Devices, Systems, and Methods for Dispensing and Analyzing Particles

Abstract

The present invention relates to a pipette tip comprising a thin holed membrane at its distal end, which is designed to be adapted with a system comprising at least an impedance analyser and a fluidic actuator to perform the dispensing and analysis of particles comprised within a conductive medium by exploiting the Coulter counter principle. The pipette tip can comprise attached or floating electrodes at its internal or external side for creating an electrical circuit. Also disclosed therein is a dispensing and analysis system, methods of using thereof and pipette tip's manufacturing methods.

Claims

1-16. (canceled)

17. A pipette tip for dispensing a conductive medium and particles, the pipette tip comprising: a proximal end; an elongated body configured to retain the conductive medium and the particles; and a distal end having a flow opening configured to dispense the conductive medium and the particles, the distal end being closed at an extremity by a membrane having an orifice, the orifice permitting a passage of the particles one at a time with the conductive medium, wherein a ratio between a diameter of the membrane and a diameter of the orifice is at least 6.32.

18. The pipette tip according to claim 17, wherein the ratio between the diameter of the membrane and the diameter of the orifice is at least 10.

19. The pipette tip according to claim 17, wherein a ratio between a surface area of the proximal end and a surface area of the distal end is at least 5.

20. The pipette tip according to claim 17, wherein a ratio between the diameter of the membrane and a thickness of the membrane is at least 5.

21. The pipette tip according to claim 17, wherein the proximal end is configured to connect to a fluidic actuator and an electrical impedance analyzer.

22. The pipette tip according to claim 17, further comprising: an electrode operatively connected to at least one of the elongated body, the distal end, the membrane, and a sterility filter, wherein the electrode is configured to be electrically activated once the pipette tip is operably connected to an electrical impedance analyzer.

23. A method of manufacturing a pipette tip, the pipette tip including a proximal end, an elongated body configured to retain a conductive medium and particles, and a distal end having a flow opening configured to dispense the conductive medium and the particles, the distal end being closed at an extremity by a membrane having an orifice, the orifice shaped to permit a passage of the particles one at a time with the conductive medium, the method comprising the step of: injecting a liquid plastic material into a mold defining walls of the elongated body and the membrane, the mold being shaped according to a desired form and thickness of both the elongated body and the membrane.

24. The method of claim 23, wherein in the step of injecting the liquid plastic material, the orifice is formed in the membrane by a shape of the mold.

25. The method of claim 23, further comprising the steps of: opening the orifice in the membrane; and sealing the membrane to the distal end of the plastic pipette tip.

26. The method of claim 23, further comprising the step of: applying an electrode on the plastic pipette tip, the electrode configured to connect to an electrical impedance analyzer.

27. A method of manufacturing a pipette tip, the pipette tip including a proximal end, an elongated body configured to retain a conductive medium and particles, and a distal end having a flow opening configured to dispense the conductive medium and the particles, the distal end being closed at an extremity by a membrane having an orifice, the orifice shaped to permit a passage of the particles one at a time with the conductive medium, the method comprising the steps of: injecting a liquid plastic material into a mold defining walls of the elongated body, the mold shaped according to a desired form and thickness of the elongated body to obtain a plastic pipette tip; temporary closing the distal end of the plastic pipette tip with a closure; depositing a film of plastic material on the walls of the elongated body and the distal end of the plastic pipette tip to define a membrane at an extremity of the distal end; removing the closure at the distal end of the plastic pipette tip; and opening an orifice on the membrane.

28. The method of claim 27, further comprising the step of: applying an electrode on the plastic pipette tip, the electrode configured to connect to an electrical impedance analyzer.

29. A system for dispensing and analyzing particles through impedance-based measurements, the system comprising: a pipette tip including a proximal end, an elongated body configured to retain a conductive medium and the particles, and a distal end having a flow opening configured to dispense the conductive medium and the particles, the distal end being closed at an extremity by a membrane having an orifice, the orifice shaped to permit a passage of the particles one at a time with the conductive medium, a ratio between a diameter of the membrane and a diameter of the orifice is at least 6.32. an electrical impedance analyzer configured to connect to the pipette tip; a fluidic actuator configured to connect to the proximal end of the pipette tip; and a controller configured to control the fluidic actuator and the electrical impedance analyzer.

30. The system according to claim 29, further comprising: a computer device operably connected to the electrical impedance analyzer for at least one of analyzing and storing impedance data.

31. The system according to claim 29, wherein the fluidic actuator includes two pressure sources and a controller for modifying a pressure inside the pipette tip.

32. The system according to claim 29, wherein the fluidic actuator includes: at least two air pumps operably connected to the pipette tip with a three-way valve such that at least one pump generates a positive pressure and at least one pump generates a negative pressure inside the pipette tip.

33. The system according to claim 29, further comprising: a hydrostatic pressure sensor located within the pipette tip and in proximity of the orifice.

34. The system according to claim 29, wherein the controller controls an activity of the fluidic actuator in response to a measurement performed by the electrical impedance analyzer.

35. A method for dispensing and analyzing particles through a dispensing system, the method comprising the steps of: providing a pipette tip filled with a conductive medium and particles before or after connection with a fluidic actuator, the pipette tip including, a proximal end, an elongated body configured to retain the conductive medium and the particles, and a distal end having a flow opening configured to dispense the conductive medium and the particles, the distal end being closed at an extremity by a membrane having an orifice, the orifice shaped to permit a passage of the particles one at a time with the conductive medium, a ratio between a diameter of the membrane and a diameter of the orifice is at least 6.32; connecting the pipette tip with both a fluidic actuator and an electrical impedance analyzer operably connected to at least two electrodes, one electrode located inside the pipette tip and another electrode located outside the pipette tip; inserting the distal end of the pipette tip inside a reservoir having a conductive medium; modifying a pressure inside the pipette tip so that the conductive medium and the particles located inside the tip are dispensed into the reservoir through the orifice; detecting a change in impedance through an electrical impedance analyzer when at least one particle passes through the orifice on the membrane; and stopping the dispensing into the reservoir of the conductive medium and the particles located inside the tip.

