ES2885001T3 - Microfluidic system for particle isolation - Google Patents

Abstract

A microfluidic system for the isolation of particles (C1) of at least one given type of a sample, the system (1) comprises: a first inlet (2), through which, during use, the sample is introduced into the system (1); a separation unit (3), which is connected to the first inlet (2), comprises a main chamber (4) and a recovery chamber (5) and is designed to transfer at least part of the particles (C1) from the given type from the main chamber (4) to the recovery chamber (5) in a substantially selective manner with respect to more particles (C2) of the sample; a first outlet (7), connected to the main chamber (4); and a second outlet (8), which is connected to the recovery chamber (5), and, in particular, through which, during use, at least part of the particles (C1) of the given type collected in the recovery chamber (5) leave the system (1); the system (1) is characterized in that it comprises: a first valve (9), arranged upstream of the main chamber (4); that is, between the first entrance (2) and the main chamber (4); a second valve (10), placed between the main chamber (4) and the first outlet (7); a third valve (11), which is connected to the recovery chamber (5) and is placed between the recovery chamber (5) and a source of a carrier liquid; and a fourth valve (12), placed between the recovery chamber (5) and the second outlet (8); the recovery chamber (5) is placed between the main chamber (4) on one side and the third and fourth valves (11, 12) on the other side; the main chamber (4) is placed between the recovery chamber (5) on one side and the first and second valves (9, 10) on the other side.

Description

DESCRIPTION

Microfluidic system for particle isolation

technical field

The present invention relates to a microfluidic system for the isolation of particles.

Antecedent technique

A device for the isolation of particles of a given type is known in the art, which comprises a main chamber, in which an operator, using a pipette, introduces a sample through an orifice of the main chamber, and a chamber from which the operator, again using a pipette, draws particles of the given type through an orifice of the recovery chamber.

EP1179585 discloses a device for separating nucleic acids from a sample. The device comprises a separation chamber containing microstructures designed, in a first step, to block nucleic acids and, in a second step (when the type of eluant is changed, which is passed through the chamber), to release nucleic acids allowing their transport to a reaction chamber. The device described in document EP1179585 is relatively complex since it needs, among other things, a large number of valves and reservoirs to contain various eluents and reagents. Document US2003073110 discloses a device for the separation of silica particles by means of a 7-Ml trap. Also in this case, the device is relatively complex.

US2006177815 discloses methods, devices and systems that use dielectrophoretic forces to separate a target species from a plurality of species in a sample.

US2003044832 discloses a microflow system for separating particles, comprising a microfabricated member having a flow channel defined therein for guiding a flow of a fluid containing the particles through the flow channel, inlet means positioned at one end of the flow channel for entering fluid into the flow channel, outlet means positioned at the other end of the flow channel for discharging fluid from the flow channel. Document US2007284254 discloses an apparatus that separates a polarizable analyte using dielectrophoresis that includes a container that includes a membrane that has a plurality of pores of nanometric to micrometric size, the membrane disposed inside the container, electrodes that generate spatially non-uniform electric fields in the nanometer to micrometer sized pores of the membrane when an AC voltage is applied to the electrodes, and a power supply that applies the AC voltage to the electrodes, wherein a cross-sectional area of the pores varies along a pore depth.

US2005196855 discloses a cartridge including a separate storage component and transport component. The storage component may be coupled to the transport component. The storage component includes one or more receptacles each containing a solution to be used in an assay. The transport component is configured to transport the solutions from the storage component receptacles to a sensor placed in the transport component.

Known devices (as well as those noted above) have various accuracy problems during the separation, introduction and retrieval steps. In addition, the results are not always reproducible, contamination of the sample sometimes occurs during the various steps and the intervention of operators with particular manual skills is often necessary.

Disclosure of the invention

The aim of the present invention is to provide a microfluidic system for the isolation of particles, which will allow the drawbacks of the current technique to be overcome, at least partially, and at the same time will be easy and economically advantageous to produce.

In accordance with the present invention, there is provided a microfluidic system as specified in the following independent claims and, preferably, in any of the claims which depend directly or indirectly on the independent claims. Unless explicitly stated otherwise, in this text the following terms have the meaning indicated below.

By "equivalent diameter" of a cross section is meant the diameter of a circle having the same area as the cross section. By "cross section" of a channel or of a duct is meant the section substantially perpendicular to the longitudinal extension of the channel (or duct), that is, to the direction of advance of the fluid in the channel (or duct).

By "equivalent diameter" of a hole is meant the diameter of a circle having the same area as the section cross section of smaller dimensions of the hole itself.

By "microfluidic system (or device)" is meant a system (or device) comprising at least one microfluidic channel (or conduit).

By "microfluidic channel (or conduit)" is meant a channel (or conduit) having a cross section with an equivalent diameter of less than 1 mm.

The dimensions of the channels or ducts can be measured in a standard way using profilometers.

In the present text, "particle" is understood to mean a corpuscle whose greatest dimension is less than 500 mm (advantageously less than 150 mm). Non-limiting examples of particles are: cells, cell debris (particularly cell fragments), cell aggregates (such as, for example, small groups of cells derived from stem cells, such as neurospheres or mammospheres), bacteria, lipospheres, microspheres (polystyrene and/or magnetic), and complex nanospheres (for example, nanospheres up to 100 nm) made up of cell-bound microspheres. Advantageously, the particles are cells.

According to some embodiments, the particles (advantageously cells and/or cell debris) have a largest dimension of less than 60 mm.

Particle dimensions can be measured in a standard way using graduated scale microscopes or normal microscopes with graduated scale slides (on which the particles are deposited).

In the present text, by "dimensions" of a particle is meant the length, width and thickness of the particle.

The term "substantially selective" is used to identify a displacement (or other analogous terms indicating a movement and/or a separation) of particles, in which the particles that are displaced and/or separated are particles for the great majority of one or more given types. Advantageously, a substantially selective displacement (or other analogous terms indicating a movement and/or a separation) foresees the displacement of particles with at least 90% (advantageously 95%) of particles of the given type or types (percentage given by the number of particles of the given type or types with respect to the total number of particles).

Brief description of the drawings

The invention is described hereinafter with reference to the attached drawings, which illustrate some non-limiting examples of embodiments thereof and in which:

Figure 1 is a schematic illustration of a system constructed in accordance with the present invention; Figure 2 is a schematic illustration of an alternate embodiment of a system constructed in accordance with the present invention;

Figure 3 is a schematic side view of the system of Figure 1;

Figure 4 is an exploded perspective view of a device that is an example useful for understanding the present invention;

Figure 5 is a top plan view of a component of the device of Figure 4;

Figure 6 is a bottom plan view of the component of Figure 5;

Figure 7 is a perspective view of the components of the device of Figure 4;

Figure 8 is an exploded perspective view of a component of the device of Figure 4;

Figure 9 is a top plan view of the component of Figure 8;

Figure 10 is a partial perspective view with elements removed for clarity of an apparatus that is an example useful in understanding the present invention;

Figure 11 is a partial perspective view with elements removed for clarity of the apparatus of Figure 10 in a different operating position;

Figure 12 is a bottom perspective view of a detail of the apparatus of Figures 10 and 11;

Figure 13 is a top plan view of a portion of the apparatus of Figures 10 and 11, with elements removed for clarity;

Figures 14 and 15 illustrate in partial cross-section a detail of the systems of Figures 1 to 3 in two different operating positions;

Figure 16 illustrates in partial cross-section a detail of the systems of Figures 1 to 3;

Figure 17 is a perspective view of a part of the detail illustrated in Figures 14 and 15;

Figure 18 is an exploded perspective view of the detail of Figure 17;

Figures 19 and 20 illustrate a detail of the Figure 4 device in various operating steps;

Figure 21 is a top plan view of a device of Figure 4;

Figure 22 illustrates photographs of tests carried out using the system of Figure 1;

Figure 23 illustrates on an enlarged scale an embodiment of a detail of Figures 1 and 2;

Figure 24 is a perspective view of a variant of the part illustrated in Figure 17;

Figure 25 is an exploded perspective view of the variant of Figure 24;

Figure 26 is a side cross-sectional view of the variant of Figure 24;

Figures 27 and 28 illustrate a detail of the device of Figure 31 in various operating steps;

Figure 29 is an exploded perspective view of a component of the device of Figure 31;

Figure 30 is a top plan view of the component of Figure 29;

Figure 31 is a top plan view of a device that is an example useful for understanding the present invention;

Figure 32 is a bottom plan view of a variant of the component of Figure 6 and, in particular, a component of the device of Figure 31; Y

Figure 33 illustrates a detail of Figure 32 on an enlarged scale.

Best mode for carrying out the invention.

microfluidic system

According to a first aspect of the present invention, a microfluidic system 1 is provided for the substantial isolation of particles C1 (schematically illustrated in Figure 20) of at least one given type of a sample. System 1 (figure 1) comprises: an inlet 2, through which, during use, the sample is introduced into system 1; a separation unit 3, which is designed to substantially selectively separate at least a part of the C1 particles of the given type from other C2 particles in the sample (schematically illustrated in Fig. 20); and an outlet 8, which is connected to the separation unit 3 and, in particular, through which, during use, at least part of the substantially selectively separated particles C1 of the given type exit the system 1. Separation unit 3 is connected to input 2.

System 1 comprises: a valve 9, arranged between inlet 2 and separation unit 3 (in particular, between inlet 2 and main chamber 4); and a valve 12, placed between the outlet 8 and the separation unit 3. In particular, the system 1 comprises a valve placed between the separation unit 3 and each opening of the system 1 to the outside.

The separation unit 3 comprises a main chamber 4 and a recovery chamber 5 and is designed to transfer at least part of the C1 particles of the given type from the main chamber 4 to the recovery chamber 5 in a substantially selective manner with respect to the other C2 particles in the sample.

The separation unit 3 further comprises a channel 5, which connects (ie allows the passage of fluid between) the chambers 4 and 5 and has dimensions (in particular, width and length) much smaller than those of both chambers 4 and 5. 5 itself.

System 1 is equipped with: an outlet 7, connected to the main chamber 4 to allow the sample to freely enter the main chamber 4, thus functioning as a vent; and the outlet 8, which is connected to the recovery chamber 5 and through which, during use, at least part of the particles C1 of the given type collected in the recovery chamber 5 leave the system 1.

