SE2050940A1 - Microfluidic device and method for manipulaiton of particles in fluids - Google Patents

Abstract

The invention relates to a method and an apparatus for manipulating fine particles in a fluid, said system comprising a High Aspect Ratio Curved, HARC, microfluidic channel with a first HARC section resulting from a revolution of a HAR cross section around an axis parallel to a long side of said cross section, said first HARC section is configured for focusing said fine particles from multiple positions when entering said first HARC section to a predetermined focus position when leaving said first HARC section, wherein particles to be focused in said first HARC section are fulfilling Q < (33,4* R*k3)/Kc Where Q is the flow rate, R the radius of curvature of the microfluidic channel, k = α/W, with a the particle effective diameter, W the width of the cross section of the HARC microfluidic channel, and Kc = —2.06 6.95/ AR, being AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel.

Description

MICROFLUIDIC DEVICE AND METHOD FOR MANIPULAITON OF PARTICLES IN FLUIDS Technical field of the lnvention The present invention relates in general to the field of microfluidics and in particular to methods and devices for manipulation of fine particles in fluids flowing in microfluidic channels.

Background of the lnvention Microfluidics is a growing field where miniaturized fluidic systems provide precise controland manipulation of fluids, enabling tasks that are normally carried out in standard laboratories tobe performed on chip; tasks like mixing, separation and concentration of particles in fluids,precise measurements and chemical analysis, to mention a few. Miniaturization provides multipleadvantages, such as the occupation of a small space, the need of tiny volumes of samples andreagents and very quick responses, but it also makes possible phenomena that do not happen inthe macro scale. |nertial focusing, the technique in this invention, is one such phenomenon. ln microfluidics, commonly surface forces dominate over body forces; e.g. inertial effectsdo not play a role and viscosity dominates the system. Under such conditions, particles simplyfollow the streamlines of the carrier fluid. There are, nevertheless, known techniques tomanipulate such particles and make them move across streamlines in a controlled manner. Theyare generally classified as active techniques if they require and external field (acoustic, electric,magnetic), or passive techniques if they only rely on the microfluidic channel geometry and thefluid flow. These techniques make possible, for instance, to move particles from one fluid toanother (for washing, reacting, mixing) or the separation and concentration of a specific target. |nertial focusing stands out amongst the other particle manipulation techniques because itis passive, which makes the systems robust and simple to operate (the phenomenon arises simplyby flowing the sample through the system), it is label free, the throughput may be very high and ithas been shown to have high resolution. High-throughput ordering and focusing of particles in afluid is a prerequisite for biomedical diagnostic operations, such as flow cytometry, cell sorting,deformability-based phenotyping studies and protein concentration analysis.

Although operating passive technique microfluidic system is simple, the prediction of thephenomenon and engineering the systems of inertial focusing is complex.

There is a need in the art for an improved understanding so that the passive microfluidic channel configuration provides for an improved particle manipulation.

Object of the lnvention The present invention aims at obviating the aforementioned problem.

A primary object of the present invention is to provide at least a portion of a microfluidicsystem for improved manipulation of fine particles in a fluid.

Another object of the present invention is to provide a method for improved manipulation of fine particles in a fluid.

Summary of the lnvention According to the invention at least the primary object is attained by means of the apparatushaving the features defined in the independent claims.

Preferred embodiments of the present invention are further defined in the dependentclaims.

According to a first aspect of the present invention it is provided a microfluidic system formanipulating fine particles in a fluid, said system comprising a High Aspect Ratio Curved (HARC)microfluidic channel having at least one inlet and at least one outlet and at least a first sectiongenerated by revolving a High Aspect Ratio (HAR) cross section around an axis parallel to the longside of said cross section, said first section downstream said at least one inlet is configured forfocusing said fine particles from multiple positions in said fluid when entering said first section toa focus position by the inner wall in said microfluidic channel when leaving said first section,wherein particles to be focused in said first section of the microfluidic channel are fulfilling Q <(33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R the radius of curvature of themicrochannel in um, k = a/W, with a the particle effective diameter in ,um, W the width of thecross section of the HARC microchannel in ,um, and KC = (-2.06 + 6.95/AR) min * ym/yL ,where AR = H/W > 1, with H the height of the cross section of the HARC microchannel in ,um.

An advantage of this embodiment is that that passive focusing of particles of different sizesinto a predetermined focus position can be made. Another advantage is that the focus will remaininvariant over a range of flow rates, making the system robust and easy to use. ln various example embodiments of the present invention the microfluidic system furthercomprising a second section, downstream said first section, configured for separating particles ofdifferent size in said fluid from said focus position in said microfluidic channel when entering saidsecond section to different positions for different sizes of particles when leaving said secondsection, wherein particles to be separated in said second section of the microfluidic channel arefulfilling Q > (33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R the radius of curvature of the microchannel in um, k = a/W, with a the particle effective diameter in ,um, W the width of the cross section of the HARC microchannel in pm, and KC = (-2.06 + 6.95/AR) min * ym/yL,where AR = H/W > 1, with H the height of the cross section of the HARC microchannel in pm.

The advantage of these embodiments is that passive separation of particles of different sizeto different positions with high resolution can be made. ln various example embodiments of the present invention said first section for focusing saidparticles from multiple positions in said fluid when entering said first section into apredetermined focus position when leaving said first section is a microfluidic channel having anAspect Ratio, AR, higher than 1 but lower than 4.

