Background art

The development of drugs is a lengthy and costly process. One of the main reasons a drug fails during clinical studies is the lack of efficacy in the required therapeutic application. Computer-Aided Drug Design, pharmacokinetics in- silico and toxicity in vitro tests are increasingly applied to deal with the costly pre-clinical steps with as much information as possible.

By virtue of the progress in cellular biology, in micro-fabrication techniques and tissue engineering, it has been possible to develop a wide range of 3D cellular culture strategies. However, many of the techniques currently available for this type of culture have low reproducibility, high costs and take time, while quick and standardized protocols are absolutely essential for this type of application. Consequently, research is increasingly moving to 3D culture systems, which allow cultures on a micro-scale, compatible with the automated screening of the high-throughput (HTS) type, enabling information to be obtained on the reaction of molecules of potential pharmacological interest in a context as close as possible to that of physio-pathological interest. Current robotic systems, needed to implement HTS methods, have different limits, including high technological costs, difficulty in obtaining a quick and precise dispensing of small volumetric amounts of liquids, rapid evaporation of the dispensed volumes.

Microfluidics deals with processing or handling small amounts of fluids (from 10 ³ to 10 ¹² liters), exploiting channels having small dimensions, in the order of hundreds of micrometers.

Microfluidic devices allow screening tests to be carried out in a continuous flow, allowing the processing and analysis, in series, on a single chip, thus presenting itself as a valid alternative to HTS.

Among the various components, comprising the microfluidic devices, valves, mixers and pumps are probably the most relevant, since they allow the introduction of reagents and samples, the movement of fluids inside the chip and the combination and mixing thereof, respectively. Micro-valves, a key element for controlling flows, prevent crossed contamination, typically via a binary mode of regulating, activating and deactivating or selectively closing specific paths when working in continuous mode. However, the efficacy thereof is ensured only in conditions of low losses, low dead volume, negligible sensitivity with contamination due to particles, low response times and linear operations, all evaluated with respect to the sizes and operating features of the system at stake.

Mechanical micro-valves are mainly divided into two categories: active and passive. Both control the flows using mechanical- and non-mechanical-type movable parts, but while the first require a certain energy consumption, the second exploit the internal pressure of the fluid to ensure the movement thereof.

Both types require a precise multi-layer micro-construction, and therefore are not suitable for rapid production; furthermore, the design options, as well as the micro-construction techniques require the valve to be made in the same way and at the same time as the system in which it is to be integrated. Thus, in the last few years, researchers have focused on developing versatile and low-cost micro-valves, which could also be used and made by non-skilled personnel. Guler et al. 2013 (Sensors Actuators, A Phys., vol. 4, pp. 1-30) describe a micro-valve comprising a rigid cylindrical rod with a through hole, which is positioned in a microfluidic channel. By rotating the rod, which aligns or misaligns the through hole, which is the valve door, the passage with the microchannel is opened or closed. The rod consists of a rigid material and is associated with a microchannel made of an elastic material.

Hickerson et al. 2013 (Sensors Actuators, A Phys., vol. 203, pp. 76-81) describe a miniaturized passive valve, comprising a body, which is a hollow cylindrical nucleus, closed at one end, with a side light and a cylindrical elastomeric sleeve placed above the central body, which covers the side light. The system operates by exerting a pressure, which varies the diameter of the core, so as to open or close a gap between this and the elastomeric sleeve. The systems of the known art, despite perfectly overcoming the problem of the opening and closing of a micro-valve, do not allow a precise control of the dispensed fluid volumes. In fact, the described devices are not capable of controlling the dispensing of a bolus, but simply have the function of a valve, which regulates between the open status and the closed status. Control of the dispensed volume is assigned to an external control system, by way of example, a syringe pump or a feedback actuator with a flowmeter. This type of control is refined, but requires components, which are not suitable for multiplication, for serving different users, in parallel, as required in an HTS context. Therefore, a microfluidic metering valve is the object of the present invention, which overcomes the problems of dispensing controlled and multiple volumes in microfluidics.

Description of the invention Description of the drawings Figure 1: a kit according to an embodiment with a body made of elastomer: (A-C) exploded; (D) assembled.

