MICROFLUIDIC CHIP

Abstract

A microfluidic chip for conducting microbiological assays, comprises a substrate in which incubation segments, a sample reservoir and microfluidic channels connecting said sample reservoir with said incubation segments are arranged. Said microfluidic chip further comprise a non-aqueous liquid reservoir for containing non-aqueous liquid wherein said reservoir is connectable via a releasable airtight and liquid-tight valve with said microfluidic channels connecting said sample reservoir with said incubation segments each incubation segment comprises an incubation well (113) connected by a gas-exchange channel (115) to an unvented gas cavity (111).

Claims

1-15. (canceled)

16. A microfluidic chip for conducting microbiological assays, comprising a substrate made of impermeable material with a first major face and a, preferably parallel, second major face, wherein the surface of the first major face is covered by a first layer of impermeable, substantially transparent material and the surface of the second major face is covered by a second layer of impermeable, substantially transparent material, and within the substrate are arranged: a plurality of incubation segments each with an inlet channel, a sample reservoir with an inlet opening and an outlet opening , and microfluidic channels connecting the outlet opening of said sample reservoir with each inlet channel to said incubation segments, wherein said microfluidic chip further comprises a non-aqueous fluid reservoir for containing non-aqueous liquid, wherein said non-aqueous fluid reservoir has an outlet opening which is connectable via a releasable airtight and liquid-tight valve with said microfluidic channels, wherein each incubation segment comprises an incubation well connected by a gas-exchange channel to an unvented gas cavity and the incubation segments are arranged in a fractal manner in which the respective microchannels connecting each of the incubation segments to the sample reservoir are substantially equally long and/or have the same resistance to flow.

17. The chip of claim 16, wherein said chip has a length of equal to or greater than 12 cm and equal to or less than 13.5 cm, a width of equal to or greater than 8 cm and less than or equal to 9 cm., and the number of incubation segments is equal to or greater than 100 incubation segments.

18. The chip of claim 16, wherein said chip is made of a polystyrene, polycarbonate, poly(methyl methacrylate), cyclic olefin polymer or cyclic olefin copolymer.

19. The chip of claim 16, wherein said valve is a heat-sensitive valve, comprising a wax valve which contains wax, which melts at a temperature greater than or equal to 37 C.

20. The chip of claim 16, wherein, the shortest distance between two adjacent incubation segments, measured along the microfluidic channels connecting these segments is less than or equal to 10 mm.

21. The chip of claim 20, wherein, the shortest distance between two adjacent incubation segments, measured along the microfluidic channels connecting these segments is less than or equal to 7 mm.

22. The chip of claim 16, wherein the volume of the sample reservoir is at least three times larger than the total volume of the incubation wells, and/or the volume of the non-aqueous liquid reservoir is at least two times larger than the total volume of the microfluidic channels leading from the sample reservoir to the incubation segments.

23. The chip of claim 16, wherein the sample reservoir is of elongated shape and its maximum longitudinal dimension is greater than or equal to 30 mm.

24. The chip of claim 16, wherein the sample reservoir has an inlet end and an outlet end, wherein the outlet end is wider than the inlet end and/or the width of the outlet end of the sample reservoir perpendicular to the longitudinal axis of the sample reservoir is greater than or equal to 5 mm, and/or the sample reservoir has at least a first side wall and an opposite second side wall, wherein at least one projection protrudes from said first side wall, wherein width of the projection measured in a direction perpendicular to said side wall is greater than or equal to 1 mm, and/or wherein the shortest distance between the projection's distal end and the opposite side wall is greater than or equal to 3 mm.

25. The chip of claim 24, wherein the sample reservoir has an inlet end and an outlet end, wherein the outlet end is wider than the inlet end and/or the width of the outlet end of the sample reservoir perpendicular to the longitudinal axis of the sample reservoir is greater than or equal to 10 mm, and/or the sample reservoir has at least a first side wall and an opposite second side wall, wherein at least one projection protrudes from said first side wall, wherein width of the projection measured in a direction perpendicular to said side wall is greater than or equal to 3 mm, and/or wherein the shortest distance between the projection's distal end and the opposite side wall is greater than or equal to 4 mm.

26. The chip of claim 16, wherein the non-aqueous liquid reservoir has an inlet end and an outlet end and the width of the outlet end is narrower than the width of the inlet end, and/or the width of the non-aqueous liquid reservoir at its widest point is greater than or equal to 4 mm.

27. The chip of claim 16, wherein the distance between the sample reservoir outlet opening and the lowest point of the sample reservoir, and the distance between the opening through which the non-aqueous liquid enters the sample reservoir and the nearest side wall of the sample reservoir are each equal to or less than 3 mm.

28. The chip of claim 27, wherein the distance between the sample reservoir outlet opening and the lowest point of the sample reservoir, and the distance between the opening through which the non-aqueous liquid enters the sample reservoir and the nearest side wall of the sample reservoir are each equal to or less than 2 mm

29. The chip of claim 16, wherein all the incubation segments are substantially identical the partial volume of all the supplying channels per a single incubation segment is constant for all the incubation segments, where the partial volume is the summation of quotients of the volumes of all the microfluidic channel sections leading from the sample reservoir to the incubation segment in question, and the number of incubation segments to which said microfluidic channel sections lead; and, all incubation segments are connected in parallel.

