MICROFLUIDIC DEVICE

Abstract

The present disclosure relates to a microfluidic device for the separation of metaphase chromosomes such that individual metaphase chromosomes may be dispensed discretely from the device. The microfluidic device comprises a flow channel including a series of expanded regions and constrictions. The present disclosure also relates to methods of separating metaphase chromosomes.

Claims

1. A microfluidic device for separating metaphase chromosomes in a metaphase chromosome-containing fluid, the microfluidic device including: a flow channel including: an inlet to receive a fluid including metaphase chromosomes; an outlet to discretely dispense individual metaphase chromosomes; a series of expanded regions; and one or more constrictions located between consecutive expanded regions in the series of expanded regions; wherein the constrictions are operable to apply sufficient shear stress to separate the metaphase chromosomes from one another; and the expanded regions are operable to disperse chromosomes from one another.

2. A microfluidic device for separating metaphase chromosomes in a metaphase chromosome-containing fluid, the microfluidic device including: a flow channel having a width of from about 10 μm to about 30 μm, the flow channel including: an inlet; an outlet; and a series of expanded regions, and one or more constrictions located between consecutive expanded regions in the series of expanded regions; wherein the plurality of expanded regions have a channel width of from about 50 μm to about 150 μm, and each constriction in the plurality of constrictions has a minimum width of from about 1 μm to about 3 μm.

3. The microfluidic device of claim 1 or claim 2, wherein the flow channel has a depth of from about 5 μm up to about 40 μm.

4. The microfluidic device of any one of the preceding claims, wherein the length of the flow channel is from about 2 mm to about 15 mm.

5. The microfluidic device of any one of the preceding claims, wherein each successive constriction from the inlet to the outlet has a smaller minimum width than a preceding constriction.

6. The microfluidic device of any one of the preceding claims, wherein one or more of the one or more constrictions has a widening tapered outlet.

7. The microfluidic device of any one of the preceding claims, wherein each of the expanded regions in the series of expanded regions has substantially the same width.

8. The microfluidic device of any one of the preceding claims, wherein the series of expanded regions includes at least 3 expanded regions and up to 20 expanded regions.

9. The microfluidic device of any one of the preceding claims, wherein the flow channel includes more than one constriction between each expanded region in the series of expanded regions.

10. The microfluidic device of any one of the preceding claims, wherein the inlet has a width of from 2 μm to 3 μm.

11. The microfluidic device of any one of the preceding claims, wherein the microfluidic device further includes cell capture and lysis structure upstream of the inlet, the cell capture and lysis structure including: a cell trap adjacent the flow channel inlet configured to receive and retain a cell from a fluid sample including the cell, the cell trap including: a viewing element to permit inspection of the cell; and an opening connected to the flow channel inlet via a passage, the opening and passage sized to impede passage of the cell therethrough; a lysis port configured to introduce a lysis buffer to the cell trap.

12. The microfluidic device of claim 11, wherein the size of the opening is from about 10 μm to 20 μm and a width of the passage is from about 2 μm to about 3 μm.

13. The microfluidic device of any one of the preceding claims, wherein the cell trap is a rectangular prism shaped hollow formation in the microfluidic device with an open face to permit entry of a cell into the cell trap.

14. The microfluidic device of any one of the preceding claims, further including a chromosome dispensing structure downstream of the outlet, the chromosome dispensing structure including: a dispensing channel defined between a channel inlet and a channel outlet, and having a port for receiving an individual chromosome from the outlet of the flow channel; wherein the channel outlet is connected to a dispensing tube configured to dispense single chromosomes from the microfluidic device in the form of a fluid droplet including the single chromosome.

15. A method for separating metaphase chromosomes in a metaphase chromosome-containing fluid, the method including: passing the metaphase chromosome-containing fluid through the microfluidic device of any one of the preceding claims at a pressure whereby the constrictions subject the metaphase chromosomes to sufficient shear stress to separate the metaphase chromosomes from one another.

16. A method for separating metaphase chromosomes in a chromosome-containing fluid, the method including: passing a chromosome-containing fluid including metaphase chromosomes through a microfluidic device, the microfluidic device having a flow channel including: a plurality of expanded regions located between an inlet and an outlet; and one or more constrictions located between one or more of the expanded regions; subjecting metaphase chromosomes, at or in the one or more constrictions, to sufficient shear stress to separate the metaphase chromosomes from one another; dispersing the separated metaphase chromosomes in the plurality expanded regions from one another.