System 1 further comprises: valve 9, placed upstream of main chamber 4; a valve 10, placed between the main chamber 4 and the outlet 7; a valve 11, connected to the recovery chamber 5; and valve 12, placed between recovery chamber 5 and outlet 8. Valve 11 is placed between recovery chamber 5 and a source of a carrier liquid.

More precisely, the recovery chamber 5 is placed between the main chamber 4 on one side and the valves 11 and 12 on the other side; main chamber 4 is placed between recovery chamber 5 on one side and valves 9 and 10 on the other side.

Valves 9 and 10 are designed to regulate the inlet flow of the sample to the main chamber 4.

Valves 11 and 12 are designed to regulate the inflow of a carrier liquid to the recovery chamber 5 and the outflow of the carrier liquid together with C1 particles of the given type from the recovery chamber 5 through the outlet 8.

If, during use, the valves 9 and 12 are closed and the valves 11 and 10 are open, a cleaning of the main chamber 4 is carried out; in other words, the other particles C2 of the sample are discharged (i.e. forced out) out of the main chamber 4.

According to specific embodiments, the carrier liquid is a buffer solution, in particular, a phosphate buffered saline (PBS) solution.

According to the embodiment illustrated in Figure 1, the system 1 comprises: a reservoir 13, which is placed between the inlet 2 and the valve 9 and is designed to collect the sample introduced through the inlet 1 itself; and a reservoir 14 for containing the carrier liquid, which is designed to fill the recovery chamber 5. In other words, the reservoir 14 functions as a source of the carrier liquid. System 1 also comprises a reservoir 7', which is placed at outlet 7 and is designed to collect the fluids coming from the main chamber 4.

The valve 9 is placed between the inlet 2 and the main chamber 4, in particular, between the reservoir 13 and the main chamber 4, and is designed to connect or isolate the inlet 2 and the main chamber 4 with respect to each other.

Advantageously, system 1 further comprises a pressure source 15 for imposing a pressure difference between reservoir 13 and main chamber 5 in a given pressure supply direction. In particular, the pressure source 15 is designed to impose a pressure from the reservoir 13 towards the main chamber 5 in a given pressure supply direction. According to some embodiments, the reservoir 13 is placed between the pressure source 15 and the valve 9.

In particular, the reservoir 13 is placed between the pressure source 15 and the main chamber 4.

According to specific embodiments, the system 1 comprises a conduit 16, which connects (ie allows the passage of fluid between) the pressure source 15 and the main chamber 4 and along which the reservoir 13 and the valve are placed. 9. Conduit 16 has an equivalent diameter cross section of less than or equal to 2mm; Advantageously, the conduit 16 has a cross section with an equivalent diameter greater than or equal to 50 mm. The conduit 16 comprises at least one section, which has a cross section with an equivalent diameter of less than or equal to 0.9 mm.

The system 1 further comprises a vibration device 17, which is designed to cause a variation in an oscillating form of the pressure exerted on the sample at least in an area from the entrance 2 to the main chamber 4 and is placed between the source of pressure 15 and reservoir 13. In this way, the sample particles C1 and C2 present in reservoir 13 and/or in conduit 16 and/or in main chamber 4 are made to vibrate; the inflow of sample particles C1 and c 2 from reservoir 13 to main chamber 4 is improved. Particles C1 and C2 have less tendency to clump together or adhere to the walls of reservoir 13 and/or conduit 16 and/or of the main chamber 4. The homogeneity of the distribution of the C1 and C2 particles inside the main chamber 4 is improved.

Advantageously, the vibration device 17 comprises an oscillating diaphragm 18, connected, in particular, by means of a T-joint 19 to the conduit 16. According to specific embodiments, the oscillating diaphragm is a micropump (Thinxxs® MDP2205) with input and output shorted.

According to some embodiments (not illustrated in figure 1), the inlet 2 is placed along the conduit 16, between the pressure source 15 (in particular, the joint 19) and the reservoir 13.

According to some embodiments, the system 1 comprises a conduit 20, which is placed between the main chamber 4 and the outlet 7 and has at least one section 20' that has a smaller cross section than the cross section of the conduit 16. In particular , the cross section of the section 20' is smaller by at least 100 mm than the cross section of the duct 16. Advantageously, the section 20' is placed immediately downstream (that is, without the interposition of additional sections or elements) of the main camera 4.

The section 20' has a width of less than 150 mm, a depth of less than 110 mm and a length of greater than 2 mm. Advantageously, the section 20' has a width greater than 100 mm, a depth greater than 30 mm and, in particular, a length of less than 6 mm.

According to some embodiments, the conduit 20 comprises a section 20", which is placed between the section 20' and the outlet 7 and has a cross section of equivalent diameter greater than that of the section 20' (in particular, substantially equal to the of duct 16).

In general, according to different embodiments, the system 1 comprises: a sensor to directly or indirectly detect when the sample starts to enter the conduit 20; a blocking device for blocking the inflow of the sample into the main chamber; and a control assembly connected to the sensor and the lockout device to actuate the lockout device as detected by the sensor (in particular, when sample begins to enter conduit 20).

According to some specific embodiments, the system 1 comprises: a pressure sensor 21 to detect pressure variations along the duct 16 and/or in the main chamber 4; and a blocking device 22 to block the inlet flow of the sample towards the main chamber 4. System 1 further comprises a control assembly 23 connected to the pressure sensor 21 and to the blocking device 22 to actuate the blocking device 22 depending on the pressure variation detected. In particular, during use, control assembly 23 actuates locking device 22 when pressure sensor 21 detects a pressure greater than a given threshold pressure.

According to other specific embodiments, the system 1 comprises, in addition to or as an alternative to the pressure sensor 21, one or more of the following elements: an optical sensor, placed at the entrance of the section 20'; an electrical conductivity detector, which is placed at the entrance of the section 20' to detect variations in conductivity due to the beginning of the passage of the sample; an electrical permittivity detector, which is placed at the entrance of the section 20' to detect permittivity variations due to the beginning of the passage of the sample; a thermal resistance detector, which is placed on the input of section 20' to detect variations in thermal resistance due to the start of the passage of the sample; and a thermal capacity detector, which is placed at the entrance of the section 20' to detect variations in thermal capacity due to the beginning of the passage of the sample. In all of these cases, the control assembly 23 is connected to the detector/detectors and is designed to actuate the locking device 20 based on variations detected by the detector/detectors. According to some embodiments, the optical sensor is an integrated photodiode or also an external video camera (possibly with an image magnification system).

With particular reference to figure 23, the blocking device 22 comprises a relief valve 22' and, advantageously, it is placed between the pressure source 15 and the reservoir 13; when the blocking device 22 is actuated, the relief valve 22' opens, and the jet of air from the pressure source 15 is released outside, and the pressure along the duct 16 drops. Relief valve 22' is hydraulically connected to conduit 16 by means of a T-joint 22”. In particular, the blocking device 22 is placed between the gasket 19 and the pressure source 15, specifically upstream of the pressure sensor 21 with respect to the direction of pressure supply to the main chamber 4.

From the above, it can be inferred that the introduction of the sample into the separation unit 3 is improved, thus reducing the risk that a significant part of the sample passes beyond the separation unit 3 itself. According to alternative embodiments , the blocking device 22 is not present, the blocking function being performed by the valve 9, which is connected to the control assembly 23. In other words, the control assembly 23, during its use, closes the valve 9 when the pressure sensor 21 detects a pressure greater than the given threshold pressure.

Advantageously, control assembly 23 is connected to valves 9, 10, 11 and 12.

The connection between the control assembly and other elements (devices, valve units, etc.) can be made by traditional electrical connections (conductive cables) or by electromagnetic waves (for example, by radio waves, microwaves, etc.).

According to some embodiments, system 1 comprises an inlet 24. Reservoir 14 is placed between inlet 24 and valve 11 and is designed to collect carrier liquid introduced through inlet 24.

Valve 11 is placed between inlet 24 and recovery chamber 5 and is designed to connect or isolate inlet 24 and recovery chamber 5 from each other. In particular, valve 11 is placed between reservoir 14 and recovery chamber 5 and is designed to connect or isolate reservoir 14 and recovery chamber 5 from each other.

According to some embodiments, the system 1 comprises a pressure source 25 to impose a pressure difference between the reservoir 14 and the recovery chamber 5. In particular, the pressure source 25 imposes a pressure from the reservoir 14 towards the recovery chamber. recovery 5.

Advantageously, the reservoir 14 is placed between the pressure source 25 and the valve 11. In particular, the system 1 comprises a conduit 26, which connects (that is, allows the passage of fluid between) the pressure source 25 and the recovery chamber 5 and along which the reservoir 14 and the valve 13 are placed.

According to some embodiments, inlet 24 is placed along conduit 26, in particular between pressure source 25 and reservoir 14.

With particular reference to figure 3, the system 1 comprises a vibration device 17a, which is similar to the vibration device 17 and is designed to cause a variation in an oscillating form of pressure in at least one area of the recovery chamber. 5. In this way, the C1 particles of the given type present inside the recovery chamber 5 are made to vibrate, and the outflow of the C1 particles of the given type itself from the recovery chamber 5 to the outlet is enhanced. The C1 particles have a reduced tendency to clump together or stick to the walls of the recovery chamber 5 and/or a conduit 27 connecting (i.e. allowing fluid passage between) the recovery chamber 5 to the outlet 8. The valve 12 is placed between the recovery chamber 5 and the outlet 8. Advantageously, the vibration device 17a comprises a diaphragm pump 18a connected, in particular by means of a T-joint 19a, to the conduit 26.

According to embodiments (not illustrated), the system for filling chamber 5 is similar to that of chamber 4. Therefore, in such cases, a blocking device (not illustrated) is placed between the pressure source 25 and the reservoir. 14; on the other hand, one or more sensors and/or detectors (not illustrated) analogous to those described above with reference to the main chamber 4 are arranged at the entrance of the duct 27 or in other appropriate positions.

According to embodiments (not illustrated), the system 1 comprises a detector (in particular, an optical or impedentiometric or ultrasound detector), which is placed on the output 8, is connected to the control assembly 23, and is designed to detect a liquid exiting through outlet 8. Control assembly 23 is designed to regulate the opening of the valve 11 and/or 12 depending on what is detected by the optical sensor. In particular, during use, when the optical sensor detects at least one drop of carrier liquid (in which at least part of the C1 particles of the given type is present), the control assembly 23 actuates a relief valve, which puts drive pressure to zero, thus blocking the flow of liquid.

