles and offers advantages through a continuous mode of sample handling and sample volume independence. Previously used methods for sub-micrometer particle handling include filters, dielectrophoresis, inertia in combination with

hydrodynamic forces, magnetophoresis, deterministic lateral displacement, surface acoustic waves (SAW), and acoustic trapping. Acoustic trapping is sometimes confused with acoustophoresis although the physical mechanisms for manipulating the particles or cells are different. In acoustic trapping of sub-micrometer particles a set of larger particles have to be preloaded in the acoustic standing wave zone where the lateral acoustic pressure gradient along the direction of flow is the source of the force that retains the large particles (seed particles) against perfusion induced viscous drag. When smaller (sub-micrometer) particles arrive to the region where the seed particles are retained the sub-micrometer particles are attracted towards the seed particles by a secondary acoustic force that is induced by the sound scattering between the seed particle and the sub-micrometer particle. In acoustophoresis no seed particles are used and secondary forces have a neglectable influence on particles in the acoustic standing wave as they move with the flow. Hence, the primary acoustic force acting on particles in acoustophoresis is the acoustic radiation force which reduces with the size of the particle to the third power.

The above described methods have been used primarily for handling bacteria and particles around 2 μπι in diameter and larger. Together, these approaches show the need for a method enabling increased throughput of crude samples without prior preparation and a continuous mode of operation not limiting the number of particles that can be processed. Acoustophoresis is a continuous flow, label-free, and gentle method, operating

independently of suspension media. The method utilizes ultrasonic standing waves to focus cells or particles in the nodal (or anti-nodal) plane of the standing wave according to their intrinsic properties size, density and compressibility.

Generally, acoustophoresis is commonly operated at a frequency of 1 to 12 MHz for focusing particles or cells. For larger particles, the acoustic particle motion is dominated by the primary acoustic radiation force. For smaller particles, however, the motion is instead dominated by the acoustic streaming induced Stokes drag, where the particles are unable to focus and instead are caught in streaming vortices. Acoustic streaming is a phenomena that occurs when an acoustic standing wave (Fig. ID), is established e.g. in a microchannel and is caused by the boundary conditions when transiting from a fluid to a solid wall boundary. In the viscous boundary layer (Fig. 1C), large shear stresses occur which drives the so called Schlichting streaming (Fig. IB), that in turn induces the so called Rayeligh streaming rolls (Fig. 1 A), that reach into the bulk volume of the channel cross-section. Figure 1 shows the Rayleigh streaming occurring as four large streaming rolls in the cross- section of a perfused microchannel excited in a λ/2 mode. At the same time as the radiation force of the acoustic standing wave drives particles into the nodal plane of the acoustic standing wave the Rayleigh vortices will strive to dislocate them from the pressure node by the viscous drag. In a one-dimensional system operated at 3 MHz the critical particle size for transition from the acoustic radiation-dominated to streaming-dominated domain is -1.4 μπι (Muller, Barnkob, Jensen, & Bruus, 2012), making focusing of most bacteria (usually with a size around 1 μιη or smaller) and other sub-micrometer particles impossible.

Thus there is a need for developing methods and systems which solve these problems and enables the possibility to focus or concentrate cells having a small size. SUMMARY OF THE INVENTION

The invention relates to acoustophoretic focusing of cells and particles smaller than 2 μιη.

The invention is based on the finding that it is possible to use two-dimensional acoustic focusing which change the known Rayleigh streaming patterns in a previously unknown way and thus enables the possibility to focus a population of cells or particles, having a size of less than 2 μπι, into an enriched stream of cells or particles.

In a first aspect the invention relates to a method to focus cells or particles having a size less than 2 μπι in a suspension, which comprises the steps of;

i) subjecting the suspension to pressure, wherein said pressure forces said suspension into one inlet and into at least one channel present in a channel having a size of

50μπι to 750 μπι,

ii) subjecting said suspension to two-dimensional acoustic force(s) directed perpendicular to the length direction of the channel, wherein the channel have square or rectangular cross-section shape, wherein the channel dimension is matched to a frequency range of from 1 MHz to 12 MHz and

iii) controlling the temperature of the suspension and

iv) focusing the cells or particles having a size less than 2 μιη into a stream of cells or particles.