30. A method of filling of the incubation wells in a microfluidic chip of claim 16, including the following steps in order: a) providing in said non-aqueous liquid reservoir a non-aqueous liquid, b) inputting a sample to the sample reservoir, c) placing a chip in a hermetically-sealed container separated from its surroundings, d) reducing the pressure in said container to a value p.sub.0 to remove gas from the microfluidic system, e) increasing the pressure in said hermetically-sealed container to a value p.sub.1, at which sample flows from a sample reservoir to the microfluidic channels connecting said sample reservoir with the incubation segments and further into said incubation segments, f) activating the valve to open a flow path from said non-aqueous liquid reservoir to said microfluidic channels connecting said sample reservoir with said incubation segments, g) further increasing the pressure in said hermetically-sealed container to a value p.sub.2, to force said non-aqueous liquid to flow into the microfluidic channels connecting the sample reservoir and said non-aqueous liquid reservoir with the incubation segments, wherein p.sub.0 ranges between $(\frac{N.Math.V_D}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}.Math..Math.\mathrm{and}.Math..Math.\begin{array}{c}N.Math.V_D\\ N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}\end{array}.Math.p_{\mathrm{atm}}.Math..Math.\mathrm{and}.Math..Math.\frac{N\left(V_D+V_C\right)}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$ p.sub.1 ranges between $\frac{N.Math.V_D}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}.Math..Math.\mathrm{and}$ $\frac{N.Math.V_D}{{\mathrm{NV}}_D+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$ and p.sub.2 ranges between $\frac{N.Math.V_D}{N\left(V_A+V_C+V_D\right)+V_{i.Math.n}}.Math.p_{\mathrm{atm}}.Math..Math.\mathrm{and}.Math..Math.p_{\mathrm{atm}.Math..Math.},$ where p.sub.atm denotes ambient atmospheric pressure outside said hermetically-sealed container, Nnumber of incubation segments in said chip, V.sub.Avolume of an inlet channel of an incubation segment, i.e. the channel connecting main microfluidic network with an incubation well, V.sub.Bvolume of an incubation well, V.sub.Cvolume of a gas exchange channel, connecting the incubation well and a gas cavity, V.sub.Dvolume of a gas cavity, V.sub.involume of an intake channel of the main microfluidic network, i.e. the channel leading from the sample reservoir to the first branch of the main microfluidic network, V.sub.totala total volume of the network of microfluidic channels leading from the sample reservoir to the incubation segments excluding the said intake channel.

31. The method of claim 30, comprising the further step of subsequently further increasing the pressure in said hermetically-sealed container to ambient atmospheric pressure P.sup.atm.

32. The method of claim 30, wherein said non-aqueous liquid has viscosity greater than or equal to 20 cP, and/or in after step g or step h, the microfluidic chip is permanently sealed and the interior of the chip separated from its surrounding.

33. The method of claim 32, wherein said non-aqueous liquid has viscosity greater than or equal to 50 cP.

34. A method for improved microbiological assays, the improvement comprising the chip of claim 16, to perform the microbiological assays.

35. The method of claim 34, wherein said microbiological assay comprises at least one of a group consisting of identification of one or more microorganisms, determination of susceptibility to an antibiotic or a combination of antibiotics, determination of a minimum inhibitory concentration (MIC), detection of a mechanism of antibiotic resistance.

Description

DETAILED DESCRIPTION OF INVENTION

[0052] Structure of the Chip

[0053] FIG. 1 shows a schematic plan view of a first major surface of a chip according to the present invention.

[0054] FIG. 2 shows a schematic plan view of the second major surface of the chip of FIG. 1.

[0055] FIG. 3 shows a schematic view of the detailed structure of the channels in the second major surface of the chip of FIG. 1.

[0056] FIG. 4 shows an enlarged schematic view of section of the first major surface of the chip including 8 incubation segments.

[0057] FIG. 5 shows a schematic representation of a chip with series connection of incubation segments.

[0058] FIG. 6 shows schematically a plan view of a section of a chip with fractal geometry including 16 incubation segments

[0059] FIG. 7 shows schematically a plan view of a section of a chip with fractal geometry including 32 incubation segments

[0060] FIG. 8 shows schematically a plan view of a section of a chip with fractal geometry including 128 incubation segments

[0061] FIG. 9 shows schematically a plan view of a chip with asymmetric fractal geometry with 640 incubation segments

[0062] FIG. 10 shows schematically a plan view of a section of the chip of FIG. 9in which the feed channels leading to two areas with different numbers of incubation segments are highlighted

[0063] FIG. 11 shows schematically stages in a method according to the present invention for using a chip in accordance with the present invention.

[0064] FIG. 12 shows a plan view of an embodiment of a sample reservoir and non-aqueous liquid reservoir in accordance with the present invention.

[0065] FIGS. 1 and 2 show schematically a first embodiment of a microfluidic chip 1 in accordance with the present invention. The chip is formed of a substrate 2. The substrate is preferably planar with a first major face 3 and a, preferably parallel, second major face 5. The substrate may be made of any liquid and vapour impermeable material, for example a polymer, metal or glass. The thickness of the substrate is preferably equal to or greater than 1.5 mm and less than or equal to 3.00 mm. The substrate may be manufactured as one part (by for example injection moulding or milling a polymer) or may be composed of two parts i.e. a base plate (with the incubation segments and the microfluidic channels) and the reservoirs which are joined together by means of bonding methods known in the state of the art. The surface of the first major face is covered by a first layer 7 of impermeable, substantially transparent material and the surface of the second major surface is covered by a second layer 9 of impermeable, substantially transparent material. Preferably each of these layers is equal to or greater than 0.05 mm thick and less than or equal to 0.15 mm thick.

[0066] These layers prevent the entry or release of unwanted gas and liquids from the structures (described later) formed in the substrate while permitting light to pass through the incubation wellsthus allowing optical examination of the samples in the incubation wells. Microfluidic chip 1 includes a sample reservoir 11 for receiving and storing a sample for analysis (for example an inoculum of bacteria), a non-aqueous liquid reservoir 13 for receiving and storing a non-aqueous liquid, and an incubation segment area 15 comprising a plurality of incubation segments 17 in each of which a portion of the sample can be cultured.

[0067] The non-aqueous liquid reservoir 13 can be supplied with a non-aqueous liquid through a non-aqueous liquid inlet passage 19 which leads through the substrate to the non-aqueous liquid reservoir from a non-aqueous liquid inlet opening 21 on the first major face.

[0068] The sample reservoir 11 can be supplied with a sample, for example an inoculum of bacteria for analysis, through a sample inlet passage 23 which leads through the substrate to the sample reservoir from a sample inlet opening 25 on the first major face.