The advantage of these embodiments is that the range in the space of variables where thefocus occurs can be customized. Such variables being the flow rate, particle size, radius andpressure for instance. ln various example embodiments of the present invention the microfluidic channel isbranched into several outlets to collect different portions of the fluid.

The advantage of these embodiments is that particles of different sizes in the fluid may becollected in separate places. ln various example embodiments of the present invention said microfluidic system furthercomprising at least one filter section arranged in between at least a first inlet of said microfluidicchannel and the first section of said microfluidic channel.

The advantage of these embodiments is that unwanted particles may be filtered out andseparated from the remaining fluid, which remaining fluid may continue to flow downstream themicrofluidic system. ln another aspect of the present invention it is provided a method for manipulating aplurality of fine particles of different sizes in a fluid, said method comprising the steps of: - providing said fluid having said fine particles into at least one inlet of amicrofluidic device, - forcing said fluid through a High Aspect Ratio Curved (HARC) microfluidicchannel of said microfluidic device having at least one inlet and at least oneoutlet, - focusing said fine particles of different sizes in said fluid in a first section thatresults from the revolution of a HAR cross section around an axis parallel tothe long side of said cross section into a predetermined focus position in saidmicrofluidic channel when leaving said first curved section, wherein particlesto be focused in said focusing section of the microfluidic channel are fulfillingQ < (33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R the radius of curvature of the microchannel in um, k = a/W, with a the particle effective diameter in pm, W the width of the cross section of the HARC microchannelin pm, and KC = (-2.06 + 6.95/AR) min * pm/pL, where AR = H/W >1, with H the height of the cross section of the HARC microchannel in pm.

A HARC microfluidic channel has a ratio of a vertical dimension of the microfluidic channeldivided by a horizontal dimension of the microfluidic channel above 1 and results from therevolution of a HAR cross section around an axis parallel to the long side of said cross section. ln still another aspect of the present invention it is provided a microfluidic focusing sectionconfigured to be used in a microfluidic system, said microfluidic focusing section comprising aHARC microfluidic channel for focusing fine particles of different sizes from multiple positions in afluid when entering said focusing section into a predetermined focus position when leaving saidfocusing section, wherein particles to be focused in said focusing section of the microfluidicchannel are fulfilling Q < (33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R the radius ofcurvature of the microchannel in pm, k = a/W, with a the particle effective diameter in pm, Wthe width of the cross section of the HARC microchannel in pm, and KC = (-2.06 + 6.95/AR)min * pm/pL, where AR = H/W > 1, with H the height of the cross section of the HARCmicrochannel in pm. ln yet another aspect of the present invention it is provided a microfluidic separatingsection configured to be used in a microfluidic system, said microfluidic separating sectioncomprising a HARC microfluidic channel for separating particles pre-focused by the inner wall in afluid when entering said separating section into multiple positions dependent on the size of saidparticles, wherein particles to be separated in said separating section are fulfilling Q >(33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R the radius of curvature of the microchannelin pm, k = a/W, with a the particle effective diameter in pm, W the width of the cross section ofthe HARC microchannel in pm, and KC = (-2.06 + 6.95/AR) min * pm/pL, where AR =H/W > 1, with H the height of the cross section of the HARC microchannel in pm.

Further advantages with and features of the invention will be apparent from the following detailed description of preferred embodiments.

Brief description of the drawings A more complete understanding of the abovementioned and other features and advantagesof the present invention will be apparent from the following detailed description of preferred embodiments in conjunction with the appended drawings, wherein: Fig. 1 depicts a schematic 2D cross section of a straight HAR microfluidic channel illustratinginertial focusing in straight microfluidic channels.

Fig. 2 depicts a schematic 3D perspective view of the straight HAR microfluidic channel infigure 1.

Fig. 3 depicts a schematic 2D cross section of an example embodiment of a HARCmicrofluidic channel according to the present invention.

Fig. 4 depicts a schematic 2D top view of a HARC microfluidic channel showing separation ofparticles.

Fig. 5 depicts a schematic 3D perspective view of the HARC microfluidic channel in figure 4.

Fig. 6 depicts a schematic 2D cross section of a HARC microfluidic channel illustrating theDean force and the predetermined focus position of particles.

Fig. 7 depicts a schematic 3D perspective view of the HARC microfluidic channel in figure 6.

Fig. 8a-d depicts a schematic 2D cross section of a HARC microfluidic channel illustratingvarious stages of inertial focusing.

Fig. 9a depicts from a top view an example embodiment of a microfluidic channel systemaccording to the present invention.

Fig. 9b depicts enlarged sections of the microfluidic channel system in figure 9a for various fluidic conditions and particle sizes.