17. A method for separating metaphase chromosomes in a chromosome-containing fluid with a microfluidic device, the method including: passing the fluid through a flow channel of a microfluidic device, the flow channel having a plurality of alternating constrictions and expansions; wherein when the fluid is passed through a constriction, the method includes applying a pressure pulse to subject the metaphase chromosomes to a shear stress sufficient to separate the metaphase chromosomes from one another; wherein when the fluid is passed through an expansion, the microfluidic device is operated at a pressure to disperse the separated chromosomes from one another.

18. The method of any one of claims 15 to 17, wherein the shear stress is from at least about 0.02 N/m.sup.2 to at least about 15,000 N/m.sup.2 as measured at walls of the minimum width of the constriction.

19. The method of any one of claims 15 to 18, wherein the method initially includes: trapping a metaphase cell in a cell trap of the microfluidic device; and introducing a lysis buffer to the metaphase cell and applying a pressure pulse to drive the metaphase cell from the cell trap and into the flow channel under sufficient shear stress to lyse the cell and provide the chromosomes in the chromosome-containing fluid.

20. The method of any one of claims 15 to 19, further including: receiving the dispensed individual chromosomes from the outlet of the flow channel into a dispensing channel of the microfluidic device; transporting the individual chromosomes to a dispensing tube; and dispense single chromosomes from the microfluidic device via the dispensing tube in the form of a fluid droplet including the single chromosome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG. 1: Schematic of a microfluidic device according to one embodiment of the invention.

[0114] FIG. 2: Schematic of the microfluidic device illustrating the constriction.

[0115] FIG. 3: Schematic of the microfluidic device illustrating the sampling port through which cells are introduced into the microfluidic device.

[0116] FIG. 4: Schematic of the microfluidic device illustrating the upstream cell trap and lysing structure.

[0117] FIG. 5: Schematic of the microfluidic device illustrating the chemically assisted shear lysing of the cell and transfer under pressure pulse from the cell trap and into the flow channel.

[0118] FIG. 6: Schematic of the microfluidic device illustrating the flow channel and expanded regions.

[0119] FIG. 7: Schematic of the microfluidic device illustrating the flow channel outlet and chromosome detection.

[0120] FIG. 8: Schematic of the microfluidic device illustrating downstream chromosome isolation for individual chromosome dispensing.

[0121] FIG. 9: Schematic illustrating the dispensing of a droplet containing a single chromosome from the microfluidic device onto a well plate.

[0122] FIG. 10: Schematic showing the pump arrangement of the microfluidic device.

[0123] FIG. 11: Close up drawing showing detail of a constriction within the flow channel of the microfluidic device.

REFERENCES

[0124] Quake et al. (Nature Methods, Vol. 11, No. 1, 2014, pp 19-21)

[0125] Dolez̆el et al. (Funct. Integr. Genomics, 2012, 12:397-416)

[0126] Fan et al. (Nat. Biotechnol., January 2011, 29(1):51-57)

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0127] The present invention relates to a microfluidic device and method for separating metaphase chromosomes from one another.

[0128] In a preferred form, the microfluidic device is configured to trap and lyse a single metaphase cell, suspend the expelled chromosomes into singulated chromosomes, detect each singulated chromosome, and then dispense each chromosome from the microfluidic device onto a receptacle (such as a glass slide or a well plate) for post processing.

[0129] Broadly, a cell is introduced into the microfluidic device where it is analysed (such as via optical microscopy) to determine whether the cell is a metaphase cell. If the cell is a metaphase cell it is trapped, then a lysis buffer is introduced into the microfluidic device accompanied with a high pressure pulse to drive the cell and its contents from the cell trap through a channel restriction and into a flow channel of the microfluidic device whilst lysing the cell via shearing on the cell membrane as it passes through the channel restriction. The chromosomes, typically in the form of one or more clusters, then emerge into the microfluidic flow channel. In the microfluidic flow channel, the one or more clusters of chromosomes are passed through an alternating series of constrictions and expansions.

[0130] The constrictions provide an impediment to the flow of the one or more clusters of chromosomes through the flow channel. A pressure pulse drives the one or more clusters of chromosomes through the constrictions, which at the same time, applies significant shear stress to the one or more clusters of chromosomes to break the one or more clusters apart.