Figure 2: a kit according to further embodiments, further comprising a support: (A) realization with 4 outlets; (B) realization with 9 outlets; (C) realization with 4 outlets, where the body comprises empty volumes; (D) realization with 9 outlets, where the body comprises empty volumes; assembled (E) and (F) exploded view of an embodiment with 9 outlets with empty volumes and rigid fins.

Figure 3: a kit according to a further embodiment, with a body made of a rigid material: (A) exploded; (B) assembled Figure 4: a kit integrated in a microfluidic system.

Figure 5: a kit integrated in a microfluidic system, comprising two circuits, a diagrammatic view of the operating steps: (A) portion-bolus in connection with a first circuit; (B) movement of the movable insert; (C) portion-bolus in connection with a second circuit; (D) movement of the movable insert. Figure 6: a kit integrated in a microfluidic system, comprising two circuits, a diagrammatic view of the operating steps: (A) portion-bolus in connection with a first circuit; (B) movement of the movable insert; (C) portion-bolus in connection with a second circuit.

Figure 7: a kit integrated in a microfluidic system, comprising two circuits, with inlet and outlet of the fluid managed in parallel in the first circuit, in series in the second circuit.

Figure 8: a kit integrated in a microfluidic system, comprising three circuits. Figure 9: (A), (B) photographs representative of two embodiments of the kit according to the present invention.

Figure 10: a kit according to a further embodiment (A) perspective view; (B) sectional view from above; (C) exploded; (D) detail of the movable insert, exploded and assembled.

A metering system for the controlled and multiple dispensing of micro-volumes of fluids is the object of the present invention.

For the purpose of the present invention, the terms “metering system” and “kit” are used indifferently.

Said metering system 1 , with reference to figure 1 , comprises:

- a movable insert 101 (figure 1A), which is a tube in which at least two separators 102, 103 (figure 1 B, which shows said separators in the phase of insertion in said tube) delimit at least a portion of said tube defined as portion- bolus 104, where said portion-bolus defines a volume, which is the volume to be dispensed, or bolus, characterized in that on the lateral surface of said portion-bolus there are two holes, an inlet hole 106 and an outlet hole 107 (figure 1 C);

- a body 110 (figure 1 C, 1 D), inside said body 110 being obtained:

- at least one channel, said dispensing channel 111 , where said dispensing channel 111 passes through said body, partially or for the whole length and has an inlet 112 and, where said dispensing channel 111 passes through said body for the whole length, an outlet 113, open towards the environment external to said body;

- at least four channels, two inlet channels 121 and two outlet channels 122, which are in fluidic communication with said dispensing channel 111 and with the environment external to said body; where said movable insert 101 moves in said dispensing channel 111 independently by sliding, by translation and/or by rotation; wherein said movable insert 101 is made of a material, which has a different stiffness to the material with which it is in contact when inserted in said dispensing channel 111 ; where said dispensing channel 111 sealably accommodates said movable insert 101.

The diameter of the movable insert 101 exceeds that of the base of the dispensing channel 111 by a difference comprised between 0 and 25% of the diameter of the dispensing channel, preferably comprised between 0 and 15%. A person skilled in the art understands that said difference between the diameters of the movable insert and the dispensing channel, able to ensure the seal, depends on the deformability of the materials. By way of example, where said movable insert were in contact, when inserted in said dispensing channel, with a highly deformable material, said difference would come close to the upper margin of the indicated range.

A person skilled in the art also knows how to correctly interpret the manufacturing tolerances so as to select the correct diameters of the movable insert in relation to the dispensing channel.

In an embodiment, said movable insert 101 comprises a portion-bolus 104. In an embodiment, said movable insert comprises two, or three, or multiple portions-bolus.

In an embodiment, advantageously, said inlet hole 106 and said outlet hole 107 are obtained in longitudinally opposite positions in said portion-bolus 104, i.e. a hole close to said separator 102, a hole close to said separator 103. In an embodiment, advantageously, said inlet hole 106 and said outlet hole 107 are obtained in diametrically opposite positions on said portion-bolus 104.

In an embodiment, said movable insert 101 is at least partially made of a semi rigid material, preferably it is made of a semi-rigid material and said body 110 is made of an elastomeric material, or said body 110 is a set of several parts, at least one of these made of an elastomeric material.

