Microfluidic device

Abstract

To provide a microfluidic device for the separation of metaphase chromosomes such that individual metaphase chromosomes may be dispensed discretely from the device.SOLUTION: A 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.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Australian Patent Application No. 2019900210, filed on January 23, 2019, which is incorporated by reference in its entirety.

[0002] FIELD OF THE PRESENT APPLICATION The present invention relates to a microfluidic device for chromosome separation. [Background technology]

[0003] 2. Background of the Invention Cells of many eukaryotic organisms, including animal and plant species, contain two or more sets of chromosomes, with a set number known as ploidy. For example, humans are diploid and possess a paired set of chromosomes (maternal and paternal copies) that form the genome. At each position or locus of a specific chromosome, an individual may have either two copies of the same sequence (such as a gene allele, mutation, marker, or epigenetic component), or two different sequences, one version for each chromosome pair. Determining whether sequence elements, such as gene alleles, mutations, markers, or epigenetic components from different loci, occur together on the same member of a chromosome pair (in cis arrangement) or on opposite members of a chromosome pair (in trans arrangement) is known as phase determination. When two or more sequences (alleles, mutations, markers, or epigenetics) occur in cis, this is known as a haplotype. Variations in the sequences of these haplotypes can result in functional differences, such as differences in gene expression, protein function, and disease. Thus, knowledge of an individual's phasing or haplotype can lead to understanding and control of biological pathways, including improved diagnostic and / or therapeutic approaches. Unfortunately, existing processes for phasing and haplotyping suffer from a number of shortcomings.

[0004] In a review by Quake et al. (Nature Methods, Vol. 11, No. 1, 2014, pp 19-21), Quake reveals that although genome "analysis has progressed from determining the reference sequence of the 'average' human genome to mass sequencing of individual genomes," difficulties remain in some aspects of genome analysis. In particular, Quake continues to complain that existing conventional techniques are not adequately adapted to haplotype measurement.

[0005] Dolezel et al. (Funct. Integr. Genomics, 2012, 12:397-416) discuss a range of approaches for the separation and isolation of individual chromosomes. One approach discussed by Dolezel includes the separation of chromosomes based on relative density, such as by gradient centrifugation; however, this approach has many drawbacks, as it only provides for the separation of large and small chromosomes and is not tailored to the separation of specific chromosomes. Another approach discussed in Dolezel is the use of magnetic beads functionalized with chromosome-specific probes; however, a drawback of this approach is that the isolated fractions are of low purity. Dolezel continues to claim that the most successful approach is the use of flow cytometry, in which droplets of dye-stained chromosomes are released from a flow chamber and passed through a laser beam in which the scattered light is analyzed to identify the chromosomes of interest in the chromosome-containing droplets according to light scattering and fluorescence, and an electric field is used to direct the droplets into a collection container. However, Dolezel considers that flow cytometry cannot resolve all chromosomes in various animal species (including humans, dogs, pigs, and chickens). Dolezel continues that many research groups have focused their efforts on improving flow cytometry to separate and isolate individual chromosomes.

[0006] Another technique is the approach taken by Fan et al. (Nat. Biotechnol., January 2011, 29(1):51-57). Fan et al. reveal that current techniques (such as mate-pair shotgun genome sequencing, various forms of polymerase chain reaction (PCR), atomic force microscopy with carbon nanotubes, fosmid / cosmid cloning, and the use of hybridized probes) all have many significant shortcomings that prevent widespread adoption. Instead, Fan et al. report the development of a microfluidic device for isolating and amplifying homologous copies of each chromosome from a single human metaphase cell. Fan et al.'s device is separated into five distinct regions according to their function. The first region involves the use of a light microscope to identify a single metaphase cell. Once a metaphase cell is identified, a series of peripheral valves are actuated to capture the cell so that it can be introduced into the second region of the device. In the second region, the metaphase cell is contacted with pepsin to digest the cell's cytoplasm and form a chromosome suspension. This suspension is then passed into a third region where it is partitioned into 48 chambers by actuating a series of valves within the device. In the fourth region, the contents of each of the 48 chambers are then individually amplified in the device through a series of different channels by treatment with trypsin, alkali, and subsequent neutralization for multiple strand displacement amplification. The fifth region of the device contains separate outlets for the collection of each amplified chromosome.

[0007] Notably, to the inventors' knowledge, the device disclosed by Fan et al. has not been adopted. The inventors have attempted to replicate the protocol reported by Fan et al. without success. The inventors speculate that the lack of reproducibility of Fan et al.'s device and process has hindered adoption. In this regard, Dolezel suggested a departure from Fan et al.'s microfluidic device and argued that "the application of flow cytogenetics may be an elegant alternative to the recently developed microfluidic approach, in which individual chromosomes from a single human metaphase are separated into different channels and amplified (Fan et al. 2011)."

[0008] Due to the shortcomings of techniques for isolating chromosomes for direct phasing, most current attempts to determine phase or infer haplotypes (e.g., for referencing bone marrow transplant patients and future donors) use indirect methods consisting of inference and assumptions, such as family segregation studies, linkage disequilibrium, or algorithms to derive phase estimates from sequenced DNA fragments.

[0009] In the metaphase situation, chromosomes consist of individual bundles of tightly folded DNA and proteins that can form connections with each other and exist in the form of clusters of chromosomes.

[0010] In view of the above, there is a need for the development of devices and / or processes for sorting and separating chromosomes to enable direct phase and haplotype determination. However, prior art approaches suffer from significant shortcomings. It is therefore an object of the present invention to address and / or ameliorate one or more shortcomings of the prior art.

[0011] The reference in the specification to any prior art is not an admission or suggestion that this prior art forms part of the general general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, i.e., considered to be relevant and / or in combination with other pieces of prior art, by a person skilled in the art. Summary of the Invention

[0012] In a first aspect of the present invention, there is provided a microfluidic device for separating metaphase chromosomes in a metaphase chromosome-containing fluid, comprising: below: an inlet for receiving a fluid containing metaphase chromosomes; an outlet for distributing individual metaphase chromosomes separately; A series of expansion areas; and one or more stenoses located between successive dilated regions in a series of dilated regions; a flow path including wherein the constriction is capable of applying sufficient shear stress to separate metaphase chromosomes from one another; and The expansion regions are operable to disperse chromosomes from one another, A microfluidic device is provided.

[0013] In a further aspect of the invention there is provided a microfluidic device for separating clustered metaphase chromosomes in a fluid, comprising: below: an inlet for receiving a fluid; an outlet for distributing separated metaphase chromosomes; one or more extension regions; and one or more stenoses having at least one dilated region downstream of the stenotic region; a flow path including wherein the constriction is capable of applying sufficient shear stress to separate clustered metaphase chromosomes; and wherein the expansion region is operable to disperse separated metaphase chromosomes. Microfluidic devices.

[0014] By "operable to" it is meant that the microfluidic device is operated under flow and / or pressure conditions such that the chromosomes are subjected to relatively high shear stress at the constriction and / or relatively slow flow rates in the expansion region (relative to the flow rate in the non-expanded region of the channel). For example, the device can be operated at a constant pressure where changes in flow rates at the constriction and expansion result in respective shear stresses and dispersion; or at fluctuating pressures such that a pressure pulse is applied to the chromosomes of interest as they pass through the constriction, and a reduction in velocity allows the chromosomes to disperse as they pass through the expansion region.