In a second aspect the invention relates to a microsystem for focusing a group of cells or particles having a size less than 2 μιη in a suspension, comprising;

i) a microchip having at least one channel, wherein the size of said channel being 50-300 μπι +/- 10 μπι, with at least one inlet and at least one outlet,

ii) means for providing pressure, wherein said pressure, forces said suspension into the first inlet,

iii) means for providing a two-dimensional acoustic force(s), wherein said two- dimensional acoustic force(s) is directed perpendicular to said at least one channel, wherein said means is one piezo and

iv) means for controlling and regulating the temperature within the micro chip.

By the invented method and system it is, for the first time, possible to focus cells or particles in two dimensions, having a size less than 2 μπι into a stream of cells or particles. The cells can then be analyzed for medical diagnosis, environmental, or food purposes, or further cultured.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1. Shows that acoustic streaming is a phenomena that occurs when an acoustic standing wave (D), is established e.g. in a microchannel and is caused by the boundary conditions when transiting from a fluid to a solid wall boundary. In the viscous boundary layer (C), large shear stresses occur which drives so called Schlichting streaming (B), that in turns induces the so called Rayeligh streaming rolls (D), that reach into the bulk volume of the channel cross-section.

Fig 2. Critical particle diameter where F_rad equals F_stream as a function of frequency in a one-dimensional focusing system, where F_rad is the primary acoustic radiation force and Fstream is the Strokes drag force induced by the Rayeligh/Schlichting streaming. Fig 3. Schematic drawing of a square microchannel cross-section with a two-dimensional acoustic standing wave actuation (A) and the net spiral shaped particle trajectory (B), induced by the acoustic radiation force and the circular acoustic streaming pattern, yielding a focused stream of particles in the channel center (C). Fig 4. The relative focusability vs Q/Upp² in the rectangular channel actuated in one dimension. Q is the flow rate in μΙ_, min^"1 and Upp is the driving voltage of the piezo transducer in Volt.

Fig 5. The relative focusability vs Upp when actuating the second transducer (vertical actuation) in the rectangular channel, hence actuating the rectangular channel with two transducers, stetting up acoustic standing waves in two dimensions. The starting conditions are the same as for the two dashed circled data points in Fig 4. Upp is the driving voltage of the vertical piezo transducer in Volt.

Fig 6. The relative focusability vs Q/Upp² in the square channel actuated in two dimensions. Q is the flow rate in μΙ_, min^"1 and Upp is the driving voltage of the piezo transducer in V.

Fig 7. Fluorescent image of 0.5 μιη particles focusing in the square cross section channel centre at a flow rate of 2 μΙ_, min^"1 and the same voltage as used for the 0.6 μιη particles in the square channel focusing experiments. The white dashed lines indicate the channel edges.

DETAILED DESCRIPTION OF THE INVENTION Method and System

The processing of smaller bio-particles is highly desirable not least for medical diagnostic purposes, or environmental, or food monitoring. The small size of many bio-particles, such as bacteria, has made them difficult to handle in acoustophoresis systems without severe losses, a problem not least prominent for rare cell processing.

Using one-dimensional focusing in acoustophoresis, the acoustic streaming will limit the possibilities to focus sub-micrometer particles (smaller than -1.4 μπι in diameter in this system) as the streaming locally counteracts the radiation force, pushing the particles outwards from the channel center instead of inwards. Theoretically, one way to overcome this limitation is to increase the frequency, as only the radiation force is dependent on this, thus increasing the radiation force while keeping the streaming constant if all other settings are kept constant. This, however, simultaneously requires a decrease in channel size, to match the shorter wavelength of the higher frequency ultrasound, and thus lower throughput, where sample processing times no longer would be relevant for medical or biological applications. The smaller channel dimensions would also induce further complications due to high back pressure in the system. To fully focus a 0.5 μιη diameter particle using one-dimensional focusing (and otherwise the same conditions as used in this application) a frequency of about 24 MHz would be needed, corresponding to a channel width of only about 30 μιη. (Fig. 2). Another way to focus small particles, as disclosed in this invention, is to use simultaneous acoustic standing wave focusing in two dimensions that does not generate streaming patterns counteracting the radiation force, thereby still allowing sufficient channel size and throughput. When actuating a rectangular or square microchannel in two dimensions the obtained streaming pattern is dominated by a circular motion in the channel cross-section, vastly different from the Rayleigh streaming rolls in the channel undergoing one-dimensional acoustic actuation. The combined effect of the two-dimensional acoustic radiation force and the acoustic streaming pattern yields a spiral shaped trajectory of particles, which focuses in the centre of the channel cross-section (Fig. 3). The consequence of this is that, when actuating a square or rectangular microchannel in two dimensions at a D/2 resonance, particles smaller than 2 μπι can be focused and the previous limiting factor of the Rayleigh streaming rolls is now eliminated, enabling continuous flow based acoustic focusing of particles.