[0069] Sample reservoir has an outlet opening 27 which leads to a channel 22 formed in the substrate on the second major face of the chip. Channel 22 leads to a passage 31 formed in the substrate in the incubation segment area 15. This passage passes through the substrate and connects channel 22 with a channel 33 formed in the first major face of the substrate. Channel 33 is connected via a further passage 34 to a network of microfluidic channels 35 which lead to incubation segments 17 formed in the substrate. Preferably the microfluidic channels have a quadratic cross-section. Preferably the microfluidic channels have a cross-sectional area which is equal to or greater than 0.05 square mm and less than or equal to 1 square mm, more preferably the microfluidic channels have a cross-sectional area which is equal to or greater than 0.16 square mm and less than or equal to 0.64 square mm.

[0070] Non-aqueous liquid reservoir has an outlet opening 39 which leads to channel 29 and further via this channel to an outlet opening 39 through which the non-aqueous liquid enters the sample reservoir. Channel 29 can be temporarily blocked by a wax valve 24 or other, preferably remotely-activated valve, located in the channel 29 which, when closed, prevents the non-aqueous liquid from flowing through channel 29. When valve 24 is open, for example by heating in the case of a wax valve, the non-aqueous liquid can flow through channel 29 and into the sample reservoir.

[0071] The chip preferably has notches 26, 26 formed in two or more edges 28, 28 to allow the chip to be hooked onto the edges of a basket (not shown) used for carrying the chips in and/or a container in an analyser device (not shown) such that the sample inlet opening 25 is above the sample reservoir outlet opening 27 during filling.

[0072] The incubation segments area includes a plurality of incubation segments 17 and microfluidic channels 35 which can lead the sample to these incubation segments. The sample is transported to the individual incubation segments by the interconnecting network of channels (22, 33, 35), also referred to as a main microfluidic network, formed on the two major faces of the substrate of the chip which improves effective use of space on the chip and hence allows more segments to be accommodated in a single chip. The chip shown in the figure has 640 independent incubation segments in which the culturing of bacteria (or other microorganisms) may take place.

[0073] During the filling process, the sample located at the beginning in the sample reservoir flows to the channel 22. The channel ends with a passage 31 through which the sample enters the channel 33 on the first major face of the chip. This channel conducts the sample to the network of microfluidic channels 35. In this embodiment of the invention the network of microfluidic channels is arranged as a fractal structure of channels leading to the incubation segments in which the sample is further divided into equal portions that enter smaller microchannel structures via branched channels. More specifically, the chip in the FIGS. 1-2 consists of two asymmetric parts with 128 and 512 incubation segments. Preferably the resistance to flow for each pathway from the sample reservoir to an individual incubation segment is substantially the same for every such pathway so that the amount of sample reaching each incubation segment will be substantially the same. FIG. 3 shows an example of one path through these connected microchannelsa portion of the sample flows down channel 36 until it reaches a T-junction 37 with a secondary channel 38. Here substantially half of the sample flows in one direction (e.g. to the left) in the first branch 38 of the secondary channel and the other half of the sample flows in the opposite direction (e.g. to the right) in the second branch 38. Each of these branches in turn leads to a T-junction 40 with a tertiary channel 41.

[0074] At the T-junction 40 substantially half of the sample flows in one direction (e.g. to the left) in the first branch 41 of the tertiary channel and the other half of the sample flows in the opposite direction (e.g. to the right) in the second branch 41. Each of these branches in turn leads to a T-junction 42 with a quaternary channel 43.

[0075] At the T-junction substantially half of the sample flows in one direction (e.g. to the left) in the first branch 43 of the quaternary channel and the other half of the sample flows in the opposite direction (e.g. to the right) in the second branch 43. Each of these branches in turn leads to a T-junction 44 with a quinary channel 45.

[0076] At the T-junction substantially half of the sample flows in one direction (e.g. to the left) in the first branch 45 of the quinary channel and the other half of the sample flows in the opposite direction (e.g. to the right) in the second branch 45. Each of these branches in turn leads to a T-junction 46 with a senary channel 47.

[0077] Here substantially half of the sample flows in one direction (e.g. to the left) in the first branch 47 of the senary channel and the other half of the sample flows in the opposite direction (e.g. to the right) in the second branch 47. Each of these branches in turn lead to a transport passage 49 which penetrates the substrate (but not the layers of impermeable material) and leads the sample to a septenary channel 51 which has two branches 51, 51 each of which extend, as shown in FIG. 4, in the surface of the first major face of the substrate, in two opposite directions from the transport passage to a T-junction 52 with a delivery channel 53.

[0078] At the T-junction 52 substantially half of the sample flows in one direction (e.g. to the left) in the first branch 53 of the delivery channel and the other half of the sample flows in the opposite direction (e.g. to the right) in the second branch 53. Each of these branches in turn leads to a T-junction 54 with a splitter channel 57.

[0079] At the T-junction 54 substantially half of the sample flows in one direction (e.g. to the right) into an inlet channel 57 of a first associated incubation segment 17 and the other half of the sample flows in the opposite direction (e.g. to the left) into an inlet channel 57 of a second associated incubation segment 17.

[0080] Each incubation segment 17 includes an incubation well 113a chamber where a subvolume of the sample is located during incubationconnected by a gas-exchange channel 115 to its associated unvented gas cavity 111comprising a chamber filled with air, or any other gas or gas mixture, necessary for microbial growth. The unvented gas cavity prevents contamination of the sample and the loss of sample or sample fluid by evaporation while providing gas which can be used by cells in the incubation chamber.

[0081] Once the sample has entered the incubation well the valve is operated to release the non-aqueous liquid, for example, the wax valve is heated and the wax melted, which releases the non-aqueous liquid from reservoir 13. This non-aqueous liquid flows via the same paths as the sample remaining in the microfluidic channels until it reaches the splitter channel 57, 57 which it at least partly fills, thereby providing a barrier which prevents gas or aqueous fluids from moving from one incubation segment to another. Preferably the viscosityat the temperature used for the loading of the chipof the non-aqueous liquid is greater than or equal to 20 cP, more preferably greater than or equal to 50 cP measured according to the ASTM method ASTM D7279.