[0015] In one form of the invention, the metaphase chromosomes are present in the form of one or more clusters of metaphase chromosomes, and the microfluidic device is for separating the one or more clusters of metaphase chromosomes into individual metaphase chromosomes. In such a case, the constriction is operable to apply a shear stress to the one or more clusters of metaphase chromosomes sufficient to separate the metaphase chromosomes from the cluster or to break the cluster into smaller clusters; and the expansion region is operable to disperse the separated metaphase chromosomes and / or the one or more clusters from each other.

[0016] "Cluster" or "clustering" refers to a grouping or collection of metaphase chromosomes that are "stuck" or tightly bound to one another. This clustering can occur as a result of many chromosome physicochemical interactions; for example, clustering can occur when metaphase chromosomes are able to form bonds with one another directly (by protein or DNA interactions) or due to the presence of materials such as the cytosol. Thus, chromosomes in a fluid may tend to stick or clump together, especially when combined with other cellular contents.

[0017] In one form of the invention, the expansion regions are operable to disperse metaphase chromosomes separated from one another.

[0018] Of course, a significant proportion, or preferably all, of the metaphase chromosomes dispensed from the outlet of the device will be separately dispensed individual metaphase chromosomes.

[0019] In one form of the invention, the metaphase chromosome-containing fluid is a lysate, or a component of a lysate, from a metaphase cell(s) contained in a fluid preparation.

[0020] As used herein, "fluid" may include dissolved materials. For example, fluids include dissolved components of a buffer, such as a lysis buffer and / or a separation buffer.

[0021] In a further aspect of the invention there is provided a microfluidic device for separating metaphase chromosomes in a metaphase chromosome-containing fluid, comprising: below: A flow channel having a width of about 10 μm to about 30 μm, comprising: entrance; Exit; and a series of dilated regions and one or more constrictions located between successive dilated regions in the series of dilated regions; a flow path including wherein the plurality of expansion regions have a channel width of about 50 μm to about 150 μm, and each constriction among the plurality of constrictions has a minimum width of about 1 μm to about 3 μm. A microfluidic device is provided.

[0022] The size of the minimum width of each constriction is required to impede the passage of chromosomes and to exert sufficient pressure to drive metaphase chromosomes through the constriction and to subject the chromosomes to shear stress that separates the metaphase chromosomes from one another.

[0023] The size of the expansion region is such that it disperses metaphase chromosomes that are separated both laterally and axially by one or more of diffusion and advection that help increase the spacing between the chromosomes as they exit the expansion. The expansion region may be of any suitable size and shape.

[0024] In one aspect of the invention, the metaphase chromosomes are present in the form of one or more clusters of chromosomes, and the microfluidic device is for separating the metaphase cluster or clusters into individual metaphase chromosomes.

[0025] In certain embodiments of the above aspects of the invention, the channel (optionally excluding the expansion region and / or constriction and / or region of the channel immediately adjacent the constriction) has a depth of from about 5 μm up to about 40 μm. Preferably, the channel depth is from about 12 μm. More preferably, the channel depth is from about 14 μm. Even more preferably, the channel depth is from about 16 μm. Most preferably, the channel depth is from about 18 μm. Alternatively or additionally, the channel depth is up to 35 μm. More preferably, the channel depth is up to about 30 μm. Most preferably, the channel depth is up to about 25 μm. In one non-limiting example, the channel depth is 20 μm ± 2 μm.

[0026] In one embodiment, the constriction has a depth shallower than the depth of the channel. Preferably, the depth of the constriction is about 5 μm to about 15 μm shallower than the depth of the channel. Preferably, the depth of the constriction can be about 5 μm to about 15 μm. A shallow depth of the constriction relative to the channel is useful for enhancing the shear that the chromosomes experience within the constriction.

[0027] In one aspect of the above embodiment, there is a step change in depth between the depth of the bulk of the channel and the depth of the constriction. Preferably, the step change in depth is from about 5 μm to about 15 μm, e.g., a constriction depth of about 5 μm when the channel has a bulk depth of 10 μm or more. It is also preferred that a region of the channel immediately adjacent to the constriction has the same depth as the constriction, such that there is a step change in depth in the channel.

[0028] In certain aspects of the above embodiments, the depth of the channel (excluding the expansion region and / or constriction and / or the region of the channel immediately adjacent to the constriction) is constant along the entire length of the channel, i.e., the depth of the channel does not vary substantially along the entire length of the channel, such as to within ±2 μm.

[0029] In certain embodiments of the above aspects of the invention, the channel (excluding the expansion region and constriction) has a width of about 10 μm to about 30 μm. Preferably, the channel width is from about 12 μm. More preferably, the channel width is from about 14 μm. Most preferably, the channel width is from about 16 μm. Alternatively or additionally, the channel width is up to 28 μm. More preferably, the channel width is up to about 26 μm. Most preferably, the channel width is up to about 24 μm. In one non-limiting example, the channel width is 20 μm ± 2 μm.

[0030] Of course, the geometry of the channels may be selected as appropriate for a particular application, for example the width of the channels may be greater than the depth of the channels or vice versa.

[0031] In certain embodiments of the above aspects of the present invention, the channel length is from about 2 mm to about 15 mm. Preferably, the channel length is from about 3 mm. Most preferably, the channel length is from about 4 mm. Alternatively or additionally, the channel length is up to 12 mm. More preferably, the channel length is up to about 10 mm. Most preferably, the channel length is up to about 8 mm. In one non-limiting example, the channel length is about 5 mm.

[0032] In one embodiment, the minimum width of the or each constriction is from about 1.00 μm to about 3.00 μm. Preferably, the minimum width is from about 1.25 μm. More preferably, the minimum width is from about 1.50 μm. Most preferably, the minimum width is from about 1.75 μm. Alternatively or additionally, the minimum width is up to about 2.75 μm. More preferably, the minimum width is up to about 2.50 μm. Most preferably, the minimum width is up to about 2.25 μm. In one non-limiting example, the minimum width is 2.00 μm ± 0.20 μm.

[0033] In one embodiment, the length of the narrowest part of the constriction is from about 4 μm to a maximum of about 16 μm. Preferably, the length is from about 6 μm. Most preferably, the length is from about 8 μm. Alternatively or additionally, it is preferred that the length is a maximum of about 14 μm. Most preferably, the length is a maximum of about 12 μm. In one non-limiting example, the length is about 10 μm.

[0034] In some embodiments, each successive constriction from the inlet to the outlet has a smaller minimum width and / or depth than the previous constriction, hi some embodiments, at least some of the successive constrictions have the same minimum width and / or depth.

[0035] In some embodiments, one or more of the one or more constrictions have an diverging tapered outlet. Preferably, the diverging tapered outlet widens to a width that is about ⅔ or less than the width of the flow passage. Preferably, the diverging tapered outlet widens to a width that is about half or less than the width of the flow passage.

[0036] In one embodiment, the extension region has a width of about 50 μm to about 150 μm. Preferably, the width of the extension region is about 60 μm or more. More preferably, the width of the extension region is about 70 μm or more. Most preferably, the width of the extension region is about 80 μm or more. Alternatively or additionally, the width of the extension region is a maximum of 140 μm. More preferably, the width of the extension region is a maximum of about 130 μm. Most preferably, the width of the extension region is a maximum of about 120 μm. In one non-limiting example, the width of the extension region is about 100 μm.