Detailed description of preferred embodiments of the invention Briefly, in straight channels with circular cross section, randomly distributed fine particleswill migrate radially across the streamlines towards an equilibrium perimeter (EP). This migrationis attributed to two main forces originated by the interaction of the fluid with the fine particlesand the walls of the microfluidic channel, the shear gradient induced lift force, which dominatesat the center of the microfluidic channel and is directed towards the walls, and the wall inducedforce, which dominates close to the walls and repels particles from them. The combination ofboth is known as the net lift force (FL) and becomes zero at the EP, where particles will find the equilibrium position. ln cross sections that are not circular, this perimeter also exists (denoted by 110a, 110b,110c, 110d) in figure 1, but FL has a minor component tangential to it (born from the asymmetryof the system) that gathers particles at the center of the faces. These two effects, radial andlateral migration, overlap in time. However, the radial force is much stronger and the radialmigration seems to occur first. The difference is such that for practical reasons the radial migration can indeed be approximated as occuring first and being followed by the lateral one, as represented in figure 2. Note that this implies that the component of the lift force that isorthogonal to the EP is very strong, while the tangential one is weak (in relative terms), which is critical for understanding systems with a secondary flow.

As we introduce curvature in the system, a secondary flow (named Dean flow; transversalto the main flow) appears, forming two symmetrical vortexes 660, 670. These vortexes 660, 670induce a drag to the particles parallel to their orbits (FD), modifying the distribution of forces and thus the equilibrium positions, see figure 6. ln a curved system one can consider FL like it is in a straight microfluidic channel and, aslong as the main flow is not deformed much by the curvature, the effect of the secondary flowcan be overlapped. What is more, the tangential FL can be neglected because it is easilysurpassed by FD, which should be, as will be discussed further below, in the range of the radial FL for inertial focusing applications.

This approximation, where we only consider the radial FL, the EP and the FD generated bythe secondary flow, enables an intuitive prediction of the phenomenon. Under thisapproximation, it is easier to engineer new systems. Here, we explain and demonstrate inventiveinertial focusing in High Aspect Ratio Curved (HARC) microfluidic channels; systems that offer very different features from what is known in the art.

The most appealing feature being a focus position by the inner wall 650, 750, which ispractically insensitive to particle size and invariant within a range of flow rates. The focus positionby the inner wall will gather particles of different sizes into equilibrium positions that are veryclose to each other. This system is ideal for concentration of particles belonging to a range ofsizes; e.g. a population of a growing bacteria species or of several species. lt is also convenient fortechnologies like FACS and flow cytometers, where otherwise a whole fluid sample is focused bymeans of a sheath and a laser is used to interrogate its contents. With the system presented here,no sheath is needed, bringing simplicity to the operation, saving reagents and multiplying thethroughput. The stability within a range of flow rates makes the system robust and non- dependent on high precision pumps/control systems. ln the following paragraphs a simplification of the phenomenon of inertial focusing incurved microfluidic channels will be disclosed, where we only consider the radial FL, the EP andthe drag by the secondary flow. The validity of this scenario is supported by the agreement of the results with the calculations. ln High Aspect Ratio (HAR) straight microfluidic channels there is a strong, radialcomponent of the lift force; particles quickly migrate to the EP. A weak component of the force, tangential to the equilibrium perimeter, brings particles towards the center of the faces. lntroducing curvature to the system, see figure 5-8, induces a secondary flow (Dean flow)that takes the shape of two vortexes. This flow generates a drag force (FD) and particles are sweptin the direction of its streamlines. ln their journey, particles pass by regions where the secondaryflow does not share direction with FL; the vectorial summation of FL and FD results in anothervector and thus particles keep following the secondary orbits. There are, however, regions wherethe secondary flow shares direction and opposes FL. lf FL is stronger than FD at these areas ofopposition, particles will be stopped, leading to their focus and concentration. lf FD is stronger, particles will keep circulating indefinitely. ln HARC microfluidic channels the secondary drag is tangential to the EP in the wholecross section except at the central part, see figure 7. Particles are easily dragged until the side ofthe inner wall, where the orbits turn and oppose FL. Provided that FL is strong enough, thehorizontal component of FD is countered out, leaving only the vertical one, which brings particles to a predetermined focus position situated at the symmetry plane, figure 7. lnterestingly and conveniently, for the practical cases in inertial focusing in HARCmicrofluidic channels (i.e. when particles are focused) the radial migration happens faster thanthe collection around the EP. Thus, one can further simplify and consider particles to first occupythe EP and then be dragged around it until the focus position at an inner wall of the microfluidic channeL ln the following paragraphs we summarize the conclusions for a proper understanding of inertial focusing.

Understanding inertial focusing in straight microfluidic channels is essential to build morecomplex, curved systems since in both cases FL plays a major role. Thus, we include a corollary extracted from the literature and our experience, which concludes: Although more sophisticated and accurate calculations are available in the literaturenowadays, a practical approximation to FL in microfluidic channels with a rectangular crosssection is FL=(CLp(Um)2a4)/W2, where p is the density of the fluid, Um is the maximum flowvelocity, a is the size of the particle, W is the smallest dimension of the cross section and CL is acoefficient that adjusts the value of the force as function of the Reynolds number (Re=(pUmW)/;1; being ,u the dynamic viscosity).

A particle migrating by FL will find an opposing drag force FD=31IpaURm (originated by the interaction with the liquid) that will settle the radia| migration speed (URm) to: URm=(CLp( lntroducing an averaged lift coefficient provides an estimation of an averaged radia|migration speed (ÜRm) that can be used to estimate the microfluidic channel length needed forparticles to reach the perimeter (LRm).

