Microfluidic structures

Abstract

A microfluidic structure for spacing out and aligning entities in an aqueous suspension is provided. The structure comprises: a channel for guiding entities in an aqueous suspension; a first comb of first inlets arranged on a first side of the channel for introducing a spacing medium into the channel; and a second comb of second inlets arranged on a second side of the channel for introducing the spacing medium into the channel; wherein the first side is opposite the second side, and wherein one of the first inlets has a corresponding, respective one of the second inlets at a substantially similar longitudinal position along the channel.

Claims

1. A microfluidic structure for spacing out and aligning entities in an aqueous suspension, the structure comprising: a main channel for guiding entities in an aqueous suspension; a first comb of first inlets arranged on a first side of said channel for introducing a spacing medium into said main channel; and a second comb of second inlets arranged on a second side of said channel for introducing said spacing medium into said channel; a first plurality of spacing fluid channels and a second plurality of spacing fluid channels for providing the spacing medium into the first comb of first inlets and the second comb of second inlets, wherein the first plurality of spacing fluid channels and the second plurality of spacing fluid channels comprise a main spacing fluid channel which joins a plurality of secondary spacing fluid channels, wherein the secondary spacing fluid channels join the first comb of first inlets and the second comb of second inlets, wherein said first side is opposite said second side, and wherein a first inlet of the first comb of first inlets has a corresponding second inlet of the second comb of second inlets at a substantially similar longitudinal position along said main channel; wherein said first comb of first inlets and said second comb of second inlets comprise a plurality of pairs of inlets joined at an acute angle to the main channel, wherein the secondary spacing fluid channels are smaller than the main spacing fluid channels, and wherein each of said plurality of first inlets and second inlets is smaller than the plurality of spacing fluid channels and the main channel.

2. A microfluidic structure as claimed in claim 1, wherein one or more of said first inlets and one or more of said corresponding second inlets each forms an angle with said main channel of less than 90 degrees.

3. A microfluidic structure as claimed in claim 1, wherein a part of a said first inlet or a said second inlet is coated with a hydrophilic coating.

4. A method of generating picodroplets from a suspension comprising a plurality of entities, the method comprising: aligning entities in a suspension in a microfluidic structure, wherein aligning entities in a suspension comprises: providing a channel on said microfluidic structure for guiding said entities in said suspension; providing a first comb of first inlets arranged on a first side of said channel for introducing a fluid into said channel; providing a second comb of second inlets arranged on a second side of said channel for introducing a said fluid into said channel; wherein said first side is opposite said second side, and wherein a first inlet of the first comb of first inlets has a corresponding second inlet of the second comb of second inlets at a substantially similar longitudinal position along said channel; the method further comprising: guiding said suspension comprising said entities through said channel; introducing said fluid into said channel from one or more of said first inlets at the same time as introducing said fluid into said channel from one or more of said corresponding second inlets to align said entities in said suspension in said channel; and forming an emulsion of picodroplets comprising said entities by providing a flow of said suspension to a picodroplet generation region of said microfluidic structure.

5. A method as claimed in claim 4, wherein said fluid is introduced into said channel with a flow rate which is higher than a flow rate of said suspension in said channel to space out said entities in said suspension in said channel.

6. A method as claimed in claim 4, wherein said fluid is introduced into said channel from a said first inlet of the first comb of first inlets with a first flow rate and from said corresponding second inlet of the second comb of second inlets with a second flow rate, wherein said first flow rate and said second flow rate are substantially the same to reduce a hydrodynamic pressure gradient in said suspension across a width of said channel from said first inlet to said corresponding, respective second inlet.

7. A method as claimed in claim 4, wherein said fluid is introduced into said channel in a flow direction of said suspension in said channel.

8. A method of generating picodroplets from a suspension comprising a plurality of entities, the method comprising: spacing out entities in a suspension, wherein spacing out entities in the suspension comprises: guiding said suspension comprising said entities through a channel of a microfluidic structure; and introducing an aqueous spacing medium into said channel from a first inlet arranged on a first side of said channel and substantially simultaneously introducing said aqueous spacing medium into said channel from a second inlet arranged on a second side of said channel to space out said entities in said suspension in said channel, wherein said first side is opposite said second side, and wherein said first inlet is arranged at a substantially similar longitudinal position along said channel as said second inlet; and forming an emulsion of picodroplets comprising said entities by providing a flow of said suspension to a picodroplet generation region of said microfluidic structure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) These and other aspects of the invention will now be further described, by way of example only, with reference to the accompanying figures, wherein like numerals refer to like parts throughout, and in which:

(2) FIG. 1 shows the percentage of a single cell per picodroplet and picodroplets containing any cells, respectively, versus ratio of total cell number to picodroplet number;