In addition or alternatively, when the optical sensor detects at least one drop of carrier liquid (in which at least part of the C1 particles of the given type is present), the control assembly 23 closes the valve 12 and/or the valve 11 .

In this way, it is possible to obtain the C1 particles in a very small volume of liquid. This facilitates subsequent analysis steps.

Figure 2 illustrates an embodiment of the system 1 which differs from the system 1 of figure 1 by the position of the duct 26 with respect to the recovery chamber 5 and in that it comprises a duct 28, which connects the recovery chamber 5 to the outlet 7 (or to another outlet, not illustrated) and along which is placed a valve 29, connected to control assembly 23. Conduit 26 connects to recovery chamber 5 between conduits 27 and 28. In particular, conduit 26 connects to recovery chamber 5 substantially in front of channel 6.

With particular reference to figures 14 and 15, according to some embodiments, at least one of the valves 9, 10, 11 and 12 (in particular, each valve 9, 10, 11 and 12) has a particular structure described below. below with reference to a particular valve V (in other words, one or more of valves 9, 10, 11 and 12 have the structure of valve V described below).

In accordance with one aspect of the present invention, a valve V is provided.

The valve V is equipped with a closing element 30, which comprises (in particular, is made of) a substantially elastic material, and is designed to pass between a blocking position (illustrated in figure 14), in which the element closing element 30 separates the two sections 31 and 32 of a respective conduit, and an open position (illustrated in figure 15), in which the closing element 30 is placed in such a way as to allow the passage of fluid between the sections 31 and 32.

Valve V comprises a diaphragm 33, which is placed between the two sections 31 and 32. When the closing element 30 is put in the blocking position, the closing element 30 itself is in contact with the diaphragm 33 to separate the sections 31 and 32. When the closing element 30 is placed in the open position, the closing element 30 itself is placed at a distance from the diaphragm 33 in order to allow the passage of fluid between the sections 31 and 32.

The valve V also comprises a respective mechanical pressure element 34, which pushes the closing element 30 towards the sections 31 and 32 (in particular, towards the diaphragm 33) to keep the closing element 30 itself in the blocking position. Advantageously, the mechanical pressure element 34 comprises (in particular, is) a spring, which is compressed when the closing element 34 passes from the blocking position to the opening position and extends when the closing element 34 passes. from the open position to the locked position.

The valve V comprises a fluid dynamic actuator 35, which in turn comprises an actuator nozzle 36 equipped with a hollow element 37, which houses the mechanical pressure element 34. The hollow element 37 has an internal channel 38 and an open end (in particular, provided with an actuator hole 39) placed in contact with the closing element 34.

The fluid dynamic actuator 35 comprises a sealing element 40, which is designed to slide fluid-tight along the internal channel 38 and is brought into a position corresponding to the actuator hole 39 in contact with the mechanical pressure element.

The fluid dynamic actuator 35 further comprises a suction unit 41, which, in turn, comprises a conduit 42 connecting the actuator nozzle 36 to a suction source 43 (illustrated in Figure 3).

During use, when the suction unit 41 is operated, the sealing element 30 is sucked in and pushes the sealing element 40, which slides inside the internal channel 38 to compress the mechanical pressure element 34. Said negative pressure pushes the closing element 30 of the diaphragm 33 in such a way that the closing element 30 reaches the open position. When the suction unit is deactivated, the mechanical pressure element 34 pushes the sealing element 40 out through the actuator hole 39. The sealing element 40 in turn pushes the closing element 30 against the diaphragm 33 of such that the closing element 30 itself reaches the locking position.

The closure element 30 (also illustrated in Figure 7) has a membrane portion 44 and a projection 45 that extends along a perimeter edge of the membrane portion 44. In other words, the closure element 30 has a greater thickness at the perimeter edge. This allows to improve the mechanical strength of the membrane and the fluid tightness between the closing element 30 and the actuator nozzle 36 (in particular, the hollow element 37). The projection 45 is annular in shape.

According to specific embodiments, the membrane portion 44 has a substantially cylindrical shape; in this case, the projection 45 has the shape of a circular ring.

According to some embodiments, the closing element 30 is made up of a single elastomeric material (ie an elastomer) or also of a combination (eg a mixture) of several elastomeric materials different from each other.

Advantageously, the elastomer comprises (in particular consists of) a silicone, in particular a silicone rubber. According to some embodiments, the silicone has the following formula:

[R ² SiO] ⁿ

where n is an integer greater than 4, each R is independently selected from the group consisting of: methyl, ethyl, propyl.

According to some embodiments, the elastomer comprises (ie is made up of) only one silicone or, alternatively, several silicones that are different from each other.

According to some embodiments, a mechanical pressure element 46 (in particular, a spring) is located below the actuator nozzle 36 for pushing the actuator nozzle 36 against the closing element 30.

According to a variant, the fluid dynamic actuator 35 has the structure illustrated in Figures 24 to 26, in which the sealing element 40 comprises two components 40' and 40'' that can be disassembled.

It should be noted that the particular structure of valve V has significant advantages over the prior art. A first advantage consists in the lower risk of contamination by gas (particularly air) of the sample. In this regard, it should be noted that the closure element 30 is normally partially permeable to gas and that in the proposed solution it is not necessary to supply an air jet to keep the closure element 30 in the locked position (by feeding the air jet air, part of the air stream would enter the duct).

A second advantage consists in the reduction of pressure drops when the suction source is working (the parts fit together to present a high fluid-tightness).

In the embodiment of Figure 3, valves 9 and 11 are schematically illustrated and have substantially the same structure as valve V described above. In this case, advantageously, the valves 9 and 11 are connected to each other to a respective suction source 43.

According to some embodiments (not illustrated), the system 1 does not comprise the pressure sources 15 and 25. In this case, the valves 9 and/or 11, instead of being single valves, comprise a plurality of valves arranged in succession along conduit 16 and/or 26. During use, the valves arranged in succession open and close in sequence to supply the sample and/or carrier liquid to the separation unit 3. In this way, the valves arranged in succession they work similar to a peristaltic pump.

It should be noted that to function as a peristaltic pump, at least three valves arranged in succession are normally necessary.

However, according to some embodiments, the valves 9 and/or 11 each comprise (in particular, consist of) two valves arranged in succession. In these cases, said valves are operated in combination with valves 10 and/or 12 to function as a peristaltic pump.

These embodiments have some advantages: they do not require the integration of cumbersome pressure sources; and allow very precise regulation of the amount of fluid that is fed to the separation unit 3.

According to the embodiment illustrated in Figure 3, system 1 comprises a dielectrophoresis system. The separation unit 3 comprises at least a part of the dielectrophoresis system. According to some embodiments, the separation unit 3 comprises the dielectrophoresis system (in its entirety).

In particular, the system 1 (specifically the dielectrophoresis system) comprises an optical sensor 47. The control assembly 23 is connected to the optical sensor 47 and to the separation unit 3. Advantageously, the optical sensor 46 comprises a video camera 48. During use, control assembly 23 actuates different active components of cameras 4 and 5 based on what is detected by optical sensor 47.

According to some embodiments, the separation unit 3 further comprises an operator interface 49 ( man/machine interface). Advantageously, operator interface 49 comprises a personal computer.

According to some embodiments, the dielectrophoresis system and/or its operation is/are as described in at least one of the patent application numbers WO0069565, WO2007010367, WO2007049120.

According to some embodiments, the system 1 (figure 2) comprises a cooling assembly 50, which is designed to cool at least part of the separation unit 3, in particular the main chamber 4 and the recovery chamber 5.

The cooling assembly 50, according to some embodiments, is a Peltier assembly and comprises: a cooling plate 51 having an active surface 52 designed to absorb heat from the separation unit 3; and a discharge surface 53 for producing heat. Advantageously, the active surface 52 has a smaller extension than the discharge surface 53.

According to some embodiments, a mat (known per se) made of a heat-conducting polymeric material is placed between the active surface 52 and the separation unit 3.

The cooling assembly 50 further comprises a heat transfer plate 54 connected to a conditioning circuit 55, which functions as a heat exchanger device.

Circuit 55 comprises: two ducts 56; a radiator 57, which is placed between the two ducts 56; a plurality of fans 58 for cooling a conditioning liquid as it flows into the radiator 57; and a pump 59 for causing the conditioning liquid to flow along the ducts 56 and through the radiator 57.

According to the embodiment illustrated in figure 3, the system 1 comprises at least one (in the case in question, four) mechanical pressure element 60 (in particular, a spring) to push the cooling assembly 50 towards the main chamber 4 and recovery chamber 5.

With particular reference to figures 16, 18, 13 and 3, system 1 further comprises two pressure supply nozzles 61 and 61a arranged between two sections of conduit 16 and conduit 26, respectively.

The pressure supply nozzle 61 comprises a hollow body 62, which is connected to a pressure device 64, and has a pressure supply port 63 placed at an opposite end with respect to the pressure device 64.

The pressure device 64 comprises a pressure unit 65, which, in turn, comprises the pressure source 15 and a conduit (in particular, a first section of the conduit 16), which connects the pressure source 15 to the nozzle. pressure supply 61.

Between the pressure supply port 63 and a second length of conduit 16 is placed a sealing ring 66 (illustrated in Figures 7, 16) comprising (in particular consisting of) a substantially elastic material.

Advantageously, the substantially elastic material is defined as described above with reference to closure element 30.

According to some embodiments, the sealing ring 66 is substantially circular and has: a substantially central hole 67; an internal portion delimiting the hole 67; and a peripheral portion having a thickness greater than an inner portion.

According to some embodiments, the system 1 further comprises at least one mechanical pressure element 68 (in particular a spring), which is positioned to push the pressure supply nozzle 63 towards (in particular against) the sealing ring 66. In this way, a lower pressure dispersion (i.e. a better seal) is obtained between the pressure supply nozzle 63, the sealing ring 66 and the second section of the conduit 16. The mechanical pressure element 68 it has the important function of compensating for any possible lack of flatness of the device and regulating the applied contact forces.

According to advantageous embodiments, the pressure supply nozzle 61a has the same structure as the pressure supply nozzle 61, is connected to the pressure device 64, and is pushed towards a respective sealing ring 66a by a corresponding mechanical pressure element. 68a.

System 1 further comprises a seat 69 (partially illustrated in Figure 13), which is designed to house a removable collector (for example, a test tube), of a type known in itself and not illustrated, and is put into operation. exit 8 .

Between the duct 27 and the seat 69 a sealing ring 70 is placed, which is designed to ensure a smaller spread (i.e. better sealing) between the duct 27 and the seat 69. The sealing ring 70 comprises (in particular, consists of) a substantially elastic material.