[0131] In the expansions, the chromosomes are subjected to a lower flow velocity and varying flow profiles which permits the chromosomes to disperse and become separated from one another. The expansions also provide the lysis buffer with an opportunity to mix with the individual chromosomes to stabilise those chromosomes, and to mix with the one or more clusters of chromosomes to chemically assist in the shear separation of chromosomes in subsequent constrictions.

[0132] The one or more clusters of chromosomes are subjected to multiple alternating constrictions and expansions until the one or more clusters of chromosomes have been broken down into separate and individual chromosomes. These individual chromosomes are detected at the outlet of the flow channel, where they are discretely dispensed and deposited onto a slide or well-plate for further analysis.

[0133] In this way, the device and method of the invention provides a mechanism for separating individual chromosomes for subsequent haplotype determination.

[0134] An embodiment of the invention is described below.

[0135] FIG. 1 is a schematic of a microfluidic device 100 for separating and dispensing single chromosomes from a chromosome suspension. In this case, the microfluidic device 100 is formed as a polydimethylsiloxane (PDMS) casting on wafer tooling. The skilled addressee will appreciate that a number of different materials may be used. The PDMS casting is capped with a glass cover slip. Again, different materials may be used. However, glass was selected as the capping material due to its optical properties (e.g. optical clarity and no autofluorescence) readily permitting observation of the components of the microfluidic device 100 and its ability to bond with PDMS via plasma activation.

[0136] The microfluidic device 100 includes a microfluidic flow channel 102 having an inlet 104 and an outlet 106. In this embodiment, the flow channel 102 has a length of 5 mm. However, different lengths could be used, such as from 3 mm to 15 mm. The flow channel 102 is divided into five zones (labelled as 1 to 5 in FIG. 1). Each of these zones includes a first flow channel portion 108 and a second flow channel portion representing an expanded portion 110. The first flow channel portion 108 has a cross-sectional area transverse to the flow direction that is less than the cross-sectional area of the expanded portion 110. In this particular case, the flow channel 102 has a depth of 20 μm, the first channel portion 108 has a width of 20 μm (e.g. a cross-sectional flow area of 400 μm.sup.2), and the expanded portion 110 has a width of 100 μm (e.g. a cross-sectional flow area of 2000 μm.sup.2). Thus, in the present embodiment, the ratio of the cross-section flow are of the first flow channel portion 108 to the expanded portion 110 is 1:5. Although this embodiment has a ratio of 1:5, the inventors are of the view that a ratio of from 1:2 to 1:10 is suitable.

[0137] Each of the first flow channel portions 108 includes a constriction 202 (see FIG. 2 and FIG. 11, expanded view). It will be appreciated that each of the first flow channel portions 108 may include multiple constrictions 202. In this embodiment, the constrictions 202 have a width of from about 1 μm to about 2 μm. Furthermore, the width of the constrictions 202 in each successive first flow channel portions 108 from the inlet 104 to the outlet 106 is less than the width of the constrictions 202 in preceding first flow channel portions 108.

[0138] FIG. 11 illustrates an embodiment of a constriction 1100 between flow channel portions 1102 and 1104. The constriction 1100 has widened tapered outlet 1106 that tapers to a width that is approximately half the width of the flow channel. In FIG. 11, the constriction has a depth that is less than flow channel portions 1102 and 1104. In this particular embodiment, the constriction has a depth of about 5 μm whereas the flow channel portions 1102 and 1104 have a bulk depth of about 20 μm. Regions of the flow channel immediately adjacent to the constriction 1100, labelled as items 1108 and 1110, have the same depth as the constriction (e.g. about 5 μm) such that there is a step change in depth within the flow channel from the depth of the constriction 1100 (e.g. about 5 μm) to the bulk depth of the flow channel (e.g. about 20 μm).