The invention relates to methods to focus cells or particles having a size less than 2 μπι in a suspension, which comprises the steps of;

i) subjecting the suspension to pressure, wherein said pressure forces said suspension into one inlet and into at least one channel present in a channel having a size of 50μπι to 750 μπι ,

ii) subjecting said suspension to two-dimensional acoustic force(s) directed perpendicular to the length direction of the channel, wherein the channel have square or rectangular cross-section shape, wherein the channel dimension is matched to a frequency ranging from 1 MHz to 12 MHz and

iii) controlling the temperature of the suspension and

iv) focusing the cells having a size less than 2 μπι into a steam of cells or particles.

The shape of the channel is either square or rectangular or essentially square or rectangular. In one specific embodiment the channel is square. The size of the channel, i.e. the width or the height may be from 50 μπι to 750 μπι, such as from 50 μπι to 350 μπι, 75 μπι to 350 μπι, or ranging from 100 μπι to 350 μπι, or ranging from 200 to 350 μπι, or being 230 μπι. The size of all the above mentioned widths and heights may vary by a factor of +/- 1-10 μπι. The size of the channel may vary depending on the material the channel is made of. The material may be glass or silicon. One example is when there is a large difference in acoustic impedance between the suspended fluid and the material. The microsystem could be designed to contain one channel. One or two ultrasonic (piezoelectric) transducers may be attached to the microchannel and are actuated by individual electric signals that can be sine, square, triangle or of any other periodic shape. One transducer is typically actuated at a frequency that gives a vertical resonance at λ/2 in the channel, but can also be multiples of this. The other transducer is typically actuated at a frequency that gives a horizontal resonance at λ/2 in the channel, but can also be multiples of this. The force being used will be in the range from 1 to 12 MHz, such as from 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6 or 3 to 5 MHz, depending on the dimensions of the channel wherein the cells are to be focused into one stream.

The cells or particles may have the size of less than 2 μπι, such as being from 0.25 to 2 μιη, 0.5 to 1.5 μιη or 0.8 to 1.2 μιη. For example the size may vary in between any of the combinations herein 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 μιη. The behavior of the cells is also dependent on the density and compressiblity of the cells, which may vary between cells even if they have the same size.

The cells or particles may be any kind of cell or particle that have a size less than 2 μιη or any of the sizes mentioned above. The cells or particles may be any cell or particle, or a cell or particle found in the tables below, such as bacteria, virus or organelles.

Examples of cells and bioparticles

Examples of bacteria

Grampositive strains Examples of classes and species

Actinobacteria Streptomyces

Firmicutes Chlostridia

Tenericutes Mykoplasma

Examples of classes and species

Gramnegativa strains

Aquificae Termosulfidibacter

Deinococcus-Thermus Vulcanithermus

Fibrobacteres-Chlorobi/Bacteroidetes Fibrobakter

Fusobacteria

Gemmatimonadetes

Nitrospirae

Planctomycetes- Verrucomicrobia/Chlamydiae

Proteobacteria E. coli, salmonella

Spirochaetes

Synergistetes

Other strains

Acidobacteria

Chloroflexi

Chrysiogenetes

Cyanobacteria

Dictyoglomi

Deferribacteres

Thermodesulfobacteria

Thermotogae

Examples of Archea 0.1-15 μηι

Strains

Crenarchaeota

Euryarchaeota

Korarchaeota

Nanoarchaeota

Thaumarchaeota The pressure that forces the suspension into the inlet of the channel may be induced by a pump or by a syringe as long as the pressure forces the suspension into the inlet of the channel and further into the channel.

The acoustic forces, which focus the cells or particles may be induced by the use of one or more piezoelectric transducer. In one embodiment solely one piezoelectric inducer is used.