(3) FIG. 2 shows a schematic of a microfluidic device according to embodiments of the present invention;

(4) FIG. 3 shows a schematic of a microfluidic structure according to embodiments of the present invention;

(5) FIG. 4 shows a video snapshot of cells in picodroplets obtained using embodiments of the present invention;

(6) FIG. 5 shows a video snapshot of cells in picodroplets obtained using embodiments of the present invention;

(7) FIG. 6 shows reinjection frequency versus picodroplet reinjection flow rate;

(8) FIG. 7 shows a video snapshot of cells in picodroplets obtained using embodiments of the present invention; and

(9) FIG. 8 shows hydrodynamic pressure versus distance.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(10) As outlined above, embodiments described herein may be used in microfluidic structures and methods for encapsulation entities, for example cells or other biological entities, and for low stress picodroplet reinjection.

(11) If picodroplets are formed from a suspension, for example an aqueous solution, whereby the suspension comprises cells, the number of cells per picodroplet generally follows a Poisson distribution.

(12) The percentage of empty picodroplets among all picodroplets, the percentage of singlets among picodroplets (i.e. the number of picodroplets containing a single cell versus the total number of picodroplets) and the percentage of singlets among cells (i.e. the number of picodroplets containing a single cell versus the number of all picodroplets containing one or more cells) are dependent on the ratio of the total cell number to the total picodroplet number, which we define as the Poisson lambda value.

(13) The following table shows the percentages of empty picodroplets among all picodroplets (second column), the percentage of singlets among picodroplets (third column) and the percentage of singlets among cells (fourth column) as defined above for Poisson lambda values ranging from 0.1 to 1.5.

(14) TABLE-US-00001 TABLE 1 Empty picodroplets among picodroplets, singlets among picodroplets, singlets among cells and cells/mL as a function of the Poisson lambda value: Empty Singlet Singlet Poisson among among among picodroplets picodroplets cells Cell/mL 0.1 90.5% 9.0% 90.5% 3.33E+05 0.2 81.9% 16.4% 81.9% 6.67E+05 0.3 74.1% 22.2% 74.1% 1.00E+06 0.4 67.0% 26.8% 67.0% 1.33E+06 0.5 60.7 30.3% 60.7% 1.67E+06 0.6 54.9% 32.9% 54.9% 2.00E+06 0.7 49.7% 34.8% 49.7% 2.33E+06 0.8 44.9% 35.9% 44.9% 2.67E+06 0.9 40.7% 36.6% 40.7% 3.00E+06 1 36.8% 36.8% 36.8% 3.33E+06 1.1 33.3% 36.6% 33.3% 3.67E+06 1.2 30.1% 36.1% 30.1% 4.00E+06 1.3 27.3% 35.4% 27.3% 4.33E+06 1.4 24.7% 34.5% 24.7% 4.67E+06 1.5 22.3% 33.5% 22.3% 5.00E+06

(15) As can be seen from table 1, the cell encapsulation quality, i.e. the one-cell-per-picodroplet (OCPD) rate, varies from 90.5% down to 22.3%. This means that more than 77% of cells generally go into picodroplets containing more than one cell.

(16) The findings of table 1 are shown in FIG. 1.

(17) When a Poisson lambda value of 0.1 is selected, a value of 90.5% of OCPD among cells is obtained, whereas only 9.0% among all picodroplets contain a cell at all. This indicates that 90% of all efforts may be spent on analysing empty picodroplets.

(18) In some examples described herein, a Poisson lambda value of 0.5 is chosen, as indicated by the row highlighted in blue in table 1. As can be seen, a Poisson lambda value of 0.5 results in 60.7% of the picodroplets being empty and 60.7% of OCPD among all cells.

(19) When the volume of each picodroplet is 300 pL, the cell concentration in the initial bulk suspension is 1.6710.sup.6 cells/mL. Even in the worst situation shown in the above table, i.e. when the Poisson lambda value is 1.5, the cell concentration in the suspension is just 510.sup.6 cells/mL.

(20) Embodiments described herein allow for approaches to cell encapsulation which may improve upon any Poisson distribution restriction in order to give OCPD quality and efficient cell encapsulation, and maintain a high cell survival rate.

(21) FIG. 2 shows a schematic of a microfluidic device or structure as generally described herein.

(22) In this example, the microfluidic structure 100 comprises three fluidic inlets in addition to the picodroplet outlet 112.

(23) A fluorinated oil reservoir 102 is provided in this example which is connected to a channel at a cross junction 110 at which discrete picodroplets are pinched off from a continuous aqueous phase at the cross junction nozzle.

