FIELD OF THE INVENTION

A first aspect of the present invention is related to a microfluidic device for exchanging a liquid medium in a mixture of liquid and particles. The device being responsible for the exchange of fluids by replacing a first fluid by a second fluid.

The present invention, a second aspect of the present invention is related to a microfluidic device for selecting particles within a range of diameters in a mixture of liquid and particles according to the diameter of the particles.

Then, a third aspect of the present invention is related to a microfluidic device for selecting a range of particles in a mixture of liquid and particles according to the electrical phenotype.

Finally, a fourth aspect of the present invention is related to a microfluidic system for selecting a range of particles in a mixture of liquid and particles according to the size and electrical phenotype of the particles.

PRIOR ART

One of the technical fields with a more intensive development is the detection, isolation and characterization of Circulating Tumor Cells, also called CTCs, in a cancer patient's peripheral blood, which would provide early detection of cancer, information to investigate the metastasis process and helping to detect cancers that are normally hard to detect. The presence of the metastasis process is relevant to the diagnosis and prognosis of a patient. Furthermore, the analysis of the CTCs, which carry the molecular and genetic fingerprint of the primary tumor, could also help defining personalized treatments.

Although CTCs are one detectable element in liquid biopsy, and their numbers and characteristics have hypothesized to be related to the tumor stage and prognosis, CTCs isolation is a challenging task because of their low numbers. More generally speaking, cancer is one of the most important cause of mortality in the world today. Additionally, its incidence continues rising while most of cancer deaths are associated with the growth of metastasis of vital organs. The cells of the original tumor, also called primary tumor, are separated from it and then migrate through the connective tissue until reaching and penetrating into blood vessels. This ultimate step is also called intravasation. While navigating though the bloodstream, these cells migrate to distant body parts and, under favorable conditions, CTCs leave the bloodstream, or so called extravasation, colonizing other non-affected areas and organs which ends up creating secondary tumors.

A tool for detecting, isolating and characterizing CTCs is especially important in some tumor types, whose complex detection implies that patients are normally diagnosed in advanced stages of the disease and where the common available treatments are not the most effective. Known treatments for now are independent steps since the detection step, the isolating step and the characterizing step can be performed separately.

As detection method, the biological methods detect the CTCs based on a characteristic antigen binding to a specific protein presents on the surface of CTCs, commonly by monoclonal antibodies for positive selection. One of the most common biological technique is based on magnetic nanoparticle separation. Alternatively, other biological methods use antigen binding to normal cellular elements of the blood, mostly leucocytes, for negative selection.

Another known detection method is based on physical properties. One example, such as microfiltration, consists in performing a filtration which manage to capture the CTC following its size and allowing sensitive and particular isolation of CTCs of the human whole blood in a few minutes. The blood is drawn and processed into an isolation device in order to capture the CTCs. Then, the captured CTCs are being characterized and finally separated based on their size and deformability. However, the filtration process of this method is known for either losing or damage the cells.

Finally, hybrid methods of detection are performed and combine physical and antibody separation, for example by gradients or applying a magnetic field, with the detection of CTCs by negative or positive selection which consists on the retrieval of all blood cells (negative separation) or CTCs (positive separation) by using a panel of antibodies as well as traditional gradient centrifugation. Following the type of cancer that has been detected, the method performed can be adapted.

The existing standard for isolation of CTCs, known as Veridex's CellSearch®, shows severe limitations, such as the viability of the detected CTCs being compromised by the use of reagents and only detects subpopulations displaying a specific antibody profile, thus discarding other subpopulations of interest.

Other commercial CTC isolation systems based on the physical properties of the cells have been developed, such as ClearCell® FX1 system or VTX-1 Liquid Biopsy system. These systems have detection limits set at 14 microns and 13 microns respectively, which neglects subpopulations made of small CTCs, in the order of 9-13 microns, which might be of interest in regards to the diagnosis or prognosis of the disease.

These limitations have led to the development of techniques based on other properties, such as techniques based on the electric phenotype. Available system of the like, that uses said property, does not constitute a definitive solution since the system works from cells enriched with the CellSearch® system or other enrichment methods, thus returning to the above-mentioned limitations.

Therefore, the present invention according to some embodiments provides a plurality of microfluidic devices for CTCs detection, isolation and characterization. Additionally, said microfluidic devices can be precisely designed, characterized and controlled, and require the use of small amounts of biological material providing a more specified populations of CTCs based on their diameter and without neglecting CTCs under 13 microns. Specifically, one aspect of the invention provides the use of a plurality of combination of microfluidic devices sequentially connected in order to enhance the detection and isolation increasing the range of the size of the sorted cells.

DESCRIPTION OF THE INVENTION

The present invention provides a first microfluidic device forexchanging a liquid medium in a mixture of liquid and particles according to claim 1, a second microfluidic device for selecting a range of particles in a mixture of liquid and particles according to the diameter of the particles according to claim 13, a third microfluidic device for selecting a range of particles in a mixture of liquid and particles according to the electrical phenotype of the particles according to claim 20, a microfluidic system for selecting a range of particles in a mixture of liquid and particles according to the diameter of the particles and their electrical phenotype according to claim 26.

First microfluidic device at the opposite side of the first microchannel; at least the first microchannel is a curved microchannel wherein the curvature is such that the second microchannel is in the concave side of the curvature of the first microchannel and the third microchannel is in the convex side of the curvature of the first microchannel, the curvature intended for focusing the particles within the first microchannel.

The present invention provides means for causing a laminar flow within a microfluidic channel under a certain curvature. Said curvature provides to the microfluidic device the ability to progressively exchange a first fluid for a liquid medium while being submitted to forces created by the curvature radio of the device.

The first microfluidic device of the invention comprises a first microfluidic plate. Said plate presents a first inlet port, a second inlet port, a first outlet port and a second outlet port. The first inlet port is configured to introduce a first fluid and particles, also called sample, which is progressively mixed with a liquid medium, also called second fluid, introduced through the second inlet port.

The second fluid introduced through the second inlet port is a new liquid medium in which particles must be surrounded until reaching the first and second outlets of the device. Also, the second outlet is also called waste outlet. Preferably, the waste outlet outputs a mixture of fluid and as less particles as possible in order to exchange as much first liquid as possible without losing particles

Both the first and the second inlet ports are connected to a first microchannel that is connected to a second microchannel and a third microchannel. The curvature of the first microchannel is such that the second microchannel is situated in the concave side of the curvature of said first microchannel and the third microchannel is situated in the convex side of the curvature of said first microchannel. Moreover, the curvature is intended for focusing the particles of the mixture within the convex side of the first microchannel, in the direction of the third microchannel.

The first fluid transports the particles focused at a point along the section of the first microchannel close to the inner wall on the side of the second microchannel. That is, between the point where the particles are focused and the outer wall there is a larger region of the first fluid than on the inner side. It is on this outer side that the first fluid exits through the second intermediate channels preventing particles from exiting through these second intermediate channels.

Likewise, the outflow of the first fluid is compensated by the inflow of the second fluid from the internal side. Since the first microchannel maintains the curvature, the inflow of the first fluid that tends to displace the particles towards the outer side is compensated by refocusing the particles towards the inner side, preventing them from exiting through the second intermediate channels.

Preferably, the first microchannel accommodates a fluid flow between 850 pL/min and 3000 pL/min. More preferably, the fluid flow of the first microchannel is 1000 pL/min in order to ensure the correct functioning of the whole device and avoid damaging both the particles of the mixture and the device itself.