[0037] In one embodiment, the length of the expansion region is from about 0.2 mm to a maximum of about 0.8 mm. Preferably, the length is from about 0.3 mm. Most preferably, the length is from about 0.4 mm. Alternatively or additionally, the length is preferably from a maximum of about 0.7 mm. Most preferably, the length is from a maximum of about 0.6 mm. In one non-limiting example, the length is about 5 mm.

[0038] In some embodiments, each expansion region in the series of expansion regions has substantially the same width.

[0039] In some embodiments, the flow path includes at least three expansion regions in a series of expansion regions. Preferably, it includes at least four expansion regions. Most preferably, it includes at least five expansion regions. Alternatively or additionally, the flow path includes up to 20 expansion regions. In preferred forms, the flow path includes a number of expansion regions selected from the following: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0040] In some embodiments, the flow channel includes one or more constrictions between each expansion region in a series of expansion regions.

[0041] In one embodiment, the inlet of the channel has a width of 2 μm to 3 μm. Preferably, the constriction has a width narrower than the inlet.

[0042] In one embodiment, the outlet of the flow channel has a width of about 1 μm to 2 μm.

[0043] In certain embodiments, the microfluidic device further comprises a cell capture and lysis structure upstream of the inlet, the cell capture and lysis structure comprising: a cell trap adjacent to the flow channel inlet configured to receive and retain cells from a cell-containing fluid sample, the cell trap comprising: Viewing window that enables cell inspection an opening connected to the channel inlet, wherein the opening is sized to prevent the passage of cells therethrough; a lysis port configured to introduce a lysis buffer into the cell trap; A cell trap, including Includes.

[0044] In one embodiment, the size of the opening and passage is about 2 μm to about 3 μm.

[0045] In certain embodiments, the cell trap is a rectangular prism-shaped hollow structure of a microfluidic device having an open face that allows entry of cells into the cell trap.

[0046] Preferably, the cell trapping opening is in a face opposite the open face.

[0047] Preferably, the cell trap has a depth substantially the same as the depth of the channel. More preferably, the cell trap has the same width and length dimensions as the depth. A preferred size is 20 μm×20 μm×20 μm (±5 μm).

[0048] The cell trap may include a valve and / or pump to isolate the opening connected to the channel inlet from the channel inlet and / or the opening that allows entry of cells into the cell trap.

[0049] In some embodiments, the microfluidic device further comprises a chromosome distribution structure downstream of the outlet, the chromosome distribution structure comprising: a distribution channel defined between the channel inlet and the channel outlet and having a port for receiving an individual chromosome from the outlet of the flow channel; Includes; wherein the channel outlet is connected to a distribution pipe configured to distribute a single chromosome from the microfluidic device in the form of a fluid droplet containing the single chromosome. Microfluidic Device In certain embodiments, the distribution channel has a depth the same as the depth of the flow path.

[0050] In one embodiment, the distribution structure further comprises a chromosome holding region downstream of the series of extensions, wherein the chromosome holding region comprises an extension region that functions to hold one or more chromosomes therein from moving downstream simultaneously as metaphase chromosomes move upstream through the flow path.

[0051] In some embodiments, the microfluidic device further comprises a detection zone, where the detection zone comprises or is associated with a device for detecting the presence of metaphase chromosomes. In some embodiments, the device is capable of detecting the presence of individual metaphase chromosomes. For example, the device may be a photodetector. Suitable outputs for detection include fluorescence.

[0052] At the detection zone, individual metaphase chromosomes can be detected downstream of the outlet of the flow channel, where they are separately dispensed and mounted onto slides or well plates for further analysis.

[0053] In a further aspect of the invention, there is provided a method for separating metaphase chromosomes in a chromosome-containing metaphase fluid, comprising the steps of: passing a metaphase chromosome-containing fluid through a microfluidic device as defined above in one or more embodiments of the present invention at a pressure whereby the constriction subjects the metaphase chromosomes to a shear stress sufficient to separate the metaphase chromosomes from one another; A method is provided that includes:

[0054] In certain embodiments of this aspect of the invention, the method further comprises separately distributing the chromosomes from the outlets.

[0055] In one form of this aspect of the invention, the metaphase chromosomes are present in the form of one or more clusters of metaphase chromosomes, and wherein the constriction subjects the one or more clusters of metaphase chromosomes to a shear stress sufficient to cause the one or more clusters of metaphase chromosomes to separate into individual metaphase chromosomes; and wherein the individual metaphase chromosomes are distributed separately from the outlet.

[0056] In one form of this aspect of the invention, the pressure is a pulsed pressure. The pulsed pressure may be applied alternately both forward and backward along the flow path, where the balance of the overall pressure applied is such that the metaphase chromosomes move gradually towards the outlet. Alternatively, the pressure may be a constant pressure.

[0057] In a further aspect of the invention there is provided a method for isolating metaphase chromosomes in a chromosome-containing fluid comprising the steps of: A chromosome-containing fluid containing metaphase chromosomes is passed through a microfluidic device, wherein the microfluidic device having the flow path performs the following: A plurality of expansion regions located between the inlet and the outlet; and one or more stenoses located between one or more dilated regions; Includes; exposing the metaphase chromosomes to shear stress sufficient to separate the metaphase chromosomes from one another at or within one or more constrictions; and dispersing metaphase chromosomes separated from one another into multiple expansion regions; A method is provided that includes:

[0058] In certain embodiments of this aspect of the invention, the method further comprises separately releasing the separated metaphase chromosomes from the microfluidic device.

[0059] In one form of this embodiment of the invention, the metaphase chromosomes are present in the form of one or more clusters of metaphase chromosomes, and wherein constriction subjects the one or more clusters of metaphase chromosomes to shear stress sufficient to cause the one or more clusters of chromosomes to separate into individual chromosomes.

[0060] In a further aspect of the invention, there is provided a method for separating metaphase chromosomes in a chromosome-containing fluid using a microfluidic device, comprising the steps of: Passing a fluid through a channel of a microfluidic device, the channel having a plurality of alternating constrictions and expansions; Includes; wherein as the fluid passes through the 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; Here, as fluid passes through the extension, the microfluidic device is operated with a pressure that disperses the separated chromosomes from one another. A method is provided.

[0061] In certain embodiments of this aspect of the invention, the method further comprises separately releasing the separated chromosomes from the microfluidic device.

[0062] In one form of this embodiment of the invention, the metaphase chromosomes are present in the form of one or more clusters of metaphase chromosomes, and wherein as fluid passes through the constriction, the pressure pulse subjects the one or more clusters of metaphase chromosomes to shear stress sufficient to cause the metaphase chromosomes to separate from the one or more clusters of metaphase chromosomes.