36. The method of claim 35, wherein the step of modifying, detecting, ad stopping are controlled by a controller programmed such that the dispensing of the conductive medium and the particles is controlled by an activity of the fluidic actuator in response to measurements performed by the electrical impedance analyzer.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0078] FIG. 1 shows a cross-section of the restricted tip;

[0079] FIG. 2 shows the restricted tip connected to the system for particle dispensing and analysis;

[0080] FIG. 3 shows the sensing tip and its multiple electrodes arrangements;

[0081] FIG. 4 shows the sensing tip connected to the system for particle dispensing and analysis;

[0082] FIG. 5 shows the connector and illustrates how it connects with the sensing tip;

[0083] FIG. 6 illustrates the sealing process of the membrane on a conventional tip;

[0084] FIG. 7 illustrates the layer deposition process using a temporary external closing element;

[0085] FIG. 8 illustrates the layer deposition process using a temporary internal closing element;

[0086] FIG. 9 shows the block diagram of one embodiment of the fluidic actuator;

[0087] FIG. 10 shows the block diagram of another embodiment of the fluidic actuator;

[0088] FIG. 11 shows the block diagram of the impedance analyser;

[0089] FIG. 12 shows the algorithm for particle dispensing and analysis;

[0090] FIG. 13 illustrates the sequence of events for dispensing and analysis of particles;

[0091] FIG. 14 shows a graph based on a simulation for a micrometric bead flowing through a cell sensor according to the invention in the form of a nozzle-like aperture or of a holed membrane. The graph highlights the high impedance sensitivity dependence on the electrodes positioning for the nozzle-like topology compared to the holed membrane;

[0092] FIG. 15 shows one embodiment of a sensing tip according to the invention;

[0093] FIG. 16 (a) shows a recording graph of the normalized impedance of 6 m-beads flowed through a sensing tip. Each peak corresponds to the passage of a particle that can be clearly distinguished; (b) Magnified impedance peak of one single bead.

[0094] FIG. 17 shows an impedance graph of the sensing tip according to the invention for different electrodes position inside the tip itself.

DESCRIPTION OF EMBODIMENTS

[0095] The present disclosure may be more readily understood by reference to the following detailed description presented in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

[0096] As used herein and in the appended claims, the singular forms a, and, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a cell includes a plurality of such cells and reference to an electrode includes reference to one or more electrodes, and so forth.

[0097] Also, the use of or means and/or unless stated otherwise. Similarly, comprise, comprises, comprising include, includes, and including are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term comprising, those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language consisting essentially of or consisting of.

[0098] Tip Structure

[0099] As used herein, a pipette tip refers to a tool (named tip) usually used in combination with a pipettor or chemical dropper, a laboratory tool used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser. A tip has an elongated, substantially tubular body that has a bottom opening (hereinafter, distal end) at the bottom end for the flow passage of a liquid, a top opening (hereinafter, proximal end) at the top end for the passage of air, and a passageway (hereinafter, elongated body) between the bottom opening and top opening for the retention of a liquid inside the tip defined by at least one delimiting wall. A pipette tip is characterized by a tubular body usually consisting of a transparent or translucent material such as glass or a (thermo)plastic material which is of a substantially cylindrical, conical or frustum (such as frusto-conical or frusto-pyramidal) shape, even if other topologies can be envisaged such as a nozzle-like design. A pipette tip can comprise at its proximal end a filter which ameliorates the sterility thereof once coupled with a fluidic actuator such a pipettor, but in the frame of the present invention it can also act as a support for placing at least one electrode as described below in more details.

[0100] A pipette tip, such as those of the invention, can be molded as a unitary, integral body of plastic from any suitable standard polymer material known in the art (e.g., polypropylene, polystyrene, polycarbonates, polysulfones, polyesters, cyclic olefins and so forth) using well-known injection molding methods. However, other materials, as well as further manufacturing methods, can be envisaged for the manufacturing of the article, such as for instance glass. The polymeric material chosen to form the tip must be nonreactive with, insoluble in, and impervious to the materials that come into contact with the pipette tip, such as cells, media such as culture media and/or further chemicals/biologic materials. Actually, a pipette tip can be made of any suitable material as long as it keep the ability of retaining and release its content on demand. Preferably, a pipette tip is a plastic pipette tip which is biocompatible, preferably also easily sterilisable (such as e.g. with irradiation by gamma-rays) and preferably also disposable, in order to avoid (cross)contamination with several samples and/or environmental pollutants.

[0101] A tip according to the present invention (FIG. 1) comprises a body 1 and a thin membrane 2 which closes the extremity of the distal end. In the frame of the present disclosure, a membrane is a blocking element which is placed at the opening of the distal end of a pipette tip and completely impedes the flow of a liquid through it. More precisely, in the present document, the membrane has to be understood as a substantially planar element, such as a disc, located within the tip distal end. The membrane surface is therefore approximatively equivalent to the internal surface of the tip distal opening. Such a membrane may be therefore defined as a floating element because its surface is not fixed to the tip body 1. The membrane is characterized by a specific ratio between its diameter and its thickness, which must be of at least 5, preferably at least 10 and most preferably at least 20. Said membrane has on its surface a small orifice 3 which is shaped in order to permit the passage, during a dispensing phase, of a liquid such as an electrical conductive medium and of particles (such as for instance cells or micro/nanobeads) comprised therein one at a time, provided that the size and the shape of the orifice 3 is such to permit the passage of said particles. Such a tip will be referred to hereinafter also as a restricted tip.