[0082] Embodiment of a chip presented in FIGS. 1-4 has overall dimensions of 12885 mm and thickness of its substrate is equal to 2.2 mm. But any sizes are possible provided that they enable accommodating of the desired number of the incubation segments and fulfilling other criteria described in this description. For example, a chip with 128 incubation segments was designed having overall dimensions of 85.549.7 mm. Also chip with larger size can be manufactured as long as it is handy for the user which is a desired property of a diagnostics chip.

[0083] Explanation of a Chip Operation

[0084] In the following description the symbol p.sub.atm refers to the ambient atmospheric pressure in the surroundings outside of a device in which the incubation segments are filled according to the method described below.

[0085] Chip with Series Connection of the Incubation Segments

[0086] A chip with a series connection between incubation segments is presented in the following. A chip 109 with a series connection of incubation segments to the sample reservoir is schematically represented in FIG. 5. For the sake of simplicity, FIG. 5 shows only one incubation segment branching off from the main microfluidic channel, although preferably many incubation segments branch off from it. They can be located on either side of the main channel. In addition, the chip can include many such main channels. A reservoir 118 includes a portion 119 for containing a sample and a reservoir 120 for non-aqueous liquid 121 which can be isolated from the portion 119 by a valve (not shown). Each incubation segment 110 comprises 4 elements i.e. a gas cavity 111 of volume V.sub.D, an incubation well 113 of volume V.sub.B, a microfluidic channel 115 of volume V.sub.c connecting these two parts, also referred to as a gas exchange channel, and a microfluidic channel 117 of volume V.sub.A, also referred to as an inlet channel of the incubation segment, leading to the incubation well from a main channel 123. Thus, the total volume of the incubation segment V.sub.ABCD is equal to V.sub.A+V.sub.B+V.sub.c+V.sub.D. The main channel 123 of volume V.sub.KG is connected to the reservoir 118 by an intake channel 125 of volume V.sub.in and connected to a vacuum chamber 129 of volume V.sub.vac via a channel 127 of volume V.sub.out. Therefore, the overall volume of the main channel V.sub.oKG is the sum of the respective volumes i.e. V.sub.oKG=V.sub.in+V.sub.KG+V.sub.out. In the case of a chip with series connection of incubation segments, the volume V.sub.in of the intake channel of the main microfluidic network is defined as a volume of the part of the main microfluidic network between the sample reservoir outlet and a point at which a first (proximal) inlet channel 117 of an incubation segment branches off from the main microfluidic network.

[0087] Chip with a Fractal Geometry

[0088] FIG. 6, FIG. 7 and FIG. 8 show schematically the parts of a chip with fractal geometry including 16, 32 and 128 incubation segments respectively. In the FIG. 6 the elements of each incubation segment are marked. These are: incubation well (62) of volume V.sub.B, gas cavity (64) of volume V.sub.D, and the microfluidic channels 61 (inlet channel of the incubation segment of volume V.sub.A) and 63 (gas exchange channel of volume V.sub.C). Preferably, the gas cavity 64 is located under the inlet channel 61 leading the sample to the incubation well and separated from the inlet channel by a predetermined thickness of substrate material. Having the inlet channel overlapping the gas cavity allows more efficient use of a space on the chip so that with chips of easily-handled sizes (e.g. standard sized microplates of approximately 128 mm85 mm), a large number of closely spaced incubation segments (up to 640 or more) can be located on the chip which improves its functionality by allowing more bacterial cultures to be conducted during a single test. It should be noted that the number of the independent reaction wells is the main factor limiting a functionality of the prior art AST test cards. The possibility of accommodating such a large number of incubation segments on a single chip enables the acquisition of comprehensive information on the drug susceptibility of the bacteria in the sample (i.e. by permitting the testing of more antibiotics or their combinations for a possible resistance, determining of a true MIC which requires conducting a bigger number of cultures than determining the antibiotic concentration break points, possible finding of a resistance mechanism). In this respect the incubation segment enables obtaining unique properties which significantly exceed the properties of the AST test cards known in the state of the art. Preferably, as shown most clearly in FIGS. 6-8, in order to utilize the area of the chip efficiently, the microfluidic channels 63 between the incubation well and the gas cavity are formed in one major face of the substrate and some or all of the other microfluidic channels formed in the opposite major face of the substrate. The channels 65, 66, 67 and 68 lead a sample to 2, 4, 8 and 16 incubation segments, respectively. Similarly, the channels 70, 81 and 82 lead a sample to 32, 64 and 128 incubation segments respectively.

[0089] Calculations

[0090] 1. Mathematical model and conditions for correct functioning of a chip: [0091] 1.1. Assumptions regarding geometry: [0092] 1.1.1. Rank of a fractal equals i means N=2.sup.i of the incubation segments V.sub.ABCD. [0093] 1.1.2. The shape of an inlet channel of an incubation segment is changed in comparison with a chip with a series connection. [0094] 1.1.3. A base cell consists of two incubation segments of volume V.sub.ABCD. A channel V.sub.k leads to the number k of incubation segments. [0095] 1.1.4. There is no vacuum chamber. [0096] 1.2. Steps during filling of the incubation segments: [0097] 1.2.1. Lowering of a pressure to the value p.sub.0.

RTn.sub.g=p.sub.0(2.sup.iV.sub.ABCD+V.sub.in+.sub.j=1.sup.i2.sup.ijV.sub.2.sub.j). [0098] 1.2.2. Causing a sample flow from the sample reservoir to the microfluidic system by changing the pressure to the exemplary preferred pressure p.sub.1. As a result there will be the following volume of a sample in the microfluidic system: 2.sup.i(V.sub.A+V.sub.B+V.sub.C)

[00004]$p_1=p_0.Math.\frac{2^i.Math.\left(V_A+V_B+V_C+V_D\right)+V_{\mathrm{in}}+{.Math.}_{j=1}^i.Math.2^{i-j}.Math.V_{2^j}}{2^i.Math.\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}+{.Math.}_{j=1}^i.Math.2^{i-j}.Math.V_{2^j}}.$ [0099] 1.2.3. Forcing a non-aqueous liquid to flow into the microfluidic channels connecting the sample reservoir and said non-aqueous liquid reservoir with the incubation segments at the exemplary preferred pressure p.sub.2. Then the channels of the main microfluidic network are fed with a volume .sub.j=1.sup.i2.sup.ijV.sub.2.sub.j of the non-aqueous liquid and the incubation segments are separated.