[0139] The operation of the components of the flow channel 102 will now be briefly described. During operation, a fluid including one or more clusters of chromosomes is introduced under pressure into the flow channel 102 via inlet 104. The fluid flows through the first flow channel portion 108 of zone 1 where it passes through a constriction 202. The constriction 202 impedes the passage of the one or more clusters of chromosomes therethrough. The approximate size of a single metaphase chromosome is from about 0.5 μm to about 3 μm; whereas a chromosome cluster can range in size from slightly larger than a single metaphase chromosome to slightly less than the size of the metaphase cell (approx. 10 μm-15 μm). In any event, fluid in the constriction is subject to increased flow velocity relative to the flow channel 102 by virtue of providing a narrow flow area, and this increased flow velocity forces the one or more clusters of chromosomes through the restriction while subjecting the one or more clusters of chromosomes to substantial shear stress such as around 0.02 N/m.sup.2 to about 1 N/m.sup.2 at the walls depending on the dimensions of the constriction, pressures applied (which in turn effects velocity) and fluid properties. This shear stress is sufficient to fragment one or more clusters of chromosomes which can result in single chromosomes being dislodged from the one or more clusters of chromosomes breaking apart into smaller chromosome clusters. The single chromosomes 203 and/or smaller chromosome clusters 204 then emerge via a widening tapered outlet of the constriction 202 of the first flow channel portion 108 of the flow channel 102 downstream of the constriction 202 before passing into the second channel portion, e.g. the expanded portion 110, of zone 1. In the expanded portion 110, the single chromosomes and/or smaller chromosome clusters are subjected to reduced flow velocity relative to the first flow channel portion 108 by virtue of the wider flow area. In this expanded portion 110, the single chromosomes and/or smaller chromosome clusters disperse in both the radial and axial directions via a combination of diffusion and advection which can result in increased spacing between the chromosomes when they exit the expanded portion to the narrower first flow channel portion 108 of zone 2. Additionally, the dispersal of chromosomes and/or smaller chromosome clusters in the expanded region 110 allows reagents that may be present in the chromosome containing fluid to mix and diffuse around the surface of the chromosomes and/or smaller chromosome clusters (e.g. stabilisers or other reagents that promote separation of the chromosomes and/or prevent or minimise aggregation).

[0140] In zone 2, the single chromosomes 203 and/or smaller chromosome clusters 204 undergo a similar process in that they pass through a first flow channel portion 108 having a constriction 202. However, in this case the constriction 202 in Zone 2 is narrower than the constriction 202 in Zone 1. The reason for this is to impede the passage of the smaller chromosome clusters, and to provide a higher flow velocity to subject the smaller chromosome clusters to higher shear stresses to further break apart the chromosome clusters and/or separate single chromosomes from the chromosome clusters. Again, after passing through this constriction, the chromosomes similarly emerge into the first flow channel portion 108 of Zone 2, before passing into the expanded region 110 of Zone 2 for further dispersal.

[0141] The above process is repeated through Zones 3, 4, and 5 whereby each constriction 202 in the first flow portion 110 of these zones decreases in width to impede the passage of and break apart smaller chromosome clusters 204; and each expanded region 110 of these zones further disperses single chromosomes 203 and/or chromosome clusters 204 from one another.

[0142] After passing through each of the zones of the flow channel 102, chromosomes then pass through the outlet 106 of the flow channel as single chromosomes spaced axially apart from one another. Because the single chromosomes are spaced axially apart, the single chromosomes can be isolated from one another for downstream purposes.

[0143] In the embodiment depicted in FIG. 1 the microfluidic device 100 includes a cell capture and lysis structure 112 upstream of the flow channel 102. The cell capture and lysis structure 112 includes a sample port 114 for introducing a fluid containing cells, a cell trap 402 (see FIG. 4, expanded view) for trapping a cell to permit interrogation of the cell, a lysis port 116 for introducing a lysis buffer to lyse the cell and release chromosomes contained therein if the cell is deemed suitable, and a waste port 118 for discharging waste reagents and cells that are deemed unsuitable. At the outlet of the flow channel, the device includes a detection zone 119 (see FIG. 7, expanded view) for detecting individual metaphase chromosomes to ensure that the chromosomes are dispensed. Downstream of the flow channel 102, the microfluidic device includes a dispensing structure 120 including a dispensing port 122, an extraction port 124 and a dispense channel 704. The cell trap 402 has dimensions of 20 μm×20 μm×20 μm which is sufficiently small to hold a single metaphase cell. The cell trap 402 includes an opening 404 to the inlet of flow channel 102. The opening 404 has a width of about 2 μm to about 3 μm to prevent a cell from passing from the cell trap 402 into the flow channel 102.

[0144] During operation, a cell sample can be provided to the microfluidic device via sample port 114.