In a further embodiment, said body 110 is at least partially made of a semi- rigid or rigid material, preferably it is made of a rigid material and said body comprises a material, which is deformable in contact with the movable insert, made of semi-rigid material. For example, with reference to figure 3A, where it is exploded, and to figure e3B, where it is assembled, said body 310 comprises a sheath 330 made of a deformable material in said dispensing channel 311. In an embodiment, said sheath 330 is integral with the body. In an alternative embodiment, said sheath 330 is integral with the movable insert.

Said semi-rigid or rigid material is, for example, PTFE.

Said elastomeric material is selected in the group that comprises TPE thermoplastic elastomers, selected, for example, from thermoplastic polyolefins, thermoplastic polyurethane, thermoplastic polyamide, SBS, SEBS or SEPS styrenic compounds, vulcanized PP/EPDM compounds; co-polyester compounds; liquid silicone rubber (LSR); PMDS.

In an embodiment, with reference to figure 2, said body 210 is accommodated in a support 225.

By way of example, said support 225 is a resin tray, where the material, forming said body 210, is poured, in which said inlet 221, outlet 222 dispensing channels 211 are preformed.

In an embodiment, figure 2C, 2D said body 210 comprises empty volumes 226 intersecting said dispensing channel through the lateral surface of the channel itself. Said empty volumes 226 are obtained in zones of said body 210 in an opportune position so as not to interfere with said inlet channels 221 and outlet channels 222. Preferably, said empty volumes are strengthened with rigid material, for example, fins made of rigid material 227. In an embodiment, said rigid strengthening material does not reach said dispensing channel 211.

In an embodiment, figure 2E, 2F, advantageously, said fins 227 are arranged on a cap 228, which resting on said support 225, allows said fins 227 to occupy said empty volumes 226 in the desired position, i.e. without interfering with said dispensing channel 211. The embodiment with empty volumes has proven to be particularly effective in reducing the friction between the movable insert 201 and the body 210 and ensuring an effective movement of said movable insert in the body. The photograph in figure 9 is representative of an embodiment of the metering system according to the present invention, inserted in a support, in the embodiment with 9 channels, with empty volumes and fins (figure 9A) or with 4 channels, with empty volumes and fins (figure 9B). Advantageously, said kit 1 is integrated in a microfluidic system. Said microfluidic system comprises at least two fluidic circuits. Said microfluidic system comprises connectors on inlet, which supply said inlet channels and connectors on outlet, which collect the fluid from said outlet channels.

With reference to figures 4 - 9, each of said inlet channels 421 , 521 , 621 , 721 , 821 obtained in said body is advantageously supplied, through an inlet connector 437, 537, 637, 737, 837 by an advantageously moved fluid. Optionally, said fluid is contained in reservoir 440, 540, 640, 740, 840. Advantageously, each of said outlet channels 422, 522, 622, 722, 822 obtained in said body is in microfluidic connection, through an outlet connector 438, 538, 638, 738, 838 with a microfluidic chip 436, 536, 636, 736, 836 and/or with a well, and/or a collector container 439, 539, 639, 739, 839.

In an embodiment, said microfluidic system comprises two circuits. In an embodiment, said microfluidic system comprises three or more circuits. Said circuits are able to manage one or more fluids, which are equal, or different to one another.

In an illustrative and non-limiting embodiment of the invention, with reference to figure 4, said system comprises two circuits: a first circuit 461 comprising a first reservoir 440, which contains a first fluid 451 , which in this particular case, is a growth medium and a second circuit comprising a second reservoir 440, which contains a second fluid 452, preferably a fluid comprising at least one active. Said first reservoir 440 is connected via an inlet connector 437 to an inlet channel 421 to the portion-bolus 404. When the metering system 1 is in the open position on said first circuit 461 , i.e. when the inlet hole is at the inlet channel 421 connected to said inlet connector 437 and the outlet hole is at said outlet channel 422, the first fluid 451 reaches a microfluidic chip 436 from said first reservoir 440. By positioning the metering system in open position on said second circuit 462, said second fluid 452 fills the portion-bolus 404, discharging said first fluid 451 in a collector container 439. By repositioning the metering system in the open position on said first circuit, said first fluid 451 pushes the bolus, i.e. the volume of the portion-bolus 404, which is occupied by the second fluid 452, in the microfluidic chip 436. In the embodiment in figure 4, said metering system manages three boluses at the same time, where said movable insert comprises three portions-bolus, thus allowing a simultaneous dispensing in three microfluidic chips. Advantageously, the number of portions-bolus is increased so as to manage, in parallel, a greater number of microfluidic chips. In a further embodiment, schematized in Figure 10, said metering system comprises:

- a movable insert 1001 , which is a tube in which at least one portion of said tube, defined as portion-bolus 1004, is delimited by two separators, which are caps 1002 and 1003, the position of which inside the movable insert 1001 is defined by two movable elements 1005 and 1008, where said portion-bolus defines a volume, which is the volume to be dispensed, or bolus, characterized in that, on the lateral surface of said portion-bolus there are two holes, an inlet hole 1006 and an outlet hole 1007;

- a body 1010, inside said body 1010 being obtained: - at least one channel, said dispensing channel 111, where said dispensing channel 111 passes through said body, partially or for the whole length and has an inlet 112 and, where said dispensing channel 111 passes through said body for the whole length, an outlet 113, open towards the environment external to said body; - at least four channels, two inlet channels 121 and two outlet channels 122, which are in fluidic communication with said dispensing channel 111 and with the environment external to said body; where said movable insert 1001 moves independently in said dispensing channel 111 by sliding, by translation and/or by rotation; where said movable insert 1001 is made of a material which has a different stiffness than the material with which it is in contact when inserted in said dispensing channel 111; where said dispensing channel 111 sealably accommodates said movable insert 1001.

The diameter of the movable insert 1001 exceeds that of the base of the dispensing channel 111 by a difference comprised between 0 and 25% of the diameter of the dispensing channel, preferably comprised between 0 and 15%. In said embodiment, the kit according to the present invention comprises, for example, two independent portions-bolus 1004 and is integrated in a microfluidic system, comprising two circuits. A first circuit 1061 with inlet connectors 1037 in parallel, a second circuit 1062 with inlet connectors 1037 in series. In this embodiment, each portion-bolus is translated by means of an actuator 1014, integrally with the movable insert and with the other portions- bolus. The fluid flow of said second circuit 1062 is managed serially, one bolus after the other, i.e. in the open position of said second circuit 1062, the second fluid fills all the portions-bolus, sequentially, through an inlet channel 1037, inter-bolus connection channels 1023, an outlet channel 1038. The first circuit 1061 manages the flow in parallel as described.

This embodiment is particularly advantageous as it allows independently managing and modulating the volumes of fluid, allowing the management of multiple combinations of fluids and different administration times.

A method for dispensing a constant and controlled volume of fluid is a further object of the present invention.

Said method comprises:

- Providing a metering system 1 according to the present invention;

- Integrating said metering system in a microfluidic system;

- Sliding, translating and/or rotating said movable insert in said dispensing channel to put at least one portion-bolus in fluidic communication with a first circuit;

- Sliding, translating and/or rotating said movable insert in said dispensing channel, to put at least one portion-bolus in fluidic communication with a second or further circuit.

With reference to figure 5, said method comprises:

- Providing a metering system 1 according to the present invention; - Integrating said metering system in a microfluidic system;

- Sliding, translating and/or rotating said movable insert 501 in said dispensing channel 511 so as to put at least one portion-bolus 504 in fluidic communication with a first circuit 561 , figure 5A;

- Sliding, translating and/or rotating said movable insert 501 in said dispensing channel 511 , figure 5B, to put at least one portion-bolus 504 in fluidic communication with a second circuit 562, figure 5C;

- Sliding, translating and/or rotating said movable insert 501 in said dispensing channel 511 , figure 5D, so as to reposition said at least one portion-bolus 504 in fluidic communication with said first circuit 561 , figure 5A.

When said portion-bolus 504 is in fluidic communication with said first circuit, figure 5A, said portion-bolus is loaded with a first fluid. When said portion-bolus is in fluidic communication with said second circuit, figure 5C, said portion- bolus is loaded with a second fluid. By repeating the movements, an advantageous release of the bolus is managed.