The temperature of the system may be controlled and being within the area of 10 to 45 °C, such as 15 to 45, 20 to 45, 30 to 40, 32 to 40 35 to 40, 35 to 38 °C. The temperature may be controlled by a Peltier controller.

Following examples are intended to illustrate, but not to limit the invention in any manner, shape, or form, either explicitly or implicitly.

EXAMPLES

MATERIAL & METHODS

Device fabrication and design: The chips were fabricated in <1 10> oriented silicon using photolithography and anisotropic wet etching in KOH (400g/L H₂0, 80 °C), to obtain vertical channel walls. Inlets and outlets were added by drilling using a diamond drill and the chips were sealed by anodic bonding to a glass lid. The two chips had one trifurcation inlet and outlet, respectively, of where only a single inlet was used and the unused one was sealed. The square cross section channel had a width and height of 230 μπι, operated at 3.19 MHz, and the rectangular cross section channel had a width of 230 μπι, operated at 3.24 MHz and a height of 150 μπι, operated at 5.09 MHz. The piezoceramic transducers (piezos), (PZ26, Ferroperm piezoceramics, Kvistgaard, Denmark) actuating the chips were glued to the chips by cyanoacrylate glue (Loctite Super Glue, Henkel Norden AB,

Stockholm, Sweden). The 3 MHz piezos were glued to the silicon and the 5 MHz to the glass lid on the middle of the chip. To control the temperature a Peltier element (Farnell, London, UK) was glue underneath the 3 MHz piezos and a PT 100/1000 resistance temperature detector (Farnell, London, UK) was glued to the glass lid.

Instrumental setup: The transducers were actuated using a dual channel function generator (AFG 3022B, Tektronix, UK Ltd., Bracknell, UK), the signals were amplified using power amplifiers (in-house build circuit board with a power amplifier, LT1012, Linear Technology Corp., Milpitas, USA) and the applied voltage was monitored using an oscilloscope (TDS 2120, Tektronix, UK Ltd., Bracknell, UK). The temperature was controlled using a Peltier-controller (TC2812, Cooltronic BmbH, Beinwil am See, Switzerland) and the temperature was set to 37 °C throughout the whole experiment.

Fluorescent microscopy images were obtained using a Hamamatsu camera (Hamamatsu Photonics K.K., Hamamatsu, Japan) connected to an Olympus microscope (BX51WI, Olympus Corporation, Tokyo, Japan).

Experimental setup: The flow rates were controlled using syringe pumps (Nemesys, Cetoni GmbH, Korbussen, Germany) mounted with glass syringes (Hamilton Bonaduz AG, Bonaduz, Switzerland) connected to the inlet and the side outlets. The center outlet was kept open and sample was collected from a short piece of tubing directly into an Eppendorf tube. When sample output flow rates were less than 10 μΕ min^"1 this outlet was also connected to a syringe pump. Sample was then collected using a 2-position 3 -port valve (MV201-C360, Lab Smith, Livermore, CA, USA) connected in series with the center outlet and the syringe pump. While the inlet and outlet flow rate were varied the ratio between the outlet flow rates were kept constant at a split ratio of 40:60 in the center and the side outlets, respectively. The flow rates and voltages used in the different experiments are summarized in table 1. To minimize errors from sedimentation in the syringes and tubings varying with the flow rate, samples collected with ultrasound either on or off for each flow rate was compared. Particles and bacteria were enumerated using a Coulter counter (Multisizer III, Beckman Coulter, Brea, USA).

Table 1. Summary of flow rates and voltages used in the different experiments.

Microparticles: Polystyrene microparticles of various sizes were used to characterize the system. 7 (7.11) μιτι, 5 (4.99) μιη and 3 (3.17) μιη diameter particles were obtained from Sigman-Aldrich (Sigma-Aldrich, Buchs, Switzerland) and 1 (0.992) μιη and 0.6 (0.591) μηι particles and 0.5 (0.49) μιη and 0.25 (0.24) μιη fluorescent particles were obtained from Kisker (Kisker Biotech GmbH & Co. KG, Germany). Particle concentrations were kept below 10⁹ mL^"1, to minimize the effect of secondary forces between the particles.