[0063] According to various of the above aspects of the invention, the shear stress is at least about 0.02 N / m2 as measured at the narrowest wall of the stenosis. 2 ~At least about 15,000N / m 2 or any value therebetween. Preferably, the shear stress is about 0.02 N / m when measured at the narrowest wall of the stenosis. 2 ~Approx. 12,500N / m 2 , about 0.02N / m 2 ~Approx. 10,000N / m 2 , about 0.02N / m 2 ~Approx. 9,000N / m 2 , about 0.02N / m 2 ~Approx. 8,500N / m 2 , about 0.02N / m 2 ~Approx. 8,000N / m 2 , about 0.02N / m 2 ~Approx. 7,000N / m 2 , about 0.02N / m 2 ~Approx. 6,000N / m 2 , about 0.02N / m 2 ~Approx. 5,000N / m 2 , about 0.02N / m 2 ~Approx. 4,000N / m 2 , about 0.02N / m 2 ~Approx. 3,500N / m 2 , about 0.02N / m 2 ~Approx. 3,000N / m 2 , about 0.02N / m 2 ~Approx. 2,500N / m 2 , about 0.02N / m 2~Approx. 2,000N / m 2 , about 0.02N / m 2 ~Approx. 1,500N / m 2 , about 0.02N / m 2 ~Approx. 1,000N / m 2 , about 0.02N / m 2 ~about 800N / m 2 , about 0.02N / m 2 ~about 600N / m 2 , about 0.02N / m 2 ~Approx. 500N / m 2 , about 0.02N / m 2 ~about 400N / m 2 , about 0.02N / m 2 ~Approx. 300N / m 2 , about 0.02N / m 2 ~about 250N / m 2 , about 0.02N / m 2 ~about 200N / m 2 , about 0.02N / m 2 ~Approx. 150N / m 2 , about 0.02N / m 2 ~Approx. 100N / m 2 , about 0.02N / m 2 ~about 80N / m 2 , about 0.02N / m 2 ~about 60N / m 2 , about 0.02N / m 2 ~Approx. 50N / m 2 , about 0.02N / m 2 ~about 40N / m 2 , about 0.02N / m 2 ~Approx. 30N / m 2 , about 0.02N / m 2 ~about 25N / m 2 , about 0.02N / m 2 ~about 10N / m 2 , about 0.02N / m 2 ~about 5N / m 2 , or about 0.02 N / m 2 ~Approx. 1N / m 2 Preferably, the shear stress is about 1 N / m2 when measured at the narrowest wall of the stenosis. 2 ~Approx. 15,000N / m 2 , about 2N / m 2 ~Approx. 12,500N / m 2, about 5N / m 2 ~Approx. 10,000N / m 2 , about 5N / m 2 ~9,000N / m 2 , about 10N / m 2 ~Approx. 8,500N / m 2 , about 10N / m 2 ~Approx. 8,000N / m 2 , about 15N / m 2 ~Approx. 7,000N / m 2 , about 15N / m 2 ~Approx. 6,000N / m 2 , about 20N / m 2 ~Approx. 5,000N / m 2 , about 20N / m 2 ~About 4000N / m 2 , from about 25N / m 2 ~Approx. 4,000N / m 2 , about 50N / m 2 ~Approx. 3,000N / m 2 , about 100N / m 2 ~Approx. 2,000N / m 2 , about 200N / m 2 ~Approx. 1,000N / m 2 , about 100N / m 2 ~Approx. 500N / m 2 , about 200N / m 2 ~about 400N / m 2 , about 5N / m 2 ~Approx. 500N / m 2 , about 10N / m 2 ~about 400N / m 2 , about 20N / m 2 ~Approx. 100N / m 2 , or about 30N / m 2 ~about 60N / m 2 Preferably, the shear stress is about 0.2 N / m2 as measured at the narrowest wall of the stenosis. 2 Super, about 1N / m 2 , about 5N / m 2 , about 10N / m 2 , about 25N / m 2 , about 30N / m 2 , about 40N / m 2 , about 50N / m 2 , about 60N / m 2 , about 80N / m 2, about 100N / m 2 , about 150N / m 2 , about 200N / m 2 , about 250N / m 2 , about 300N / m 2 , about 400N / m 2 , about 500N / m 2 , about 600N / m 2 , about 800N / m 2 , approx. 1,000N / m 2 , approx. 1,500N / m 2 , approx. 2,000N / m 2 , approx. 2,500N / m 2 , approx. 3,000N / m 2 , approx. 3,500N / m 2 , approx. 4,000N / m 2 , approx. 5,000N / m 2 , about 6,000N / m 2 , about 7,000N / m 2 , approx. 8,000N / m 2 , about 8,500N / m 2 , approx. 9,000N / m 2 , about 10,000N / m 2 , or about 12,500 N / m 2 Preferably, the shear stress is about 1 N / m2 when measured at the narrowest wall of the stenosis. 2 , about 5N / m 2 , about 10N / m 2 , about 25N / m 2 , about 30N / m 2 , about 40N / m 2 , about 50N / m 2 , about 60N / m 2 , about 80N / m 2 , about 100N / m 2 , about 150N / m 2 , about 200N / m 2 , about 250N / m 2 , about 300N / m 2 , about 400N / m 2 , about 500N / m 2 , about 600N / m 2 , about 800N / m 2 , approx. 1,000N / m 2 , approx. 1,500N / m 2 , approx. 2,000N / m2 , approx. 2,500N / m 2 , approx. 3,000N / m 2 , approx. 3,500N / m 2 , approx. 4,000N / m 2 , approx. 5,000N / m 2 , about 6,000N / m 2 , about 7,000N / m 2 , approx. 8,000N / m 2 , about 8,500N / m 2 , approx. 9,000N / m 2 , about 10,000N / m 2 , approx. 12,500N / m 2 , or about 15,000 N / m 2 is less than.

[0064] According to the various above aspects of the invention, pressure is preferably applied across the flow path. The pressure applied across the flow path is from about 0 mbar to about 10,000 mbar or any value therebetween. Preferably, the pressure applied is from about 2 mbar to about 7,000 mbar, from about 30 mbar to about 5,000 mbar, from about 50 mbar to about 2,500 mbar, from about 100 mbar to about 1,000 mbar, from about 250 mbar to about 1,000 mbar, from about 300 mbar to about 1,000 mbar, or from about 400 mbar to about 700 mbar. Preferably, the applied pressure is greater than about 0 mbar, about 2 mbar, about 30 mbar, about 50 mbar, about 100 mbar, about 250 mbar, about 300 mbar, about 400 mbar, about 700 mbar, about 1,000 mbar, about 2,500 mbar, about 5,000 mbar, or about 7,000 mbar. Preferably, the applied pressure is less than about 2 mbar, about 30 mbar, about 50 mbar, about 100 mbar, about 250 mbar, about 300 mbar, about 400 mbar, about 700 mbar, about 1,000 mbar, about 2,500 mbar, about 5,000 mbar, about 7,000 mbar, or about 10,000 mbar.

[0065] According to various of the above aspects of the invention, a method comprises: Capturing metaphase cells within a cell trap in a microfluidic device; and introducing a lysis buffer to the metaphase cells and applying a pressure pulse to drive the metaphase cells out of the cell trap and into the flow channel under sufficient shear stress to lyse the cells and provide metaphase chromosomes in the chromosome-containing fluid; may include:

[0066] In various of the above aspects of the invention, the method comprises the steps of: receiving the distributed individual chromosomes from the outlet of the flow path of the microfluidic device into a distribution channel; Transporting individual chromosomes to distribution tubes; Dispensing the single chromosome from the microfluidic device via a distribution tube in the form of a fluid droplet containing the single chromosome; It may further include.