Advantageously, the substantially elastic material is defined as described above with reference to closure element 30.

According to some embodiments, the sealing ring 70 is substantially circular and has: a substantially central hole 71; an internal portion delimiting the hole 71; and a peripheral portion having a thickness greater than an inner portion.

According to some examples useful for understanding the invention, the system 1 comprises two separable portions: a substantially fixed apparatus 72 (an embodiment of the apparatus 72 is partially illustrated in Figures 10 and 11) and a device 73 (an embodiment of the device 73 illustrated in top plan view in Figure 21 and in exploded view in Figure 4).

According to a particular aspect of the present invention, a microfluidic system is provided for the isolation of C1 particles of at least one given type from a sample, the system 1 comprises: a first inlet 2, through which, during its use, the sample is introduced into system 1; a separation unit 3, comprising a main chamber 4 and a recovery chamber 5 and designed to transfer at least part of the C1 particles of the given type from the main chamber 4 to the recovery chamber 5 in a substantially selective manner with relative to more C2 particles in the sample; a first outlet 7, connected to the main chamber 4; and a second outlet 8, which is connected to the recovery chamber 5, through which, during use, at least part of the particles C1 of the given type collected in the recovery chamber 5 leave the system 1; system 1 is characterized in that it comprises: a first valve 9, arranged upstream of main chamber 4; a second valve 10, placed between the main chamber 4 and the first outlet 7; a third valve 11, arranged upstream of the recovery chamber 5; and a fourth valve 12, placed between the recovery chamber 5 and the second outlet 8. According to some embodiments, the system comprises one or more of the features described above according to the first and second aspects that are useful examples to understand the present invention.

In use, system 1 (in accordance with one or more of the aforementioned aspects of the invention) is used in accordance with the method described below.

Method

Provided as an example useful for understanding the invention is a method for isolating C1 particles of at least one given type from a sample by means of a microfluidic system. The microfluidic system is system 1 as described above according to the present invention. In any case, for reasons of simplicity in the following description of the method, the microfluidic system and its parts will be identified with the reference numerals used above to identify system 1 and similar or identical parts, respectively.

The method comprises: a step of introducing the sample into a system 1 through an inlet 2 of the system 1; a separation step, during which at least part of the C1 particles of the given type are separated from other C2 particles within a separation unit 3 of the system 1; a first supply step, which at least partially precedes the separation step and during which at least part of the sample is fed to the separation unit 3 (the first supply step is schematically illustrated in Figures 19e-19i); and a recovery step, which is at least partially subsequent to the separation step and during which at least part of the particles C1 of the given type separated in a substantially selective manner flow from the separation unit 3 through an outlet 8 of the system 1 (the recovery step is schematically illustrated in Figures 20c and 20d).

According to some embodiments, the system 1 comprises: a valve 9 placed between the inlet 2 and the separation unit 3; and a valve 12 placed between outlet 8 and separation unit 3. During the separation step, valves 9 and 12 are kept closed.

In particular, the system 1 comprises a set of valves between each opening (for example, inlets and/or outlets) of the system 1 to the outside and the separation unit 3. During the separation step, each of these valves is maintained closed.

According to some embodiments, the recovery step is entirely subsequent to the separation step.

According to some embodiments, the separation step is entirely subsequent to the first supply step.

According to some embodiments, during the separation step, particles C1 of the given type are transferred from a main chamber 4 to a recovery chamber 5 of separation unit 3 in a substantially selective manner with respect to other particles C2 of the sample (the end of the separation step is illustrated in Figure 20a).

According to some embodiments, during the separation step, using a system as described in Figure 2, particles C1 of various given types are transferred from a main chamber 4 to a particular area of the recovery chamber 5 isolated by means of the fluid resistance of the rest of the recovery chamber.

An outlet 7 and an outlet 8 of system 1 connect to main chamber 4 and recovery chamber 5, respectively.

Advantageously, the system 1 comprises: the valve 9, which is arranged upstream of the main chamber 4; a valve 10, arranged downstream of the main chamber 4; a valve 11, arranged upstream of the recovery chamber 5; the valve 12, arranged downstream of the recovery chamber 5. During the separation step, the valves 9, 10, 11, 12 are closed, in particular to isolate the main chamber 4 and the recovery chamber 5 with respect to the outside .

The method further comprises: the first supply step, which at least partially precedes the separation step and during which at least part of the sample is fed into the main chamber 4 (the first supply step is schematically illustrated in Figures 19e -19i); and a second supply step, which at least partially precedes the separation step and during which the carrier liquid is fed to the recovery chamber 5 (the second supply step is illustrated in Figures 19a-19d).

The method further comprises the recovery step, during which the carrier liquid, together with at least part of the particles C1 of the given type, flows from the recovery chamber 5 through the outlet 8 (the recovery step is schematically illustrated in Figures 20c and 20d).

According to some embodiments, during the introduction step, at least part of the sample is introduced into a reservoir 13 of the system 1.

Advantageously, the separation step occurs by dielectrophoresis. At least during the separation step, the separation unit 3 cools down.

According to some embodiments, at least one or both of the first and second supply steps completely precede the separation step.

According to some embodiments, the second delivery step at least partially precedes the first delivery step. Advantageously, the second supply step is completely prior to the first supply step.

According to some embodiments, a first pressure is established to supply the sample to the main chamber 4.

In particular, the first pressure pushes the sample from the reservoir 13 towards the main chamber 4.

According to some embodiments, the first pressure is exerted at least before and during the first delivery step.

Advantageously, during the recovery step, at least part of the particles C1 of the given type are subjected to vibration; in particular, they are subjected to a pressure that varies in an oscillating way (the vibration frequency is between 2 Hz and 80 Hz, advantageously from 5 Hz to 40 Hz). Advantageously, during the first supply step, a valve 9 of system 1 is opened, said valve 9 is placed before the main chamber 4, and a valve 10 of system 1, said valve 10 is placed between the main chamber 4 and outlet 7. In particular, during the first supply step, the sample passes through valve 9.

According to some embodiments, during the first supply step, the sample is subjected to vibration; in particular, it is subjected to a pressure that varies oscillatingly (the vibration frequency is between 2 Hz and 80 Hz, advantageously from 5 Hz to 40 Hz).

According to particular embodiments, the system 1 comprises: a conduit 16 to connect the inlet 2 to the main chamber 4; and a conduit 20, which is placed between the main chamber 4 and the outlet 7 and has a smaller cross section, in particular by at least 100 mm, than the cross section of the conduit 16; during the first supply step, the sample pressure is detected; the supply of the sample is blocked according to the detected pressure, in particular, when a pressure greater than a given value is detected.

According to further embodiments, in addition to or as an alternative to pressure detection, one or more of the following detections are performed: optical detection of sample passage between chamber 4 and conduit 20; detection of the variation of the electrical conductivity in a connection area between the chamber 4 and the conduit 20 due to the beginning of the passage of the sample; detection of the variation of the electrical permittivity in a connection area between the chamber 4 and the conduit 20 due to the beginning of the passage of the sample; detection of the variation of the thermal resistance in a connection area between the chamber 4 and the conduit 20 due to the beginning of the passage of the sample; and detection of the variation of the thermal capacity in a connection area between the chamber 4 and the conduit 20 due to the beginning of the passage of the sample.

In all of the above cases, the inflow of sample 40 is blocked when it is found that the sample begins to enter conduit 20.

According to some embodiments, during the second supply step, a valve 11 of system 1 is opened, said valve 11 being arranged upstream of the recovery chamber 5, and a valve 12, said valve 12 being arranged between the recovery chamber. recovery 5 and exit 8.

Advantageously, a second pressure is established to supply the carrier liquid to the recovery chamber 5. In particular, the second pressure pushes the carrier liquid from a reservoir 14 of the system 1 towards the recovery chamber 5. During the second step of supply, the carrier liquid passes through valve 11.

According to some embodiments, the second pressure is exerted at least before and during the second delivery step.

During the recovery step, valves 11 and 12 open.

According to some embodiments, the method comprises a discharge step, which is at least partially subsequent to the separation step and at least partially prior to the recovery step and during which at least part of the other C2 particles in the sample are made flow away from main chamber 4 through outlet 7; the discharge step is schematically illustrated in Figures 20b and 20c. Advantageously, the discharge step is completely subsequent to the separation step and/or completely prior to the recovery step.

During the discharge step, the valves 10 and 11 open to supply the carrier liquid to the main chamber 4. Carrying out the discharge step allows to reduce the risks that part of the other particles C2 that are removed from the main chamber 4 by the flow of the carrier liquid through the recovery chamber 5 pass, during the recovery step, through the outlet. 8.

In practice, according to some embodiments, valves 11 and 12 open to fill recovery chamber 5 with carrier liquid. At this point, valves 9 and 10 are opened, so that the sample fills the main chamber 4. Next, valves 9, 10, 11 and 12 are closed, and the C1 particles of the given type are put into a substantially selectively from main chamber 4 to recovery chamber 5. At this point valves 11 and 10 open to cause at least part of the other particles C2 to flow out of chamber 4.

According to some embodiments, the particles C1 of the given type are arranged inside the recovery chamber 5 in such a way that, during the discharge step, they remain at least in part inside the recovery chamber 5 itself. In particular, the C1 particles of the given type are arranged laterally with respect to (i.e. not in front of) a channel 6 for the connection between the main chamber 4 and the recovery chamber 5. Specifically, the C2 particles are arranged between the channel 6 and a conduit 26 for connection to outlet 8.

According to some embodiments, the system 1 comprises a valve 29 (figures 2 and 27), which is placed between the recovery chamber and the outlet 7 (or an additional outlet not illustrated). Chamber 5 comprises: a first area 5' which is hydraulically connected to conduit 27 (and thus to valve 12); a second area 5”, hydraulically connected to the conduit 28 (and therefore to the valve 29) and an additional area, which defines a terminal section of the conduit 26 (and therefore connects to the valve 11).

During the second filling step, valves 12 and 11 open to fill the first area 5' of recovery chamber 5, which connects valves 12 and 11; valves 11 and 29 open to fill the second area 5” of recovery chamber 5 that connects valves 11 and 29.

According to specific embodiments, valves 12, 11 and 29 open to fill the first area 5' (figures 27b and 27c); at this point, the valve 12 closes and the second area 5'' is filled (figure 27d).

