Nanopore array microfluidic device for the analysis of liquid compounds

Abstract

Microfluidic device (D) for the analysis of liquid compounds, containing a plurality of drops (Ga, Gb, …, Gn), comprising a first layer (1) and a second layer (2) facing each other and capable of defining a through-channel (C) for said plurality of drops (Ga, Gb, …, Gn), wherein said first layer (1) comprises a supporting base (11), covered by a first coating element (15), along the entire length and width of said supporting base (11), comprising a common fluidic space (12), arranged on said supporting base (1), in which a space electrode (EC) is arranged, a membrane (13), arranged on said fluidic space (12), in which a plurality of electrodes (13a, 13b, …, 13n) is obtained, in each electrode (13n) a corresponding nanopore (14n) being obtained, thus forming a corresponding plurality of nanopores (14a, 14b, …, 14n), said second layer (2) comprises a main electrode (21), and a second coating element (22), arranged in contact with said main electrode (21), so as to face said first coating element (15), so that, in operation, by applying an electrical voltage between said main electrode (21) and said plurality of electrodes (13a, 13b, …, 13n), said plurality of drops (Ga, Gb, …, Gn) is capable of flowing independently of each other, until each nanopore (14n) or a group of nanopores of said plurality of nanopores (14a, 14b, …, 14n) is occupied by a corresponding drop (Gn) of said plurality of drops (Ga, Gb, …, Gn) for detecting the ionic or electrical current associated with the entry of each drop (Gn) into the corresponding nanopore (14n), by said plurality of electrodes (13a, 13b, …, 13n) and by said space electrode (EC).

Description

[0001] Title: Nanopore array microfluidic device for the analysis of liquid compounds

[0002] DESCRIPTION

[0003] Field of application

[0004] The present invention relates to a microfluidic device for the analysis of liquid compounds.

[0005] In particular, the present invention relates to a microfluidic device having a nanometre-sized pore array structure, also called nanopores. More in detail, the invention relates to a device of the above type, designed and particularly made to detect the presence of nanoparticles dispersed within a liquid compound, in particular a biological fluid, such as DNA fragments, but which can be used for detecting any nanometre-sized analyte.

[0006] Herein after the description will be addressed to a device for detecting nanoparticles dispersed in a biological fluid, having a nanopore array structure, but it is clear that it should not be considered limited to this specific use.

[0007] Prior art

[0008] It is currently known that liquid compound analysis devices are arrays of nanopores, i.e. two-dimensional arrays of nanopores arranged on a single membrane, usually made on silicon oxide substrates, according to a geometric lattice.

[0009] To be used as sensors, nanopores must be filled with a conductive liquid solution, typically an electrolyte such as potassium chloride (KC1), and must put two spaces adapted to house small amounts of liquid in fluidic communication, while being separated by the membrane in which the nanopore is present.In turn, each liquid space must have an electrode capable of reacting with the ions present in the electrolytic solutions, so as to convert the ionic currents present in the nanopore into electrical currents and capable of providing an electrical polarisation.

[0010] Configured in this way, the nanopores operate as sensors capable of detecting the presence of nanoparticles, DNA fragments or other analytes having a nanometric size commensurate with the nanopore size, since the partial occlusion caused by the transit of the analyte through the nanopore produces a modulation of the ionic current flowing through the nanopore, which can be measured by means of measurement systems connectable to the electrodes.

[0011] It is essential to ensure a proper electrical and fluidic insulation between all of the liquid spaces present on at least one side of the membrane containing the nanopores, so as not to compromise the detection capacity of the single nanopores.

[0012] In particular, the more the miniaturisation of the microfluidic system is exaggerated, the more it is necessary to ensure electrical and fluidic insulation between a measurement site and the adjacent ones.

[0013] The drawbacks of known devices therefore concern the ability of ensuring electrical and fluidic insulation between a measurement site and the adjacent sites, which is essential for performing correct measurements.

[0014] Insulation is closely related to the size of the nanopore arrays. The larger the size, the more difficult it is to maintain electrical and fluidic insulation.

[0015] Furthermore, for arrays containing hundreds of nanopores per membrane or more, in order to direct the fluid onto each nanopore present on the membrane and to arrange the electrodes to stimulate and measure the currents modulated by the nanopores, it is necessary to make microfluidic systems with very complex circuits that requiremultiple electrical contacts, which may develop parasitic currents and crosstalk phenomena that alter the measurement.

[0016] In light of the drawbacks and unsolved issues by the prior art, an object of the present invention is to provide a nanopore array microfluidic device for the analysis of liquid compounds that allows ensuring a proper electrical and fluidic insulation even in the presence of a large number of nanopores.