[0067] Preferably, the fluid droplet has a volume of about 100 nL up to about 500 nL, more preferably about 100 nL up to about 400 nL, and even more preferably 100 nL up to about 300 nL.

[0068] In one or more aspects and embodiments thereof described above, the chromosome-containing fluid comprises a lysis buffer such that the method is a method for chemically assisted shearing separation of metaphase chromosomes.

[0069] Of course, a lysis buffer is a buffer that supports the lysis of cells. A separation buffer is a buffer that supports the separation of chromosomes. A lysis buffer may contain a separation buffer and vice versa.

[0070] In one or more aspects and embodiments thereof described above, a lysis buffer is introduced such that cells are lysed by the chemical or combined chemical and physical action of the lysis buffer. A separation buffer, either incorporated in the lysis buffer or subsequent to the lysis buffer, is introduced to the inlet of the flow channel prior to or together with the introduction of metaphase chromosomes in the chromosome-containing fluid.

[0071] In one or more aspects and embodiments thereof of the invention described above, the chromosome-specific label and / or DNA stain may be added prior to introduction of the cells into the inlet of the device. In one or more aspects and embodiments thereof of the invention described above, metaphase cells are fixed and permeabilized to facilitate hybridization of the chromosome-specific label and / or DNA stain prior to introduction of the cells into the inlet of the device.

[0072] Further aspects of the invention and further embodiments of the aspects mentioned in the preceding paragraphs will become apparent from the following description, given by way of example, and from reference to the accompanying drawings, in which: [Brief description of the drawings]

[0073] [Figure 1] 1 is an illustration of a microfluidic device according to one embodiment of the present invention.

[0074] [Diagram 2] Schematic of a microfluidic device illustrating constriction.

[0075] [Diagram 3] Schematic of a microfluidic device illustrating a sampling port through which cells are introduced into the microfluidic device.

[0076] [Figure 4] Schematic of a microfluidic device illustrating the upstream cell trapping and lysis structure.

[0077] [Diagram 5] Schematic of a microfluidic device illustrating chemically assisted shear lysis of cells and transfer under pressure pulses out of a cell trap and into a channel.

[0078] [Figure 6] Schematic of a microfluidic device illustrating channels and expansion regions.

[0079] [Figure 7]Schematic of the microfluidic device illustrating channel outlets and chromosome detection.

[0080] [Figure 8] Schematic of a microfluidic device illustrating downstream chromosome segregation for individual chromosome segregation.

[0081] [Figure 9] Schematic illustrating the dispensing of single chromosome-containing droplets from a microfluidic device onto a well plate.

[0082] [Figure 10] Schematic showing pump arrangement of a microfluidic device.

[0083] [Figure 11] A close-up view showing details of a constriction in a channel of a microfluidic device.

[0084] References Quake et al. (Nature Methods, Vol. 11, No. 1, 2014, pp 19-21)

[0085] Dolezel et al. (Funct. Integr. Genomics, 2012, 12:397-416)

[0086] Fan et al. (Nat. Biotechnol., January 2011, 29(1):51-57) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] Detailed Description of the Invention Detailed Description of the Embodiments The present invention relates to a microfluidic device and method for separating metaphase chromosomes from each other.

[0088] In a preferred form, the microfluidic device is configured to capture and lyse single metaphase cells, suspend excluded chromosomes in single chromosomes, detect each single chromosome, and then dispense each chromosome from the microfluidic device into a container (such as a glass slide or well plate) for downstream processing.

[0089] Roughly speaking, a cell is introduced into a microfluidic device where it is analyzed (such as by optical microscopy) to determine whether the cell is a metaphase cell. If the cell is a metaphase cell, it is captured, and then a lysis buffer is introduced into the microfluidic device with a high pressure pulse to drive the cell and its contents out of the cell trap, through the channel restriction and into the flow path of the microfluidic device, while simultaneously lysing the cell by shear against the cell membrane as it passes through the channel restriction. The chromosomes then emerge into the microfluidic flow path, typically in the form of one or more clusters. In the microfluidic flow path, the one or more clusters of chromosomes pass through a series of alternating constrictions and expansions.

[0090] The constriction provides an obstacle to the flow of one or more clusters of chromosomes through the channel. A pressure pulse drives the one or more clusters of chromosomes through the constriction, which simultaneously exerts a large shear stress on the one or more clusters of chromosomes to break the one or more clusters into pieces.

[0091] In spreading, the chromosomes are exposed to a low flow rate and a changing flow profile that causes the chromosomes to disperse and become separated from one another. Spreading also provides a lysis buffer that has the opportunity to mix with individual chromosomes to stabilize them, as well as with one or more clusters of chromosomes to chemically assist in the shear separation of the chromosomes during subsequent constriction.

[0092] The cluster or clusters of chromosomes are subjected to multiple alternating constrictions and expansions until the cluster or clusters of chromosomes are broken down into separate and individual chromosomes that are detected at the outlet of the flow channel where they are separately dispensed and placed onto slides or well plates for further analysis.

[0093] Thus, the devices and methods of the present invention provide a mechanism for isolating individual chromosomes for subsequent haplotyping.

[0094] Embodiments of the present invention are described below.

[0095] FIG. 1 is a diagram of a microfluidic device 100 for separating and distributing single chromosomes from a chromosome suspension. In this case, the microfluidic device 100 is formed as a polydimethylsiloxane (PDMS) casting by wafer tooling. Those skilled in the art will appreciate that many dissimilar materials may be used. The PDMS casting is capped with a glass cover slip. Again, dissimilar materials may be used. However, glass is chosen as the capping material due to its optical properties (e.g., no autofluorescence despite optical clarity) that allow easy observation of the components of the microfluidic device 100, and its ability to bond with PDMS by plasma activation.

[0096] The microfluidic device 100 includes a microfluidic channel 102 having an inlet 104 and an outlet 106. In this embodiment, the channel 102 has a length of 5 mm. However, different lengths may be used, e.g., from 3 mm to 15 mm. The channel 102 is divided into five zones (labeled 1 to 5 in FIG. 1 ). Each of these zones includes a first channel portion 108 and a second channel portion representing an extension portion 110. The first channel portion 108 has a smaller cross-sectional area cross section in the flow direction than the cross-sectional area of ​​the extension portion 110. In this case, the channel 102 has a depth of 20 μm and the first channel portion 108 has a width of 20 μm (e.g., 400 μm). 2100 μm) and the extension 110 has a width of 100 μm (e.g., 2000 μm 2 10. Thus, in this embodiment, the ratio of the first flow channel section 108 to the cross flow channel of the expansion section 110 is 1:5. Although this embodiment has a ratio of 1:5, the inventors are of the opinion that a ratio of 1:2 to 1:10 is preferred.

[0097] Each of the first flow path portions 108 includes a constriction 202 (see FIGS. 2 and 11, close-up). It is understood that each of the first flow path portions 108 may include multiple constrictions 202. In this embodiment, the constrictions 202 have a width of about 1 μm to about 2 μm. Furthermore, the width of the constriction 202 of each successive first flow path portion 108 from the inlet 104 to the outlet 106 is narrower than the width of the constriction 202 of the previous first flow path portion 108.