The movement of said movable insert is obtained with methods known to a person skilled in the art. For example, the translation is carried out through screw precision positioners or it is carried out automatically by using a linear actuator, managed with a closed loop system having a webcam or sensor system. In figure 5, it is obtained with a handler 514, which is a linear actuator. With reference to the specific example in figure 5, which should not be considered limiting of the scope of the invention, when said first circuit 561 is open, position in figure 5A, the fluid from said reservoir 540, which is the first fluid 551 , completely fills the volume of the portion-bolus 504 until it comes out into the outlet channel 522 and reaches the microfluidic chip 536. Moving by translation, the movable insert results in the opening of the second circuit 562, figure 5C. The second fluid 552, which, in the case in the example, is fluid comprising at least one active, fills the volume of the portion-bolus, replacing the first fluid 551 , which is pushed into the collector container 539. By bringing the movable insert back into such a position as to allow the closing of the second circuit 562 and the opening of the first circuit 561 , the first fluid 551 , flowing through said portion-bolus, brings said bolus into said microfluidic chip. With reference to figure 6, a kit is shown according to the present invention integrated in a system comprising two circuits in the operating steps thereof. The movable insert 601 is controlled by a handler 614, which causes it to move by rotation. Figure 6A shows the first circuit open position, figure 6B the rotation of the movable insert, figure 6C the second circuit open position. Advantageously, the inlet channels 621 and the outlet channels 622 are obtained in said body so as to allow the connection with said inlet holes 606 and outlet holes 607. In particular, said inlet channel 621 of said first circuit is in a diametrically opposite position to said dispensing channel of said inlet channel 621 of said second circuit.

In a further embodiment, with reference to figure 7, the kit according to the present invention comprises three portions-bolus 704 and is integrated in a microfluidic system comprising two circuits. A first circuit 761 with inlet connectors in parallel, a second circuit 762 with inlet connectors in series. In this embodiment, each portion-bolus is translated integrally with the movable insert and with the other portions-bolus. The fluid flow of said second circuit is managed serially, one bolus after another, i.e. in the open position of said second circuit, the second fluid fills all the portions-bolus, sequentially, through an inlet channel 721, inter-bolus connection channels 723, an outlet channel 722. The first circuit manages the flow in parallel, as described.

This embodiment is particularly advantageous since it allows minimizing the volumes of fluid managed, using the volume of fluid of the boluses, minimizing waste. A further embodiment, with reference to figure 8, shows a kit integrated in a system with three circuits, which, advantageously, simultaneously allows managing three different fluids.

In said embodiment, said at least one portion-bolus 804 allows opening said first circuit 861 , said second circuit 862 or said third circuit 863, thus allowing three different fluids contained in the three reservoirs 840 to be dispensed, each of these in fluidic connection with one of the three circuits. Advantageously, the method and kit according to the present invention allow managing multiple combinations of fluids and different administration times. Advantageously, the control of the volume of an active is regulated exclusively by the volume of said portion-bolus, thus allowing the dispensing to be managed precisely and simply. The kit is inserted in any microfluidic system without any particular technological requirements.

A person skilled in the art understands how the metering system of the present invention, integrated in microfluidic systems, can advantageously manage the dispensing of defined doses of an active, without any limitations, for example, repeated in time, or of different actives, or washing steps, or again the sowing of controlled volumes of a cellular suspension.

Numbering

1 = metering system, or kit 101, 201, 401, 501, 601, 1001 = movable insert 102, 103 = separators

104, 404, 504, 704, 804, 1004 = portion-bolus

106, 406, 606, 1006 = inlet hole

107, 407, 607, 1007 = outlet hole 110, 210, 310, 1010 = body 111, 211, 311, 411, 511 = dispensing channel

112 = dispensing channel inlet

113 = dispensing channel outlet

121, 221, 421, 521, 621, 721, 821 = inlet channels

122, 222, 422, 522, 622, 722, 822 = outlet channels 225, 1025 = support

226 = empty volumes

227 = fins

228 = fin holder cap 330 = sheath 436, 536, 636, 736, 836 = microfluidic chip

437, 537, 637, 737, 1037 = inlet connectors

438, 538, 638, 738, 838, 1038 = outlet connectors

439, 539, 639, 739, 839 = collector container

440, 540, 640, 740, 840 = reservoir 451, 551 = first fluid

452, 552, 752 = second fluid

461, 561, 661, 761, 861, 1061 = first circuit

462, 562, 662, 762, 862, 1062 = second circuit 863 = third circuit 514, 614, 714, 814, 1014 = handler

723, 1023 = inter-bolus connection channels

1002, 1003 = caps

1005, 1008 = movable elements