[0102] For the sake of clarity, a dispensing phase as used herein refers to a process of releasing a conductive medium contained within the tip of the invention through the application, as will be detailed later on, of a positive pressure inside the tip, allowing the flow and therefore the release of at least a part of its content. However, for dispensing phase is also herein meant, mutatis mutandis, the opposite process of aspiring a conductive medium, possibly comprising particles dispersed therein, from a container or a reservoir into the body 1 of the tip, through application of a negative pressure within the tip itself. A skilled person would easily envisage how to adapt the system and the methods according to the invention in order to obtain the desired dispensing phase, either in releasing or aspiring mode. The use of the wording dispensing, dispensing phase and the like is exclusively used for conciseness and ease of description, but should be intended to refer to both meaning.

[0103] As used herein, an orifice is any opening, hole or vent that completely crosses a physical body so that the volumes at the two sides of said body are brought into communication among them. Particularly, an orifice 3 according to the present disclosure completely crosses the membrane 2 at the tip's distal end by creating a channel that is substantially cylindrical or conical in shape so that the internal volume of the pipette tip and the outside are fluidically connected. In its simplest embodiment, an orifice is a cylinder sized and shaped so that a liquid and a particle, such as a cell, comprised therein can passed through it. However, several variants of such an orifice can be envisaged, some of which are particularly useful in order to avoid the clogging thereof and/or to avoid the reentry of a particle once it has been dispensed, such as a slotted hole, a funnel or a diaphragm-like shape. In preferred embodiments, the orifice of the membrane's pipette tip has a diameter comprised between 1 and 1000 m, which is a suitable size for most of the particles (such as cells) usually analyzed via impedance-based means.

[0104] The addition of a holed membrane at the extremity of a pipette tip represents the core inventive concept behind the present invention. In fact, a such-designed tip is particularly suitable and optimized to perform highly reliable and accurate measurements, preferably impedance measurements, once said tip is coupled with a system for dispensing, and analysing the characteristics of (e.g. an impedance analyser), the particles comprised in a conductive medium, especially up to a single-particle level. This approach has huge potential advantages and applications for examples in single cell research or protein production for medicament manufacturing, where selecting a single cell, possibly with precise features, is crucial for a good outcome.

[0105] The membrane 2 is a key element in the structure and design of the pipette tip of the invention. The structure of the membrane 2 and of the orifice 3 thereon simplifies the positioning of the electrodes (one inside and one outside the tip, both at least partially emerged in conductive media) when the tip is intended to be coupled with an impedance-based system for analysing particles flowing throughout it. In fact, for its intrinsic characteristics, such a design permit not to place the electrodes in a precise and particular arrangement, such as on the body tip and/or the membrane, and in any case not very closed to the orifice 3. Moreover, the membrane 2 defines the frontier of the sensing area, giving no incertitude between what is detected and what is dispensed in e.g. a medium-filled reservoir, allowing high confidence measurements particularly for single cell dispensing and analysis.

[0106] Impedance analyses, and particularly the sensibility thereof, are influenced by the electrical current density at the orifice's 3 level. In fact, the orifice 3 represents the frontier between the conductive medium placed inside the tip and a medium in an external reservoir fluidically connected with the pipette tip, thus creating a passage through which the electrical current is forced to pass in order to close the electrical circuit established by the activated electrodes. In such a way, a particle traversing the orifice 3 will interrupt or in any case alter the electrical circuit, thus providing a detectable signal. In a first approximation, the detectable signal is proportional to the variation of resistance in the orifice divided by the sum of the resistances in series in the electrical circuit including the resistance of the conductive medium in and out of the sensing tip. The sensitivity can be approximated by the following formula:

[00001] $sensitivity = \frac{.Math. .Math. R_{orifice}}{R_{orifice} + R_{inside} + R_{outside}}$

[0107] Consequently, the sensitivity is improved when the resistance inside and outside the restricted tip are low compared to the resistance at the orifice. The resistance of a resistor depends upon its length and cross sectional area. This is regulated by the resistivity law:

[00002] $R = .Math. \frac{l}{a}$

[0108] where l is the length of the conductor (in this case, the liquid height inside the tip), a is the cross-sectional area (of the membrane; for a round conductor a=r.sup.2 if r is radius) in units of meters squared, and is the resistivity of the conductor (in this case, the conductive medium). According to this formula, the resistance is thus mostly influenced by the cross-sectional area of the distal end.

[0109] In order to achieve a good sensitivity, it is then best to design the pipette tip with a large cross-sectional area at the distal end, a large membrane 2 and a small orifice 3 with a diameter in the magnitude of the particle size. In addition, choosing such a design gives some freedom on the placement of the electrodes, because the length l has a linear impact in the equation and is thus much less influent than the cross section, which has a quadratic influence. Therefore, the greater the membrane area will be, the least the electrode placement will be important.

[0110] In the attempt of improving the pipette tip design for impedance particle analyses, the inventors have been able to define that the optimal membrane diameter/orifice diameter ratio should be of at least 6.32, preferably of at least 10. Coulter et al. proposed a design of a Coulter counter at the distal part of a nozzle (U.S. Pat. No. 3,714,565A). This solution is known to allow cell detection with characteristic length between inner electrode and an aperture in the range of the cell size. However, to obtain such a topology, an expensive microfabrication process including photolithography is necessary. Avoiding photolithography, imposes an increase in the fabrication tolerance. In this context, the electrode position can change in a range spanning from 50 to 500 m (one order of magnitude); therefore, in order to have similar performance than that proposed by the Coulter counter nozzle, the electrical resistance inside the tip above the membrane should remain unchanged.