(24) The aqueous fluid comprising cells or particles is provided in this example in a reservoir 106 which allows introducing the cells or particles to the cross junction (picodroplet generation region) 110 at which discrete picodroplets are pinched off from the aqueous fluid using the fluorinated oil from reservoir 102.

(25) An additional aqueous spacing medium (for example a culture medium, which may have a different viscosity than water) is provided in this example in reservoir 104. The aqueous spacing medium may be introduced into the main channel in which the cells or particles are guided in the aqueous fluid from the reservoir 106 towards the cross junction 110.

(26) First and second combs 108a, 108b of inlets are provided on opposing sides of the main channel at a longitudinal position between the reservoir 106 and the cross junction 110 at which the fluorinated oil is used to pinch off discrete droplets from the aqueous fluid comprising cells or particles.

(27) In this example, the additional aqueous spacing medium inlets between the fluorinated oil inlet and the aqueous fluid inlet at reservoir 106 connects with a pair of 2.sup.n flow splitting microfluidics which face each other at each side of the aqueous microfluidic main channel for cell or particle suspension.

(28) The combs 108a, 108b thereby allow for spacing out cells or particles from each other and aligning cells or particles, in this example, in the middle of the aqueous microfluidic main channel before being punch off into discrete picodroplets.

(29) In this example, n=4, resulting in 16 fluidic open mouths or nozzles at each side. However, it will be appreciated that n may be a different number, or alternatively 3 pairs, 5 pairs or any other integer number of pairs of nozzles may be provided via combs 108a 108b.

(30) In this example, the fluidic flow rates from the nozzles are identical which assures that there is no (or no significant) flow gradient within this spacing region.

(31) FIG. 3 shows a close-up of the schematic of the microfluidic structure of FIG. 2 as indicated in the rectangle in FIG. 2.

(32) As can be seen, in this example, a first comb 108a of first inlets and a second comb 108b of second inlets are arranged on opposing sides of the main channel 302. A spacing fluid channels provide the spacing medium into the first comb 108a of first inlets and the second comb 108b of second inlets. The spacing fluid channels include a main spacing fluid channel 316 that joins secondary spacing fluid channels 318. A first inlet 314a of the first comb 108a of first inlets has a corresponding second inlet 314b of the second comb 108b of second inlets at a similar longitudinal position along the main channel 302.

(33) In fluid dynamics, laminar flow (or streamline flow) occurs when a fluid flows in parallel layers, with no disruption between the layers. At low velocities, the fluid tends to flow without lateral mixing, and adjacent layers slide past one another like playing cards. There are no cross-currents perpendicular to the direction of flow, nor eddies or swirls of fluids. In laminar flow, the motion of the particles of the fluid is very orderly with all particles moving in straight lines parallel to the pipe walls. Laminar flow is a flow regime characterised by high momentum diffusion and low momentum convection.

(34) In this example, the inlets of the first and second combs 108a, 108b are at an acute angle to the main channel 302 such that, when the spacing medium is introduced into the main channel 302 via the inlets of combs 108a, 108b, the aqueous cell or particles suspension is less disturbed when guided through the main channel 302 while the cells or particles are aligned and/or spaced out. This reduces the risk of cell or particle deformation while the cells or particles are aligned within the main channel 302 and/or spaced out.

(35) In this example, the main channel 302 has a width of approximately 50 um.

(36) Two preliminary experiments were carried out in this example using 2.5 um Latex beads.

Experiment 1

(37) This experiment started with a concentration of 2.510.sup.7 beads/mL. The bead suspension flow rate was set at 50 uL/hr and the spacing fluid water rate was 500 ul/hr, which gave a final bead concentration of 2.2710.sup.6 beads/mL. The fluorinated oil was 5% Pico-Surf--1 in Novec-7500 at a flow rate of 1000 uL/hr.

(38) FIG. 4 shows a video snapshot of cells in picodroplets obtained using the above parameters.

(39) In the snapshot (which shows the microfluidic structure only at the cross junction where picodroplets were pinched off from the aqueous cell or particle suspension), 21 OCPD and 1 doublet (a picodroplet containing two cells) were counted. This indicates a higher OCPD rate (95.5%) than that from an encapsulation of a similar final concentration (210.sup.6 beads/mL in table 1) of an ideal suspension on a conventional Pico-Gen biochip (54.9%).

Experiment 2

(40) In this experiment, a concentration of 2.510.sup.8 beads/mL, circa 100-fold higher, was used. The bead suspension flow rate was set at 20 uL/hr and the spacing fluid water rate at 500 ul/hr, which gave a final bead concentration 9.610.sup.6 beads/mL. The fluorinated oil was 5% Pico-Surf-1 in Novec-7500 at flow rate of 1000 uL/hr.