[00005]$p_2=p_1.Math.\frac{2^i.Math.\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}+{.Math.}_{j=1}^i.Math.2^{i-j}.Math.V_{2^j}}{2^i.Math.\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}}.$ [0100] 1.2.4. Causing further flow into the microfluidic system of the non-aqueous liquid at the pressure p.sub.3=p.sub.atm. This allows compressing air in the gas cavities V.sub.D and filling the entire incubation wells with a sample.

[00006]$p_3=p_2.Math.\frac{2^i.Math.\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}}{2^i.Math.\left(V_D+\frac{1}{2}.Math.V_C\right)}.$ [0101] 1.3. Summary: [0102] 1.3.1. Exemplary preferred volume formulas for gas V.sub.air, sample V.sub.w and non-aqueous liquid V.sub.o contained in the microfluidic structure of the chip after filling at the temperature T.sub.1=20 C.:

V.sub.air=2.sup.iV.sub.D,

V.sub.w=2.sup.i(V.sub.A+1V.sub.B+V.sub.C),

V.sub.o>2.sup.iV.sub.A+V.sub.in+.sub.j=1.sup.i2.sup.ijV.sub.2.sub.j. [0103] 1.3.2. The volume formulas at the temperature T.sub.2=37 C.:

V.sub.air=1.07.Math.2.sup.iV.sub.D2.sup.i(V.sub.D+V.sub.C),

V.sub.w=2.sup.i(V.sub.A+1V.sub.B+V.sub.C),

V.sub.o2.sup.i(V.sub.A)+V.sub.in+.sub.j=1.sup.i2.sup.ijV.sub.2.sub.j. [0104] 1.3.3. Optimal pressure values:

[00007]$p_0=p_3.Math.\frac{2^i.Math.\left(V_D+\frac{1}{2}.Math.V_C\right)}{2^i.Math.\left(V_A+V_B+V_C+V_D\right)+V_{\mathrm{in}}+{.Math.}_{j=1}^i.Math.2^{i-j}.Math.V_{2^j}},.Math.p_1=p_3.Math.\frac{2^i.Math.\left(V_D+\frac{1}{2}.Math.V_C\right)}{2^i.Math.\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}+{.Math.}_{j=1}^i.Math.2^{i-j}.Math.V_{2^j}},.Math.p_2=p_3.Math.\frac{2^i.Math.\left(V_D+\frac{1}{2}.Math.V_C\right)}{2^i.Math.\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}},$ [0105] where: [0106] .sub.j=1.sup.i2.sup.ijV.sub.2.sub.j is a sum of the volumes of all channels leading from the sample reservoir to the incubation segments (with the exception of the intake channel V.sub.in); [0107] V.sub.in is a volume of an intake channel of the main microfluidic network, i.e. the channel leading from the sample reservoir (and non-aqueous liquid reservoir) to a fractal microfluidic structure of the chip, i.e. to the first branching point; [0108] Therefore V.sub.in+.sub.j=1.sup.i2.sup.ijV.sub.2.sub.j is the sum of the volumes of all channels leading from the sample reservoir to the incubation segments; [0109] p.sub.3 is the atmospheric pressure. [0110] 1.4. Input data:

p.sub.3=p.sub.atm,

V.sub.A=0.197 L,

V.sub.B=2.45 L,

V.sub.C=0.174 L,

V.sub.D=1.17 L,

i=7,

V.sub.2=1.Math.0.5.Math.0.5 L=0.25 L,

V.sub.4=2.Math.0.5.Math.0.5 L=0.5 L,

V.sub.8=4.Math.0.5.Math.0.5 L=1 L,

V.sub.16=4.Math.0.5.Math.0.5 L=1 L,

V.sub.32=8.Math.0.5.Math.0.5 L=2 L,

V.sub.64=8.Math.0.5.Math.0.5 L=2 L,

V.sub.128=16.Math.0.5.Math.0.5 L=4 L,

.sub.j=1.sup.i2.sup.ijV.sub.2.sub.j=72 L,

V.sub.in=4 L. [0111] 1.4.1. Resultsthe optimal values of pressure which allow correct functioning of the chip with the above volumes and ratios between the volumes of the different sections:

p.sub.3=P.sub.atm=1013.25 mbar,

p.sub.0=0.274p.sub.3=278 mbar,

p.sub.1=0.645p.sub.3=653 mbar,

p.sub.2=0.906p.sub.3=918 mbar.

[0112] Chip with a Fractal Geometry and Asymmetric Branches [0113] 1.1. Geometry of the chip

[0114] An example of an asymmetric fractal chip is shown in FIG. 9. The chip includes 640 incubation segments which consists of the following structures [0115] i) 4 parts with 128 incubation segments (described above)shown by 91 in FIG. 9, [0116] ii) 2 parts with 64 incubation segmentsshown by 92 in FIG. 9. [0117] 1.2. Mathematical and design assumptions: [0118] 1.2.1. All incubation segments are identical and indistinguishable (from a model point of view). [0119] 1.2.2. A partial volume of all supplying channels per a single incubation segment (quotient of a sum of the volumes of all channels leading to an incubation segment, including channel V.sub.in, and a number of the incubation segments which share these channels) is constant. This is calculated for each incubation segment by adding up, for each microfluidic channel section leading from the sample reservoir to the incubation segment in question, a quotient of the microfluidic channel section's volume and a number of incubation segments to which it leads. [0120] 1.2.3. All incubation segments are connected in parallel. This means that there can be only forks of the channels (or, more generally, each channel can only split into two branches at junctions) without any series connections.