[0017] A further object of the present invention is to provide a nanopore array microfluidic device for the analysis of liquid compounds that it is economical and simple to make and maintain.

[0018] Finally, an object of the present invention is to provide a nanopore array microfluidic device for the analysis of liquid compounds capable of analysing different types of liquid compounds.

[0019] Therefore, the technical problem underlying the present invention is to ensure electrical and fluidic insulation between different measurement sites in arrays containing a high number of nanopores.

[0020] Summary of the invention

[0021] The idea underlying the present invention is to provide a nanopore array microfluidic device for the analysis of liquid compounds that allows for maximum miniaturisation, while ensuring electrical and fluidic insulation between the various measurement sites in arrays containing a large number of highly miniaturised nanopores.

[0022] Therefore, an object of the present invention is a microfluidic device for the analysis of liquid compounds, containing a plurality of drops, comprising a first layer and a second layer facing each other and capable of defining a through-channel for said plurality of drops, wherein said first layer comprises a supporting base, covered by a first coating element, along the entire length and width of said supporting base, said device comprises a common fluidic space, arranged on saidsupporting base, in which a space electrode is arranged, it further comprises a membrane, arranged on said fluidic space, in which a plurality of electrodes is obtained, in each electrode a corresponding nanopore being obtained, thus forming a corresponding plurality of nanopores, said second layer comprises a main electrode, and a second coating element, arranged in contact with said main electrode, so as to face said first coating element, so that, in operation, by applying an electrical voltage between said main electrode and said plurality of electrodes, said plurality of drops is capable of flowing independently of each other, until each nanopore or a group of nanopores of said plurality of nanopores is occupied by a corresponding drop of said plurality of drops for detecting the ionic or electrical current associated with the entry of each drop into the corresponding nanopore, by said plurality of electrodes and by said space electrode.

[0023] Preferably according to the invention, in said first coating element through-holes are obtained, which are arranged at said nanopores, to allow the passage of said drops and the contact of said drops with said electrodes.

[0024] Further according to the invention, said device is connectable to measurement systems capable of measuring said ionic currents.

[0025] Still according to the invention, each electrode of said plurality of electrodes is capable of polarizing separately from the other electrodes, by applying said electrical voltage, and of measuring the ionic current determined by the passage of each drop.

[0026] Still according to the invention, said membrane is a bidimensional structure extending along a first axis and a second axis, orthogonal to each other.

[0027] Preferably according to the invention, each drop of said plurality of drops is capable of flowing in said through-channel according to a first direction, parallel to said first axis, and / or according to a seconddirection, parallel to said second axis.

[0028] Further according to the invention, said membrane is made of silicon or silicon dioxide or silicon nitride.

[0029] Still according to the invention, each electrode of said plurality of electrodes is arranged at a micrometric distance from the preceding electrode and / or from the subsequent electrode.

[0030] Still according to the invention, said micrometric distance is filled with insulating material.

[0031] Preferably according to the invention, said first and second coating elements are made of hydrophobic material.

[0032] Brief description of the drawings

[0033] In the drawings:

[0034] figure 1 shows a side schematic view of the nanopore array microfluidic device for the analysis of liquid compounds in a position of two drops of the fluid, object of the present invention;

[0035] figure 2 shows a schematic view of the device shown in figure 1, in another position of the drop of the fluid;

[0036] figure 3 shows a top view of a portion of the device shown in figure 1; and

[0037] figure 4 shows a side schematic view of a detail of the device shown in figure 1.

[0038] Detailed description

[0039] With reference to figure 1, a nanopore array microfluidic device for the analysis of liquid compounds is an object of the present invention.

[0040] The microfluidic device D comprises a plurality of layers that allowmoving a plurality of drops Ga, Gb, ■■■, Gnof the liquid compound to be analysed, by means of the well-known dielectrophoresis or digital microfluidics - DMF technique.

[0041] According to the DMF digital microfluidics technique, it is possible to move or fragment drops of a fluid into nanolitre quantities, on an electrode array structure, covered with a hydrophobic surface, based on the polarisation of the underlying electrode.

[0042] The microfluidic device D comprises a first layer 1 and a second layer 2, facing each other, and capable of defining a through-channel C, through which the plurality of drops Ga, Gb, ..., Gnof said liquid compound to be analysed is capable of flowing.