Additionally, the first microchannel is defined by two parameters: its height and its width. In particular, the width of the first microchannel is greater than its height.

The second microchannel is intended for entering the second fluid in operative manner and connects to the first microchannel thanks to a plurality of first intermediate channels. At the same time, the third microchannel is intended for outputting the first fluid in operative manner and connects to the first microchannel thanks to a plurality of second intermediate channels.

Furthermore, the microfluidic plate also comprises a first outlet port which is intended for outputting a new mixture of the second liquid and particles previously introduced through the second inlet port, that is to say, the fluid of the first outlet port is a mixture of the second fluid and the remaining particles which have not been extracted through the second intermediate channels in the operative manner. Preferably, the fluid of the first outlet port is a mixture of the second fluid and all the particles primarily introduced in the device through the first inlet port.

Also, the microfluidic plate comprises a second outlet port, which is connected to the first microchannel thanks to the third microchannel. Said outlet port is intended to outputting the first fluid and at least part of a population of particles. Preferably, the second outlet port is intended to only outputting the first fluid in the preferred example where all the particles are maintained in the mixture with the second fluid. Additionally, the second microchannel and the third microchannel are located at different sides of the first microchannel and, more preferably, the second microchannel is located at one side of the first microchannel and the third microchannel is located at the other side of the first microchannel so that both the second microchannel and the third microchannel do not enter in contact.

Furthermore, the second microchannel is located in the concave side of the curvature of the first microchannel and are both connected thanks to the plurality of first intermediate channels. In the opposite, the third microchannel is located in the convex side of the curvature of the first microchannel and are both connected thanks to the plurality of second intermediate channels.

Additionally, and preferably, the first intermediate channels and the second intermediate channels are rectangular shaped channels.

Also preferably, the first fluid, or sample, is blood or diluted blood presenting a plurality of particles characterized as CTCs.

In a preferred embodiment, said curvature presents a balanced point which is localized towards the third microchannel, that is to say, in the concave side of the curvature of the first microchannel.

In another preferred embodiment, the microfluidic device presents a plurality of balanced points, responsive to the size of each particle, localized all along the interior part of the curvature of the first microchannel. Additionally, the plurality of balanced points provides the ability to send the first fluid and the particles towards the optimal sorting area which is the concave side of the curvature.

In a preferred embodiment, the first outlet port is intended for outputting the second fluid and at least a portion of particles being the second fluid a biological-friendly fluid. More preferably, the first outlet port is intended for outputting the second fluid and the totality of particles.

Additionally, the flow is laminar in order to protect all particles from being damaged. In a preferred embodiment, the first inlet port can be connected to any size of tubing, preferably, the tubing is a 20G tube.

In a preferred embodiment, the radius of the first microchannel is comprised between 3mm and 9mm. More preferably, the radius of the first microchannel is 6mm.

In another preferred embodiment, the section of the first microchannel is a rectangular section of dimensions 300 microns in width by 90 microns in height.

Advantageously, the introduced liquid medium is dielectric in order to further provide polarization to the particles and for these to be adapted for responding to dielectrophoresis.

In a particular embodiment, the second microchannel, the third microchannel or both microchannels, are curved microchannels wherein the convexity of the curved microchannels is the convexity of the first microchannel.

The second microchannel or the third microchannel, or both the second and third microchannels are curved wherein the convexity of the curved microchannel is the convexity of the first microchannel which provides the ability to set a plurality of first intermediate channels between the first and the second microchannel and a plurality of second intermediate channels between the first and the third microchannel.

In a preferred embodiment, the radius of the second microchannel is comprised between 2mm and 8mm, more preferably 5.075mm, and the radius of the third microchannel is comprised between 4 and 10mm, more preferably 7.125mm.

In a particular embodiment, the first intermediate channels merge into the first microchannel with a direction showing an acute merging angle in respect to the direction of said first microchannel, the direction being measured according to the flow direction in each microchannel in operative manner.

The plurality of first intermediate channels are connecting the first microchannel to the second microchannel in order to progressively introduce the second fluid in operative manner. Both fluids, the first fluid in the first microchannel and the second fluid in the second microchannel are in the same vectorial direction which provides pushing forces to the second fluid responsible for the progressive introduction of said second fluid.

Additionally, and with respect to the point of contact of each first intermediate channel with the first microchannel, each first intermediate channel flow direction can be represented by a vector which presents an acute merging angle with respect to the vector representation of the direction of the flow accommodated inside the first microchannel. Thus, the acute angle is responsible for introducing, in an optimal manner, the medium fluid inside the mixture already circulating inside the first microchannel since the main flow in the first microchannel and the merging flow of the first intermediate channels merging the first fluid into the main flow shows the same longitudinal direction; that is, the direction of the flow of the first microchannel preventing a pressure drop.

In a preferred embodiment, the device presents eight first intermediate channels connecting the first microchannel to the second microchannel.

In a particular embodiment, the second intermediate channels departure from the first microchannel with a direction showing an obtuse departure angle in respect to the direction of said first microchannel, the direction being measured according to the flow direction in each microchannel in operative manner.

The plurality of second intermediate channels are connecting the first microchannel to the third microchannel in order to progressively output the mixture made of the first fluid and at most a quantity of particles in operative manner. Preferably, the plurality of second intermediate channels are connecting the first microchannel to the third microchannel in orderto progressively output the mixture made of the first fluid without particles in operative manner.

Additionally, and with respect to the point of contact of each second intermediate channel with the first microchannel, each second intermediate channel flow direction can be represented by a vector which presents an obtuse departure angle regarding the vector representation of the direction of the flow accommodated inside the first microchannel. Thus, the obtuse departure angle of each second intermediate channel is responsible for outputting the first fluid through a flow direction opposite to the flow direction of the first microchannel.

The outflow of the first fluid through the second intermediate channels must be such as to avoid the outflow of particles since these must be preserved in the main flow of the first microchannel. The obtuse angle condition causes the flow through the second intermediate channels to have the longitudinal component of the flow direction with respect to the flow in the first microchannel in the opposite direction causing particles not to enter such second intermediate channels.

In a preferred embodiment, the device presents eight second intermediate channels connecting the first microchannel to the third microchannel.

In a more preferred embodiment, the device presents eight first intermediate channels and eight second intermediate channels.

In a particular embodiment, the first microchannel comprises a first focusing portion, this first focusing portion being curved and located between the first inlet port and the portion wherein the first intermediate channels and the second intermediate channels show a connection with the first microchannel, the first focusing portion intended for focusing the particles within the first microchannel.

The microfluidic device presents a first focusing portion intended for focusing the particles within the microchannel, said first focusing portion preferably presents a plurality of balanced points which are the location of concentration of the particles contained in the first fluid, or also called sample. Also preferably, said location of concentration of particles is the concave side of the curvature of the first microchannel.

In a preferred embodiment, the first focusing portion is a curvilinear loop having a section of 300 microns by 85 microns.

In a particular embodiment, the first focusing portion is configured as a combination of convex and concave portions, preferably in a sinuous form.

The focusing portion is configured as a combination of convex and concave portions in order to increase the ratio of particles sent on the concave side of the first microchannel.

Preferably, the focusing portion is a series of 4 to 12 combinations of convex and concave portions enhancing the efficiency of the device while orienting and selecting particles. Both the concave and convex sections give rise to a focusing effect of the particles, the latter section being the one that establishes towards which wall the focused particles are positioned.