[0111] Assuming that the length L between the suspended (floating) membrane and an inner electrode is at least 10 time less controllable in a manufacturing process than in the Coulter's nozzle design:

L.sub.suspended membrane=10*L.sub.nozzle

[0112] There is the need to keep the same electrical resistance inside the tip for the cell sensor of the present invention to work

[00003] $R_{inside} = \frac{.Math. .Math. L_{nozzle}}{S_{nozzle}} = \frac{.Math. .Math. L_{suspended .Math. .Math. membrane}}{S_{suspended .Math. .Math. membrane}} = \frac{* 10 * L_{nozzle}}{S_{suspended .Math. .Math. membrane}}$

[0113] The surface of the suspended membrane is therefore:

[00004] $= .Math. > .Math. S_{suspended .Math. .Math. membrane} = \frac{* 10 * L_{nozzle}}{R_{inside}} = \frac{* 10 * L_{nozzle}}{\frac{.Math. .Math. L_{nozzle}}{S_{nozzle}}} = 10 * S_{nozzle}$

[0114] And its radius is therefore:

[00005] $= .Math. > .Math. r_{suspended .Math. .Math. membrane} = \sqrt{\frac{S_{suspended .Math. .Math. membrane}}{}} = \sqrt{\frac{10 * S_{nozzle}}{}} = \sqrt{\frac{10 * r_{nozzle}^{2} *}{}} = \sqrt{10 * r_{nozzle}^{2}} = \sqrt{10} * r_{nozzle} = 3.16 * r_{nozzle}$

[0115] As described in the above-mentioned U.S. Pat. No. 3,714,565A, the Coulter design gives

r.sub.nozzle=2*r.sub.aperture

r.sub.suspended membrane=2*3.16*r.sub.nozzle=6.32*r.sub.aperture

[0116] And therefore:

d.sub.suspended membrane=2*3.16*d.sub.nozzle=6.32*d.sub.aperture

[0117] This result justifies the choice of a precise ratio between the membrane diameter and the orifice size, which must be of at least 6.32, for having enough freedom in electrode positioning while keeping an excellent performance in terms of sensitivity, and without being linked to micromanufacturing processes. However, as shown in FIG. 14, a surprising effect is particularly given when said ratio is of at least 15, even better with a ratio of 20 (FIG. 14). This effect is further experimentally demonstrated in FIG. 17; here, the plain curve shows the case of an inner electrode placed at 1 mm from the aperture, and the dotted curve the case of electrode placed at 5 mm from the aperture. The difference of impedance in the resistive plateau if less than 20% (37 kOhm for the 5 mm distance and 31 kOhm for the 1 mm distance with measurement at 50 kHz). The figure clearly demonstrates that even a large electrode displacement in the tip does not change the order of magnitude of the tip impedance.

[0118] A continuous calculation based on the above mentioned considerations and on a theoretical model developed by the inventors was obtained in the case of an orifice placed in a membrane and of an aperture at the end of a nozzle. FIG. 14 shows the simulation results of impedance variation due to the passage of 15 m polymeric beads through an impedance sensor in function of the distance between the aperture and an electrode placed inside an aperture tube for two different embodiments. In the depicted image, a 30 m (d) hole has been included in a 500 m (D) membrane (D/d=16.6) and a 30 m (d) hole has been included at the end of a 60 m (D) nozzle (D/d=1.66). This figure highlights that the effect of the electrode placement in terms of particle sensitivity becomes significant in the case of small D/d ratio. The proposed design with larger D/d ratio has the advantage of more flexibility for electrode placement with limited effect on particle sensing sensitivity.

[0119] The membrane 2 can be made of any suitable material, which is preferably a (thermo)plastic polymeric material commonly used for pipette tips such as polypropylene, polystyrene, polycarbonate, and the like. Preferably, the membrane 2 of the pipette tip has a thickness comprised between 1 and 1000 m and a membrane's diameter/membrane's thickness ratio of at least 5. These parameters have been established by the inventors as the optimal ones in order to obtain an excellent impedance readout while maintaining a good resistance of the membrane 2 to hydrostatic and/or applied pressures.

[0120] At the same time, the surface of the proximal end of the pipette tip should be bigger than the surface of the distal end, preferably with a ratio of a least 5. For this reason, in a particular embodiment of the invention (such as the one shown in FIG. 1), the pipette tip of the invention has a substantially frustum three-dimensional volume shape. As used herein, a frustum is a portion of a solid, such as a cone or a pyramid, which lies between two parallel planes cutting it. Each plane section is a floor or base of the frustum. A frustum is circular if it has circular bases; it is right if the axis is perpendicular to both bases, and oblique otherwise. The height of a frustum is the perpendicular distance between the planes of the two bases. For example, in one embodiment, the pipette tip comprises a single rounded wall connecting the proximal and the distal ends, and a bottom distal end surface represented by the holed membrane 2, the height of the pipette tip being the distance between the proximal end opening and the bottom membrane surface area. Accordingly, the tip is substantially frusto-conical in shape. In an alternative embodiment, the pipette tip comprises three or more wall faces, and the volumetric shape may therefore be substantially frusto-pyramidal.

[0121] The restricted tip of the invention has been particularly conceived and adapted in order to best perform in the frame of impedance analyses once coupled with a system comprising at least an impedance analyser and a fluidic actuator, as discussed below in more details.

[0122] For example, as shown in FIG. 2, a restricted tip can be operably connected to a system comprising a fluidic actuator 6 and an impedance analyser 4 via e.g. a tip connector 7 having at least two electrodes by locking the tip so that one of the electrodes (9) falls inside the body of the tip while the other (10) lies outside the tip. In such an arrangement, the two electrodes 9, 10 are so called floating or connecting electrodes, adapted to be at least partially emerged into the conductive media comprised in the tip and in a reservoir fluidically connected with the tip's inside.