During the separation step, at least part of the C1 particles of the given type and at least part of the C3 particles of at least one second given type are transferred to the recovery chamber 5 (figures 28a and 28b) (in particular, to the second area 5"). The recovery step comprises a first recovery sub-step, during which at least part of the C1 particles of the given type is placed in a substantially selective manner in the first area 5' (FIG. 28d) and, subsequently, at least part of the particles C1 of the given type is made to flow from the first area 5' through the outlet 8 by supplying the recovery chamber 5 with more carrier liquid (figure 28e).The recovery step comprises a second recovery sub-step, during which at least part of the particles C3 are caused to exit recovery chamber 5 through outlet 8 by supplying recovery chamber 5 with more carrier liquid.

Advantageously, during the second recovery step, at least part of the C3 particles are placed in the first area 5' (Figures 28e and 28f) and subsequently at least part of the C3 particles are made to flow out of the area 5'. first area 5' through outlet 8 (FIG. 28g).

According to some embodiments, the method comprises a washing step, during which the other particles C2 present in the main chamber 4 are removed from the channel 6. During the washing step, valves 11 and 10 open (figure 28c) . Advantageously, during the washing step, the valve 29 is closed and the particles C1 and C3 are arranged in the second area 5”. Advantageously, during the washing step, the valve 12 is closed. Advantageously, during the washing step, the valve 9 is closed.

Advantageously, the washing step is at least partially (in particular, completely) subsequent to the recovery step and at least partially (in particular, completely) before the recovery step.

According to some embodiments, during the recovery step, the first drop of carrier liquid is detected to exit the outlet 8; when the first drop is detected, the flow out of the recovery chamber 5 is blocked. According to some embodiments, several recovery stages follow one another, changing the containers arranged in the vicinity of the outlet 8 whenever at least one drop is detected. a drop.

According to some embodiments, carbon dioxide is fed to system 1. In this way, the presence of oxygen within system 1 is reduced or eliminated. The presence of oxygen within the system can cause bubble formation during the various steps of the method.

According to alternative embodiments, the carrier liquid (and/or possibly the sample) is degassed by means of ultrasound before its introduction into the system 1 (or into the separation unit 3).

Advantageously, the sample and the carrier liquid are used at a temperature greater than 20°C, in particular greater than 25°C. Furthermore, this reduces the risk of bubble formation. According to some embodiments, the method is applied using a defined system 1 according to the claims.

According to some examples useful for understanding the invention, the system 1 comprises two separable portions: a substantially fixed apparatus 72 (an embodiment of the apparatus 72 is partially illustrated in Figures 10 and 11) and a device 73 (an embodiment of the device 73 illustrated in top plan view in Figure 21 and in exploded view in Figure 4). The device 73 is advantageously disposable and is designed to be connected to the apparatus 72. According to some embodiments, only a part of the sample is placed in the main chamber 4. In practice, the sample is subjected to a plurality of successive partial separations .

microfluidic device

In accordance with an example useful for understanding the present invention, device 73 for isolating C1 particles of at least one given type from a sample is provided. Device 73 comprises: inlet 2, through which, during use, the sample is introduced into device 73; and the separation unit 3, comprising a main chamber 4 and a recovery chamber 5. The separation chamber 3 (in particular, the main chamber 4) is connected to the inlet 2. In particular, the separation unit 3 comprises part of the dielectrophoresis system.

In use, when the device 73 is mounted inside the apparatus 72, the separation unit 3 is designed to transfer at least part of the particles C1 of the given type from the main chamber 4 to the recovery chamber 5 in a substantially selective manner. relative to other C2 particles in the sample. According to some embodiments, device 73 comprises outlet 7 connected to main chamber 4; outlet 8 connects to recovery chamber 5.

Through the outlet 8, during use, at least part of the particles C1 of the given type collected in the recovery chamber 5 leave the device 73.

The outlet 7 is designed to allow the sample to freely enter the main chamber 4, thus functioning as a vent.

Device 73 further comprises; a valve portion 74, which is disposed upstream of the main chamber 4 (in particular, between the main chamber 4 and the inlet 2); and a valve portion 75, which is placed between the main chamber 4 and the outlet 7.

Valve portion 74 is designed to be part of valve 9. Valve portion 75 is designed to be part of valve 10.

The device also comprises: a valve portion 76, which is connected to the recovery chamber 5; and a valve portion 77, which is placed between the recovery chamber 5 and the outlet 8.

In particular, the recovery chamber 5 is placed between the main chamber 4 on one side and the third and fourth valve portions 76, 77 on the other side; the main chamber 4 is placed between the recovery chamber 5 on one side and the first and second valve portions 74 and 75 on the other side.

Valve portion 75 is designed to be part of valve 11. Valve portion 76 is designed to be part of valve 12.

According to some embodiments, at least one of the valve portions 74, 75, 76 and 77 comprises the closing element 30, which is designed to pass between a locked position, in which the closing element 30 is arranged to separating two sections of a respective channel of the device 73, and an open position, in which the closing element 30 is arranged in such a way that the two sections are connected to each other. Advantageously, each valve portion 74, 75, 76 and 77 comprises a respective closure element 30.

Advantageously, the closing element 30 is defined as described above in relation to system 1. In particular, the closing element 30 has a membrane part, which it comprises, in particular, is made of a substantially elastic material.

According to some embodiments, at least one, in particular each of the valve portions 74, 75, 76 and 77, comprises the diaphragm 33, which is placed between the two sections of the conduit of the device 73. In the locked position , the closing element 30 is in contact with the diaphragm 33; in the open position, the closing element 30 is placed at a distance from the diaphragm 33.

At least one, in particular each of the valve portions 74, 75, 76 and 77 comprises at least one hole in a channel of the device 73. In particular, each closing element 30 is placed at a point corresponding to two holes of a corresponding channel, said holes are separated from each other by a respective diaphragm 33. Each of these holes has a diameter ranging from 0.1 to 0.7 mm. According to specific embodiments, each hole has a diameter of approximately 0.5 mm.

According to some embodiments, each valve portion 74, 75, 76 and 77 corresponds to a portion of the valve V described above without the fluid dynamic actuator 35.

At least one of the closing elements 30 can be actuated by an actuator external to the device 73; in particular, the external actuator forms part of the device 72. More specifically, each of the closing elements 30 can be actuated by a respective actuator external to the device 73; in particular, the external actuators form part of the apparatus 72.

At least one, in particular each of the closure elements 30 is at least partially exposed and facing outwards. In this way, the possibility of coupling the closure element 30 with the respective external actuator and an interaction thereof becomes more convenient.

According to some embodiments, the device 73 further comprises: the reservoir 13, which is placed between the inlet 2 and the valve portion 74 and is designed to collect the sample introduced through the inlet 2; and a channel 78, which connects the reservoir 13 to the main chamber 4 and along which the valve portion 74 is placed. In particular, the channel 78 constitutes a part of the conduit 16.

Advantageously, channel 78 has an equivalent diameter cross section ranging from 0.9mm to 50mm. In particular, the channel 78 has a width ranging from 0.7 to 0.1 mm and a depth ranging from 1.00 to 0.15 mm. In accordance with specific embodiments, channel 78 has a width of about 0.5 mm and a depth of about 0.25 to about 0.5 mm. The particular paths of the channel 78 help to reduce the risk of air entering the device 73.

Advantageously, the reservoir 13 has a volume of 5 ml to 100 ml, in particular a width of 3 to 0.8 mm and a depth of 1.5 to 0.25 mm.

According to specific embodiments, the reservoir 13 has a volume of approximately 35 ml, a width of approximately 1 mm and a depth of approximately 0.5 mm.

Advantageously, the valve portion 74 is placed between the reservoir 13 and the main chamber 4.

According to some embodiments, the device 73 comprises a supply port 79. In particular, the supply port 79 is placed at the inlet 2. The reservoir 13 is placed between the supply port 79 and the main chamber 4. The channel 78 connects supply port 79 to main chamber 4.

According to some embodiments, the device 73 comprises the sealing ring 66, which surrounds the delivery port 79 outwards.

Advantageously, the sealing ring 66 is defined as described above in relation to system 1 and, in particular, is designed to mate with a respective pressure supply nozzle 61.

According to some embodiments, the device 73 comprises a channel 80 (corresponding to part of the conduit 20), which is placed between the main chamber 4 and the outlet 7 and comprises the section 20'. The section 20' has a smaller cross section, in particular by at least 100 mm, than the cross section of the channel 78 (the section 20' is illustrated more clearly in Figure 9).

Advantageously, the section 20' has a width of less than 150 mm, a depth of less than 110 mm and a length greater than 2 mm. Advantageously, the section 20' has a width greater than 100 mm, a depth greater than 30 mm and, in particular, a length of less than 6 mm.

According to some embodiments, the device 73 comprises the reservoir 14, which is designed to contain the carrier liquid.

Advantageously, the reservoir 14 has a volume ranging from 10 ml to 100 ml, a width ranging from 5 to 0.8 mm, and a depth ranging from 1.5 to 0.25 mm.

In accordance with specific embodiments, reservoir 14 has a volume of approximately 150 ml, a width of approximately 1 mm, and a depth of approximately 0.5 mm.

The device 73 comprises a channel 81, which connects the reservoir 14 to the recovery chamber 5 and along which the valve portion 76 is placed.

In particular, the channel 81 constitutes a part of the duct 26.

Advantageously, channel 81 has an equivalent diameter cross section ranging from 0.9mm to 200mm. In particular, the channel 81 has a width ranging from 0.7 to 0.25 mm and a depth ranging from 0.7 to 0.15 mm. According to specific embodiments, the channel 81 has a width of approximately 0.5 mm and a depth of approximately 0.25 mm.

The particular paths of the channel 81 help to reduce the risk of air entering the device 73.

According to some embodiments, the valve portion 76 is placed between the reservoir 14 and the recovery chamber 5. According to some embodiments, the device 73 comprises a supply port 82. The reservoir 14 is placed between the supply port 82 and recovery chamber 5, channel 81 connecting supply port 82 to recovery chamber 5.

According to some embodiments, the device 73 comprises the sealing ring 66a, which surrounds the delivery port 82 outwards.

Advantageously, the sealing ring 66a is defined as described above in relation to system 1 and, in particular, is designed to mate with a respective pressure supply nozzle 61a.

The device 73 comprises electrical connectors 83 (illustrated in figure 3) for the electrical connection of the device 73 itself to the apparatus 72. Advantageously, the electrical connectors 83 comprise (in particular, consist of) at least one electrical circuit, in particular, a electrical printed circuit (PCB) connection.