[0043] Said first layer 1 comprises a supporting base 11 , made of a material suitable for microfluidic systems, such as glass or plastic polymers. On said supporting base 11 the common fluidic space 12 is arranged, which extends by the entire length of said supporting base 11. Said fluidic space 12 is filled with an electrolytic solution. In said fluidic space 12 a space electrode Ec is arranged, which is arranged in contact with said electrolytic solution.

[0044] Said first layer 1 further comprises an array or membrane structure 13, arranged on said fluidic space 12, in which a non-polarized plurality of electrodes 13a, 13b, ..., 13nis obtained. Said membrane 13 is made of silicon or silicon dioxide (SiO2) or silicon nitride (Si3N4). The membrane 13 may be also made of other similar materials.

[0045] Said membrane 13 extends in a plane defined by an X axis and an Y axis, orthogonal to each other.

[0046] The electrodes of said plurality of electrodes 13a, 13b, ..., 13nmay be polarised individually and independently of each other.

[0047] Each electrode 13nof said plurality of electrodes 13a, 13b, ..., 13nisarranged at a micrometric distance from the preceding electrode 13n-i and / or from the subsequent electrode 13n+i. The space between each electrode 13nand the preceding electrode 13n-i and / or the subsequent electrode 13n+i is filled with insulating material I, so as to ensure the electrical insulation among electrodes.

[0048] Each electrode of said plurality of electrodes 13a, 13b, 13nis perforated to obtain a nanopore 14n. Therefore, in said membrane, a plurality of electrodes 13a, 13b, ..., 13nand a corresponding plurality of nanopores 14a, 14b, ..., 14nare arranged.

[0049] Said first layer 1 further comprises a first coating element 15, arranged over said membrane 13 and thus over said plurality of electrodes 13a, 13b, ..., 13n, along the entire length and width of said supporting base 11.

[0050] Said first coating element 15 is a hydrophobic coating, such as polytetrafluoroethylene (PTFE), in which through-holes 15a, 15b, ..., 15nare obtained, which are arranged at the nanopores 14a, 14b, ..., 14n. Said second layer 2 comprises a main electrode 21 extending by the entire length and width of said supporting base 11, and a second coating element 22, arranged in contact with said main electrode 21, so as to face said first coating element 15.

[0051] Even said second coating element 22 is a coating of hydrophobic material, such as polytetrafluoroethylene - PTFE.

[0052] The surfaces of said first 15 and second 22 coating elements define the through-channel C, through which the liquid compound to be analysed flows, in the form of a plurality of drops Ga, Gb, ..., Gn.

[0053] The operation of the nanopore array microfluidic device for the analysis of liquid compounds, object of the present invention, is the following. When a liquid compound is to be analysed, this is inserted into channelC defined by the surfaces of said first 15 and second 22 coating elements, in the form of pluralities of drops Ga, Gb, ..., Gn.

[0054] By applying an electrical voltage between the main electrode 21 and said plurality of electrodes 13a, 13b, ..., 13n, it is possible to act on the physical features of the single drop Gn. In particular, by activating and de-activating the voltage, each electrode 13nis individually polarised, allowing the movement of a single drop Gnin the channel C, independently of the other drops Gn.

[0055] The drops Gnare capable of flowing along a first direction dx, parallel to the X axis, and along a second direction dy, parallel to the Y axis, in one direction and in the opposite one.

[0056] The application of the voltage will continue until the liquid is distributed over the first layer 1, so that each nanopore 14nof said plurality of nanopores 14a, 14b, ..., 14nis occupied by a respective drop Gnof said plurality of drops Ga, Gb, ..., Gn.

[0057] It is also possible for the drops Ga, Gb, ..., Gnto only occupy a group of nanopores 14a, 14b, ..., 14n.

[0058] In particular, when a drop Gnis positioned over a single through-hole 15nof said first coating element 15 and thus over a single electrode 13n, thus generating an electrical contact with it, and as a result over a single nanopore 14n, the nanopore 14nis filled thanks to its hydrophilic properties, or under the action of other agents and / or forces, thus creating a fluidic path between the drop Gnand the solution present in the fluidic space 12, thus putting the drop Gnin fluidic communication with the opposite fluidic space, underneath the membrane 13, i.e. with the fluidic space 12.

[0059] Inside the single nanopore 14na ionic current measurable by said plurality of electrodes 13a, 13b, ..., 13nand by said space electrode Ec is established, and the variation of this ionic current corresponds to the passage of particles present in the fluid. Said plurality of electrodes 13a,13b, 13nand said space electrode Ec are connectable to a detection system that amplifies these currents.