[0123] Alternatively, the restricted tip can be implemented by physically connecting or attaching at least one electrode to a portion of the elongated body, the distal end, the tip membrane and/or a filter in a manner to activate said at least one electrode once the tip is operably connected to the electrical impedance analyser 4 through a tip connector 7. Such an implemented restricted tip is referred to hereinafter as a sensing tip 17 (FIG. 3).

[0124] Also in this case, in view of the inventive design of the pipette tip of the invention, many alternatives concerning the placement of the electrodes can be envisaged, thus providing not only a great operational freedom so that many diverse impedance analyser can be used for particle analyses, but also the possibility to adapt the manufacture process in order to be the more comfortable as possible, depending on the needs and the tools at one's disposal. As depicted in FIG. 3 for instance, several different arrangements of the electrodes can be imagined, wherein the electrodes lie along the internal or external walls of the tip's body (13, 14), remain floating (15, 16) or combinations thereof. The electrodes can also be physically connected to the tip's walls via an external (11) or external (12) connection pad that may facilitate the coupling with the impedance analyser 4 through a tip connector 18 comprising connector pads (19, 20) (FIGS. 4 and 5). Additionally or alternatively, floating electrodes can be locked within a filter provided at the proximal end of the sensing tip 17. Additionally or alternatively, the internal electrode can be coupled with or embedded within a further tip element such as a plunger or a capillary piston included inside the tip itself, so that the sensing tip can work as a positive displacement pipette tip such as the ones described e.g. in EP0494735.

[0125] The electrodes can be shaped to have any form and can be made of any suitable electrical conductive material, including but not limited to metals such as Au, Pt, Al, Cu and the like, liquid metals such as Hg or Ga, as well as any alloy or oxide thereof, conductive powders or adhesives as well as any combination of the foregoing. In a preferred embodiment, the electrodes are made of non-toxic and biocompatible materials.

[0126] For the sake of clarity, in the frame of the present disclosure, the expression operably connected reflects a functional relationship between the several components of the system or the sensing tip among them, that is, the term means that the components are correlated in a way to perform a designated function. The designated function can change depending on the different components involved in the connection; for instance, the designated function of an electrical impedance analyser 4 operably connected to a restricted tip or a sensing tip is the detection of an impedance signal once a particle flows together with a conductive medium through the orifice 3. Similarly, the designated function of a fluidic actuator 6 operably connected to a restricted tip or a sensing tip is the regulation of the pressure inside said tip. A person skilled in the art would easily understand and figure out what are the designated functions of each and every component of the system of the invention, as well as their correlations, on the basis of the present disclosure.

[0127] Tip Fabrication Process

[0128] According to another aspect of the invention, it is provided a manufacturing method for the production of the restricted or the sensing tip described above.

[0129] The restricted tip has a very simple structure that is easy to manufacture at large scale while maintaining minimal costs. The machines used for these processes can host batches of hundreds of tips, and in mass production the process can be automated with digital processing software. Generally speaking, in preferred embodiments of the invention, a variety of polymeric plastic materials may be used for manufacturing the pipette tips body 1 and/or membrane 2, including but not limited to standard thermoplastic polymers such as polypropylene, polystyrene, polycarbonate, and the like.

[0130] In one embodiment, a one-step manufacturing process foresees the creation of a pipette tip having its distal end closed with the membrane 2 having an orifice 3 in the form of a monolithic piece of a polymeric plastic material. In this case, the monolithic tip may be manufactured e.g. by plastic injection moulding techniques using ad hoc molds.

[0131] In another, two-steps monolithic manufacturing process embodiment, the pipette tip including the membrane 2 is produced by plastic injection moulding techniques. Subsequently, the orifice is opened through the membrane by any suitable means such as laser ablation, punching, etching, drilling and the like. In a preferred embodiment, the orifice is opened through the membrane by an excimer laser (LightShot, OPTEC).

[0132] In a three-steps layer deposition manufacturing process embodiment (FIG. 6), the pipette tip is first produced by plastic injection moulding techniques (A). In a currently developed process, commercially available pipette tips 21 are taken as the basis of the manufacturing of the restricted tip (RatioLab Colorless E, 200 uL). In a second step (B), the membrane 2, a film-like plastic material such as polypropylene, polystyrene, polycarbonate, and the like, is sealed to the tip's distal end by e.g. welding or gluing techniques. In a third step (C), the orifice 3 is opened through the membrane 2 by any suitable means (laser ablation, punching, etching, drilling and the like). In a preferred embodiment, the orifice is made by an excimer laser (LightShot, OPTEC).

[0133] In another embodiment, an indirect layer deposition manufacturing process is used. The tip is first produced by plastic injection moulding techniques. In a second step, the membrane is a film-like plastic materials such as polypropylene, polystyrene, polycarbonate, and the like and then an orifice is opened through the membrane by suitable means as described above. The membrane including the orifice is later sealed to the tip by suitable means as described above ultrasonic (e.g. welding or gluing techniques). In a currently developed process, the orifice opening is made by an excimer laser.

[0134] In a five-steps layer deposition manufacturing process embodiment (FIG. 7), the tip 21 is first produced by plastic injection moulding techniques (A) and is later temporary closed at the aperture's level (B) with an external closing element 22 such as a film of e.g. polypropylene, polystyrene, polycarbonate and the like on the external side. In a currently developed process, Parafilm M (Bemis NA) has been chosen because it has an optimal adherence on the tip. Subsequently (C), the membrane 23 is coated on the internal side of the tip and of the temporary external closing element by using any suitable technique such as physical or preferably chemical vapour deposition (CVD) process so that the polymer closes the opening of the tip 21. In a fourth step (D), the temporary external closing element 22 is mechanically detached. Finally (E), the orifice 3 is opened through the membrane by suitable means as described above.