Advantageously, the combination of convex and concave portions of the focusing portion provide a first microfluidic device having a smaller and more compact size.

In a particular embodiment, the combination of convex and concave portions is in a sinuous form and comprises at least four combinations.

In a particular embodiment, for all microchannels, the dimensions according to the crosssection dimensions are the same.

D _h is the hydraulic diameter of the first microchannel, u is the velocity of the fluid,

L is the characteristic length of the first microchannel,

R is the radius of curvature of the first microchannel, p is the density of the fluid, p is the dynamic viscosity of the fluid, is in operative manner in the range [5.6 - 16], more preferably in the range [7.3 - 15], more preferably in the range [8.9 - 14].

The Dean coefficient is linking geometries aspects of the operative manner of the microfluidic device, such as the curvature radius, the height and width of the first microchannel, with aspects of the fluid. In a particular embodiment, the minimum length of the first microchannel is in the range [88 mm - 772 mm], more preferably in the range [91 mm - 681 mm], more preferably in the range [95 mm - 617 mm], more preferably about 324 mm.

In a particular embodiment, the first microchannel is adapted to operate when in operative mode with an inlet particulate Hguidflow Qi introduced through the first inlet port and the second microchannel is adapted to operate when in operative mode with an inlet replacement Hguidflow 0.2 introduced through the second inlet port, wherein the ratio between O1/O2 is less than 2.5, and more preferably less than 1.6, and more preferably less than 1.3, and more preferably less than 1.0, and more preferably less than 0.95, and more preferably less than 0.93, and most preferably less than 0.9.

In a particular embodiment, the fluid flow introduced in the first inlet port in in the range [700 l/min - 1500 l/min], more preferably in the range [900 l/min - 1400 l/min], more preferably in the range [llOOpl/min - 1300pl/min], and more preferably about 1200 .l/min.

Second microfluidic device

In a second inventive aspect, the invention provides a microfluidic device for selecting particles within a range of diameters in a mixture of ligu id and particles according to the diameter of the particles, the device comprising:

- a second microfluidic plate comprising a third inlet port, a third outlet port and a fourth outlet port, the third inlet port intended for entering the mixture of liguid and particles, the third outlet port intended for outputting a new mixture with the selected range of particles and the fourth outlet port intended for outputting a mixture ofliguid and discarded particles, when the device is in operative manner;

- a main microchannel connecting the third inlet port and at least the third outlet port and the fourth outlet port; wherein

- the main microchannel comprises a first splitting portion comprising at least two outlets, a main outlet wherein the main microchannel and the third outlet port are connected and a secondary outlet wherein the main microchannel and the fourth outlet port are connected; - the main microchannel comprises a second focusing portion located upstream of the first splitting portion, this second focusing portion being curved and intended for focusing the particles within the main microchannel; at least the main microchannel and the connections between the main microchannel and the two outlets show a rectangular cross-section wherein the width dimension (w) is parallel to the first microfluidic plate and the height dimension (h) is perpendicular to the first microfluidic plate and wherein the width dimension (w) is greater than the height dimension (h);

- the main microchannel showing a section comprising a first subsection and a second subsection, the first subsection being the section of the microchannel connecting to the third outlet port downstream of the first splitting portion and, the second subsection being the section of the microchannel connecting to the fourth outlet port downstream of the first splitting portion, the first subsection being in the concave side in respect to the curvature of the second focusing portion and, the second subsection being in the convex side in respect to the curvature of the second focusing portion;

- the first subsection and the second subsection having a dividing line between the two subsections, transversal to the plate, located at a predetermined distance (di)from the inner wall of the microchannel located in the concave side;

- the microfluidic device is adapted to operate with a fluid flow in the main microchannel having a predetermined mean velocity (u);

- the predetermined distance (di) being the distance that implements the cut-off separation value for those particles above a predetermined diameter <t>i is in the range [7pm - 15pm], more preferably in the range [7.5pm - 12pm], more preferably in the range [8.5 pm - 9.5 pm], more preferably <t>i about 9 pm; and, wherein the third outlet port is intended for outputting a mixture of liguid and particles with a diameter greater than <t>i.

The second microfluidic device of the invention comprises a second microfluidic plate. Said second plate presents a third inlet port, a third outlet port and a fourth outlet port.

The third inlet port is intended for entering the mixture of a liquid and particles. The third inlet port is connected to the only input of a second focusing portion. Advantageously, any type of tube can be connected to the third inlet port of this second microfluidic device. Preferably, the tube is a 20G tubing. In a preferred embodiment, the liquid of the mixture entering the third inlet port is whole blood, or diluted blood, and the particles of the mixture entering the same third inlet are blood cells.

In a more preferred embodiment, the particles of the mixture entering the third inlet comprises blood cells, and cancerous cells, also called CTCs.

The third outlet port is intended for outputting a new mixture of fluid with a selected range of particles. Said third outlet port is connected to one of the at least two outlets of the first splitting portion in order to link the main microchannel to the third outlet port.

In a preferred embodiment, the selected range of particles is chosen in order to optimally target CTCs contained in the mixture of liquid and particles previously introduced in the third inlet port. Preferably, the range of diameter of selected particles is comprised between [7 pm - 20 pm]. More preferably, all particles presenting a range of diameter from [9 pm - 20 pm] are to be considered in the range of diameter of selected particles.

The fourth outlet port is intended for outputting a mixture with the discarded particles when the device is in an operative manner. Said fourth outlet port is connected to one outlet of the at least two outlets of the first splitting portion in order to link the main microchannel to at least the third outlet port and the fourth outlet port.

In a preferred embodiment, the discarded particles are all particles presenting a diameter lower than 9 pm and previously contained in the mixture introduced in the third inlet port.

The second plate of the second microfluidic device also comprises a main microchannel, which is responsible for connecting the third inlet port with at least the third and fourth outlet ports. Said microchannel comprises the second focusing portion located upstream of the first splitting portion in order to focus the particles within the main microchannel. Preferably, the microchannel of the second focusing portion presents a rectangular section. More preferably, said second focusing portion section presents a width in the range of 200 to 500 microns and a height in the range of 50 to 200 microns. Even more preferably, the second focusing portion presents a rectangular section of 300 microns by 85 microns.

Moreover, the width of the splitting portion section increases. Preferably, the width of the splitting portion section increases up to 500 microns. Said width increases and influences the separation of particles and can be adapted following the type of performed operation.

Additionally, the main microchannel is curved. Therefore, the particles contained in the mixture of liquid and particles are focused in the concave side of the curvature within the main microchannel.

In a preferred embodiment, the particles are focused in a plurality of balanced points located along the concave side of the curvature of the main microchannel.

The main microchannel of the second microfluidic device presents a first and a second subsection where the first subsection is the one connecting to the third outlet port downstream of the first splitting portion and where the second subsection is the one connecting to the fourth outlet port downstream of the first splitting portion. A dividing line divides both subsections.

Advantageously, the first subsection redirects the selected range of particles of the mixture, throughout the third outlet port and previously introduced in the third inlet port. Also advantageously, the second subsection redirects the unwanted particles of the mixture, previously introduced in the third inlet port, towards the fourth outlet port.

According to other embodiments, at the same location of a main microchannel exists two or more channels departing from a main channel resulting in three subsections and two dividing lines.

Also, the first subsection is located in the concave side of the curvature of the second focusing portion of the main microchannel and is proximal to the first splitting portion connected to the third outlet port.

In the contrary, the second subsection is located in the convex side of the curvature of the second focusing portion of the main microchannel and is proximal to the first splitting portion connected to the fourth outlet port.