(41) FIG. 5 shows a video snapshot of cells in picodroplets obtained using the above parameters.

(42) In the snapshot (which shows the microfluidic structure only at the cross junction where picodroplets were pinched off from the aqueous cell or particle suspension), 41 OCPD and 3 doublets were counted. This indicates a much higher OCPD rate (93.2%) than that from an encapsulation of a similar final concentration (510.sup.6 beads/mL in table 1) of an ideal suspension on a conventional Pico-Gen biochip (22.3%).

Further Applications

(43) Such a pair of, in this example, 2.sup.n flow splitting microfluidics may be used for picodroplet reinjection on Pico-Sort designs.

(44) FIG. 6 shows the correlation between the picodroplet reinjection flow rate and the reinjection frequency.

(45) As can be seen in FIG. 6, a linear correlation between the picodroplet reinjection flow rate and the reinjection frequency was observed with a slope of 0.85.

(46) The following table outlined the experimentally observed parameters.

(47) TABLE-US-00002 TABLE 2 Correlation between the picodroplet reinjection flow rate and the reinjection frequency: Novec7500 Picodroplet t1 t2 dt No. of Frequency (uL/hr) (uL/hr) (ms) (ms) (ms) Picodroplet (Hz) 3000 300 4603.6 4526.5 77.1 20 259.4 4000 400 3982.6 3921.6 61.0 20 327.9 5000 500 5225.1 5181.1 44.0 20 454.5 6000 600 1654.6 1614.5 40.1 20 498.8 7000 700 3362.6 3328.6 34.0 20 588.2

(48) Such picodroplet reinjection microfluidics was challenged with a very high flow rate of 1,000 uL/hr for picodroplets (300 pL) and 10,000 uL/hr for the re-injection oil (5% Pico-Surf 1 in Novec7500).

(49) FIG. 7 shows a video snapshot of cells in picodroplets obtained using the above parameters.

(50) From the video (of which FIG. 7 shows a single snapshot), it was observed that the elongation of picodroplets was minor or negligible, as the picodroplets experienced much less stress compared to that experienced in a conventional cross junction. No broken picodroplets were observed at such a high re-injection frequency (850 Hz).

(51) These observations prove that the cells experience less stress during picodroplet generation, resulting in a higher survival rate of cells contained in the picodroplets.

(52) As outlined above, the flow splitting microfluidic structure may allow for generating a local region (i.e. the area between two corresponding inlets on either side of the main channel) of homogeneous pressure environment. This may assure minimum stress which may be exerted onto fragile cells, entities or picodroplets.

(53) FIG. 8 shows hydrodynamic pressure versus distance.

(54) The blue line shows a side way comb design which generates a pressure which is higher at the side at which the spacing medium inlet is arranged. The pressure decreases constantly with increasing distance to the spacing medium inlet and the lowest pressure is observed at the opposite side of the channel at which no inlet is arranged.

(55) In a middle way comb design (red line in FIG. 8), a higher pressure is generated in the middle of the channel which is close to the spacing medium inlet, and the pressure decreases to both sides away from the middle way comb. Such a pressure gradient may still result in a stretching force which may be exerted onto the cells or picodroplets, which may cause elongation of cells which may cause a potentially irrevocable destruction of the cells. Equally, the picodroplets may break up into satellite picodroplets.

(56) The brown line in FIG. 8 represent the hydrodynamic pressure across the width of the (main) channel from a first inlet on a first side of the main channel to a corresponding, respective second inlet on the opposite side of the channel. As can be seen, the hydrodynamic pressure is, in this schematic illustration, constant across the width of the channel.

(57) The split fluidic flow, which spreads one big flow stream into multiple small and, in this example, equal flow streams, and homogeneous hydrodynamic pressure within spacing regions ensure the formation of a laminar flow which can align cells in the middle of the microfluidic channel and facilitate single cell encapsulation, in particular as the cells are spaced out within the channel prior to pinching off picodroplets from the suspension to encapsulate a single cell in a single droplet, thereby increasing the OCPD rate beyond that expected from Poisson statistics.

(58) Although aspects and embodiments of the invention described throughout the specification refer to picodroplets (which may be defined as droplets having a volume of less than one nano-litre), the skilled person will appreciate that aspects of the invention and embodiments generally as described herein may equally be used for droplets with other sizes, for example droplets having a volume of 1-1000 nano-litres.

(59) No doubt many other effective alternatives will occur to the skilled person. It will be understood that the invention is not limited to the described embodiments and encompasses modifications apparent to those skilled in the art lying within the spirit and scope of the claims appended hereto.