[0135] In an alternative embodiment of the five-steps layer deposition manufacturing process (FIG. 8) the tip 21 is first produced by plastic injection moulding techniques (A). Then (B) the tip 21 is temporary filled with an internal closing element 24 such as a sacrificial plug made of a soluble polymer, for instance poly(ethylene-glycol) which is soluble in water. In a currently developed process, PEG1000 (Sigma Aldrich) is chosen because it is liquid at 50 C. and then can be easily loaded inside the tip 21. It is also easily removable because of its water solubility. In a third step (C), a membrane 25 is coated on the external side of the tip's distal end closed with the internal closing element and possibly also on the external side of the pipette's wall by using any suitable technique such as physical or preferably chemical vapour deposition (CVD) process. The coating can be for instance a conformal polymer such as poly(p-xylylene) (tradename Parylene) deposited with accurate thickness ranging from 50 nm to 1 mm depending on the application. In a current embodiment, the membrane is made of a biocompatible polymer (Parylene USP class VI). After the coating, the polymer closes the opening of the tip. In a fourth step (D), the internal closing element 24 is removed for example by dissolution in a solvent. Thus, the polymer forms a membrane that closes the pipette tip 21. In a last step (E), the orifice 3 is opened through the membrane by any suitable method, as those described above.

[0136] The same fabrication processes of the restricted tip described above are applicable to the manufacture of the sensing tip according to the invention. In addition to the described steps, (a) last step(s) of positioning and physically applying at least one electrode in the internal and/or external side of the pipette tip is performed (see for instance FIG. 3), together with an optional final step of positioning an air filter. As extensively stated throughout the description, the physical application of electrodes on a restricted tip to obtain a sensing tip can be attained with a great variety of designs and combinations, as well as with various manufacturing techniques, in view of the relative importance of the electrodes' position.

[0137] In one embodiment, at least one external electrode is deposited outside of the tip's tubular body 1. The external electrode comprises or consists of e.g. a metal or other suitable conductive material and may be deposited on the surface of the tip by suitable methods such as sputtering or any other deposition method such as metal evaporation. In a currently developed manufacturing method, the external electrode is made of a thin (about 100 nm) gold layer that is deposited by evaporation on the external surface of the tip. In mass production, large batch of tips can be placed in the evaporator. The gold layer has been chosen for its excellent adhesion on the tip, particularly plastic pipette tips, and moreover it is well suited for handling biological samples because it is biocompatible. The external electrode can also be for example a simple conductive wire or a conductive adhesive.

[0138] In one embodiment, at least one internal electrode is deposited inside the tip's tubular body. Similar considerations as for the external electrode can be applied to the internal electrode, particularly in terms of used materials and deposition methods. In a currently developed manufacturing method, the internal electrode is a simple metal wire placed into the tip and hold in place by an air filter located at the tip's proximal end (floating electrode). Wires made of medical-grade metal may be used, such as stainless steel 316 L which is biocompatible and has no electrical insulator outside.

[0139] Fluidic Actuator

[0140] Another important feature of the invention reside in the particular design of the fluidic actuator 6, which is adapted to control the conductive medium and particles' retention or their passage through the tip (FIG. 9).

[0141] In its simplest embodiment, the fluidic actuator 6 is any kind of device able to apply a pressure on a tip operably connected thereto. In this embodiment, the fluidic actuator 6 commonly works by exerting, upon activation, a negative pressure change inside a tip connected thereto to aspire a fluid, and selectively releasing said fluid to draw up and dispense it according to a preferred volume by applying a positive pressure change. A syringe or syringe-like device could be suitable for this purpose. In a preferred, alternative embodiment, devices such as a manual or electronic pipettor, as commercially available ones, could be used. Alternatively, the use of a pipettor compatible with positive displacement pipettes can be envisaged.

[0142] In another embodiment, the fluidic actuator 6 comprises at least two pressure sources (one positive and one negative) and a regulator capable of applying a controllable negative pressure (also referred to hereinafter as holding pressure) and a controllable positive pressure (also referred to hereinafter as dispensing pressure).

[0143] In another embodiment (FIG. 9), the fluidic actuator 6 comprises at least one air pump 26 for generating the pressure inside the restricted or sensing tip. In this configuration, the fluidic actuator 6 works by only exploiting the hydrostatic pressure of the internal conductive medium as a dispensing pressure, and the actuator 6 typically applies a negative pressure (also referred to hereinafter as holding pressure) towards the proximal end of the restricted or sensing tip 17 that sucks a smaller flow compared to the dispensing flow to ensure that no particle can leave the restricted or sensing tip. In this embodiment, the fluidic actuator 6 typically comprises one air pump 26 as for example mircofabricated piezo actuated membrane pump operably connected to the restricted or sensing tip. In order to better perform and rapidly invert the pressure imposed to the conductive medium inside the restricted or sensing tip, the micropump need to be able to establish at least 2 mbar (or 2 mbar) in the tip's internal volume by adding or removing air from it. This delay (e.g. 100 ms) need to be fulfilled in order to obtain a system sufficiently reactive to be able to stop the particles' containing conductive medium dispensing just after the chosen number of particles of interest (e.g. one single cell) has been detected, and without dispensing more than that number.

[0144] In another embodiment (FIG. 10), the system comprises at least two air pumps 26, 27 adapted so that at least one pump 26 generates a positive pressure and at least one pump 27 generates a negative pressure inside the restricted or sensing tip 17. In this embodiment, the fluidic actuator typically comprises two air pumps as for example microfabricated piezo actuated membrane pumps with capability to pump external air and operably connected with the pipette tip 17 of the invention through e.g. a 3 ways valve 28. In order to better perform and rapidly invert the pressure imposed to the conductive medium inside the pipette tip 17, the valves 28 need to be able to switch very quickly, preferably in less than 100 ms, and the micropump(s) need to be able to establish at least 2 mbar (or 2 mbar) in the tip's internal volume by adding or removing air from it. This delay (e.g. 100 ms) need to be fulfilled in order to obtain a system sufficiently reactive to be able to stop the particles' containing conductive medium dispensing just after the chosen number of particles of interest (e.g. one single cell) has been detected, and without dispensing more than that number.