The first subsection and the second subsection present a dividing line which is transversal to the second plate located at a predetermined distance (d _± ) from the inner wall of the main microchannel located at the concave side. This dividing line is materialized in most of the examples of realization in an edge that establishes the division between two output channels.

Preferably, the third outlet port dimension is a microchannel having a section of 103 microns in width by 85 to 90 microns in height, the fourth outlet is a microchannel having a rectangular section of 410 microns by 85 to 90 microns and the third inlet is a microchannel having a rectangular section which presents the same section as the main microchannel which is a microchannel having a rectangular section of 300microns by 85 to 90 microns.

In addition, the predetermined distance is the distance providing the cut-off separation value for those particles having a diameter (p ₁ greater than the predetermined value. Preferably, the cut-off separation value for those particles above a predetermined diameter (p ₁ is in the range [7 //m - 15 //m], more preferably in the range [7.5 //m - 12 /zm], more preferably in the range [8.5 //m - 9.5 //m], more preferably (p ₁ about 9 //m.

In a preferred embodiment, the particles having diameter greaterthan (p ₁ are unhealthy particles, also called CTCs mixed with remaining blood cells larger than the cut-off diameter.

Hence, the particles having a diameter (p ₁ greater than the predetermined value are localized on the concave side of the curvature of the main microchannel and outputted through the third outlet port.

In another preferred embodiment, the splitting portion presents 2 or more outlet ports. Preferably, the second microfluidic device is able to accommodate a flow rate between 850 pL/min and 3000 pL/min. More preferably, the optimal flow rate in the second microfluidic device is 1150 pL/min.

Advantageously, designing the microfluidic channel of the second microfluidic device following the particles size criteria, replicating the real behavior of the present system through a mathematical model, enhance the performance of selection of particles of the mixture.

Additionally, the present mathematical representation takes into account the pressure equilibrium at the outlet of the device so that the particles of the mixture follow an ideal trajectory implementing lower redirections of the mixture and the particles compared to existing devices.

In a particular embodiment, the second focusing portion shows a spiral shape.

The second focusing portion shows a spiral shape which provides to the second microfluidic device a smaller and more compact size and shape with respect to the already existing solutions.

Also, the spiral shape of the second microfluidic device enhances the ability to concentrate the particles on the concave side of the main microchannel which improve the selection of particles when exiting the first focusing part and entering the first splitting portion.

Additionally, the spiral shape of the second focusing portion helps sorting the particles following their diameter by intending to focus the largest particles of the mixture along the concave side of the main microchannel and the smallest particles of the mixture along the convex side of the main microchannel.

In a particular embodiment, the second focusing portion shows two nested spiral subsections arranged consecutively, a first spiral section portion for transporting the flow from the outer part of the spiral to the inner part of the spiral and a second spiral section portion for transporting the flow from the inner part of the spiral to the outer part of the spiral providing a compact shape of the second focusing portion. The two-nested spiral subsections arranged consecutively provide to the device a more compact shape and enhance its ease of use. Additionally, with this configuration both the input and output are set on the outside facilitating connections to other devices on the main board plane.

In a particular embodiment, the microfluidic device further comprises a fifth outlet port wherein: the first splitting portion comprises further a third outlet located at the opposite side of the outlet connected to the fourth outlet port in respect to the microchannel connected to the third outlet port; the first splitting portion further having a predetermined second distance d ₂ adapted to define a second cut-off value for those particles having a diameter greater than a predetermined value (p ₂ , being (p ₂ > (p _± ; wherein

The microfluidic device of the second inventive aspect further comprises a fifth outlet port and presents a predetermined second distance d ₂ adapted to define a second cutoff value for those particles having a diameter greater than a predetermined value ( 2, being ( 2 > By presenting said second distance d ₂ , the device provides an even more precise way of selecting particles.

D _h is the hydraulic diameter, u is the velocity of the fluid, L is the characteristic length of the first microchannel,

R is the radius of curvature of the first microchannel, p is the density of the fluid, p is the dynamic viscosity of the fluid, is in operative manner in the range [4.6 - 39], more preferably in the range [5.3 - 34], more preferably in the range [5.7- 30].

In a particular embodiment, the minimum length of the second microchannel is in the range [90 mm - 772 mm], more preferably in the range [95 mm - 772 mm], more preferably in the range [101 mm - 697 mm], more preferably in the range [300 mm - 400 mm], more preferably about 324 mm.

In a preferred embodiment, the minimum length of the second microchannel is about 324 mm in order to provide optimal geometric aspect for the device in operative manner.

In a particular embodiment, the fluid flow introduced in the third inlet port in the range [700 l/min - 1500pl/min], more preferably in the range [800pl/min - 1300pl/min], more preferably in the range [860pl/min - 1150pl/min], more preferably in the range [lOOOpl/min - 1150pl/min], and more preferably about HOOpl/min.

Third microfluidic device

In a third inventive aspect, the invention provides a microfluidic device for selecting a range of particles in a mixture of liguid and particles according to the electrical phenotype, the device comprising:

- a third microfluidic plate comprising a fourth inlet port, a fifth inlet port, a sixth outlet port and a seventh outlet port, the fourth inlet port intended for entering the mixture of liguid and particles, the fifth inlet port intended for entering liguid, the sixth outlet port intended for outputting a new mixture with the selected range of particles and the seventh outlet port intended for outputting a mixture with discarded particles, when the device is in operative manner;

- a feeding microchannel connecting the fourth inlet port and at least the sixth outlet port and the seventh outlet port, the feeding microchannel comprising at least a third focusing portion intended for being under the influence of an electrical field; - at least the feeding microchannel and the connections between the feeding microchannel with the inlets and the outlets show a rectangular cross-section wherein the width dimension (w) is parallel to the third microfluidic plate (P3) and the height dimension (h) is perpendicular to the third microfluidic plate (P3), and wherein the width dimension (w) is greater than the height dimension (h);

- at least two electrodes located at both sides of the third focusing portion adapted to cause a transversal alternating electrical field into said third focusing portion;

- a first merging portion wherein the fifth inlet port is connected to the feeding microchannel and adapted to merge the flow inputted through the fifth inlet port into the feeding microchannel at one side of the feeding microchannel to displace the particles to the opposite side and, wherein the fifth inlet port and sixth outlet port are connected to the feeding microchannel at the opposite sides of said feeding microchannel respectively;

- a second splitting portion adapted to split the flow of the feeding microchannel into at least a first flow with the focused particles and into a second flow with at least part of the fluid without the focused particles, the first flow directed to the sixth outlet port and the second flow directed to the seventh outlet port.

The third microfluidic device of the invention comprises a third microfluidic plate. Said plate presents a fourth inlet port and a fifth inlet port, a sixth outlet port and a seventh outlet port. Firstly, the fourth inlet port is intended for entering a mixture of liquid and particles and the fifth inlet port (15) intended for entering liquid. Then, the sixth outlet port is intended for outputting a new mixture of liquid with a selected range of particles. Finally, the seventh outlet port is intended for outputting a mixture of liquid and discarded particles. All four ports, the fourth inlet port, the fifth inlet port, the sixth outlet port and the seventh outlet port are under operative manner.

In a preferred embodiment, the mixture is a fluid comprising particles which are a combination of blood cells, and unhealthy cells, or CTCs. Additionally, the selected range of particles is predetermined in order to target unhealthy cells. Furthermore, the rest of particles refers to the discarded particles which present a particular electrical phenotype.