Bacteria: For biological evaluation of the system Escherichia coli (E. coli) DH5-a (containing a plasmid that carries the ampicillin resistance gene) were used. E. coli were cultured in liquid LB-medium or LB-plates, containing 10 g L^"1 tryptone (T1332, Saveen & Werner, Limhamn, Sweden), 5 g L^"1 yeast extract (Hy- Yeast 412, Sigma-Aldrich, Buchs, Switzerland), 10 g L^"1 NaCl (Sigma-Aldrich, Buchs, Switzerland) and 100 mg L^"1 ampicillin (A9518-5G, Sigma-Aldrich, Buchs, Switzerland) or agar (Agar bacteriology grade A0949, Saveen & Werner, Limhamn, Sweden).

EXAMPLE 1

One-dimensional focusing in a rectangular channel

Figure 4a show the relative focusability, the ratio of particles moved by the ultrasound to the center outlet, when the flow rate has been varied and the voltage kept constant for a range of particles sizes. To be able to compare the data obtained for the different particles sizes it has been plotted normalized for energy and size. Larger polystyrene particles with diameters of 7 μπι, 5 μιη and 3 μιη can all be fully focused. The smaller polystyrene particles with diameters of 1 μιη and 0.6 μπι, however, cannot be fully focused under the same conditions, and a relative focusability of only about 0.50 could be obtained, (dashed circles Fig 4). The relative focusability will never reach unity for these particles because of the acoustically induced streaming. The smaller the particles diameter the more influence the streaming will have in comparison to the primary acoustic radiation force. The effect of the streaming can be seen by the smaller particles (1 μιη and 0.6 μιη) relative focusability deviating from the fully focusable particles (7 μπι, 5μιη and 3 μιη) in Figure 4. The 1 μιη diameter particles are just starting to deviate and the 0.6 μιη diameter particles are deviating even more.

EXAMPLE 2

Two-dimensional focusing in rectangular channel

By adding a second piezo ceramic transducer to the rectangular channel also focusing the particles in the horizontal plane the focusability of the smaller particles (1 μιη and 0.6 μιη) was increased (Fig. 5). Increasing the voltage of the second piezo ceramic transducer while keeping the voltage of the first piezo ceramic transducer and the flow rate constant at the same settings as used for the data points obtained at the lowest flow rate in Figure 4 (dashed circles), increased the focusability for these smaller particles. At the chosen flow rate the focusability did not fully reach unity. Increasing the voltage more would have resulted in higher focusability, however in the current set-up, it also caused the system temperature to rise above what the temperature regulator could control. It was visually determined that these particles indeed could be fully focused at lower flow rates where the voltage power could be lowered, avoiding over heating of the system. The ability to improve the focusing of these smaller particles by the addition of the second piezo ceramic transducer was attributed to a change in the acoustic streaming velocity field pattern, leading to a reduced counteraction of the primary radiation force than in the streaming patterns induced by a one-dimensional resonance. EXAMPLE 3

Two-dimensional focusing in square channel

The easiest way to generate two-dimensional focusing in an acoustophoresis microchannel is by using square cross section geometry. In this way, the same piezo ceramic transducer (operated at a fixed frequency) can be used to generate both the vertical and horizontal standing waves. In the square channel actuated in two dimensions the larger particles with diameters 7 μπι, 5μπι and 3 μπι could be fully focused, however, in this channel also the smaller particles with diameters 1 μπι and 0.6 μπι were fully focused (Fig. 6). This is seen by the fact that all the different particles sizes in Figure 6 now are collected on the same line as compared to in Figure 4, were 1 and 0.6 μπι particles display a significantly lower focusability.

It is a more simple procedure to use only one piezo ceramic transducers to produce two- dimensional focusing, as in the square cross section channel, than to use two different ones as in the rectangular cross section channel. First, the use of two different piezo ceramic transducers creates the need for more electronic driving equipment, adding both costs and complexity to the system. Second, two transducers place a higher demand on the temperature regulator and are more likely to cause overheating leading to optimal frequency shift and poor focusability performance. Third, compared to a square channel cross section area, the rectangular configuration will always be smaller giving lower throughput when having the same retention time of the particles in the channel. Using two different piezo ceramic transducers could, however, be useful if the channel fabrication method is difficult to control and the square channel cross section geometry is difficult to obtain. If the channel is not a perfect square, using two different transducers will allow for separate tuning of the frequencies to find both horizontal and vertical optima.