[0098] FIG. 11 illustrates an embodiment of a constriction 1100 between flow channel portions 1102 and 1104. The constriction 1100 has an enlarged tapered exit 1106 that tapers to a width that is approximately half the width of the flow channel. In FIG. 11, the constriction has a shallower depth than flow channel portions 1102 and 1104. In this particular embodiment, the constriction has a depth of approximately 5 μm, yet flow channel portions 1102 and 1104 have a bulk depth of approximately 20 μm. Regions of the flow channel immediately adjacent to the constriction 1100, labeled items 1108 and 1110, have the same depth as the constriction (e.g., approximately 5 μm), such that there is a gradual change in depth within the flow channel from the depth of the constriction 1100 (e.g., approximately 5 μm) to the bulk depth of the flow channel (e.g., approximately 20 μm).

[0099] The operation of the components of the channel 102 will now be briefly described. In operation, a fluid containing one or more clusters of chromosomes is introduced under pressure into the channel 102 via the inlet 104. The fluid flows through a first channel portion 108 in zone 1 where it passes through a constriction 202. The constriction 202 obstructs the passage of one or more clusters of chromosomes therethrough. Whereas the approximate size of a single metaphase chromosome is about 0.5 μm to about 3 μm; chromosome clusters can range in size from slightly larger than a single metaphase chromosome to slightly smaller than the size of a metaphase cell (about 10 μm to 15 μm). Regardless, the fluid within the constriction is subjected to an enhanced flow velocity relative to the channel 102 by providing a narrow flow area, and this enhanced flow velocity propels the one or more clusters of chromosomes through the restriction, at the same time impinging the one or more clusters of chromosomes with a force of about 0.02 N / m at the wall, which depends on the dimensions of the constriction, the applied pressure (which in turn affects the velocity) and the fluid properties. 2 ~Approx. 1N / m 2Such shear stress is sufficient to fragment the cluster or clusters of chromosomes, which may result in detached single chromosomes, which disperse from the cluster or clusters of chromosomes into smaller chromosome clusters. The single chromosomes 203 and / or smaller chromosome clusters 204 then emerge through an expanded tapered outlet of the constriction 202 in the first channel portion 108 of the channel 102 downstream of the constriction 202, before entering a second channel portion, e.g., the expansion portion 110 in Zone 1. In the expansion portion 110, the single chromosomes and / or smaller chromosome clusters are subjected to a reduced flow rate relative to the first channel portion 108 due to the wider channel area. In this expansion portion 110, the single chromosomes and / or smaller chromosome clusters are dispersed both radially and axially by a combination of diffusion and advection, which may result in increased spacing between the chromosomes as they exit the expansion into the narrower first channel portion 108 in Zone 2. Furthermore, dispersion of the chromosomes and / or smaller chromosome clusters in the extension 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., stabilizers or other reagents that promote chromosome separation and / or prevent or minimize clumping).

[0100] In zone 2, single chromosomes 203 and / or smaller chromosome clusters 204 undergo a similar process in which they pass through the first 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 rate to expose the smaller chromosome clusters to higher shear stresses to further fragment the chromosome clusters and / or separate the single chromosomes from the chromosome clusters. Meanwhile, after passing through this constriction, the chromosomes similarly emerge in the first channel portion 108 of zone 2 and then enter the expansion region 110 of zone 2 for further dispersion.

[0101] The above process is repeated through zones 3, 4, and 5, whereby each constriction 202 in the first flow portion 110 of these zones shrinks in width to impede the passage of chromosome clusters and disrupt smaller chromosome clusters 204; and each expansion region 110 of these zones further disperses single chromosomes 203 and / or chromosome clusters 204 from one another.

[0102] After passing through the flow channel 102 in each zone, the 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 are isolated from one another for downstream purposes.

[0103] In the embodiment shown 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, close-up) for capturing cells and allowing cell examination, a lysis port 116 for introducing a lysis buffer to lyse the cells and release the chromosomes contained therein if the cell is deemed suitable, and a waste port 118 for discharging waste reagents and cells deemed unsuitable. At the outlet of the flow channel, the device includes a detection zone 119 for detecting individual metaphase chromosomes and ensuring that the chromosomes are distributed (see FIG. 7, close-up). Downstream of the flow channel 102, the microfluidic device includes a distribution structure 120 including a distribution port 122, an extraction port 124, and a distribution channel 704. The cell trap 402 has dimensions of 20 μm×20 μm×20 μm, small enough to hold a single metaphase cell. The cell trap 402 includes an opening 404 to the inlet of the channel 102. The opening 404 has a width of about 2 μm to about 3 μm to prevent cells from passing from the cell trap 402 into the channel 102.

[0104] During operation, a cell sample is provided to the microfluidic device via sample port 114 .

[0105] Figure 3 shows the addition of a fluid sample containing cells 403 via sample port 114. In Figure 3, sample port 114 is operated at high pressure, waste port 118 is operated at low pressure, lysis port 116 and dispense port 122 are operated at base pressure, and extraction port 124 is closed. Given this configuration, the sample flows through sample transfer channel 126 to waste port 118.

[0106] FIG. 4 illustrates the capture of a cell 403 in a cell trap 402 for investigation. In FIG. 4, the sample port 114, the lysis port 116, and the waste port 118 are operated at a reference pressure; the dispense port 122 is operated at a low pressure; and the extraction port 124 is closed. The effect of this arrangement is to provide a pressure difference that maintains the cell 403 within the cell trap, e.g., there is a suction effect that biases the cell in the cell trap 402 against the opening 404. However, the cell 403 cannot pass through the opening 404. Once the cell 403 is held within 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.

[0107] If the cell 403 is not a metaphase cell, then the cell 403 is flushed out of the cell trap 402, such as by applying back pressure through the distribution port 122 and expelling the cell through the waste port 118. That is, the distribution 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 base pressure; and the extraction port 124 is closed.

[0108] If the cell is in metaphase, then the cell is subjected to a lytic process to rupture the cell membrane and release the chromosomes from within the cell. This process is illustrated in Figure 5. Lysis buffer 405 is added by operating lysis port 116 at a higher pressure (e.g., 40-45 mbar) and distribution port 122 at a lower pressure (e.g., below the base pressure of 35 mbar, such as less than 30 mbar); sample port 114 and waste port 118 are operated at the base pressure (e.g., 35 mbar), and extraction port 124 is closed. The effect of this is that lysis buffer 405 flows from lysis port 116 through lysis channel 502 where it contacts the cells it is lysing.

[0109] A pressure pulse is then used to force the cells through the opening and along the constriction which lyses the cells by shearing the cell membrane, and pass the cell contents (including the cluster or clusters of chromosomes) into the channel 102 via the inlet 104. In this pressure configuration, the sample port 114, waste port 118, and lysis port 116 are operated under pulsed pressure; the distribution port 122 is operated at low pressure, and the extraction port 124 is closed.