Then, the third microfluidic plate also comprises a feeding microchannel which connects the fourth inlet port and at least the sixth and seventh outlet ports in order to provide flowing speed to the mixture inside the microfluidic device. Also, the feeding microchannel presents at least a third focusing portion intended for being under the influence of an electrical field. Particularly, it is applied a transversal alternating electrical field to the third focusing portion thanks to at least two electrodes located on both sides of the third focusing portion.

In a preferred embodiment, the feeding microchannel section is getting narrower where the transversal alternating electric field is applied in order to improve the efficiency of the electrodes and enhance the influence of the electric field applied on the particles located inside the feeding microchannel.

Additionally, and preferably, the applied electric field is transversal in order to ensure that the particles are carried at a predetermined and preferred side of the feeding microchannel.

Finally, the third microfluidic plate also presents a second splitting portion adapted to split the flow of the feeding microchannel. Said splitting portion separates the flow in at least a first flow containing the fluid with the focused particles, that is the CTCs, and at least into a second flow with the at least part of the fluid without the focused particles, so called healthy cells. In order to maintain the separation of both the fluid with the focused particles and the fluid without the focused particles, where said fluid comprising the focused particles is directed to the sixth outlet port and said fluid comprising the rest of particles, which are the healthy cells, said second flow is directed to the seventh outlet port.

In a preferred embodiment, the mixture of liquid and particles contained inside the feeding microchannel is a dielectric medium with low conductivity which permits applying low frequencies of electric field and lower the risk of damaging the particles. In addition, the particles are dielectrics in order to be responsive to the electric field and ensure high performance ratio in the particle selection operation.

Advantageously, the third microfluidic device differentiates subpopulations of particles comprised in a mixture of liquid using electrical phenotype and consequently sort the selected range of particles, that is the CTCs, from the rest of particles of the mixture, that is the blood cells, preferably leucocytes, by applying the optimal frequency and voltage through the electrodes. Preferably, the voltage is comprised between [1 V- 100 V], More preferably, the voltage applied is set around 20 V.

Preferably, the medium liquid of the mixture presents conductivity favorable to the appliance of the electrical field and can be adapted in order to enhance performances of the present device.

Then, the second splitting portion of the third microfluidic plate is adapted to split the flow contained in the feeding microchannel into at least a first flow comprising the fluid and the selected particles and at least a second flow comprising at least part of the fluid without the focused particles.

In another preferred embodiment, the focused particles, are CTCs and present an electrical phenotype comprised in a predetermined range.

In the same preferred embodiment, the rest of particles, having a different electrical phenotype than the predetermined range of the focused particles, are healthy cells or leucocytes.

Additionally, the first flow is directed to the sixth outlet port and the second flow is directed to the seventh outlet port. Each of the two flows is directed to a different outlet port in order to ensure the optimal separation of the selected particles from the unselected particles.

Furthermore, and in another preferred embodiment, the mixture is adapted to be a biological support medium for the cells.

In a particular embodiment, the electrodes extend over a portion of the surface parallel to the third microfluidic plate leaving inside the channel a portion without electrodes with a width narrower than the channel width.

Advantageously, this specific geometry aspect provides to the device a higher precision while applying the electric voltage from the electrodes and enhances the performance of selecting particles comprised in the flow of the microchannel. In a particular embodiment, at least part of the inner surface of the microchannel has a passivation layer.

First, second or third microfluidic device

In a particular embodiment of the first and/or second inventive aspect, the confinement ratio a/h, the relationship between the particle diameter and the height of the crosssection of the microchannel is in the range [0.01 - 1], more preferably in the range [0.01 - 0.2], more preferably in the range [0.01 - 0.07].

In a particular embodiment of the first and/or second inventive aspect, the height of the cross-section of the microchannel is in the range [60 pm - 200 pm], more preferably in the range [70 pm - 120 pm], more preferably in the range [80 pm - 90 pm], more preferably about 88 pm.

In the particular embodiment of the third inventive aspect, the height of the cross - section of the microchannel is preferably in the range [20 pm - 200 pm], more preferably in the range [30 pm - 150 pm], more preferably in the range [40 pm - 110 pm], more preferably about 50pm.

In a particular embodiment of the first and/or second inventive aspect, the aspect ratio between the height (h) and the width (w) of the cross-section of the microchannel is in the range [0.2 - 1 ], more preferably in the range [0.2 - 0.7], more preferably in the range [0.25 - 0.35], more preferably about 0.3.

In the particular embodiment of the third inventive aspect, the aspect ratio between the height (h) and the width (w) of the cross-section of the microchannel is preferably in the range [0.001 - 0.8], more preferably in the range [0.01 - 0.4], more preferably in the range [0.05 - 0.25], more preferably about 0.06.

A microfluidic system

In a fourth inventive aspect, the invention provides a microfluidic system for selecting a range of particles in a mixture of liguid and particles according to the electrical phenotype of the particles, the system comprising: a first module comprising a device according to the first inventive aspect of the invention; a second module comprising a device according to the third inventive aspect of the invention; wherein the first outlet port of the device of the first module is connected to the fourth inlet port of the device of the second module; the first inlet port of the device of the first module is intended for entering the mixture ofliguid and particles; the fifth inlet port of the device of the second module is intended for entering liguid; the sixth outlet port of the device of the second module is intended for outputting a mixture with the selected range or particles; the second inlet port of the device of the first module is intended for feeding a replacemen t fluid; the remaining outlet ports are intended for outputting wasted fluids.

The microfluidic system for selecting a range of particles in a mixture of liquid and particles according to the electrical phenotype of the particles is a combination of a first module being an embodiment of the first microfluidic device of the present invention and a second module being an embodiment of the third microfluidic device of the present invention.

In that particular embodiment, the first outlet port of the device of the first module is connected to the fourth inlet port of the device of the second module. The first inlet port of the device of the first module is intended for entering the mixture of liquid and particles, preferably blood sample comprising a combination of healthy and unhealthy cells. Also, the sixth outlet port of the device of the second module is intended for outputting a mixture of fluid and particles within a predetermined selected range of particles following their electrical phenotype.

Then, the second inlet port of the device of the first module is intended for feeding a replacement fluid, preferably conductive, in order to progressively being introduced and mixed with the mixture presents in the microchannel which consequently enhance the reactivity of the particles to an electric field. Finally, the remaining outlet ports are intended for outputting wasted fluids in order to avoid contamination of the mixture with the mixture containing the unselected particles and extract the unwanted particles as soon as possible. The mixture containing the fluid and the particles that are part of the selected range is, preferably, biological friendly.

In addition, in that combination of devices, the first microchannel of the first microfluidic device and the feeding microchannel of the third microfluidic device of the present invention are merged into a one and only microchannel being responsible for the mixture flowing inside the whole microfluidic system from the first inlet port to at least the second, the sixth and the seventh outlet port.

Also in that combination of devices, the selection of particles is conditioned by the electric field applied on a narrowed section of the microchannel, the merged first microchannel and feeding microchannel of the two devices of the invention, which ensures a high attraction of the selected particles.

In a particular embodiment, the microfluidic system further comprises:

- a third module comprising a device according to the first inventive aspect of the invention; wherein the sixth outlet of the device of the second module is connected to the first inlet port of the device of the third module; the second inlet port of the device of the third module is intended for inputting a replacement fluid, the replacement fluid adapted to be compatible with the stability of the particles; the first outlet port of the third module is intended for outputting a mixture with the selected range of particles; the second outlet port is intended for outputting wasted fluid.