[0145] FIG. 3 shows the addition of a fluid sample containing cells 403 via the sample port 114. In FIG. 3, the sample port 114 is operated at high pressure, the waste port 118 is operated at low pressure, the lysis port 116 and dispensing port 122 are operated at datum pressure, and the extraction port 124 is closed. Given this arrangement, the sample flows through sample transfer channel 126 to waste port 118.

[0146] FIG. 4 illustrates the capture of a cell 403 in the cell trap 402 for interrogation. In FIG. 4 the sample port 114, lysis port 116, and waste port 118 are operated at datum pressure; the dispensing port 122 is operated at low pressure; and the extraction port 124 is closed. The effect of this arrangement is to provide a pressure differential that maintains the cell 403 in the cell trap, e.g. there is a suction effect that biases the cell in the cell trap 402 against opening 404. However, the cell 403 is unable to pass through opening 404. Once the cell 403 is held in the cell trap 402, visual inspection is possible through the glass coverslip. The purpose of the visual inspection is to confirm that the cell 403 is a metaphase cell—and thus suitable for obtaining a chromosome suspension.

[0147] If the cell 403 is not a metaphase cell, then the cell 403 is flushed from the cell trap 402, such as by applying a back pressure via the dispensing port 122 and discharging the cell through the waste port 118. That is, the dispensing port 122 is operated at high pressure; the waste port 118 is operated at low pressure, the sample port 114 and the lysis port 116 are operated at datum pressure; and the extraction port 124 is closed.

[0148] If the cell is a metaphase cell, then the cell is subjected to a lysing process to rupture the cell membrane and release the chromosomes from within the cell. This process is shown in FIG. 5. In FIG. 5, lysis buffer 405 is applied by operating the lysis port 116 at a higher pressure (e.g. 40-45 mbar), the dispensing port 122 is operated at low pressure (e.g. below the datum pressure of 35 mbar, such as less than 30 mbar); the sample port 114 and the waste port 118 are operated at datum pressure (e.g. 35 mbar); and the extraction port 124 is closed. The effect of this is that a lysis buffer 405 flows from the lysis port 116 through the lysis channel 502 where it contacts the cell to be lysed.

[0149] A pressure pulse is then used to force the cell through the opening and along a constriction which lyses the cell by shearing the cell membrane, and passing the contents of the cell (including one or more clusters of chromosome) into the flow channel 102 via the inlet 104. In this pressure configuration, the sample port 114, the wasteport 118, and the lysis port 116 are operated under a pulse pressure; with the dispense port 122 being operated at low pressure and the extraction port 124 being closed.

[0150] The lysis buffer is an aqueous solution that can include Type 1 ultrapure water, 2 v/v % acetic acid, 5 w/v % triton X-100 also known as Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (a non-ionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group), 0.1 w/v % pepsin, 75 mM potassium chloride. In this buffer; the acetic acid fixes and preserves the chromosome morphology, the triton X-100 solubilise/lyse the cell membrane components and the hydrophobic proteins and has a secondary role in releasing chromosomes, the pepsin releases individual chromosomes from their clusters and aids cell lysis and removes cellular proteins, and the potassium chloride is a salt used to swell the cells via osmotic pressure and enhances pepsin solubility. Alternatively, the buffer may include 0.1% w/v pepsin, 1 mM EDTA, 73 mM potassium acetate buffer, 2 mM magnesium sulphate, buffered to pH 5 with acetic acid. Alternatively, the fixative role of acetic acid in either of these buffers could be performed by fixative formaldehyde. A person skilled in the art would appreciate that other buffer compositions known in the art would also be suitable for use as the lysis and/or separation buffer.

[0151] FIG. 2 illustrates the use of a pressure pulse to drive chromosome clusters through the constrictions 202. This is achieved by applying a high pressure pulse through the lysis port 116 (e.g. 300-1000 mbar). Under this mode of operation, the sample port 114 and the waste port 118 are operated at a datum pressure (e.g. 35 mBar); the dispensing port 122 is operated at low pressure (e.g. <30 mbar); and the extraction port 124 is closed. The additional pressure from the lysis port 118 drives impeded clustered chromosomes 204 (e.g. one or more clusters of chromosomes that may have become trapped at the narrow opening of the constriction 202) through the constriction 202 subjecting the one or more clusters of chromosomes to high shear stress conditions to break the one or more clusters of chromosomes 204 into single chromosomes 203 and/or smaller chromosome clusters. This process is repeated for the various zones.