As we introduce curvature to the High Aspect Ratio microfluidic channel, a secondary flowarises; Fig. 7. ln absence of other forces, particles quickly accelerate and follow the streamlineswith the same speed of the flow. We refer to this force as the secondary drag, FD=31maUD*;where UD* is the relative speed of the particle compared to speed of the secondary flow (UD);being UD*=0 if a particle freely follows the streamlines, and UD*=UD when a particle does notmove; case for particles stopped at the equilibrium position.

We divide the study of the effect of this flow into two subsections: a migration around theEP and an opposition of FL and FD at the center, by the side of the inner wall.

Figure 3 depicts a schematic 2D cross section of an example embodiment of a HARCmicrofluidic channel 300 according to the present invention. A HARC microfluidic channel isdefined as having a base W which is smaller than the height H. A curved microfluidic channel 300is best represented in an angular coordinate system where the base W is along the Z-axis, theheight H is along the Y-axis and the curvature is defined by the distance R from the center of thecross section of said curved microfluidic channel 300 to an imaginary rotational axis 310 in parallelto the Y axis. When the HAR Cross Section H*W is revolved around the rotational axis 310 parallelto the Y axis, a High Aspect Ratio Curved (HARC) microfluidic channel is generated. A HARmicrofluidic channel is defined as H/W>1. The HARC microfluidic channel 300 may be curvedaround a single axis, to form a microfluidic channel with homogeneous curvature, but the axis canalso be displaced, achieving a microfluidic channel with different radii of curvature in differentsections, enabling the formation of several loops which otherwise would make the microfluidicchannel collide with itself after one loop, namely a spiral. The revolution can also have atranslational component in the Y-axis, bringing the microfluidic channel out of a single plane,forming a 3D spiral.

Figure 1 and figure 2 depicts a schematic 2D cross section of a straight HAR microfluidicchannel 100 and a schematic 3D perspective view of the straight HAR microfluidic channel respectively, illustrating inertial focusing in straight microfluidic channels. ln a straight HAR microfluidic channel 100, particles 110 in a microfluidic channel will focus into four positions 110a,110b, 110c, 110d. Due to distribution of forces, the majority reach the position by the middle of thelong faces H 110b, 110d. The arrows 120 represent a direction and also a magnitude of a Lift Force,FL. The smooth line 130 represents the Equilibrium Perimeter (EP); a region where the Lift force FLis almost zero (there is a small component of the Lift tangential to it). Particles 110 quickly fall tothat perimeter, followed by a slow migration to the four positions 110a, 110b, 110c, 110d.

Figure 2 depicts a schematic 3D perspective view of the straight HAR microfluidic channel100 in figure 1. Figure 2 depicts the evolution of |nertial Focusing in a straight HAR microfluidicchannel 100.

Figure 6 depicts a schematic 2D cross section of a HARC microfluidic channel 600illustrating Dean Force or Drag Force. Dean Force FD is a drag force originated by the secondaryflow coming from the curvature in a microfluidic channel. The arrows 640 represent the directionand also the magnitude of the Dean Forces FD in the curved HAR microfluidic channel 600. TheDean Force FD appears together with the Lift Force FL like in the straight microfluidic channel. Atthe same time that particles fall into the Equilibrium Perimeter EP, the Dean Force FD moves themfollowing the vortexes. Where the vortex moves from an inner wall towards an outer wall, theDean Force FD is in opposition with the Lift Force FL. Provided that the Lift Force FL is stronger thanthe Dean Force FD, particles lose their horizontal component of the speed, remaining only thevertical one that brings all together 650 to the symmetry plane. The smooth line 630 indicateswhere the lift force is practically zero within the curved HAR microfluidic channel 600.

Figure 7 depicts a schematic 3D perspective view 700 of the curved microfluidic channelin figure 6. Figure 7 depicts the evolution on |nertial Focusing in HARC microfluidic channels 700.The arrows represent 740 represent the Dean Force FD. Particles 710 in numerous positions in afluid provided at an inlet 702 of the HARC microfluidic channel 700 will successively be focusedinto a predetermined focus position 750 at an outlet 704 of said HARC microfluidic channel 700.

Figure 8a-8d depicts a schematic 2D cross section of a HARC microfluidic channel 800illustrating various stages of inertial focusing. Below each schematic 2D cross section is theobservation of said various stages of the |nertial Focusing in HARC microfluidic channels under themicroscope. ln figure 8a, an early stage shortly after the beginning of the HARC microfluidic channel,particles 810 have fallen to the Equilibrium Perimeter by the Lift Force FL but still occupy most ofit. Two bright lines 860, 870 are observed under the microscope. ln figure 8b, the secondary flow FD has moved the particles 810 around the Equilibrium Perimeter (EP), increasing the concentration on the side of the inner wall 880 of the HARC microfluidic channel 800. The line close to the outer wall 890 of the HARC microfluidic channel 800loses intensity. ln figure 8c all particles 810 have reached the side close to the inner wall 880 and the lineat the outer wall 890 disappears under a fluorescent microscope. We refer this stage as Half DeanLoop. At this point, particles 810 form a plane, which is seen as a single line 870 close to the innerwall 880 of the curved HAR microfluidic channel 800 under the microscope. The Half Dean Loopstage may be used to collect the particles 810.