[0145] Moreover, the fluidic actuator 6 needs to work by pumping external air as such a system does not tolerate a liquid contact with the particle media and the pump. For instance, one pump is arranged in a way to pump air from atmosphere into the tip (increasing, positive pressure) and the other pump is arranged so to extract air from the tip's inside into the atmosphere.

[0146] With this configuration, the fluidic actuator 6 applies two different pressures within the internal side of the tip, namely a positive pressure (also referred to hereinafter as dispensing pressure) that imposes the liquid inside the tip to flow out the tip 17 and makes the particles leaving the sensing tip, and a holding pressure as described above.

[0147] The dispensing pressure is chosen to avoid a too fast passage of particles through the sensing or restricted tip's membrane that would prevent the particles' detection. The holding pressure is chosen to counter act the hydrostatic pressure imposed by the particles' containing medium and creates a minimal flow entering the tip to avoid any particle leaving the sensing or restricted tip 17. At the same time, the chosen holding pressure is such that it avoids the aspiration of particles from the outside of the sensing or restricted tip towards the inside.

[0148] In a preferred embodiment, the pumps 26, 27 used for the fluidic actuator are microfabricated piezo-actuated membrane pump. This kind of micropumps work by transferring a volume of fluid from one of their outlet to the other by moving a membrane. Their dead volume is small compared with the displaced volume and the membrane movement can be quick as it is piezo actuated. The combination of those element allows a precise control of the pressure in the sensing or restricted tip 17 as well as a fast switching of the fluidic actuator described above and required by the application. For example, it allows to switch from 2 mbar to 2 mbar in the sensing or restricted tip in less than 100 ms.

[0149] When the system is operating, during the dispensing of the particles' comprising conductive medium located inside the pipette tip 17 of the invention towards an external reservoir filled with a conductive medium, also the inner hydrostatic pressure is consequently modified according to the change in total internal medium volume. As a consequence, both dispensing and holding pressures need to be adapted time after time based on the actual hydrostatic pressure.

[0150] Different solutions may be proposed to measure or estimate the hydrostatic pressure at each time point. For instance, in one embodiment, a pressure sensor 29 is placed in the proximity of the pipette tip membrane 3 in order to measure the inner hydrostatic pressure. In an alternative or additional embodiment, easier to fabricate, the internal electrode is not fully immersed into the particles' comprising conductive medium, and measuring its capacitance allow the determination of the internal medium height (and therefore, the internal hydrostatic pressure). In a further alternative or additional embodiment, by knowing beforehand, after a calibration of the restricted or sensing tip, the dispensed volume of the internal conductive medium over time (e.g., dispensed microliters of particles' containing medium per second), the measurement of the time spent in the dispensing and holding state allows to estimate the tip's internal conductive medium volume. In an additional embodiment, a fluidic actuator 6 of any kind can be operated and regulated by an external controller, such as a pressure flow controller, a vacuum flow controller, a mechanical flow controller and the like, which can be embedded in a higher robotic system and possibly handled by a computer-like device.

[0151] Impedance Analyser

[0152] In order to produce an impedance signal, possibly interpretable by a controller 5, the impedance has to be read from the restricted or sensing tip of the invention (FIG. 11). For this purpose, an impedance analyser 4 is operably connected to the restricted or sensing tip through a tip connector. In a particular case and to allow the integration of the full system in a handheld device, this impedance analyser can be constituted of e.g. commercial integrated circuits following the well-known lock-in demodulator principle with the following block: a) an excitation source 30 at a single frequency or at multiple (at least two) frequencies for discrimination between e.g. two different particle types (e.g. cells and other particles) or different parameters of the same particle type such as for example cell viability; b) a preamplifier 31 to amplify the current received from the restricted or sensing tip 17; c) a demodulator 32 to extract the impedance signal from the modulated signal and d) a filter 33 to remove the harmonic created during the demodulation as well as any unwanted frequency from the impedance signal such as variation of medium conductivity.

[0153] One of the main features and advantages of the proposed system is actually the use of an impedance analyser 4 for recording an impedance signal, and store said information in an associated computer-like device as for example a log file. In cell dispensing procedures into multi-well plates for cell culture, this possibility has huge implications. In fact, saving in a file the impedance records together with the well index is an outstanding tool for process traceability. The traceability is a key feature of the system of the invention, which can be exploited to perform a quality control of the single-cell dispensing. Therefore, after a dispensing process, the impedance files of each well are retrieved and analysed by post-processing on a computer. If one single impedance peak is detected, the well is considered to have one single cell with high confidence.

[0154] Controller

[0155] In at least some embodiments of the system of the invention, particularly those embodiments in which the system is intended for complete or semi-automatization, an optional, additional controller element 5 can operate the full system. In its simplest embodiment, a controller 5 is a computer-like device or an embedded micro-computer e.g. microcontroller operably connected to a fluidic actuator 6 and an impedance analyser 4 of the disclosed system, which is programmed with an algorithm in order to manage all the actions of the system.

[0156] Ideally, a controller 5 receives an impedance signal coming from the impedance analyser 4 and applies post-processing treatment on this signal that consists in a high pass filtering to remove the mean value of the signal. The controller 5 then performs a peak detection to reveal the event of particle's passage through the orifice 3 defined in the membrane 2 of the restricted or sensing tip. For example, in one embodiment the method used to perform this peak detection on the impedance signal is the comparison of the impedance signal with a given threshold representing the size of a particle. In another embodiment, the peak detection is performed by a correlation between the filtered impedance signal and signal representing the passage of a particle. In one embodiment, the controller 5 can furthermore receive instructions from the user in order to start different operational such as fluid aspiration or particle dispensing. Finally, the controller 5 drives the fluidic actuator according to a method suited for different operation such as cells or particles dispensing and this down to the resolution of one single cell.