[0060] It is essential that a high level of electrical insulation is maintained among the various drops Gn, in order not to alter the measurement of the ionic current that flows in the liquid path between membrane 13 and the fluidic space 12. The insulation among the drops Gnis the result of the relation between the area and the geometry of the electrode 13n, the volume of the drop Gnthat occupies it and the spacing between the various electrodes 13a, 13b, ..., 13n. Since the electrodes 13a, 13b, ..., 13nare covered with hydrophobic material 15, there will be no liquid among the various electrodes 13a, 13b, ..., 13nbut only air.

[0061] As is evident from the above description, the advantage of the microfluidic device D lies in the possibility of analysing single drops of a liquid compound, avoiding electrical and fluidic interference with the other drops present.

[0062] Obviously a person skilled in the art, in order to meet contingent and specific needs, may make several changes and variants to the above described support, all of them falling within the scope of protection of the invention as defined by the appended claims.

Claims

CLAIMS1. Microfluidic device (D) for the analysis of liquid compounds, containing a plurality of drops (Ga, Gb, Gn), comprising a first layer (1) and a second layer (2) facing each other and capable of defining a through-channel (C) for said plurality of drops (Ga, Gb, ..., Gn), wherein, said first layer (1) comprises a supporting base (11), covered by a first coating element (15), arranged along the entire length and width of said supporting base (11), said device (D) being characterized in that it comprises a common fluidic space (12), arranged on said supporting base (1), in which a fluidic space electrode (Ec) is arranged, in that it comprises a membrane (13), arranged on said fluidic space (12), wherein a plurality of electrodes (13a, 13b, ..., 13n) is obtained, in each electrode (13n) a corresponding nanopore (14n) being obtained, thus forming a corresponding plurality of nanopores (14a, 14b, ..., 14n), in that said second layer (2) comprises:a main electrode (21) and a second coating element (22), arranged in contact with said main electrode (21), so as to face said first coating element (15), so that, in operation, by applying an electrical voltage between said main electrode (21) and said plurality of electrodes (13a, 13b, ..., 13n), said plurality of drops (Ga, Gb, ..., Gn) is capable of flowing independently of each other, until each nanopore (14n) or a group of said plurality of nanopores (14a, 14b, ..., 14n) is occupied by corresponding drops (Gn) of said plurality of drops (Ga, Gb, ..., Gn) for detecting the ionic or electrical current associated with the entry of each drop (Gn) into the corresponding nanopore (14n), by said plurality of electrodes (13a, 13b, ..., 13n) and by said fluidic space electrode (Ec).

2. Microfluidic device (D) according to the preceding claim, characterized in that in said first coating element (15) through-holes (15a, 15b, ..., 15n) are obtained, which are arranged at said nanopores (14a, 14b, ..., 14n), to allow the passage of said drops (Ga, Gb, ..., Gn)and the contact of said drops (Ga, Gb, Gn) with said electrodes (13a, 13b, ..., 13n).

3. Microfluidic device (D) according to any one of the preceding claims, characterized by being connectable to measurement systems capable of measuring said ionic or electrical current.

4. Micro fluidic device (D) according to the preceding claim, characterized in that each electrode (13n) of said plurality of electrodes (131, 132, ..., 13n) is capable ofpolarizing separately from the other electrodes (131, 132, ..., 13n), by applying said electrical voltage, andmeasuring said ionic or electrical current determined by the flowing of each drop (Gn).

5. Microfluidic device (D) according to any one of the preceding claims, characterized in that said membrane (13) is a bidimensional structure, extending along a first axis (X) and a second axis (Y) , orthogonal to each other.

6. Microfluidic device (D) according to any one of the preceding claims, characterized in that each drop (Gn) of said plurality of drops (Ga, Gb, ..., Gn) is capable of flowing in said through-channel (C) along a first direction (dx), parallel to said first axis (X), and / or along a second (dy) direction, parallel to said second axis (Y).

7. Micro fluidic device (D) according to any one of the preceding claims 5 or 6, characterized in that said membrane (13) is made of silicon or silicon dioxide or silicon nitride.

8. Microfluidic device (D) according to any one of the preceding claims, characterized in that each electrode (13n) of said plurality of electrodes (13a, 13b, ..., 13n) is arranged at a micrometric distance from the preceding electrode (13n-l) and / or from the subsequent electrode(13n+l).

9. Micro fluidic device (D) according to the preceding claim, characterized in that said micrometric distance is filled in with insulating material (I) .

10. Microfluidic device (D) according to any one of the preceding claims, characterized in that said first (15) and second (22) coating elements are made of hydrophobic material.

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