I figure 8d the Dean Loop is complete, Full Dean Loop, and all particles reach apredetermined focus position 850. lt cannot be distinguished from the Half Dean Loop stageunder a microscope with a normal focal length. lt takes double microfluidic channel length thanthe Half Dean Loop to achieve this stage (or same channel Length but double Flow Rate). ln thisstage the focus is very good. Further microfluidic channel length has in practicality no function,perhaps the quality of the focus may be improved a bit if the microfluidic channel is somewhatlonger, but this improvement is difficult to measure. Particles initially situated at the horizontalsymmetry plane are in an unstable equilibrium line and do not follow either of the vortexes.Therefore, although not detected under the microscope, it is expected to find a very small fractionof particles at the outer wall even after a full Dean loop.

The formula to know the length of a microfluidic channel required for the Dean Loop to becompleted has been calculated with COMSOL and it is an approximation: ' p Umaxw Re (loops) The other equation expresses when the Dean Force FD becomes stronger than FL and theparticle will be able to go through the Lift force FL, remaining unfocused. lt gives the upper limit of Q where the focus will occur:Q < SÉJR låKC Where Q is the flow rate in pL/min, R the radius of curvature of the microchannel in um,k = a/W, with a the particle effective diameter in ,um, W the width of the cross section of theHARC microchannel in ,um, and KC = (-2.06 + 6.95/AR) min * ym/yL, where AR = H/W >1, with H the height of the cross section of the HARC microchannel in ,um.

These two equations are the key to define the phenomenon. The latter equation stronglydepends on the particle size. Small particles go more easily through the barrier in the middlegenerated by the Lift force FL. This equation must be fulfilled when aiming at concentration. The first equation defines the length of the microfluidic channel and it is valid if particles are initially 11 randomly distributed. ln the situation where two fluids come into the microfluidic channel andtherefore particles only occupy half of it (the half closer to the inner wall), the microfluidic channelcould be shorter and still achieve focus.

Regarding the separation, particles should initially be confined to a predetermined focusposition being close to a single position 450, 550, 650, 750, 850 close to the inner wall 480, 580,780, 880. Then, for those particles 420, 430, 520, 530 that will migrate towards the outer wall achieving the separation, the equation must be fulfilled but in reverse: >3&4Rk3QKC Where Q is the flow rate in pL/min, R the radius of curvature of the microchannel in um, k =a/W, with a the particle effective diameter in ,um, W the width of the cross section of the HARCmicrochannel in ,um, and KC = (-2.06 + 6.95/AR) min * ym/yL, where AR = H/W > 1, withH the height of the cross section of the HARC microchannel in ,um.ln various example embodiment of the present invention it is provided a microfluidic system for manipulating fine particles in a fluid comprising a High aspect Ration Curved (HARC)microfluidic channel having at least one inlet and at least one outlet and at least a first section hatresults from the revolution of a HAR cross section around an axis parallel to the long side of saidcross section, said first section downstream said at least one inlet is configured for focusing saidfine particles from multiple positions in said fluid when entering said first section to apredetermined focus position when leaving said first section, wherein particles to be focused in said focusing section of the microfluidic channel are fulfilling <3&4Rk3QKC Where Q is the flow rate in pL/min, R the radius of curvature of the microchannel in um,k = a/W, with a the particle effective diameter in ,um, W the width of the cross section of theHARC microchannel in ,um, and KC = (-2.06 + 6.95/AR) min * ym/yL, where AR = H/W >1, with H the height of the cross section of the HARC microchannel in ,um. ln various example embodiments of the present invention it is provided a microfluidicfocusing section configured to be used in a microfluidic system, said microfluidic focusing sectioncomprising a HARC microfluidic channel for focusing fine particles of different sizes from multiplepositions in a fluid when entering said focusing section into a focus position by the inner wallwhen leaving said focusing section, wherein said particles to be focused in said focusing section are fulfilling 12 < 33.4R kgQ KC This microfluidic section may be used as a building block in a microfluidic system togetherwith other building blocks such as filter sections, separation sections, measuring sections,collecting sections, etc. ln various example embodiments of the present invention it is provided a microfluidicseparating section configured to be used in a microfluidic system, said microfluidic separatingsection comprising a HARC microfluidic channel for separating particles pre-focused in a positionby the inner wall when entering said separating section into multiple positions dependent on the size of said particles, wherein said particles to be separated in said separating section are fulfillingQ > SÉJR låKC Where Q is the flow rate in pL/min, R the radius of curvature of the microchannel in um,k = a/W, with a the particle effective diameter in ,um, W the width of the cross section of theHARC microchannel in ,um, and KC = (-2.06 + 6.95/AR) min * ym/yL, where AR = H/W >1, with H the height of the cross section of the HARC microchannel in ,um.

The pre-focused particles may be focused using the above-mentioned focusing section,however the focusing may also be performed by other means such as sheath flow focusing.

The Aspect Ratio in the HARC microfluidic channel may be between 1-4. ln anotherembodiment said HARC microfluidic channel has an Aspect Ratio between 1,2-3,5. ln yet anotherexample embodiment the Aspect Ratio is between 1,5-3. ln various example embodiments saidsize of the particles in said fluid is smaller than 100um. ln various example embodiments saidparticle size is smaller than 50um. ln various example embodiments said particle size is smallerthan 25um. The microfluidic system may comprise at least one filter section for filtering out andcollecting unwanted items to further flow downstream the microfluidic system. The microfluidicsystem may comprise a plurality of outlets for collecting different portions of the fluid havingtravelled through a portion of or the full microfluidic system. ln various example embodimentsdifferent outlets may be designed to receive different sizes of particles in the fluid passingthrough the microfluidic system. By using a suitable design of a separation section together with aplurality of outlets different sizes of the particles may be collected at different outlets.