EVALUATION OF THE SYSTEMS

Further comparison between the three systems

Fluorescence microscopy was used to further compare the three systems as smaller particles than 0.6 μπι in diameter not could be enumerated in the Coulter counter. It could visually be seen that 0.5 μπι fluorescent polystyrene particles could be focused in both the square and the rectangular cross section channels using two-dimensional focusing. Fig. 7 show focusing of 0.5 μπι particles in a square channel. Using one-dimensional focusing in the rectangular cross section channel could, however, not focus the particles fully in consistence with previously obtained results.

Bacteria in the form of E. coli were used to biologically evaluate the systems (Table 2.). The bacteria showed a relative focusability of 0.95 ± 0.35 in the square channel whereas the relative focusability for the rectangular channel using one-dimensional focusing was 0.40 ± 0.13. At higher samples concentrations the inter particles forces become significant. These secondary forces, that attract particles to each other, are caused by sound waves that are scattered by other particles and does only come in to play when the particle to particle distance is very small. In the examples above the particles and bacteria concentrations were deliberately kept below the limit where these forces become significant.

Table 2. Highest relative focusability achievable for E. coli, 1 μπι, and 0.6 μπι

polystyrene.

Particle ID 2D 2D Square

Rectangular Rectangular

E. coli 0.40±0.13 0.95± 0.35

1 μπι 0.52 ± 0.17 0.87± 0.099 0.95± .081

0.6 μπι 0.48± 0.071 0.92±0.34 1.04± .097

CONCLUSION

The examples above discloses the use of acoustophoresis to manipulate sub-micrometer cells and particles. The use of two-dimensional focusing is shown to enable focusing of E. coli and particles as small as 0.5 μπι in diameter with only minute losses, something that could not be done using one-dimensional focusing only. These results are attributed to a previously unknown change in the streaming velocity field that no longer counteracts the sub-micrometer particle focusing as compared to the streaming field generated by a one- dimensional ultrasound standing wave. Visual observation of particle trajectories at different depth in the channel also confirmed the rotational streaming pattern.

Claims

1. A method to focus cells or particles having a size less than 2 μιη in a suspension, which comprises the steps of;

i) subjecting the suspension to pressure, wherein said pressure forces said suspension into one inlet and into at least one channel present in a channel having a size of 50μιη to 750 μιη ,

ii) subjecting said suspension to two-dimensional acoustic force(s) directed perpendicular to the length direction of the channel, wherein the channel have square or rectangular cross-section shape, wherein the channel dimension is matched to a frequency range from 1 MHz to 12 MHz and

iii) controlling the temperature of the suspension and

iv) focusing the cells or particles having a size less than 2 μιη into an enriched stream of cells or particles.

2 The method according to claim 1, wherein the channel in i) is either rectangular, or square.

3. The method according to claim 2, wherein the channel is square.

4. The method according to claims 1-3, wherein the channel have the size of 50 to 350 μιη.

5. The method according to claim 4, wherein the channel have the size of 230 μιη +/- 1-10 μηι.

6. The method according to any of preceding claims, wherein the frequency is

approximately in the range of 3 to 5 MHz.

7. The method according to any of the preceding claims, wherein the temperature in the system is kept within a range of 10 to 45 °C.

8. The method according to any of the preceding claims, wherein the cells or particles are selected from the group consisting of bacteria, virus or organelles.

9. The method according to claim 8, wherein said cells or particles are selected from the group consisting of mitochondria, endoplasmic reticulum, lysosomes, chloroplasts centromers, small vesicles, vacuoles, Gram positive, Gram negative bacteria, viruses and Archae.

10. A microsystem for focusing a group of cells or particles having a size less than 2 μιη in a suspension, comprising;

i) a microchip having at least one channel, wherein the size of said channel being from 50 to 300 μιη +/- 10 μπι, with at least one inlet and at least one outlet,

iii) means for providing a two-dimensional acoustic force, wherein said two- dimensional acoustic force is directed perpendicular to said at least first channel, wherein said means is one piezo and

iv) means for controlling and regulating the temperature within the micro chip.

11. The micro system according to claim 10, wherein said microchip has one channel.