[0157] For an exemplary purpose only, an operation that can be performed by the system of the invention is the dispensing of N particles (with N1) (FIG. 12), such as a cell, in an external reservoir comprising a conductive medium and fluidically connected to the pipette tip of the system.

[0158] As depicted in FIG. 13, in a first step that is triggered by the user, particles in a conductive medium are loaded inside the restricted or sensing tip that is later operably connected to the fluidic actuator 6, preferably through a tip connector. For this step, the controller 5 activates the fluidic actuator 6 to generate a negative pressure for a given time to fill the sensing tip with a particles containing conductive medium. The controller 5 then places the system in an idle state (A) with the fluidic actuator 6 applying a holding pressure matching at least the hydrostatic pressure represented by the liquid within the restricted or sensing tip to avoid the exit of the medium with particles outside of the tip. Once the user has placed the restricted or sensing tip in the medium containing reservoir where the particle has to be dispensed, so that the pipette tip and the reservoir are fluidically and electrically connected, and the user has commanded the particles' dispensing (B), the controller 5 drives the fluidic actuator 6 so to generate a positive dispensing pressure to the restricted sensing tip. The conductive medium and the contained particles then start to flow out of the restricted or sensing tip through the orifice 3. Once the controller 5 detects the passage of a particle based on the signal received from the impedance analyser (D), the controller 5 waits a certain delay to make sure that the particle is not re-aspirated and then operates so that a negative holding pressure is applied again by the fluidic controller (E). The system restarts the dispensing process (B) to reach the number N of particles to be dispensed and then waits again in the idle state (F) to receive further commands for particle dispensing.

[0159] As will be evident to a person skilled in the art, the inclusion of a controller 5 renders the entire system of the invention easily automated/automatable. In this context, additional elements such as robotic arms, displaceable platforms, optical sensors and the like can be included in the system; for instance, in cell dispensing procedures into multi-well plates for cell culture, a fluidic actuator 6 in the form of a pipettor, equipped with sensing or restricted tips according to the invention, can suitably be coupled with a robotic arm that moves according to a dispensing path along the wells of the plate, and based on the instructions provided by the controller 5. The robotic arm moves and displaces the pipettor 6, inserting and removing the tip from a first well, and repeating the operation cycle for any other well of the well-plate. Alternatively, the well plate can be moved along a pre-set path through a displaceable platform support, and the robotic arm simply inserts/removes the sensing tip into the wells by lifting and lowering the coupled pipettor 6.

EXAMPLES

[0160] FIG. 15 shows one embodiment of a sensing tip according to the invention. In this exemplary form, a 15 m Parylene C membrane is deposited by chemical vapour deposition (CVD) (C-30-S, Comelec, CH) on a commercial plastic pipette tip having a distal end (D) of 500 m in diameter (200 l colorless, RatioLab, GE). A 100 nm thick gold outer electrode is subsequently deposited on the tip by evaporation (LAB 600H, Leybold Optics, CH). An orifice is then micromachined by excimer laser (LSV3, Optec, BE) on the previously deposited membrane. This is designed like a channel across a thin membrane restricting the tip opening. In order to optimize the sensor sensitivity and to avoid clogging, the orifice diameter (d) is of 30 m, giving a D/d ratio of 16.6.

[0161] The depth of the orifice, which is equivalent to the membrane thickness, is of 15 m. An inner electrode placed within the body of the pipette tip consists of a stainless steel wire. For sterility purposes and to block the internal electrode at a fixed height inside the tip, a high-density polyethylene filter (TipOne 200 L, StarLab GE) is added to the tip from its top. The precise placement of the electrode being not critical, it is even possible to place the electrode manually, so that all the elements (the tip, the filter and the electrode) can be manually assembled under laminar flow.

[0162] To test whether the so-obtained sensing tip is capable of detecting particles, a suspension of 10.sup.4 beads/mL as a surrogate of small cells was loaded in the tip (6-m polystyrene beads). Once loaded, the sensing tip was placed in a tube containing fresh medium. The system was then set to flow some beads out for a period of 10 s, while recording of the impedance was performed. FIG. 16 shows the impedance measurements graph acquired with the system of the invention implemented with a sensing tip.

Devices, Systems, and Methods for Dispensing and Analyzing Particles

Inventors

Cpc classification

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B01L3/0275

PERFORMING OPERATIONS; TRANSPORTING

Classification Explorer

G01N15/12

PHYSICS

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G01N2015/1493

PHYSICS

Classification Explorer

B01L2200/12

PERFORMING OPERATIONS; TRANSPORTING

Classification Explorer

B01L2300/0851

PERFORMING OPERATIONS; TRANSPORTING

Classification Explorer

G01N33/48721

PHYSICS

Classification Explorer

G01N2015/1029

PHYSICS

Classification Explorer

G01N2015/1488

PHYSICS

Classification Explorer

G01N2015/135

PHYSICS

Classification Explorer

G01N15/1459

PHYSICS

Classification Explorer

B01L2300/044

PERFORMING OPERATIONS; TRANSPORTING

Classification Explorer

B01L2300/0681

PERFORMING OPERATIONS; TRANSPORTING

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G01N2015/1006

PHYSICS

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B01L2300/0645

PERFORMING OPERATIONS; TRANSPORTING

International classification

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B01L3/02

PERFORMING OPERATIONS; TRANSPORTING

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G01N33/487

PHYSICS

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G01N15/12

PHYSICS

Abstract

Claims

Description