Figure 4 depicts a schematic 2D top view of a HARC microfluidic channel 400 showing separationof particles and figure 5 depicts a schematic 3D perspective view of the curved microfluidic channel in figure 4. ln the separation section 400, 500 particles of different sizes are pre-focusedinto a predetermined focus position 450, 550 when entering an inlet 402, 502 of said separation section 400, 500. As the fluid passes through the separation section 400, 500, large particles 410, 13 510 will remain close to the inner wall 480, 580 whereas small particles 430, 530 will be separatedtowards the outer wall 490, 590. Medium size particles 420, 520 will be separated to a position inbetween said large particles 410, 510 and said small particles 430, 530. When the particles reachthe outlet 504 of said separation section 400, 500 different sizes of particles are provided atpredetermined positions within the microfluidic channel allowing them to be measuredindependently of each other and/or collected independently of each other by branching saidoutlet 404, 504 of the separation section 400, 500 into a plurality of end collection each collectingdifferent sizes of particles. During the separation, because FL is weaker than FD and the result ofthe addition of both forces depends on the particle size, particles of different sizes move atdifferent speeds, enabling their separation provided they start from said predetermined focusposition where all particles are grouped together in a confined volume more or less attachingeach other. Small particles will travel faster than large particles.

To validate the presented theory on inertial focusing in HARC systems, we used devices withcurved microfluidic channels (R 400 um; 2 loop) with AR 2 and 2.6 (6x12 um and 6x16 um (WxH),respectively). Minimum flow rate for particles to complete a Dean loop (QDL) can be calculated. FLdoes not grow as fast as FD as the flow rate increases. Therefore, for a given device, increasing theflow rate will eventually lead to FD surpassing FL and the device will lose the focusing capabilities.We refer to this upper limit as Qmax and can be calculated using the hereinabove mentionedequations.

Our devices can safely stand 200 bar, which was set as an upper limit of pressure, leading toanother upper limit of Q (Om bar). The pressure limit due to manufacturing choice of the microfluidic channels should not limit the use of the invention above 200 bar.

Table 1: 6 12 2 2 4006 16 2.6 2 400 14 (Th and Exp)35 (Th and Exp) 26 (Exp)67 (Exp) (Th)56 (Th) ln table 1, dimensions of the devices used to validate the presented theory related to HARCmicrofluidic channels, predictions using the proposed equations (denoted by (Th) by the side ofthe number) of the critical conditions to achieve focus, and obtained experimental values of suchconditions (denoted by (Exp) by the side of the number). The critical conditions are: minimum flow rate for particles to complete a Dean loop (QDL), maximum flow rate for particles not to cross 14 to the outer wall (Qmax) and an upper limit of Q where the pressure reaches 200 bar and thedevices may break (QZOO bar).

The experiments agreed with the hypothesis presented for the working mechanism ofinertia| focusing in HARC microfluidic channels.

We also validated the existence of an upper limit in flow rate for the concentration ofparticles (Qmax); FD surpassed FL at Qmwf26 uL/min, where the microfluidic channels lost thefocusing capacities. lnterestingly and contrary to what happens in prior art low aspect ratio curved microfluidicchannels, the focus position in HARC microfluidic channels was practically insensitive to the flowrate. The fact that the position is independent of a range of flow rates makes the system morerobust than when other cross sections are used, where a drift in the pump may lead tofluctuations in the focus positions and therefore diminished performances.

Once the phenomenon with AR=2 was proved, we used COMSOL to investigateperformance of systems with higher AR. Maintaining Um and H gives a similar distribution andstrength of FL. The secondary flow FD still has the shape of two vortexes, but its local strengthover the cross section is highly affected. For instance, while maintaining a comparable speed forthe collection around the EP, the secondary flow in AR = 2.6 has half the strength than in AR = 2 atthe focus position. Therefore, AR = 2.6 should be able to focus the same particles up to muchhigher flow rates (or allow a reduction in R and thus alleviate the demand of pressure for thesame flow rate). At the same time, these conditions are favourable to focus smaller particles.

We fabricated the same device, 4 um deeper (6x16 um (WxH); AR 2.6) and compared theperformance. The superiority of the system was evident from the experiment. For 1 um particles,while QDL demanded a bit higher pressure (60 bar instead of 50 bar), Qmax was more thandouble (67 uL/min instead of 26 uL/min). We included more particles in the system (0,7um,0,79um and 0,92um) and found, in agreement with the proposed theory, that all sizes focusedclose to the inner wall, at a very similar position. What is more, QDL was independent over a rangeof sizes a, further supporting the fact that the collection around EP is the limiting factor in thefocusing. On the other hand, we verified Qmax to be dependent on a for 67, 60, 45 and 32 uL/minfor 1, 0.92, 0.79 and 0.7 um particles, respectively.

From the COMSOL simulations, we estimate that the microchannels lose the focusingcapacity for AR much higher than those presented here; the secondary flow becomes very slow atthe central regions and it likely does not have the capacity to collect the particles.