[0110] The lysis buffer is an aqueous solution containing 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 with hydrophilic polyethylene oxide chains (on average, it is 9.5 ethylene oxide units) and aromatic hydrocarbon lipophilic or hydrophobic groups), 0.1% w / v pepsin, and 75 mM potassium chloride. In this buffer; acetic acid fixes and preserves chromosome shape, Triton X-100 solubilizes / dissolves cell membrane components and hydrophobic proteins and has a second role in releasing chromosomes, pepsin releases individual chromosomes from their clusters and aids in cell disintegration and cell protein removal, and potassium chloride is a salt used to osmotically swell cells and increase pepsin solubility. Alternatively, the buffer may comprise 0.1% w / v pepsin, 1 mM EDTA, 73 mM potassium acetate buffer, 2 mM magnesium sulfate, buffered to pH 5 with acetate. Alternatively, the fixative role of the acetate in any of these buffers may be performed by the fixative formaldehyde. Those skilled in the art will appreciate that other buffer compositions known in the art would also be suitable for use as lysis and / or separation buffers.

[0111] FIG. 2 illustrates the use of pressure pulses to drive chromosome clusters through the constriction 202. This is accomplished by applying a high pressure pulse (e.g., 300-1000 mbar) through the lysis port 116. Under this mode of operation, the sample port 114 and waste port 118 are operated at a base pressure (e.g., 35 mBar); the distribution port 122 is operated at a low pressure (e.g., <30 mbar); and the extraction port 124 is closed. The additional pressure from the lysis port 118 drives the clustered chromosomes 204 (e.g., one or more clusters of chromosomes that may become trapped at the narrow opening of the constriction 202) obstructed by the constriction 202 and subjects 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.

[0112] In an alternative approach, a high pressure pulse is applied via the sample port 114 (e.g., 250-950 mBar or 250-1,000 mBar), waste port 118 (e.g., 250-950 mBar or 250-1000 mBar), and lysis port 116 (e.g., 300-1000 mBar), and a low pressure is applied to the dispense port 122 (e.g., 0 mBar) and the extraction port 124 is closed.

[0113] Under the pressure regime described, the shear stress through the constriction zone is approximately 0.02 N / m, depending on the size of the constriction and the pressure applied. 2 ~15,000N / m 2 The range is as follows:

[0114] The combination of the lysis buffer and the differential pressure between the lysis port 116 and the distribution port 122 induces a chemically assisted shear lysis process that causes the cell membrane to break up and forces the cell contents into the channel 102 through the opening 404. The cell contents include one or more clusters of chromosomes 204 (and potentially a single chromosome 203). The cluster or clusters of chromosomes are then subjected to a shearing process in the channel 102 as described previously herein to separate the chromosomes.

[0115] FIG. 6 provides an illustration of the operation of the microfluidic device 100 in which, after the cells are lysed, the chromosomes 205 are expelled into the channel 102 and move through the expansion region 110 of one of the zones. In FIG. 6, the sample port 114, the lysis port 116, and the waste port 118 remain set to the lysis buffer application setting; the distribution port 122 is operated at the reference pressure; and the extraction port 124 is closed. Thus, a pressure differential exists across the channel 102 driving the chromosomes from the inlet 104 of the channel 102 towards the outlet 106 of the channel. The expansion section shows a single chromosome 203 dispersed and separated through the expansion region 110 of zone 1. The expansion region 110 may include a mixing device, such as a herringbone mixer, to aid in the dispersion of the single chromosomes.

[0116] Once the chromosomes are separated, they are detected at the outlet 106, such as by live recording of the fluorescent signal. Each detection event triggers the activation of the distribution system. Figures 7 and 8 illustrate the detection and counting of chromosomes and the transfer of a single chromosome out of the microfluidic device 100 via the extraction port 124. Figure 7 illustrates the detection of a single chromosome 203 at the outlet 106 using a photodetector 702. The detection limit 703 ensures that the chromosomes are present in a single file. Upon detection of a chromosome, the distribution system is activated. A flow of neutralizing buffer (to stop the degradation of the chromosome shape from pepsin activity, if present) is provided via the distribution port 122 to capture and distribute the detected chromosome from the microfluidic device 100. Each chromosome is released from the microfluidic device in the form of a droplet. The droplet is distributed onto a container (e.g., a glass slide or a special well plate). More specifically, once a chromosome is detected, the valve of the extraction port 124 is switched from a closed position (shown in FIG. 7) to an open position, and the pressure of the distribution port 122 is increased to provide a neutralizing agent. This increased flow rate 709 (shown in FIG. 8) results in the single chromosome 203 being distributed from the outlet 106 to the distribution channel 704 where it is subsequently deposited onto a well plate or glass slide. The increased flow rate 709 also results in a flow reversal in the flow channel 102, helping to maintain the separated chromosomes.

[0117] As an example, during detection, sample port 116 and waste port 118 are operated at 0 mBar; lysis port 116 is operated at 2-5 mBar; and distribution port 122 is operated at 2 mBar. As an alternative example, during detection, sample port 116 and waste port 118 are operated at 10 mBar; lysis port 116 is operated at 20 mBar; and distribution port 122 is operated at 2 mBar. This low pressure differential indicates flow through flow channel 102 to allow detection of chromosomes at outlet 106. Once chromosomes are detected, the pressure at distribution port 122 is increased to 15 mBar to distribute the chromosomes from outlet 106 to distribution channel 704.

[0118] 9 illustrates the deposition of a 200 nL droplet 900 containing a single chromosome 203 into a well plate 902 that travels from the outlet of a distribution channel 704 through a distribution tube 705. This process can be repeated until each chromosome is deposited on the well plate 902, such as for an array of 46 individual droplets, each containing a single chromosome, for example, chromosomes taken from human cells. A hydrophobic coating 706 on the distribution tube ensures that the droplets do not adhere to the distribution tube 705. FIG. 9 also illustrates the distribution channel 704 in relation to a cartridge 707 and a glass coverslip 708.

[0119] 10 shows a pump arrangement according to one embodiment of the invention using reference pressures. In this embodiment, the sample port 114 is configured to use a 69 mBar pressure pump with a reference pressure set at 35 mBar; the lysis port 116 is configured to use a 1000 mBar pressure pump with a reference pressure set at 35 mBar; the waste port 118 is configured to use a 70 mBar pressure pump with a reference pressure set at 35 mBar; the dispense port 122 is configured to use a 345 mBar pressure pump with a reference pressure set at 35 mBar; and the extraction port 124 is normally closed.

[0120] Generally as described above, operation of the microfluidic device 100 is performed by connecting the various fluid ports of the microfluidic device to pressure / flow controllers. A 345 mbar pressure pump is connected to the sample port 114 and waste port 118 because they are used to control cell movement during cell screening and capture, which requires high resolution of pressure changes to create 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 cells held in the trap. 69 mBar is connected to the distribution port 122 to allow a pressure drop in the distribution channel for chromosome transfer. The distribution channel 704 has a valve (a tube mounted on a gasket) on the extraction port 124 that is normally closed during operation except when dispensing droplets. All pressure regulators are initially set to a base pressure of 35 mBar, from which each pressure line may be raised or lowered depending on the desired directionality of flow in the microfluidic device 100.

[0121] Alternatively, in this embodiment, the sample port 114 is configured to use a 1000 mBar pressure pump with a base pressure set at 35 mBar; the dissolution port 116 is configured to use a 1000 mBar pressure pump with a base pressure set at 35 mBar; the waste port 118 is configured to use a 1000 mBar pressure pump with a base pressure set at 35 mBar; the dispense port 122 is configured to use a 345 mBar pressure pump with a base pressure set at 35 mBar; and the extraction port 124 is normally closed.