[0152] An alternative approach is to apply a high pressure pulse via the sample port 114 (e.g. 250-950 mBar or 250-1,000 mBar), the waste port 118 (e.g. 250-950 mBar or 250-1000 mBar), and the lysis port 116 (e.g. 300-1000 mBar); low pressure at the dispensing port 122 (e.g. 0 mBar); and the extraction port 124 is closed.

[0153] Under the pressure regimes described, the shear stress through the constriction zones range from about 0.02 N/m.sup.2 to 15,000 N/m.sup.2 depending on the dimensions of the constriction and pressures applied.

[0154] The combination of the lysis buffer and the pressure differential between the lysis port 116 and the dispensing port 122 induces a chemically-assisted shear lysing process which causes the cell membrane to rupture and forces the contents of the cell through opening 404 and into the flow channel 102. The contents of the cell include one or more clusters of chromosomes 204 (and potentially single chromosomes 203). The one or more clusters of chromosomes are then subjected to the shear treatment process in channel 102 as hereinbefore described to separate the chromosomes.

[0155] FIG. 6 provides an illustration of the operation of the microfluidic device 100 after the cell has been lysed and the chromosomes 205 expelled into the flow channel 102 and moving through the expanded region 110 of one of the zones. In FIG. 6 the sample port 114, lysis port 116, and waste port 118 maintain the lysis buffer application settings; the dispensing port 122 is operated at datum pressure; and the extraction port 124 is closed. Thus, a pressure differential exists across the flow channel 102 that drives the chromosomes from the inlet 104 of the flow channel 102 toward the outlet 106 of the flow channel. The expanded section shows single chromosomes 203 dispersing and being separated through the expanded region 110 of zone 1. The expanded region 110 may include a mixing apparatus, such as a herringbone mixer to assist the dispersal of the single chromosomes.

[0156] Once the chromosomes are separated they are detected at the outlet 106, such as by live recording of fluorescent signals. Each detection event triggers the dispense system to activate. FIGS. 7 and FIG. 8 illustrate the detection and count of chromosomes, and the transfer of single chromosomes out of the microfluidic device 100 via extraction port 124. FIG. 7 illustrates the detection of a single chromosome 203 at the outlet 106 using a photodetector 702. The detection restriction 703 ensures that chromosomes are in single file. On detection of a chromosome, the dispensing system is activated. A flow of neutralisation buffer (to stop the degradation of chromosome morphology from Pepsin activity, if present) is provided via dispensing port 122 to capture the detected chromosome and dispense it from the microfluidic device 100. Each chromosome is discharged from the microfluidic device in the form of a droplet. The droplet is dispensed onto a receptacle (e.g. a glass slide or specialised well plate). In more detail, once the chromosome is detected, a valve on the extraction port 124 is switched from the closed position (shown in FIG. 7) to the open position and the pressure of the dispensing port 122 is increased to provide the neutralisation agent. This increased flow 709 (shown in FIG. 8) results in the single chromosome 203 being dispensed from the outlet 106 and into dispensing channel 704 where it is subsequently deposited onto a well plate or glass slide. The increased flow 709 also results in a flow reversal in the flow channel 102, helping to keep the chromosomes separated.

[0157] By way of example, during detection the sample port 116 and waste port 118 are operated at 0 mBar; the lysis port 116 is operated at from 2-5 mBar; and the dispensing port 122 is operated at 2 mBar. As an alternative example, during detection the sample port 116 and waste port 118 are operated at 10 mBar; the lysis port 116 is operated at at 20 mBar; and the dispensing port 122 is operated at 2 mBar This low pressure differential slows the flow through the flow channel 102 to permit detection of chromosomes at the outlet 106. Once a chromosome has been detected the pressure at the dispensing port 122 is increased to 15 mBar to dispense the chromosome from the outlet 106 and into the dispensing channel 704.

[0158] FIG. 9 illustrates the deposition of a 200 nL droplet 900 including a single chromosome 203 from an outlet of dispensing channel 704 onto a moving well plate 902 through dispense tube 705. This process may be repeated until each chromosome has been deposited onto the well plate 902 such as in an array, e.g. for chromosomes taken from a human cell, there will be 46 discrete droplets each including a single chromosome. The hydrophobic coating 706 of the dispense tube ensures that the droplet does not stick to the dispense tube 705. FIG. 9 also illustrates the dispense channel 704 in relation to the cartridge 707 and the glass coverslip 708.