According to the embodiment illustrated in figure 31, the device 73 comprises an additional valve portion 29' designed to form part of the valve 29. In this case, the valve portion 29' is placed between the recovery chamber 5 and an output of device 73 (ie system 1). Said outlet may be an additional outlet with respect to outlets 7 and 8 described above or may coincide with outlet 7 or outlet 8 (in the embodiment illustrated in Figure 31, said outlet corresponds to outlet 7).

Therefore, according to some embodiments, the device 73 comprises an additional outlet; valve portion 29' is placed between recovery chamber 5 and further outlet; optionally, the additional output corresponds to output 7.

The device 73 further comprises the conduit 28, which hydraulically connects the chamber 5 (in particular the second area 5'') to the additional outlet. The valve portion 29' is placed in a position corresponding to the conduit 28.

In these cases, the chamber 5 comprises: the first area 5', which is hydraulically connected to the conduit 27 (and thus to the valve portion 77); the second area 5", which is hydraulically connected to the conduit 28 (and therefore to the valve portion 29'); and the additional area, which defines an end section of the channel 81 (that is, of the conduit 26) (and thus it connects to the valve portion 76).

Conduit 28 has an equivalent diameter cross section ranging from 0.9mm to 200mm. In particular, the conduit 28 has a width of 0.7 to 0.25 mm and a depth of 0.7 to 0.15 mm. In accordance with specific embodiments, conduit 28 has a width of approximately 0.5 mm and a depth of approximately 0.25 mm.

The device 73 of Figure 31 is designed to be part of the system 1 illustrated in Figure 2 and to function accordingly. according to what is illustrated in figures 27 and 28.

Apparatus

In accordance with an example useful in understanding the present invention, an apparatus 72 for isolating C2 particles of at least one given type from a sample is provided.

Apparatus 72 comprises: a seat 84 (shown open in Figure 11 and closed in Figure 10) for housing a microfluidic device (in particular, device 73) for isolation of C1 particles of the given type from the sample; electrical connectors 85 (illustrated in figures 3 and 13) for the electrical connection of the apparatus 1 to the microfluidic device; and control assembly 23, connected to electrical connectors 85. According to some embodiments, apparatus 72 comprises part of the dielectrophoresis system.

In accordance with some embodiments, apparatus 72 comprises a hatch 86, which is illustrated in a raised position in Figure 11 and in a lowered position in Figure 10. The underside of hatch 86 is illustrated in Figure 12.

Apparatus 72 comprises: at least four fluid dynamic actuators 35, each of which is designed to form part of a respective valve and comprises a respective actuator nozzle 36 (see, in particular, Figure 13), having an actuator port 39 respective; and at least two pressure supply nozzles 61 and 61a, each of which has a respective pressure supply port 63 and 63a.

Each fluid dynamic actuator 35 is designed to move a respective closing element 30 external to the apparatus 72, which belongs, in particular, to said microfluidic device 73. In particular, each fluid dynamic actuator 35 is designed to engage (come into contact) with an element closing 30 respective.

The apparatus comprises: at least pressure device 64, connected to pressure supply nozzles 61 for determining a pressure at pressure supply ports 63 and 63a; and at least one pressure device 87, which is connected to the actuator nozzles 36 (figure 3) and is designed to cause suction in a region corresponding to at least one of the actuator holes (figures 14 and 15).

When the hatch 86 is in a raised position, the seat 84 opens and is accessible from the outside (FIG. 11); in particular, when the hatch 86 is in a raised position, the microfluidic device (in particular, the device 73) can be inserted below the hatch 86 itself. In use, once the microfluidic device has been inserted below the hatch 86, the hatch 86 is lowered (FIG. 10) and the microfluidic device is placed in the seat 84. This is done by rotating the handles 88, which have cam profiles 89 at one of their ends. The cam profiles 89, by turning them, they push the hatch 86 downwards, overcoming the resistance of springs (which are themselves known and not illustrated), which tend to hold the hatch 86 in a raised position.

According to what is illustrated in figure 12, the hatch 86 comprises holes 86a to inspect the valves 9, 10, 11 and 12 and an opening 86b to make the chambers 4 and 5 visible.

According to some embodiments, apparatus 72 comprises a pressure assembly 90 (FIG. 3), comprising pressure devices 64 and 87. Pressure assembly 90 comprises at least one pump.

According to some embodiments, the pressure device 64 comprises the pressure unit 65 and at least one pressure unit 65a, each of which is connected to the respective pressure supply nozzle 61 and 61a. The pressure units 65 and 65a can be operated separately and each is designed to define a pressure (in particular an air jet through) at the corresponding pressure supply port 63 and 63a.

According to some embodiments, pressure device 64 comprises at least one pressure source 15 (and/or 25) (FIGS. 1, 2, and 3). At least one of pressure unit 65 and pressure unit 65a comprises a corresponding conduit (in particular, for pressure unit 65, a first section of conduit 16; for pressure unit 65a, a first section of conduit 26). ), which connects the pressure source 15 and/or 25 to the respective pressure supply nozzle 61 and/or 61a.

Apparatus 72 comprises: pressure sensor 21 for detecting pressure along the aforementioned conduit; and the blocking device 22, which is designed to interrupt the transmission of pressure to the respective pressure supply nozzle 61 and/or 61a. Control assembly 23 connects to pressure sensor 21 and lockout device 22 to actuate lockout device 22 based on sensed pressure.

According to some embodiments, pressure sensor 21 is placed in a position corresponding to pressure device 64.

Advantageously, the blocking device 22 comprises a relief valve, which is arranged, in particular, along ^{the aforementioned conduit (a first section of the conduit 16 and/or a first section of the conduit} 26 ^).

According to the embodiments illustrated in figures 1 and 2, the blocking device 22 is placed along a first section of the conduit 16, and the pressure sensor 21 is designed to detect the pressure within the conduit 16 itself.

In accordance with some embodiments (not illustrated), apparatus 72 comprises a pressure sensor for sensing pressure in conduit 26 and a blocking device. The pressure sensor and lockout device are defined and arranged similarly to what has been described above with reference to pressure sensor 21 and lockout device 22. According to some embodiments, apparatus 72 comprises at least one vibration device 17 and/or 17a, which is placed along the aforementioned conduit (a first section of conduit 16 and/or a first section of conduit 26) and is designed to cause a variation in an oscillating form of pressure defined by the pressure source 15 and/or 25 at the respective pressure supply port 63 and/or 63a (FIG. 3).

Advantageously, the vibration device 17 and/or 17a comprises a diaphragm pump.

Advantageously, the apparatus 72 comprises two vibration devices 17 and 17a, which are arranged along a first section of the duct 16 and a first section of the duct 26, respectively. The vibration devices 17 and 17a are designed to cause an oscillating variation of the pressure defined by the corresponding pressure sources 15 and 25 in the pressure supply ports 63 and 63a, respectively.

According to some embodiments, the pressure device 87 comprises at least four suction units 41, each connected to a respective actuator nozzle 36. The suction units 41 can be operated separately from one another and each is designed to perform at least one suction operation on a corresponding actuator port 39.

Advantageously, the pressure device 87 comprises at least one suction source 43. At least one of the suction units 41 comprises: a respective conduit 42, connecting the suction source 43 to the respective actuator nozzle 36; and a blocking device (itself known and not illustrated), which is designed to interrupt the transmission of suction to said respective actuator nozzle 36.

Advantageously, said blocking device comprises an element chosen from a group consisting of: a valve arranged along the duct 42 and a pressure source 43 drive, said drive being designed to activate or deactivate the pressure source 43 itself .

According to some embodiments, at least one of the actuator nozzles 36 comprises (FIGS. 14 and 15) a corresponding mechanical pressure element 34, which is designed to exert a pressure through the respective actuator hole 39 outwards.

Advantageously, the mechanical pressure element 34 comprises a spring, at an external end of which the sealing element 40 is placed.

Advantageously, one or more of the actuator nozzles 36 comprises a hollow element 37 to house the mechanical pressure element 34 and to connect the respective suction unit 41 to the corresponding actuator hole 39. The hollow element 37 is equipped with one end having the corresponding actuator hole 39.

According to some embodiments, the apparatus 72 comprises at least one mechanical pressure element 46 for urging one or more of the actuator nozzles 36 toward the microfluidic device. Advantageously, the mechanical pressure element 46 comprises (in particular, is constituted by) a spring.

Advantageously, apparatus 72 comprises a plurality of mechanical pressure elements 46, each for urging a respective actuator nozzle 36 toward the microfluidic device. In particular, each mechanical pressure element 46 is designed to push a respective actuator nozzle towards (against) a corresponding closing element 30.

According to some embodiments, one or more (in particular, all) of the fluid dynamic actuators 35 are defined as described above in relation to system 1.

According to some embodiments, apparatus 72 comprises at least one mechanical pressure element 68 and/or 68a for urging at least one respective pressure supply nozzle 61 and/or 61a toward (in particular, against) the microfluidic device.

According to some embodiments, apparatus 72 comprises cooling assembly 50, which is designed to cool at least part of the microfluidic device. In particular, the part of the microfluidic device from which heat is absorbed is the separation unit 3.

Advantageously, the cooling assembly 50 is defined as described in relation to system 1.

In particular, the cooling assembly 50 comprises: a cooling plate 51 having an active surface 52 designed to absorb heat from the microfluidic device; and a discharge surface 53 for producing heat. The active surface 52 has smaller dimensions than the discharge surface 53.

Advantageously, the cooling assembly 50 comprises a Peltier and a heat exchanger device (in particular, the conditioning circuit 55) connected to the Peltier.

Advantageously, apparatus 72 comprises at least one mechanical pressure element 60 (in particular, a plurality thereof) for urging cooling assembly 50 toward the microfluidic device.

According to one embodiment (not illustrated), the apparatus 72 comprises at least one additional actuator nozzle 36 and a corresponding additional suction unit 41.

According to some embodiments, control assembly 23 is connected to pressure devices 64 and 87 to regulate pressure and/or suction at each actuator nozzle 36 and/or each pressure supply nozzle 61 independently of each other.

According to some embodiments, the apparatus 72 comprises a collection unit for collecting a carrier liquid containing at least part of the C1 particles of the given type. In particular, the collection unit comprises a seat 69 (FIG. 13), which is designed to house a removable collector (for example, a test tube), of a type known per se and not illustrated, and is placed in the exit 8.