[0121] The above-mentioned conditions lead to a fractal distribution of incubation segments and a uniform partition of a sample and non-aqueous liquid. [0122] 1.3. Mathematical model. [0123] The assumptions from point 1.2 do not change the values of pressure derived in the previous subsection because they depend only on the volumes occupied by a sample and air in the incubation segments which are the same. Hence, we obtain the following values of a pressure: [0124] i) Optimal initial pressure:

[00008]$p_0=p_3.Math.\frac{N\left(V_D+\frac{1}{2}.Math.V_C\right)}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}},$ [0125] Where V.sub.total means a sum of the volumes of all channels leading from the sample reservoir to the incubation segments without the intake channel V.sub.in and N means total number of the incubation segments. [0126] ii) The optimal pressure required for causing the flow of sample into the incubation segments:

[00009]$p_1=p_3.Math.\frac{N\left(V_D+\frac{1}{2}.Math.V_C\right)}{N\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.$ [0127] iii) The optimal pressure required for separating the incubation segments with a non-aqueous liquid:

[00010]$p_2=p_3.Math.\frac{N\left(V_D+\frac{1}{2}.Math.V_C\right)}{N\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{\mathrm{in}}}.$ [0128] 1.4. Conclusionsan optimization of a geometry of the asymmetric fractal chip is necessary to ensure its correct functioning: [0129] 1.4.1. In the first fork 100 (in FIG. 10), the volume of a sample that flows into the incubation segments at the right side should be correspondingly larger than the volume of a sample which flows into the incubation segments at the left side. A ratio of these volumes is equal to the ratio of the numbers of the incubation segments at the right side and the left side.

[00011]$\frac{V_{{\mathrm{sample}}_{\mathrm{right}}}}{V_{{\mathrm{sample}}_{\mathrm{left}}}}=\frac{N_{\mathrm{right}}}{N_{\mathrm{left}}}.$ [0130] 1.4.2. Assumption above imposes an analogous ratio of a volume of air and therefore of a volume of the channels at the right side and the left side.

[00012]$\frac{V_{{\mathrm{channels}}_{\mathrm{right}}}}{V_{{\mathrm{channels}}_{\mathrm{left}}}}=\frac{N_{\mathrm{right}}}{N_{\mathrm{left}}}.$ [0131] There are the following symmetries in this case: [0132] i) The channels 101-104 and the channels 105-107 in FIG. 10 must have a volume proportional to N.sub.right and N.sub.left respectively which can be written as the following equation:

[00013]$\frac{V_{{\mathrm{channels}}_{101.Math.\text{-}.Math.104}}}{V_{{\mathrm{channels}}_{105.Math.\text{-}.Math.107}}}=\frac{N_{\mathrm{right}}}{N_{\mathrm{left}}}.$ [0133] ii) The other channels are shared by the same number of incubation segments.

[0134] Hence all such channels at both sides of the forking A are identical and their number scale up with the N.sub.right and N.sub.left.

[0135] Conclusions [0136] 1.1. A chip with fractal geometry does not need any vacuum chamber 129 for even filling of all wells. Therefore, the required sample volume is equal to a sum of the volumes of the incubation wells with possibly a small reserve. [0137] 1.2. Uniform filling of the incubation segments does not require any specific controlling of a liquid flow. It is achieved by a thermal equilibrium and a pressure balance. [0138] 1.3. The required values of pressures can be easily applied with the use of vacuum pump. A filling deviation equal to or less than 5% does not affect the correct functioning of a chip. [0139] 1.4. The above derivation shows the optimal values of pressures. However, a pressure can be applied with some tolerance resulting from different occupation of the elements of the incubation segment (V.sub.A-V.sub.D) by an air, a sample and a non-aqueous liquid. These different configurations should ensure that a sample must not enter a gas cavity and that a gas and a non-aqueous liquid must not enter an incubation well. But such conditions leave certain margins for the p.sub.0, p.sub.1 and p.sub.2 values according to the following formulas and specification:

[00014]$p_{0.Math.\min }=\frac{N.Math.V_D}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities only);

[00015]$p_{0.Math..Math.\mathrm{opt}}=\frac{N\left(V_D+\frac{1}{2}.Math.V_C\right)}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities and a half of each gas exchange channel);

[00016]$p_{0.Math.\max }=\frac{N\left(V_D+V_C\right)}{N\left(V_A+V_B+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities and gas exchange channels);

[00017]$p_{1.Math.\min }=\frac{N.Math.V_D}{N\left(V_A+V_C+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$

(gas fills only the gas cavities, sample fills only the incubation wells);

[00018]$p_{1.Math..Math.\mathrm{opt}}=\frac{N\left(V_D+\frac{1}{2}.Math.V_C\right)}{N\left(\frac{1}{2}.Math.V+\frac{1}{2}.Math.V+V_D\right)+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities and half of each gas exchange channel, sample fills incubation wells and halves of all gas exchange and inlet channels);

[00019]$p_{1.Math.m.Math..Math.x}=\frac{N.Math.V_D}{N.Math.V_D+V_{i.Math.n}+V_{\mathrm{total}}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities, sample fills the incubation wells and inlet and gas exchange channels);

[00020]$p_{2.Math.\min }=\frac{N.Math.V_D}{N\left(V_A+V_C+V_D\right)+V_{i.Math.n}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities, sample fills incubation wells and gas exchange channels, non-aqueous liquid fills inlet channels (or their partsdepending on pa) and the channels leading to the incubation segments);

[00021]$P_{2.Math.o.Math.p.Math.t}=\frac{N\left(V_D+\frac{1}{2}.Math.V_C\right)}{N\left(\frac{1}{2}.Math.V_A+\frac{1}{2}.Math.V_C+V_D\right)+V_{i.Math.n}}.Math.p_{\mathrm{atm}}$

(gas fills the gas cavities and halves of gas exchange channels, sample fills incubation wells, halves of gas exchange channels and part of each inlet channel (depending on pa), non-aqueous liquid fills part of each inlet channel (depending on pa) and the channels leading to the incubation segments); [0140] p.sub.2max=p.sub.atm (gas fills the gas cavities and gas exchange channels, sample fills incubation wells and part of each intake channel (depending on p.sub.1), non-aqueous liquid fills part of each inlet channel (depending on p.sub.1) and the channels leading to the incubation segments).