We explain and demonstrate the phenomenon of inertia| focusing in high aspect ratiocurved microfluidic channels; novel systems where microparticles are focused in a predetermined focus position, largely independent of the flow rate and particle size, by the side of the inner wall of said microfluidic channel. Combining the state of art knowledge in the field with our experienceand simulations with COMSOL, we describe how the migration and focus of particles occurs. Wealso provide the governing equations, which strongly agree with our experiments, and enable thedesign of the systems.

Both the theory and the experiments show that, keeping the rest of the dimensionalvariables fixed, a microfluidic channel with higher the aspect ratio offers a better performance.lndeed, the higher the aspect ratio, the weaker the secondary flow becomes at the equilibriumposition. This translates into higher flow rates until the secondary flow dominates for a fixedparticle size and suitability to focus smaller particles or a possible reduction in radius andtherefore of the length and pressure needed to run the system. However, a too high aspect ratio(e.g. AR 4) has such a low secondary flow at the central part that it can be expected to lose thefocusing capabilities.

Figure 9a depicts from a top view an example embodiment of a microchannel system 900according to the present invention. The microfluidic channel system comprises an inlet 930, anoptional filter section 932, a focusing section 934, a separation section 938 and an outlet 942. ln afirst expanded section between the focusing section 934 and the separation section 938 it isillustrated that particles of different sizes will focus into a predetermined focus position 936 ofthe microfluidic channel. ln a second expanded section between the separation section 938 andthe outlet 942 particles of different sizes are separated into different positions 940. ln figure 9b itis depicted enlarged views of the first expanded portion 910 and the second expanded portion920. ln the first expanded portion 910 it is clear from figure 9b that particles of different sizes arefocused into one and the same position of the microfluidic channel. ln the second expandedportion 920 it is illustrated separation of particles of three different sizes namely 0,92um, 0,79umand 0,70um and different flow rates/pressure. For lower pressure, here illustrated as 80bar, tothe left of the second expanded portion 920 all particles have been moved from the inner wall950 towards the center of the channel. The separation at 80bar is distinct but particles with slightdifference in size will still appear close to each other. The second expanded portion illustrateshow the three different sizes of particles will move towards the outer wall 952 as the flowrate/pressure increases. At 160bar the three different particles chosen in this experiment areseparated around the center of the microfluidic channel. As the pressure further increase in themicrofluidic channel the particles move further towards the outer wall of the microfluidic channel.|rrespective of pressure there is a clear distinction of placement of particles of different sizes indifferent positions. The distance separating the particles can be amplified modifying the variables of the separation section; e.g. R or number of loops. 16 The microfluidic channels exemplified in the figures have been illustrated with sharpcorners, however, the invention will also work with microfluidic channels having rounded corners.

We believe that this work, presenting, explaining and demonstrating inertia| focusing inhigh aspect ratio microfluidic channels, paves the road to a new branch of inertia| focusing devices with a lot of unexplored potential.

Feasible modifications of the |nvention The invention is not limited only to the embodiments described above and shown in thedrawings, which primarily have an illustrative and exemplifying purpose. This patent application isintended 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 equivalentsthereof. Thus, the equipment may be modified in all kinds of ways within the scope of theappended claims.

Throughout this specification and the claims which follows, unless the context requiresotherwise, the word "comprise", and variations such as "comprises" or "comprising", will beunderstood 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.

Claims (11)

1. A microfluidic system for manipulating fine particles in a fluid, said system comprising aHigh Aspect Ratio Curved, HARC, microfluidic channel having at least one inlet and at leastone outlet and at least a first HARC section resulting from a revolution of a HAR crosssection around an axis parallel to a long side of said cross section, said first HARC sectionis configured for focusing said fine particles from multiple positions in said fluid whenentering said first HARC section to a focus position by an inner wall of the microfluidicchannel when leaving said first HARC section, wherein particles to be focused in said firstsection of the microfluidic channel are fufilling Q < (33,4*R*k3)/Kc, where Q is the flowrate in pL/min, R is the radius of curvature of the microfluidic channel in pm, k = a/W,with a the particle effective diameter in pm, W the width of the cross section of the HARCmicrofluidic channel in pm, and KC = (-2.06 + 6.95/AR) min * pm/pL, where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in pm.

2. The microfluidic system according to claim 1, further comprising a second HARC sectionresulting from a revolution of a HAR cross section around an axis parallel to a long side ofsaid cross section, downstream said first section, configured for separating particles ofdifferent size in said fluid from said focus position by the inner wall in said microfluidicchannel when entering said second section to different positions for different sizes ofparticles when leaving said second section, wherein particles that will migrate towards anouter wall achieving the separation in said separating section are fulfilling Q >(33,4*R*k3)/Kc, where Q is the flow rate pL/min, R is the radius of curvature of themicrofluidic channel in pm, k = a/W, with a the particle effective diameter in pm, W thewidth of the cross section of the HARC microfluidic channel in pm, and KC = (-2.06 +6.95/AR) min * pm/pL, where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in pm.

3. The microfluidic system according to any one of claim 1-2, wherein said first section forfocusing said particles from multiple positions in said fluid when entering said first sectioninto said predetermined focus position when leaving said first section is a microfluidic channel having an Aspect Ratio, AR, higher than 1 but lower than 4.

4. The microfluidic system according to any one of claim 1-3, wherein the microfluidic channel is branched into several outlets to collect different portions of the fluid.