Advantageously, the apparatus 72 comprises a detector (eg, a video camera itself known and not illustrated) for detecting when a drop of said carrier liquid enters the collection unit.

Said detector is connected to the control assembly 23. During use, when the detector detects the passage of a drop, the control assembly interrupts the outflow of the carrier liquid from the recovery chamber 5.

According to some embodiments, parts of the device 73 and/or of the apparatus 72 are defined, even only with respect to some aspects taken separately from the others, as similar parts of the system 1 and/or vice versa.

microfluidic device

In accordance with an example useful in understanding the present invention, a device 73 for the substantial isolation of C1 particles of at least one given type from a sample is provided. Device 73 comprises: inlet 2, through which, during use, the sample is introduced into device 73; a separation unit 3, which is designed to substantially selectively separate at least a part of the C1 particles of the given type from other C2 particles in the sample; and an outlet 8, which is connected to the separation unit 3 and through which, during use, at least part of the substantially selectively separated particles C1 of the given type exit the device 73.

The device 73 comprises: a valve portion 74, arranged between the inlet 2 and the separation unit 3; and a valve portion 77, arranged between the outlet 8 and the separation unit 3. In particular, the device 73 comprises a valve portion arranged between each opening of the device 73 to the outside and the separation unit 3.

According to some embodiments, the device 73 is defined, even only with respect to some aspects taken separately from the others, as according to the fourth aspect of the present invention.

Advantages

It should be noted that the present invention has several advantages, in addition to those mentioned above, compared to the state of the art. Among these we mention the following.

Particles C1 of the given type can be introduced into the recovery chamber 5 while the separation unit 3 is isolated from the outside (valves 9-10 are closed). In this way, evaporation of both the liquid part of the sample and the carrier liquid is substantially prevented. By avoiding evaporation, there are no return currents from the recovery chamber 5 to the main chamber 4 (or vice versa), thus reducing the risk that the C1 and/or C2 particles move in an uncontrolled way (specifically, the particles C1 put in the recovery chamber 5 do not return to the main chamber 4, likewise the other cells C2 are not recovered in the recovery chamber 5). Furthermore, by avoiding evaporation, there is no increase in the concentration of the salts in the separation unit 3. The increase in the concentrations of the salts can cause increases in conductivity and local power dissipation with the corresponding problems (damage to possible electrodes present in chambers 4 and 5).

The various steps are controlled by the control assembly 23. This determines a higher degree of reproducibility (according to the state of the art, an operator had to introduce the sample and the recovery liquid by a pipette).

The presence of carbon dioxide inside the device 73 before loading the sample reduces the risk of bubble formation inside chambers 4 and 5. The bubbles reduce the volume of the analyzed sample and can prevent the recovery of C1 particles of the type given especially if they occupy a region that corresponds to channel 6.

The sample contacts only the device 73, which is advantageously disposable. In this way, it is not necessary to wash the different parts of the system 1, and the risks of contamination between successive samples are drastically reduced. In this regard, it should also be emphasized that most of the active components, which are more complex and expensive, are disposed in apparatus 72, which is not disposable. System 1 has extremely small unused volumes because the sample is held in reservoir 13 in close proximity to separation unit 3. Reservoirs 13 and 14 are part of device 73.

This patent application claims priority from an Italian patent application (specifically, document BO2009A000154). Other characteristics of the present invention will emerge from the subsequent description of some merely illustrative and non-limiting examples of the realization of the device 73 and the operation of the system 1. Example 1

This example describes the production of a silicon chip 91 from the separation unit 3. The chip is shown more clearly in Figure 9 and in an exploded view in Figure 8.

The chip is 19.9mm wide, 24.5mm long and 1.2mm thick and comprises: a silicon substrate 92 (thickness: 600mm); a spacer element 93, which delimits the chambers 4 and 5 and the channel 6 (thickness: 90 mm); and a transparent cover 94, in particular, made of borosilicate or quartz (500 mm).

Substrate 92 was made using standard CMOS (complementary metal oxide semiconductor) technologies. A layer of photopolymer (Dryresist® in particular, Ordyl SY300, Elga Europe) (thickness: 90 mm) was laminated at 90° onto the substrate 92. The photopolymer layer was then partially protected with a photolithographic mask (a transparent slide printed at 24000 DPI resolution) and subjected to UV radiation (150 W) for 15 seconds to polymerize the areas of the exposed photopolymer layer (i.e. , not covered by the dark parts of the mask). After selective polymerization was complete, the unpolymerized portion was removed by immersing the laminated substrate in a developer (BMR developer - xylene blend, 2-butoxyethyl acetate, mixed isomers).

At this point, the substrate 91 with the corresponding spacer element 93 thus obtained was put in an oven at 50°C for one hour to obtain drying.

Lid 94 (made of glass and 500 mm thick) was obtained by grinding. The holes in the lid 94 were frustoconical in shape, with the bottom having a diameter of 700mm and the top having a diameter of 1200mm.

The cap 94 was pressed against the spacer element 8 for 80 minutes at a temperature of 95°C to obtain a thermal bond.

Example 2

This example describes a PCB (printed circuit board) 95 illustrated partially in perspective in Figure 4. PCB 95 consisted of four layers of copper prepared by photolithographic techniques of known type (see, for example, the example above).

PCB 95 had a main body made of epoxy polymer and fiberglass composite material. The shape of the PCB 95 illustrated in Fig. 4 was obtained by milling the main structure.

The copper layers were embedded in the main structure and exposed outwards with four hundred pads oriented (in Figure 4) upwards (i.e. towards chip 91) and arranged (two hundred, in the area indicated by arrow 96 and two hundred, in the area indicated by arrow 97) on opposite sides of an opening 98 in the PCB 95 itself. These pads were electrically connected to four hundred other pads facing (in Figure 4) downwards; two hundred of the additional pads were arranged on one edge of the PCB in the area indicated by arrow 99; two hundred of the additional pads were arranged in a position corresponding to an edge of PCB 95 in the area indicated by arrow 100.

Pads arranged in areas 96 and 97 were gold plated designed to electrically connect PCB 95 to chip 91.

Pads arranged in areas 98 and 99 were plated with gold and functioned as electrical connectors for the electrical connection of device 73 to apparatus 72 and, in particular, control assembly 23.

PCB 95 had a thickness of approximately 1.6 mm.

Example 3

This example describes the connection between chip 91 and PCB 95.

Chip 91 was aligned to the center of PCB 95 using a "pick and place" device and glued onto PCB 95 itself with an adhesive.

The four hundred PCB 95 pads arranged in areas 96 and 97 were connected to the 91 chip by known wire bonding techniques with aluminum wires, each of which was connected to a respective pad on either the 101 or 101a side of the chip. 95 . The wires were then coated with an epoxy resin that was made to polymerize in order to protect the wires themselves.

At this point, a 1 ml quantity of silver-containing paint was introduced into each of the four chambers 102 (FIG. 9) that were arranged at the corners of the chip 91 through four of the holes in the lid 94 (said holes are designated in figures 8 and 9 by the number 103). The paint was used to create an electrical connection between the silicon substrate 92 and the cap 94.

Figures 29 and 30 illustrate a variant of the chip 91. Such a variant can be produced and assembled to obtain the device 73 illustrated in Figure 31 in a similar manner to that described in Examples 1 to 3.

Example 4

This example describes the production of an intermediate plate 104 made of PMMA, a top plate 105 made of Plexiglas and a support plate 106 made of Plexiglas (FIG. 4).

Plates 104, 105 and 106 had a thickness of about 1 mm and were obtained by grinding. After milling, a satin finish was made to eliminate burrs derived from milling. Plates 104, 105 and 106 were then washed in an ultrasonic bath.

Plate 104 is illustrated in a top plan view in Figure 5 and in a bottom plan view in Figure 6. As can be readily seen, part of the channels (for example, 78, 80, 81), reservoirs (for example, 13, 7' and 14) and holes (for example, 79, 82, 107, 107', 107" and 108) of the device 73. Holes 107 were through holes passing through the thickness of plate 104 and were pairwise component elements of valve portions 74, 75, 76 and 77 (FIG. 21). Port 108 was provided at outlet 8; Through the hole 108, during use, the carrier liquid flows together with the particles C2. Holes 107' and 107" were through holes for connection to chip 91.

On the lower surface of the plate 104 around each pair of holes 107 and around the hole 108 a respective cavity 109 was provided. Each cavity was annular in shape and had a diameter of approximately 0.5 mm and a depth of approximately 0.25 mm. The presence of cavity 109 reduced the area that closure elements 30 and sealing ring 70 had to be pressed to remain in fluid-tight contact (with respect to closure elements 30 to close holes 107).

Plates 104, 105 and 106 had respective through holes 109. During assembly of device 75, plates 104, 105, and 106 were arranged such that fixed linear rods extended through holes 110; in this way, it was possible to align the plates 104, 105 and 106 precisely. Plates 104, 105 and 106 had respective central openings through which, once device 73 was assembled, it was possible to view the contents of chambers 4 and 5.

Plate 106 had openings 111 which, once device 73 was assembled, allowed closure elements 30 and sealing rings 66 and 70 to be exposed to the outside. Around each opening 111 there was a respective annular cavity, which allowed a better positioning and a better sealing of the closing elements 30 and of the sealing rings 66 and 70. In practice, said cavities functioned as housings for the closing elements. closure 30 and sealing rings 66 and 70.

Likewise, the aforementioned channels, deposits, cavities, openings and orifices were obtained by micromilling.

Figures 32 and 33 illustrate a variant of the plate 104. In this case, each cavity 109 has a perimeter channel 119, which is, in particular, substantially circular. For each valve portion 74, 75, 76 and 77, a hole 107 is provided at a position corresponding to the channel 119 and a hole 107 is provided in the cavity 109 outside the channel 119. This particular configuration allows the reduction of the disturbances (in particular, movement - suction - of fluid) during the opening of the valve V (figures 14 and 15). The opening of valve V is relatively gradual and therefore the negative pressure that is created in valve V itself during opening is relatively low.

Example 5

Closing elements 30, sealing rings 66 and 70, and a connecting element 112 were obtained by injection molding techniques known per se. The material used was treated with Elastosil® to obtain a degree of hardness of 60 Shore for the closure elements, 30 and 50 Shore for the sealing rings 66 and 70 and the connection element 112.