[0159] FIG. 10 shows the pump arrangement according to one embodiment of the invention with datum pressures. In this embodiment, the sample port 114 is configured to use a 69 mBar pressure pump with the datum pressure set to 35 mBar; the lysis port 116 is configured to use a 1000 mBar pressure pump with the datum pressure set to 35 mBar; the waste port 118 is configured to use a 70 mBar pressure pump with the datum pressure set to 35 mBar; the dispensing port 122 is configured to use a 345 mBar pressure pump with the datum pressure set to 35 mBar; and the extraction port 124 is normally closed.

[0160] As generally described above, operation of the microfluidic device 100 is carried out by connecting the various fluid ports of the microfluidic device to pressure/flow controllers. The 345 mbar pressure pumps are connected to the sample port 114 and the waste port 118 because they are used to control cell motion during cell screening and trapping, which requires high resolution in pressure change to generate and maintain low flow rates. One 1000 mBar pressure pump is connected to the lysis port 116 to provide high pressure pulses to induce shear in the cell that is held in the trap. A 69 mBar is connected to the dispensing port 122 to allow pressure drop in the dispense channel for chromosome transfer. The dispensing channel 704 will have a valve (seated tube on a gasket) on the extraction port 124 that will normally be closed during operation except when dispensing droplets. All the pressure controllers will initially be set to a datum pressure of 35 mBar, from this datum pressure, each pressure line can either be raised or dropped depending on the required direction of flow within the microfluidic device 100.

[0161] Alternatively, in this embodiment, the sample port 114 is configured to use a 1000 mBar pressure pump with the datum pressure set to 35 mBar; the lysis port 116 is configured to use a 1000 mBar pressure pump with the datum pressure set to 35 mBar; the waste port 118 is configured to use a 1000 mBar pressure pump with the datum pressure set to 35 mBar; the dispensing port 122 is configured to use a 345 mBar pressure pump with the datum pressure set to 35 mBar; and the extraction port 124 is normally closed.

[0162] As generally described above, operation of the microfluidic device 100 is carried out by connecting the various fluid ports of the microfluidic device to pressure/flow controllers. The 1000 mbar pressure pumps are connected to the sample port 114 and the waste port 118 because they are used to control cell motion during cell screening and trapping, which requires high resolution in pressure change to generate and maintain low flow rates. One 1000 mBar pressure pump is connected to the lysis port 116 to provide high pressure pulses to induce shear in the cell that is held in the trap. A 345 mBar is connected to the dispensing port 122 to allow pressure drop in the dispense channel for chromosome transfer. The dispensing channel 704 will have a valve (seated tube on a gasket) on the extraction port 124 that will normally be closed during operation except when dispensing droplets. All the pressure controllers will initially be set to a datum pressure of 35 mBar, from this datum pressure, each pressure line can either be raised or dropped depending on the required direction of flow within the microfluidic device 100.

[0163] Dispensing is conducted by dispensing droplets from the microfluidic device 100 via a dispensing tube 705 (for example, the dispensing tube of the present embodiment has an outer diameter 0.79 mm, an inner diameter 0.15 mm, and a length of 7 mm), where each droplet contains a chromosome. This is done by creating a higher pressure at the dispensing port 122 and opening the valve at the extraction port 124. Fluid then travels due to the pressure drop through the dispensing channel 704, through the dispensing tube, and out of a dispensing tube tip. Once a correct droplet size is generated (droplet size is varied via varying the pressure drop but an example size is 200 nL), each droplet will be dispensed onto a glass slide or a specially designed well plate. The pressure from the dispensing port 122 will then return to datum pressure. The droplet attaches to the receptacle by the surface tension of the formed droplet. To dispense each droplet in an array and onto the receptacle, an automated mechanism that holds the receptacle is utilised. The mechanism moves independently to the cartridge in three axes, such as along x and y axes to create the array of droplets on the receptacle and in the z axis to attach each droplet to the receptacle.

[0164] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. For instance, it will be understood that alternative topologies of the individual features described above constitute alternative aspects of the invention.