The microfluidic system of the present invention further comprises a third module being an embodiment of the first microfluidic device. Therefore, the microfluidic system presents a circuit in series made of a first microfluidic device of the invention coupled with a third microfluidic device of the invention coupled with a new iteration of a first microfluidic device of the invention. In another preferred embodiment, the microfluidic system can present a plurality of the present embodiment mounted in parallel in order to improve the quantity of liquid inputted in the system and lower working time in operative manner.

In that particular embodiment, the sixth outlet of the second module is connected to the first inlet port of the third module. At the same time, the second inlet port of the third module is intended for inputting a replacement fluid which provide stability to the particles contained in the mixture of fluid and particles initially introduced in the first module.

Also, the first outlet port of the third module in intended for outputting a mixture with the selected range of particles and the second outlet port is intended for outputting wasted fluid which contains the fluid with the rest of the mixture with unhealthy particles.

In addition, in that embodiment of combination of devices, the first microchannel of the first microfluidic device, the feeding microchannel of the third microfluidic device and the other first microchannel of the third module, being another embodiment of the first microfluidic device, are merged into a one and only microchannel being responsible for the mixture flowing inside this specific microfluidic system from the first inlet port of the first module to the first outlet port of the third module.

In a particular embodiment, the microfluidic system further comprises:

- a fourth module comprising a device according to the second inventive aspect of the invention; wherein In that embodiment of the microfluidic system, a fourth module is placed before any combination of the previous combinations cited. Therefore, the fourth module, being an embodiment of the second microfluidic device of the present invention, is placed upstream of the first module. Hence, the third outlet of the fourth module is connected to the first inlet port of the first module.

Advantageously, connecting the fourth module upstream to the first module provides to said first module an entry of the mixture made of liquid and particles, preferably a blood sample, flowing through the feeding microchannel of the fourth module and then through the first microchannel of the first module, carrying the mixture of liquid and particles to the following modules of any type of combination previously cited of the whole microfluidic system.

Additionally, the third outlet port and the fifth outlet port of the fourth module are intended for outputting fluids containing the mixture comprising fluid and particles having a diameter greater than a predetermined value, resulting in focusing, preferably, on the particles that have been previously characterized as unhealthy cells, or CTCs and large leucocytes.

In particular, the fifth outlet port of the device of the fourth module is intended for outputting a mixture with a selected range of particles which is an even finer range of selected particles than the previously performed outputting of the mixture.

In a particular embodiment, before at least one inlet port of a device of any of the modules the system comprises a pump for causing a flow in operative manner.

Preferably, before at least one inlet port of a device of any of the modules of the system excepting the modules comprising a device according to the third aspect of the invention, the pump provide a fluid flow introduced in the range [700^1/min - lBOO^I/min], more preferably in the range [800^1/min - 1300^1/min], more preferably in the range [860^1/min - 1150 il/min], more preferably in the range [lOOO^I/min - 1150^1/min], and more preferably about llOO^I/min. In a particular embodiment, one or more modules comprises a plurality of devices in parallel for increasing the total amount of flow being processed.

By mounting a plurality of devices in parallel of any type of embodiment of the system of the present invention, it increases the total of flow processed by the microfluidic system and permit gaining time by lowering processing time.

In a particular embodiment, the particles are cells, preferably circulating tumor cells (CTC).

In a particular embodiment, the replacement fluid adapted to be compatible with the stability of the particles is a fluid adapted to be a biological support medium for the cells.

The replacement fluid is adapted to be compatible with the stability of the cells contained in the mixture and is adapted to be a biological support medium for the cells in order to ensure maintaining the integrity of the cells, and moreover, avoiding these cells to be damaged. In that way, the mixture flowing out through the outlet port of the last module of the combination of the microfluidic system is operational for being reintroduced, and implemented, as a biological friendly fluid.

DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the invention will be seen more clearly from the following detailed description of a preferred embodiment provided only by way of illustrative and non-limiting example in reference to the attached drawings.

Figure 1 A This figure shows an schematic representation of the first microfluidic device according to an example of realization of the invention.

Figure IB This figure depicts the angles measured according to the direction of the flows in the first microfluidic device shown in figure 1A.

Figure 1C This figure depicts a sectional view of the first microchannel identifying the dividing line position depending on the position of the center or curvature. Figure ID This figure depicts the curvature criterion depending on the location of the center of curvature for identifying the convex and the concave side.

Figure 2 This figure shows an schematic representation of the third microfluidic device according to an example of realization the invention.

Figure 3 This figure shows an schematic representation of the second microfluidic device according to an example of realization the invention.

Figure 4 This figure shows an schematic representation of the system comprising embodiments of the first, second and third aspect of the invention sequentially connected.

DETAILED DESCRIPTION OF THE INVENTION

As will be appreciated by one skilled in the art, aspects of the present invention may be embodied as a system, method or computer program product

Some of the figures have been represented without curvature in order to ease the identification of channels and other elements of the realizations of the devices of the invention.

Figure 1A depicts a first microfluidic plate (Pl) of a first microfluidic device for exchanging a liquid medium in a mixture of liquid and particles. The first microfluidic plate (Pl) presents a first inlet port (11) in which it is injected a mixture of liquid and particles. The liquid of the mixture is also called first liquid. Once injected through the first inlet port (11), the mixture circulates inside the device through a first microchannel (CHI).

The first microchannel (CHI) presents a rectangular cross-section wherein the width dimension (w) is parallel to the first microfluidic plate (Pl) and the height dimension (h) is perpendicular to the first microfluidic plate (Pl) and the height dimension (h) is parallel to the direction transverse to the first microfluidic plate (Pl), and wherein the width dimension (w) is greater than the height dimension (h);

Also, said first liquid with particles, previously injected through the first inlet port (11), is flowing inside a first portion of the first microchannel (CHI) of the microfluidic plate (Pl) called the first focusing portion (FP1). Then, the fluid reaches a second portion where a first intermediate channel (Ini) and a second intermediate channel (I n2) present a first connection with the first microchannel (CHI).

Preferably, the first microchannel accommodates a fluid flow between 850 /zl/min and 3000 /zl/min. More preferably, the fluid flow of the first microchannel is 1100 /zl/min in order to ensure the correct functioning of the whole device and avoid damaging both the particles of the mixture and the device itself.

The plurality of first intermediate channels (Ini) are responsible for connecting the first microchannel (CHI) to a second microchannel (CH2). Respectively, the plurality of second intermediate channels (In2) are responsible for connecting the first microchannel (CHI) to a third microchannel (CH3).

In a preferred embodiment, the second microchannel (CH2) and the third microchannel (CH3) are located at different sides of the first microchannel (CHI). In particular, the second microchannel (CH2) is located at one side and the third microchannel (CH3) is located at the opposite side of the first microchannel (CHI);

In the same preferred embodiment, the first microchannel (CHI) is a curved microchannel and its curvature is such that the second microchannel (CH2) is in the concave side of the curvature of the first microchannel (CHI) and the third microchannel (CH3) is in the convex side of the curvature of the first microchannel (CHI).

Additionally, the first focusing portion (FP1) is intended for focusing the particles within the first microchannel (CHI) and, particularly, in the curvature or said first microchannel (CHI). More particularly, the focusing part (FP1) of the first microchannel (CHI) is the concave part of the curvature of the first microchannel (CHI). In a preferred embodiment, the device presents eight first intermediate channels (Ini) and eight second intermediate channels ( I n2).