[0122] Generally as described above, operation of the microfluidic device 100 is performed by connecting the various fluid ports of the microfluidic device to pressure / flow controllers. 1000 mbar pressure pumps are connected to the sample port 114 and waste port 118 because they are used to control cell movement during cell screening and capture, which requires high resolution of pressure changes to create 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 cells held in the trap. 1000 mBar is connected to the distribution port 122 to allow a pressure drop in the distribution channel for chromosome transfer. The distribution channel 704 has a valve (a tube mounted on a gasket) on the extraction port 124 that is normally closed during operation except when dispensing droplets. All pressure regulators are initially set to a base pressure of 35 mBar, from which each pressure line may be raised or lowered depending on the desired directionality of flow in the microfluidic device 100.

[0123] Dispensing is performed by dispensing droplets from the microfluidic device 100 through the distribution pipe 705 (for example, the distribution pipe in this embodiment has an outer diameter of 0.79 mm, an inner diameter of 0.15 mm, and a length of 7 mm), where each droplet contains a chromosome. This is done by inducing a higher pressure at the distribution port 122 and opening the valve at the extraction port 124. The flow is then moved through the distribution channel 704, through the distribution pipe, and by the pressure drop outside the distribution pipe tip. Once the appropriate droplet size is generated (droplet size can be changed by changing the pressure drop, but one example size is 200 nL), each droplet is dispensed onto a glass slide or a specially designed well plate. The pressure from the distribution port 122 is then returned to the base pressure. The droplets attach to the containers due to the surface tension of the formed droplets. An automated mechanism that holds the containers is used to dispense each droplet onto the array and the containers. The mechanism moves independently in three axes to the cartridge, eg, along the x and y axes to create an array of droplets on the reservoir, and in the z axis to attach each droplet to a reservoir.

[0124] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or apparent from the text or drawings, all of which various combinations constitute various alternative aspects of the invention. For example, it will be understood that the alternative topologies of the individual features described above constitute alternative aspects of the invention.

Claims

1. 1. A microfluidic device for separating metaphase chromosomes in a metaphase chromosome-containing fluid, comprising: below: an inlet for receiving a fluid containing metaphase chromosomes; an outlet for distributing individual metaphase chromosomes separately; A series of expansion regions; and one or more stenoses located between successive dilated regions in a series of dilated regions; a flow path including wherein the constriction is capable of applying sufficient shear stress to separate metaphase chromosomes from one another; and The expansion regions are operable to disperse chromosomes from one another, Microfluidic devices.

2. 1. A microfluidic device for separating metaphase chromosomes in a metaphase chromosome-containing fluid, comprising: below: A channel having a width of about 10 μm to about 30 μm, comprising: entrance; Exit; and a series of dilated regions and one or more constrictions located between successive dilated regions in the series of dilated regions; a flow path including wherein the plurality of expansion regions have a channel width of about 50 μm to about 150 μm, and each constriction in the plurality of constrictions has a minimum width of about 1 μm to about 3 μm. Microfluidic devices.

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

4. The microfluidic device according to any one of claims 1 to 3, wherein the length of the channel is from about 2 mm to about 15 mm.

5. The microfluidic device of any one of claims 1 to 4, wherein each successive constriction from the inlet to the outlet has a minimum width narrower than the previous constriction.

6. The microfluidic device of any one of claims 1 to 5, wherein one or more of said one or more constrictions have an enlarged tapered outlet.

7. The microfluidic device of any one of claims 1 to 6, wherein each expansion region in the series of expansion regions has substantially the same width.

8. The microfluidic device of any one of claims 1 to 7, wherein the series of expansion regions comprises at least 3 expansion regions and at most 20 expansion regions.

9. The microfluidic device of any one of claims 1 to 8, wherein the flow path comprises two or more constrictions between each expansion region in a series of expansion regions.

10. The microfluidic device of any one of claims 1 to 9, wherein the inlet has a width of between 2 μm and 3 μm.

11. The microfluidic device further comprises a cell capture and lysis structure upstream of the inlet, the cell capture and lysis structure comprising: A cell trap adjacent to the flow channel inlet configured to receive and retain cells from a cell-containing fluid sample, the cell trap comprising: Viewing window that enables cell inspection an opening connected to the channel inlet, wherein the opening is sized to prevent passage of a cell therethrough; a lysis port configured to introduce a lysis buffer into the cell trap; A cell trap, including The microfluidic device according to any one of claims 1 to 10, comprising:

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

13. The microfluidic device of any one of claims 1 to 12, wherein the cell trap is a rectangular prism-shaped hollow structure of the microfluidic device having an open face to allow entry of cells into the cell trap.

14. The microfluidic device further comprises a chromosome distribution structure downstream of the outlet, the chromosome distribution structure comprising: a distribution channel defined between the channel inlet and the channel outlet and having a port for receiving an individual chromosome from the outlet of the flow channel; Including; 14. The microfluidic device of claim 1, wherein the channel outlet is connected to a distribution pipe configured for dispensing a single chromosome from the microfluidic device in the form of a fluid droplet comprising a single chromosome.

15. 1. A method for separating metaphase chromosomes in a chromosome-containing metaphase fluid, comprising: passing a metaphase chromosome-containing fluid through the microfluidic device of any one of claims 1 to 14 at a pressure whereby the constriction subjects the metaphase chromosomes to a shear stress sufficient to separate the metaphase chromosomes from one another; The method includes:

16. 1. A method for isolating metaphase chromosomes in a chromosome-containing fluid, comprising: A chromosome-containing fluid containing metaphase chromosomes is passed through a microfluidic device, wherein the microfluidic device has a flow path for: A plurality of expansion regions located between the inlet and the outlet; and one or more stenoses located between one or more dilated regions; Including; exposing the metaphase chromosomes to shear stress sufficient to separate the metaphase chromosomes from one another at or within one or more constrictions; dispersing metaphase chromosomes separated from one another into multiple expansion regions; The method includes:

17. 1. A method for separating metaphase chromosomes in a chromosome-containing fluid using a microfluidic device, comprising: Passing a fluid through a channel of a microfluidic device, the channel having a plurality of alternating constrictions and expansions; Including; wherein as the fluid passes through the 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; Here, as fluid passes through the extension, the microfluidic device is operated with a pressure that disperses the separated chromosomes from one another. method.

18. The shear stress is at least about 0.02 N / m2 when measured at the narrowest wall of the stenosis. 2 ~ At least 15,000N / m 2 The method according to any one of claims 15 to 17, wherein

19. The method comprises the steps of: Capturing metaphase cells in a cell trap of a microfluidic device; and introducing a lysis buffer to the metaphase cells and applying a pressure pulse to drive the metaphase cells out of the cell trap and into the flow channel under sufficient shear stress to lyse the cells, and providing the chromosomes in a chromosome-containing fluid; The method according to any one of claims 15 to 18, comprising:

20. The method is as follows: receiving the distributed individual chromosomes from an outlet of a flow path of the microfluidic device into a distribution channel; transporting individual chromosomes to distribution tubes; Dispensing the single chromosome from the microfluidic device via a distribution tube in the form of a fluid droplet containing the single chromosome; The method of any one of claims 15 to 19, further comprising:

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