[0141] The table below presents the exemplary values of a pressure defined as above which were calculated for a chip with asymmetric branches and 640 incubation segments accommodated. The following volumes are used V.sub.A=0.36 l, V.sub.B=2.26 l, V.sub.C=0.26 l, V.sub.D=1.06 l, V.sub.in=0, and V.sub.total=668.2 l. Furthermore, p.sub.atm is assumed to be equal to 1013.25 mbar.

TABLE-US-00001 p.sub.0min 215.50 mbar p.sub.0opt 241.92 mbar p.sub.0max 268.35 mbar p.sub.1min 435.88 mbar p.sub.1opt 499.48 mbar p.sub.1max 510.46 mbar p.sub.2min 639.31 mbar p.sub.2opt 880.12 mbar p.sub.2max 1013.25 mbar

[0142] FIGS. 11 A to 11G show schematically lateral views of the steps of filing one incubation well of a chip.

[0143] FIG. 11A shows schematically a portion of a chip. The chip comprises a sample reservoir for receiving and storing a sample for analysis 11, a non-aqueous liquid reservoir 13 for receiving and storing a non-aqueous liquid, a non-aqueous liquid inlet opening 21 leading to the non-aqueous liquid reservoir, a sample inlet passage 23 which leads to the sample reservoir, a sample reservoir outlet opening 27 leading to a passage 22, non-aqueous liquid reservoir outlet opening 39 leading to the passage 22, wax valve 24 which can prevent the non-aqueous liquid from leaving the non-aqueous liquid reservoir and entering the sample reservoir, a network of channels leading from the sample reservoir to the inlet channel 57 of an incubation segment 17, an incubation well 113 connected by a gas-exchange channel 115 to its associated unvented gas cavity 111. The ambient pressure around and inside the chambers and channels in the chip is atmospheric pressure p.sub.atm.

[0144] In FIG. 11B the non-aqueous liquid reservoir has been partly filled with a non-aqueous liquid NAL and the inlet opening 21 sealed at atmospheric pressure.

[0145] In FIG. 11C the sample reservoir has been partly filled with a sample SAM. The inlet opening remains open to ambient pressure. Interplay between capillary and surface tension forces present at the entrance to the network of channels and the back-pressure of gas contained in the closed (unvented) microfluidic system downstream of the sample reservoir prevents the sample from entering the network of channels. The chip is placed in a chamber in which the pressure is then reduced to a pressure p.sub.o which is below atmospheric pressure. This causes expansion of the gas in the microfluidic system and causes some of the gas in the chip to flow out of the gas cavity, gas-exchange channel, incubation well, inlet channel and network of channels (the main microfluidic network) and to pass through the sample in the sample reservoir to the exterior of the chip until the pressure inside the microfluidic system is equal to p.sub.o.

[0146] In FIG. 11D the pressure has been raised to p.sub.1 which is higher than p.sub.0. This pressure difference between upstream and downstream of the sample causes the sample to be pushed out of the sample reservoir into the microfluidic system, until the pressure inside the gas cavity, gas-exchange channel, incubation well, inlet channel and network of channels is substantially equal to p.sub.1.

[0147] In FIG. 11E the valve between the non-aqueous liquid reservoir and the sample reservoir has been opened. As the pressure in the non-aqueous liquid reservoir is initially greater than p.sub.1, the non-aqueous liquid in the non-aqueous liquid reservoir will flow from the non-aqueous liquid reservoir into the sample reservoir until the pressure in the non-aqueous liquid reservoir drops to p.sub.1. Preferably the density of the non-aqueous liquid is greater than that of the sample, so that the excess sample floats on top of the non-aqueous liquid.

[0148] In FIG. 11F the ambient pressure is raised to p.sub.2 which can be less than atmospheric pressure or equal to atmospheric pressure. The non-aqueous liquid is sucked into the network of channels and reaches the inlet channel 57 of an incubation segment 17, thereby preventing cross-contamination (cross-talk) between the incubation segment 17 and any neighbouring incubation segment.

[0149] FIG. 11G shows the continued penetration of non-aqueous liquid into the incubation segment if the pressure p.sub.2 was less than atmospheric and which would occur when the chip is subjected to an ambient pressure equal to atmospheric pressure p.sub.atm.