Also preferably, the convexity of the curvature of the second and third microchannel (CH2, CH3) is the convexity of the first microchannel (CHI).

Then, as shown in Figure 1A, the first microfluidic plate (Pl) presents a second inlet port (12) where it is introduced a second fluid, said second fluid being a liquid medium, preferably low-conductive or dielectric, about to be exchange in the first fluid with particles already circulating inside the first microchannel (CHI). Thanks to the curvature of the device, the second fluid is progressively introduced inside the first microchannel (CHI) from the second microchannel (CH2) and, the first fluid is progressively transferred from the first microchannel (CHI) to the third microchannel (CH3). Once the first fluid is totally exchanged by the second fluid, the mixture presents a fluid made of liquid medium which completely surrounds the particles contained in the mixture.

In particular, and thanks to the plurality of first intermediate channels (Ini), the second fluid is progressively introduced from the second microchannel (CH2) towards the first microchannel (CHI). On the opposite, the plurality of second intermediate channels (In2) is intended for progressively outputting the first fluid towards the third microchannel (CH3) in operative manner.

On one hand, the first microchannel (CHI) and the second microchannel (CH2) are connected to the first outlet port (01) in order to output the new mixture made of the second fluid and particles off the device. On the other hand, the third microchannel (CH3) is connected to the second outlet port (02) in order to output the first fluid which has been replaced from the initial mixture off the device.

In a preferred embodiment, said curvature presents a balanced point which is localized towards the second microchannel (CH2), that is to say, in the concave side of the curvature of the first microchannel (CHI).

In another preferred embodiment, the first microfluidic device presents a plurality of balanced points localized all along the interior part of the curvature of the first microchannel (CHI). Additionally, the plurality of balanced points provides the ability to send the first fluid and particles towards the optimal sorting area which is the concave side of the curvature.

In a preferred embodiment, the first inlet port (11) can be connected to any size of tubing, preferably, the tubing is a 20G tube.

In another preferred embodiment, the section of the first microchannel is a microchannel of dimensions ranging from 80 to 600 microns by 50 to 170 microns.

Figure IB shows a portion of the first microchannel (CHI) where said first microchannel (CHI) is in connection with a first intermediate channel (Ini) and a second intermediate channel (In2). As above mentioned, the first intermediate channel (Ini) is then connected to the second microchannel (CH2, not shown in this figure) and the second intermediate channel (I n2) is then connected to the third microchannel (CH3, not shown in this figure).

Each channel (CHI, Ini, I n2) is represented with an arrow symbolizing the direction of the fluid circulating in each of said channel. Said direction of the fluid can be related to a vector direction. The projection of the vector from the first intermediate channel (Ini) forms an angle ai with respect to the vector representing the direction of the flow inside the first microchannel (CHI). Following the same reasoning, the vector from the second intermediate channel (I n2) forms an angle a? with respect to the vector representing the direction of the flow inside the first microchannel (CHI).

Additionally, the angle ai is an acute angle providing pushing forces to the second fluid for exchanging the first fluid circulating in the first microchannel (CHI). Therefore, said angle ai is also characterized as an acute merging angle since both vectors of the first microchannel (CHI) and the first intermediate channel (Ini) are pushing the fluid flow of the first microchannel (CHI) in the same longitudinal direction of the first microchannel (CHI).

Also, the angle a? is an obtuse angle which characterizes the second intermediate channel (I n2) as outputting progressively the first fluid initially contained in the first microchannel (CHI). Said direction of the flow, and vector, of the second intermediate channel (In2) is oriented counterflow with respect to the flow circulating in the first microchannel (CHI). Therefore, said angle a? is an obtuse departure angle since it is responsible for the outputting of the first fluid from the mixture and avoiding any reversion of the fluid thanks to the effect of the curvature of the device of the first microfluidic plate (Pl).

Figures 1C presents a sectional view of a portion of the first microchannel (CHI) where the first microchannel (CHI) shows a first subsection and a second subsection. The first subsection is the section of the first microchannel (CHI) located in the concave side of said first microchannel (CHI), closer to the center of curvature (CC). The second subsection is the section of the first microchannel (CHI) located in the convex side of said first microchannel (CHI), farer to the center of curvature (CC).

The first subsection and the second subsection present a dividing line which is predetermined by the predetermined distance (d _± ) from the inner wall of the first microchannel (CHI) located in the concave side of said first microchannel (CHI). The dividing line is transversal to the first microfluidic plate (Pl) and the predetermined distance d _± is the distance that implements the cut-off separation value forthe particles above a predetermined diameter (p ₁ (not represented in operative manner).

The first subsection is connected to the third outlet port (03) and is intended for outputting a mixture of liquid and particles larger than (p ₁ . Furthermore, the second subsection is connected to the fourth outlet port (04) and is intended for outputting the rest of the mixture of liquid and particles smaller than (p _± .

Figure ID shows an schematic representation of the curvature of the main microchannel (M) with respect to a center of curvature (CC) where the plurality of arrows correspond to the direction of the flow of a mixture of liquid and particles previously introduced in said main microchannel (M). Also, the main microchannel (M) shows a curvature radius and consequently presents a concave side, located on the interior of the main microchannel (M), and a convex side, located on the exterior of the same main microchannel (M).

Said curvature radius of the main microchannel (M) is intended for focusing the particles of a diameter greater than a predetermined diameter on the concave side of said main microchannel (M). Finally, the main microchannel (M) is divided into two outlets ports (03, 04), the third outlet port (03) and the fourth outlet port (04).

The third outlet port (03) outputs a new mixture with the selected range of particles from the mixture initially introduced in the microfluidic device and, on the opposite, the fourth outlet is intended for outputting a mixture with the discarded particles from the mixture initially introduced in the main microchannel (M).

Then, Figure 2 depicts a third microfluidic plate (P3) of the third microfluidic device of the invention which comprises a first merging portion (MGl). Said first merging portion (MGl) shows a fifth inlet port (15) connected to the fourth inlet port (14) of a feeding microchannel (FM) and adapted to merge the flow inputted through the fifth inlet port (15) into the feeding microchannel (FM) at one side of the feeding microchannel (FM) in order to displace the particles to the opposite side and, wherein the fifth inlet port (15) and sixth outlet port (06) are connected to the feeding microchannel (FM) at the opposite sides of said feeding microchannel (FM) respectively.

Inside the fourth inlet port (14), it is previously introduced a mixture of liquid and particles and the device presents a plurality of outlet ports (06, 07) intended for outputting different mixtures. The merging portion (MGl) and the plurality of outlet ports (06, 07) are connected through a feeding microchannel (FM) having a section which is getting narrower while entering into a third focusing portion (FP3).

The third microfluidic plate (P3) also shows two electrodes (E) located on both sides of the third focusing portion (FP3) which provide the ability to apply a transversal alternating electric field onto said third focusing portion (FP3). These electrodes (E) are both connected to a source of alternating current (S).

Once the electric field has been applied to the feeding microchannel (FM) while in the third focusing portion (FP3), the mixture enters in the second splitting portion (SP2) and is split into at least a first flow with the focused particles, and a second flow with at least part of the fluid without the focused particles.

Particularly, the first flow with the focused particles is directed towards the sixth outlet port (06), the second flow is directed towards the seventh outlet port (07). More particularly, the seventh outlet ports (07) is intended to output the waste part of the mixture being the fluid with discarded particles.