5. The microfluidic system according to claim 4, wherein particles of at least a first size are guided to a first outlet and particles of at least a second size are guided to a second outlet.

6. The microfluidic system according to any one of the preceding claims, further comprising a filter section arranged in between at least a first inlet of said microfluidic channel and the first section of said microfluidic channel.

7. A method for manipulating a plurality of fine particles of different sizes in a fluid, said method comprising the steps of: providing said fluid having said fine particles into an inlet of a microfluidicdevice, forcing said fluid through a High Aspect Ratio Curved, HARC, microfluidicchannel of said microfluidic device having at least one inlet and at least oneoutlet, focusing said fine particles of different sizes in said fluid in a first HARC sectionresulting from a revolution of a HAR cross section around an axis parallel to along side of said cross section into a focus position by an inner wall of themicrofluidic channel when leaving said first HARC section, wherein particles tobe focused in said first HARC section of the microfluidic channel are fufilling Q< (33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R is the radius ofcurvature of the microfluidic channel in um, k = a/W, with a the particleeffective diameter in ,um, W is the width of the cross section of the HARCmicrofluidic channel in ,um, and KC = (-2.06 + 6.95/AR) min * mn/yL,where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in ,um.

8. The method according to claim 7, further comprising the steps of: forcing said focused fine particles from said first HARC section into a secondHARC section, separating said fine particles of different sizes in said fluid from said focusposition by the inner wall in said microfluidic channel when entering saidsecond HARC section to different positions for different sizes of particleswhen leaving said second HARC section, wherein the second HARC section is a HARC microfluidic channel resulting from a revolution of a HAR cross section around an axis parallel to a long side of said cross section, said second HARCmicrofluidic channel having an Aspect Ratio, AR, greater than 1 and whereinparticles that will migrate towards an outer wall achieving the separation insaid separating section are fulfilling Q > (33,4*R*k3)/Kc, where Q is the flowrate in pL/min, R is the radius of curvature of the microfluidic channel in pm,k = a/W, with a the particle effective diameter in pm, W is the width of thecross section of the HARC microfluidic channel in pm, and KC = (-2.06 +6.95/AR) min * pm/pL, where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in pm.

9. The method according to claim 8, further comprising the steps of - guiding different portions of the fluid to different outlets.

10. The method according to claim 8 or 9, further comprising the steps of- guiding particles of a first size to a first outlet via a first microfluidic channelbranch, and- guiding particles of at least a second size to a second outlet via a second microfluidic channel branch.

11. A method for manipulating a plurality of fine particles of different sizes in a fluid, saidmethod comprising the steps of: - forcing focused fine particles into a separation section, - separating said fine particles of different sizes in said fluid from a focusposition by an inner wall of a microfluidic channel when entering saidseparating section to different positions for different sizes of particles whenleaving said separation section, wherein the separation section is a HARCmicrofluidic channel resulting from a revolution of a HAR cross section aroundan axis parallel to a long side of said cross section, said HARC microfluidicchannel having an Aspect Ratio, AR, greater than 1 and wherein particles thatwill migrate towards an outer wall achieving the separation in said separatingsection are fulfilling Q > (33,4*R*k3)/Kc, where Q is the flow rate in pL/min, Rthe radius of curvature of the microfluidic channel in pm, k = a/W, with athe particle effective diameter in pm, W is the width of the cross section of the HARC microfluidic channel in pm, and KC = (-2.06 + 6.95/AR) min * mn/yL, where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in ,um. The method according to claim 11, further comprising the steps of - guiding different portions of the fluid to different outlets. The method according to claim 11 or 12, further comprising the steps of- guiding particles of a first size to a first outlet via a first microfluidic channelbranch, and- guiding particles of at least a second size to a second outlet via a second microfluidic channel branch. A microfluidic focusing section configured to be used in a microfluidic system, saidmicrofluidic focusing section comprising a HARC microfluid channel for focusing fineparticles of different sizes from multiple positions in a fluid when entering said focusingsection into a focus position by an inner wall of the microfluidic channel when leaving saidfocusing section, wherein particles to be focused in said focusing section of themicrofluidic channel are fulfilling Q < (33,4*R*k3)/Kc, where Q is the flow rate in pL/min,R is the radius of curvature of the microfluidic channel in um, k = a/W, with a theparticle effective diameter in ,um, W is the width of the cross section of the HARCmicrofluidic channel in ,um, and KC = (-2.06 + 6.95/AR) min * ,um/;1L, where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in ,um. A microfluidic separating section configured to be used in a microfluidic system, saidmicrofluidic separating section comprising a HARC microfluidic channel resulting from arevolution of a HAR cross section around an axis parallel to a long side of said crosssection, said separating section for separating particles pre-focused by an inner wall ofsaid microfluidic channel when entering said separating section into multiple positionsdependent on the size of said particles, wherein particles that will migrate towards the anouter wall achieving a separation in said separating section are fulfilling Q >(33,4*R*k3)/Kc, where Q is the flow rate in pL/min, R is the radius of curvature of themicrofluidic channel in um, k = a/W, with a the particle effective diameter in ,um, W isthe width of the cross section of the HARC microfluidic channel in ,um, and KC = (-2.06 + 6.95/AR) min * ym/yL, where AR = H/W > 1, with H the height of the cross section of the HARC microfluidic channel in ,um.