[0150] FIG. 12 shows a plan view of an embodiment of a sample reservoir 141 according to an embodiment of the present invention. The following paragraphs together with FIG. 12 describe preferred conditions for the proper operation of the sample and non-aqueous liquid reservoir according to the present invention. When the ambient pressure initially is decreasing, the gas from the microfluidic structure is evacuated from the interior of the chip by a pathway which flows through the sample in the reservoir. Optionally, loss of sample can be prevented by covering the sample inlet opening by a material which is impermeable for liquids after the sample has been loaded. Preferably this material is permeable to gas to allow gas to vent from the sample reservoir. As the sample inlet opening 25 need not necessarily be covered by a material impermeable for liquid during incubation segments filling (in fact it can even be open), it is desirable to provide means that help to ensure that the sample should not be lost through this opening. This can be achieved by making the vertical height h.sub.s of the sample reservoir large enough to give enough distance between the surface of a sample in the reservoir and the inlet opening to prevent leakage in normal use when the inlet opening is arranged higher than the outlet opening of the sample reservoir. Preferably the chip is loaded with sample and operated with the major surfaces of the substrate perpendicular to the horizontal and the inlet opening of the sample reservoir higher than the outlet of the sample reservoir. The height h.sub.s is preferably greater than or equal to 30 mm, more preferably greater than or equal to 40 mm, even more preferably greater than or equal to 50 mm. Furthermore, the prevention of sample leakage by being entrained by gas bubbles during the exiting of gas through the sample reservoir can be enhanced by providing the sample reservoir with one or more internal projections 131 and 132. These projections protrude from the side walls of the reservoir and prevent a sample from being pushed up by the gas bubbles flowing upward. They should be large enough to modify the cross-section of a channel through which the gas may flow and to change the shape of the bubbles. However, the distance between distal ends of the projections and the opposite side wall should also be greater than the distance across which capillary forces cause liquid flow, to prevent capillary forces affecting the sample there. Therefore, the width w.sub.s of the sample reservoir is preferably greater than or equal to 6 mm, more preferably greater than or equal to 7 mm, even more preferably greater than or equal to 9 mm. The width of the projection w.sub.p is preferably greater than or equal to 1 mm, more preferably greater than or equal to 2 mm, even more preferably greater than or equal to 3 mm. The width of the gap between the distal tip of the projection and the opposite side wall w.sub.g is preferably greater than or equal to 3 mm, more preferably greater than or equal to 4 mm. The sample reservoir can be broader at its lower (i.e. opposite to the sample inlet opening) end. This facilitates the evacuation of a gas and, for a given volume of sample, increases the distance from the exposed surface of the sample to the sample inlet opening compared to a narrow sample reservoir's lower end, which is helpful to prevent leakage. All width dimensions (i.e. perpendicular to a longitudinal axis of the sample reservoir) are relevant for these effects. Preferably the width, perpendicularly to the plane of the substrate, of the sample reservoir in the proximity of the lower end is greater than or equal to 5 mm, more preferably greater than or equal to 7 mm. Preferably the width in the plane of the substrate of the lower portion of the sample reservoir is greater than or equal to 10 mm. Those conditions significantly affect a volume of the sample reservoir. Its minimal value is equal to the product of the volume of single incubation well and the number of the incubation segments in the chip. Such a minimal volume is equal to or greater than 1.5 ml for a chip with 640 segments but is smaller for a smaller number of segments (for example about 0.29 ml for a chip with 128 segments). However, as can be seen from the above considerations, the volume of the sample reservoir should be larger. Preferably it is equal to or greater than twice the total volume of the incubation wells of all the incubation segments of the chip to which it is connected and more preferably it is equal to or greater than three times the total volume of said incubation segments.

[0151] The non-aqueous liquid (NAL) reservoir 143 should also have a volume which is larger than a minimal volume of NAL which is equal to the total volume of the microfluidic channels leading from the sample reservoir to all incubation segments. When a NAL flows to the sample reservoir after activation of a valve, the gas over the liquid should change its pressure from p.sub.atm to p.sub.1 where p.sub.1 is the pressure in the microfluidic chip when the sample flows into the incubation segments. In order to push out substantially the whole volume of NAL from the reservoir, its volume should not be smaller than

[00022]$\frac{p_{\mathrm{atm}}}{p_{\mathrm{atm}}-p_1}.Math.V_{N.Math.A.Math.L}$

where V.sub.NAL the minimal volume of NAL mentioned above. Since p.sub.1 can be about 0.67 p.sub.atm or less (it generally decreases with an increasing number of incubation segments), the NAL reservoir preferably has a volume equal to or greater than two times the total volume of the channels leading from the sample reservoir to all incubation segments, more preferably it is equal to or greater than three times said total volume. This reservoir should be also wide enough so that capillary forces do not prevent NAL flow to the sample reservoir. The reservoir width w.sub.n is preferably greater than or equal to 4 mm, more preferably greater than or equal to 5 mm, even more preferably greater than or equal to 6 mm. As a NAL enters the sample reservoir through the NAL outlet opening 39 and leaves it through the sample reservoir outlet opening 27, their proper positioning is important to minimize the dead volume of NAL. It is possible that the volume of the part of the sample reservoir enclosed between these openings, V.sub.act, is greater than the minimal volume of NAL as defined above. The opening 27 preferably should be also located close to the lowest point of the sample reservoir, preferably at a distance d.sub.1 which is smaller than or equal to 3 mm. It is also advantageous when a NAL entering the sample reservoir flows down the side of the sample reservoir. For this purpose, a distance d.sub.2, which is smaller or equal to 3 mm, is preferred.

[0152] An embodiment of a chip according to the present invention has a substrate with a length of from 12 to 13.5 cm, preferably 12.8 cm length and a width of from 8 cm to 9 cm, preferably 8.5 cm, as described previously on page 14. Most preferably it has footprint dimensions of a microplate as specified in ANSI SLAS 1-2004 (R2012) Footprint dimensions for microplates, namely 127.76 mm (with a tolerance of 0.5 mm)85.48 mm (with a tolerance of 0.5 mm). Preferably it has a depth of from 0.19 to 0.22 cm, preferably 0.20 cm. Preferably, if the substrate is made of two parts joined together as described previously on page 9, then the base plate may have a thickness which is equal to or greater than 0.19 cm and less than or equal to 0.22 cm, and the reservoir portion a greater thickness, for example equal to or greater than 1 cm and equal to or less than 1.5 cm. A total of 640 incubation segments can be formed in this chip by choosing appropriate dimensions of the chambers and microchannels, and disposing them on both major faces of the substrate, e.g. as shown most clearly in FIGS. 1-2 and 6-9. Preferably, the volume of the sample reservoir is equal to or greater than 4 ml, less than or equal to 6 ml and more preferably 5 millilitres. Preferably, the volume of the non-aqueous reservoir is equal to or greater than 2.0 ml, less than or equal to 3.0 ml and more preferably 2.5 ml. Preferably, the total volume of the microfluidic channels is equal to or greater than 500 less than or equal to 900 ,l and more preferably about 668 l. Preferably, each incubation segment has a volume which equal to or greater than 3.0 l, less than or equal to 5.0 l and more preferably about 3.94 l. Preferably each incubation well has a volume equal to or greater than 2.0 l, less than or equal to 2.50 l and more preferably about 2.26 l. Preferably each gas-exchange channel has a volume equal to or greater than 0.2 l, less than or equal to 0.3 l and more preferably about 0.26 l. Preferably the unvented gas cavities each have a volume equal to or greater than 0.75 l, less than or equal to 1.25 l and more preferably a volume of about 1.06 l.