Figure 3 shows an embodiment of the second microfluidic device of the invention for selecting a range of particles in a mixture of liquid and particles having a second microfluidic plate (P2) comprising a third inlet port (13), a third outlet port (03), a fourth outlet port (04), and a fifth outlet port (05).

A main microchannel (M) is connecting the third inlet port (13), where a mixture of liquid and particles is initially introduced, to the plurality of outlet ports (03, 04, 05), which are intended for outputting either a new mixture, preferably through the third outlet port (03) and/or the fifth outlet port (05) with the selected range of particles; and, the fourth outlet port (04) being intended to output the waste part of the mixture being the fluid with discarded particles.

The plurality of outlet ports (03, 04, 05) are located at the end of the main microchannel (M) and branch out in an area called first splitting portion (SP1). In between the third inlet port (13) and the first splitting portion (SP1), the main microchannel (M) presents a second focusing portion (FP2) intended for focusing the particles within the main microchannel (M). Said second focusing portion (FP2) is curved and correspondingly presents a convex side and a concave side as described in Figure ID.

Preferably, the second focusing portion (FP2) shows a spiral shape which provides to the second microfluidic plate (P2) a more compact and smaller size.

Also preferably, the second focusing portion (FP2) shows two nested spiral subsections arranged consecutively, a first spiral section for transporting the flow from the outer part of the spiral to the inner part of the spiral and a second spiral section for transporting the flow from the inner part of the spiral to the outer part of the spiral providing a compact shape of the second focusing portion (FP2).

Additionally, the first splitting portion (SP1) presents a predetermined second distance which is adapted to define a second cut-off value for focusing on those particles having a diameter (p ₂ bigger than a predetermined value outputted throughout the fifth outlet port (05). The second cut-off value provides the ability to sort the focusing particles in two groups, CTCs that present a diameter greater than (p ₂ and CTCs that present a diameter smaller than <p ₂ . Correspondingly, the third outlet port (03) is provided to output the mixture of fluid with said focusing particles being in the range of diameter [ i, ₂ ]- The fourth outlet port (04) outputs those particles having a diameter smaller than (f) ₁ .

Finally, Figure 4 depicts a preferred embodiment of the microfluidic system of the invention for selecting a range of particles in a mixture of liquid and particles according to the diameter of the particles and the electrical phenotype.

Said preferred embodiment of the microfluidic system is a combination of modules (MDl, MD2, MD3, MD4), each module being an embodiment of a microfluidic plate (Pl, P2, P3) of the present invention, that is either the first, the second or the third microfluidic device.

As shown in Figure 4, the present embodiment is a combination of a fourth module (MD4) placed upstream of the microfluidic system, a first module (MDl), a second module (MD2) and a third module (MD3) located downstream of the microfluidic system.

The fourth module (MD4) is an embodiment of the second microfluidic device for selecting particles within a range of diameters in a mixture of liquid and particles according to the diameter of the particles. Said mixture is previously introduced throughout the third inlet port (13) and circulates through the focusing portion (FP2) and the first splitting portion (SP1) where the liquid with selected particles of predetermined diameter is directed towards the third outlet port (03) and the rest of liquid with the discarded particles are directed towards the fourth outlet port (04) According to a more specific configuration there are two ranges of particles, a first range of particles having a diameter within the range [(p _lt (p ₂ ] being outputted by the third outlet port (03) and, the device further comprises the fifth outlet port (05) intended for outputting the second range or particles; that is, those having a diameter greater than (f> ₂ .

In that embodiment of the microfluidic system shown in Figure 4, the microfluidic system then presents a forth module (MD4) which is an embodiment of the second microfluidic device. Furthermore, the third outlet port (03) is connected to the first inlet port (11) of the first module (MDl), e.g. through the intermediary of a driving pump, where, after entering and exiting the first focusing portion (FP1), the liquid with selected particles introduced into the first module (MDl) enters in a second portion where a second liquid is introduced through the second inlet port (12). Said second liquid is introduced inside the second microchannel (CH2) which is connected to the first microchannel (CHI) thanks to a plurality of first intermediate channels (Ini) permitting a progressive introduction of said second liquid inside the first microchannel (CHI).

The second liquid progressively expulses the liquid of the first liquid and replace it. Said first liquid is sent towards the third microchannel (CH3) through a plurality of second intermediate channels (I n2). Then, the third microchannel (CH3), where the liquid of the first fluid is circulating, is directed towards the second outlet port (02). Preferably, the first fluid is blood without particles. On the opposite, the new mixture, made of the second fluid and particles from the first fluid which has been maintained, is directed towards the first outlet port (01) thanks to the first microchannel (CHI)

Then, the first outlet port (01) of the first module (MDl) is connected to the second module (MD2) which is an embodiment of the third microfluidic device and where the first outlet port (01) of the first module (MDl) is directed towards the fourth inlet port (14) of the second module (MD2), e.g. through the intermediary of a driving pump. Once the mixture of fluid with selected particles is introduced into the fourth inlet port (14), it enters in the first merging portion (MG1) where it merges with the flow of fresh liquid, preferably fresh second fluid, inputted through the fifth inlet port (15) into the main microchannel at one side of said main microchannel in order to displace the particles to the opposite side of the channel.

Once exiting the first merging portion (MG1), the mixture of fluid and particles enters in the third focusing portion (FP3) where the particles are submitted to an electric field thanks to a plurality of electrodes (E) applied on the external surface of the main microchannel. Said electric field permits selecting a specific range of particles thanks to the electrical phenotype. By applying said electric field and due to the polarization of the particles, which as considered dielectric and therefore polarizable when subjected to an electric field, part of the mixture made of liquid and the selected particles are directed towards the sixth outlet port (06). On the opposite side, the rest of the mixture made of liquid and non-selected particles is directed towards the seventh outlet port (07). Finally, the mixture exiting the second module (MD2) is introduced inside a third module (MD3) which is another iteration of an embodiment of the first microfluidic device. The sixth outlet port (06) of the second module (MD2) is connected to the first inlet port (11) of the third module (MD3). Said third module (MD3) proceed to another exchange of fluid replacing the fluid from the introduced mixture coming from the first inlet port (11) of the third module (MD3) by the fluid introduced throughout the second inlet port (12) of the third module (MD3).

As repeating the same steps, the final mixture made of the fluid introduced throughout the second inlet port (12) of the third module (MD3) and selected particles is directed towards the first outlet port (01). On the opposite, the replaced fluid is directed towards the second outlet port (02).

In that embodiment, the plurality of inlet ports (13, 11, 14, 11) are responsible for introducing the mixture into respectively the fourth module (MD4), the first module (MDl), the second module (MD2) and the third module (MD3) respectively. Then, the plurality of outlet ports (03, 01, 05, 06) are responsible for outputting the mixture made of liquid with selected particles. These outlet ports (03, 01, 05, 06) direct said mixture towards the next module of the microfluidic system respectively to the first module (MDl), the second module (MD2) and the third module (MD3). The first outlet port (01) is responsible for outputting the final mixture made of liquid with selected particles.

Moreover, the remaining outlet ports (04, 02, 07, 02) of the whole microfluidic system are intended for outputting wasted fluids.

In another preferred embodiment, the microfluidic system comprises a plurality of series of devices, such as depicted in Figure 4, mounted in parallel for increasing the total amount of flow being processed.

Preferably, the particles are cells, preferably circulating tumor cells or CTC and blood cells. Also preferably, the replacement fluids of both the first and the third modules (MDl, MD3) are adapted to be compatible with the stability of the particles and are fluids adapted to be a biological support medium for the cells.