The connecting element 112 had a central opening 113 and through holes 114 which, once the device 73 was assembled, connected the chip 91 to the board 104. In particular, the holes 114 connected the holes 107' with the holes 115 of the substrate 92. and the holes 107" with the holes 116 of the substrate 92.

Example 6

This example describes the assembly of the various components described above to obtain the device 73. As already mentioned, fixed linear rods were used to align the various components.

Plates 104 and 105 were connected with ethanol linkage. A bi-adhesive layer 117 (Duplobond® manufactured by Elcom SpA - thickness: 0.325 mm) was applied to the upper face of the plate 106. The bi-adhesive layer 117 suitably configured (in particular, with a central opening and corresponding holes to the holes 110) was obtained, for example, from a continuous strip, which was cut by means of a laser or a punching machine.

Closure elements 30, sealing rings 66 and 70, and connecting element 111 were mounted on plate 106. At this point, an organosilane layer was deposited on the lower surface of plate 104 and was selectively removed by means of of plasma to form a bond only when necessary (a method for selective bonding between silicone elements and PMMA - polymethylmethacrylate is described in patent application number IT B02007A000588, the contents of which are fully retrieved herein for completeness of description) . In particular, the organosilane was removed or not applied in the areas corresponding to delivery ports 79 and 82 and ports 107, 107' and 107''. Closing elements 30, sealing rings 66 and 70 and connecting element 111 were activated by means of plasma. Plates 104 and 106 were brought into contact and pressed against each other.

At this point, a new bi-adhesive layer 118 (Duplobond® manufactured by Lohmann S.p.A. - thickness: 0.325 mm) was applied to the underside of plate 106.

The board 106 was then pressed against the top face of the PCB 95, the chip 91 having already been mounted on that face.

Example 7

This example describes the tests performed to optimize the operation of system 1.

The sedimentation of the particles C1 and/or C2 represents one of the causes of adhesion of the particles in the tank 13 and/or in the duct 78.

Normally, before the sample is introduced into the chamber 4, the sample itself remains in the reservoir 13 for a long time (in particular, about half an hour). During this period, the C1 and C2 particles settle to the bottom of the tank. To detach the particles from the background, a strong force is usually necessary. Furthermore, the C1 and C2 particles generally travel more slowly than the liquid portion of the sample within chamber 4. Therefore, the C1 and C2 particles enter chamber 4 when chamber 4 has already been occupied at least partly by the liquid part of the sample and they manage to distribute themselves only in the central part of chamber 4 and not uniformly (they do not manage to reach the peripheral corners of chamber 4). It should be noted that there is also a marked risk that not all C1 and C2 particles reach chamber 4.

The non-uniform distribution of the C1 and C2 particles within the chamber 4 makes the separation of the C1 particles from the C2 particles and the transfer of the C1 particles themselves to the chamber 5 more problematic.

Therefore, two load tests of the chamber 4 were carried out keeping the vibration device 17 (comprising a short-circuited Thinxxs® MDP2205 micropump) switched off during the first test and operating (at a frequency of 30 Hz) during the second. proof.

The samples used were prepared using a K562 cell culture (sample concentration was approximately 1250 particles/ml) labeled with DAPI.

The results of the tests with the vibration device 17 deactivated are illustrated in the photographs in the left column of figure 22. The results of the tests with the vibration device 17 activated are illustrated in the photographs in the right column of figure 22. The photographs were taken under different optical conditions.

As can be easily seen, when the vibration device 17 was activated, the particles C1 and C2 were distributed in a more homogeneous way also within the corners of the chamber 4, which led to a significant reduction of the unused volumes.

Claims (20)

1. A microfluidic system for the isolation of particles (C1) of at least one given type from a sample, the system (1) comprises: a first inlet (2), through which, during use, the sample in system (1); a separation unit (3), which is connected to the first inlet (2), comprises a main chamber (4) and a recovery chamber (5) and is designed to transfer at least part of the particles (C1) from the given type from the main chamber (4) to the recovery chamber (5) in a substantially selective manner with respect to more particles (C2) of the sample; a first outlet (7), connected to the main chamber (4); and a second outlet (8), which is connected to the recovery chamber (5), and, in particular, through which, during use, at least part of the particles (C1) of the given type collected in the recovery chamber (5) leave the system (1); the system (1) is characterized in that it comprises: a first valve (9), arranged upstream of the main chamber (4); that is, between the first entrance (2) and the main chamber (4); a second valve (10), placed between the main chamber (4) and the first outlet (7); a third valve (11), which is connected to the recovery chamber (5) and is placed between the recovery chamber (5) and a source of a carrier liquid; and a fourth valve (12), placed between the recovery chamber (5) and the second outlet (8); the recovery chamber (5) is placed between the main chamber (4) on one side and the third and fourth valves (11, 12) on the other side; the main chamber (4) is placed between the recovery chamber (5) on one side and the first and second valves (9, 10) on the other side. 2. The system according to claim 1, and comprising a dielectrophoresis system; the separation unit (3) comprises at least part of the dielectrophoresis system; the first and second valves (9, 10) are designed to regulate the inlet flow of the sample to the main chamber (4); the third and fourth valves (11, 12) are designed to regulate the inlet flow of the carrier liquid to the recovery chamber (5) and the outlet flow of the carrier liquid together with the particles (C1) of the given type from the chamber recovery (5) through the second outlet (8). 3. The system according to one of claims 1 or 2, and comprising a channel (6), connecting the main chamber (4) and the recovery chamber (5) and having dimensions smaller than those of the main chamber (4) and the recovery chamber (5). 4. The system according to any of the preceding claims, wherein the first valve (9) is arranged between the first inlet (2) and the main chamber (4), in particular, between the first reservoir (13) and the main chamber (4), and is designed to connect or isolate the first entrance (2) and the main chamber (4) with respect to each other. 5. The system according to any of the preceding claims, and comprising a first tank (13), which is arranged between the first inlet (2) and the first valve (9) and is designed to collect the sample introduced by the first entry (2); and a second reservoir (14) to contain the carrier liquid, which is designed to fill the recovery chamber (5). 6. The system according to any of the preceding claims, and comprising: a first tank (13), which is arranged between the first inlet (2) and the first valve (9) and is designed to collect the sample introduced by the first input (2); and a second tank (14) to contain the carrier liquid, which is designed to fill the recovery chamber (5); the system (1) comprises a first pressure source (15) to impose a pressure difference between the first reservoir (13) and the main chamber (4), and a second pressure source (25) to impose a pressure difference between the second tank (14) and the recovery chamber (5). 7. The system according to claim 6, and comprising: a first conduit (16), which connects the first pressure source (15) to the main chamber (4) and next to which the first reservoir (13) is arranged ) and the first valve (9); and a second conduit (26), which connects the second pressure source (25) to the recovery chamber (5) and next to which the second tank (14) and the third valve (11) are arranged. 8. The system according to claim 6 or 7, and comprising a vibration device (17), which is designed to impose a vibration on the sample at least in an area from the first entrance (2) to the main chamber (4) and is disposed between the first pressure source (15) and the first reservoir (13). 9. The system according to any of the preceding claims, and comprising: a first tank (13), which is arranged between the first inlet (2) and the first valve (9) and is designed to collect the sample introduced by the first input (2); a first conduit (16) for connecting the first reservoir (13) to the main chamber (4); and a third conduit (20), which is arranged between the main chamber (4) and the first outlet (7) and has a smaller cross-section, in particular by at least 100 mm, than the cross-section of the first conduit (16). ). The system according to claim 9, and comprising: a sensor for directly or indirectly detecting when the sample begins to enter the third conduit (20); a blocking device (22; 9) to block the inflow of the sample towards the main chamber (4); and a control assembly (23), connected to the sensor and to the blocking device (22; 9) to actuate the blocking device (22; 9) depending on what has been detected by the sensor. The system according to claim 10, and comprising a first pressure source (15) for imposing a pressure from the first reservoir (13) towards the main chamber (4); the first reservoir (13) is disposed between the first pressure source (15) and the first valve (9); the blocking device (22) comprises a relief valve (22'), in particular arranged between the first pressure source (15) and the first reservoir (13), said relief valve (22') is actuated by the assembly (23) and is designed, when actuated, to put the pressure source (15) in communication with the outside world to substantially zero the pressure from the first reservoir (13) to the main chamber (4). 12. The system according to any preceding claim, and comprising: a third outlet; and a sixth valve (29), which is arranged between the recovery chamber (5) and the third outlet; optionally the third output matches the first output (7). 13. The system according to any of the preceding claims, comprising a vibration device (17), which is designed to cause an oscillating variation of the pressure at least in an area from the first inlet (2) to the main chamber (4). 14. The system according to any of the preceding claims, wherein at least one of the valves (9; 10; 11; 12) comprises: a diaphragm (33), which is arranged between two sections (31,32) of a conduit; a closure element (30), which comprises a substantially elastic material and is designed to pass between a locking position, where the closure element (30) is in contact with the diaphragm (33) to separate the two sections (31 , 32), and an open position, in which the closing element (30) is arranged at a distance from the diaphragm (33) and the two sections are connected to each other; a first mechanical pressure element (34) to push the closure element (30) towards the diaphragm (33) to maintain the closure element (30) in the locking position; and a fluid dynamic actuator (35) to bring the closing element from the blocking position to the open position. The system according to claim 14, wherein the fluid dynamic actuator (35) comprises a suction unit (41), in particular it comprises a pump (43). The system according to any of the preceding claims, and comprising a cooling assembly (50), which is designed to cool at least part of the separation unit (3). The system according to claim 16, wherein the cooling assembly (50) comprises a cooling plate (51) having: an active surface (52) designed to absorb heat from the separation unit (3); and a discharge surface (53) for producing heat; the active surface (52) has smaller dimensions than the discharge surface (53). The system according to claim 16 or claim 17, and comprising a third mechanical pressure element (60) to push the cooling assembly (50) towards the separation unit (3). 19. The system according to any of the preceding claims, comprising a control assembly (23) connected to said valves (9, 10, 11, 12) to open and close the valves themselves (9, 10, 11, 12 ); a collection unit for collecting the carrier liquid containing at least part of the particles (C1) of the given type coming out of the second outlet; and a detector for detecting when at least one drop of said carrier liquid enters the collection unit; The control assembly (23) connects to the detector and is designed to close the third valve (11) and/or the fourth valve (12) when the drop is detected. 20. The system according to any of the preceding claims, comprising a second vibration device (17a), which is designed to cause an oscillating variation of the pressure in at least one area of the recovery chamber (5) .