U.S. patent application number 16/704175 was filed with the patent office on 2020-10-29 for single-sheath microfluidic chip.
The applicant listed for this patent is Genus plc. Invention is credited to Gopakumar Kamalakshakurup, Zheng Xia.
Application Number | 20200338557 16/704175 |
Document ID | / |
Family ID | 1000004520592 |
Filed Date | 2020-10-29 |
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United States Patent
Application |
20200338557 |
Kind Code |
A1 |
Xia; Zheng ; et al. |
October 29, 2020 |
SINGLE-SHEATH MICROFLUIDIC CHIP
Abstract
Microfluidic devices and methods for focusing components in a
fluid sample are described herein. The microfluidic device has at
least one flow focusing channel where the components are focused or
re-oriented by the geometry of the channel. From an upstream end of
the flow focusing channel to a downstream end of the flow focusing
channel, at least a portion of the flow focusing channel has a
reduction in height and at least a portion of the flow focusing
channel narrows in width, thereby geometrically constricting the
flow focusing channel. The devices and methods can be utilized in
sex-sorting of sperm cells to improve performance and increase
eligibility.
Inventors: |
Xia; Zheng; (DeForest,
WI) ; Kamalakshakurup; Gopakumar; (DeForest,
WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genus plc |
DeForest |
WI |
US |
|
|
Family ID: |
1000004520592 |
Appl. No.: |
16/704175 |
Filed: |
December 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16396138 |
Apr 26, 2019 |
10532357 |
|
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16704175 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/0636 20130101;
G01N 15/1484 20130101; B65G 51/00 20130101; B01L 3/502776 20130101;
G01N 15/1404 20130101; B01L 2400/0487 20130101; G01N 2015/1409
20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 15/14 20060101 G01N015/14 |
Claims
1. A microfluidic chip (100) comprising at least one flow focusing
channel (120), wherein from an upstream end (127) of the flow
focusing channel to a downstream end (128) of the flow focusing
channel, at least a portion of the flow focusing channel (120) has
a reduction in height and at least a portion of the flow focusing
channel (120) narrows in width, thereby geometrically constricting
the flow focusing channel (120).
2. The microfluidic chip (100) of claim 1, wherein the height
reduction of the portion of the flow focusing channel (120) is
caused by a ramp, a step, or constriction disposed on a bottom
surface (121) of the flow focusing channel (120), a top surface
(122) of the flow focusing channel (120), or both.
3. The microfluidic chip (100) of claim 1, wherein the flow
focusing channel (120) has a first sidewall (125) and a second
sidewall (126) opposite the first sidewall (125), wherein the first
and second sidewalls (125, 126) taper the flow focusing channel
(120) to form the portion of the flow focusing channel (120) that
narrows in width.
4. The microfluidic chip (100) of claim 1, wherein at least a
portion of the flow focusing channel (120) has a shape of a cone
with a larger diameter of the cone biased towards the upstream end
(127) and a smaller diameter of the cone biased towards the
downstream end (128).
5. The microfluidic chip (100) of claim 1, wherein the portion of
the flow focusing channel (120) with the height reduction and the
portion of the flow focusing channel (120) narrowing in width are
simultaneous or overlapping.
6. The microfluidic chip (100) of claim 1, wherein the portion of
the flow focusing channel (120) with the height reduction and the
portion of the flow focusing channel (120) narrowing in width are
sequential.
7. The microfluidic chip (100) of claim 1, wherein the geometrical
constriction of the flow focusing channel (120) is configured to
focus a material in a fluid that is flowing through the flow
focusing channel (120).
8. A microfluidic chip (100) comprising: a. a first micro-channel
(110); and b. at least one flow focusing region (120) fluidly
coupled to the first micro-channel (110), said flow focusing region
(120) formed by a bottom surface (121), a top surface (122), a
first sidewall (125) and a second sidewall (126) opposite the first
sidewall (125), wherein from an upstream end (127) of the flow
focusing region to a downstream end (128) of the flow focusing
region, at least a portion of the bottom surface (121) is raised,
at least a portion of the top surface (122) is lowered, and at
least a portion of the first and second sidewalls (125, 126) taper
the flow focusing region (120), thereby reducing a cross-sectional
area (129) of the flow focusing region at the downstream end (128)
relative to the upstream end (127).
9. The microfluidic chip (100) of claim 8 further comprising one or
more sheath fluid micro-channels (130) intersecting the first
micro-channel (110) upstream of the flow focusing region (120).
10. The microfluidic chip (100) of claim 9, wherein the one or more
sheath fluid micro-channels (130) are configured to flow a sheath
fluid.
11. The microfluidic chip (100) of claim 8, wherein the first
micro-channel (110) comprises an inlet (112) through which a sample
fluid enters the first micro-channel (110).
12. The microfluidic chip (100) of claim 8 further comprising one
or more output micro-channels (140) fluidly coupled to the first
micro-channel (110) downstream of the flow focusing region (120),
the one or more output micro-channels (140) configured to output a
fluid from the first micro-channel (110).
13. The microfluidic chip (100) of claim 8, wherein the bottom
surface (121) includes a ramp (123) having a positive slope that
raises the portion of the bottom surface.
14. The microfluidic chip (100) of claim 8, wherein the entire
bottom surface (121) is a ramp (123) that gradually increases a
height of the bottom surface from the upstream end (127) to the
downstream end (128).
15. The microfluidic chip (100) of claim 8, wherein the top surface
(122) includes a ramp (124) having a negative slope that lowers the
portion of the top surface.
16. The microfluidic chip (100) of claim 8, wherein the entire top
surface (122) is a ramp (124) that gradually decreases a height of
the top surface from the upstream end (127) to the downstream end
(128).
17. The microfluidic chip (100) of claim 8, wherein the entire
first and second sidewalls (125, 126) are angled so as to taper the
flow focusing region (120) from the upstream end (127) to the
downstream end (128).
18. The microfluidic chip (100) of claim 8, wherein at least two of
the raised portion of the bottom surface (121), the lowered of the
portion of the top surface (122), and the sidewall tapering occur
simultaneously.
19. The microfluidic chip (100) of claim 8, wherein at least two of
the raised portion of the bottom surface (121), the lowered portion
of the top surface (122), and the sidewall tapering are
overlapping.
20. The microfluidic chip (100) of claim 8, wherein the raised
portion of the bottom surface (121), the lowered portion of the top
surface (122), and the sidewall tapering occur in a pre-determined
sequence.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation and claims benefit of
U.S. Non-Provisional application Ser. No. 16/396,138 filed Apr. 26,
2019, the specification(s) of which is/are incorporated herein in
their entirety by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to a microfluidic chip design,
in particular, to a microfluidic chip for isolating particles or
cellular materials using laminar flow from a single sheath and
geometric focusing.
Background Art
[0003] Microfluidics enables the use of small volumes for preparing
and processing samples, such as various particles or cellular
materials. When separating a sample, such as the separation of
sperm into viable and motile sperm from non-viable or non-motile
sperm, or separation by gender, the process is often a
time-consuming task and can have severe volume restrictions.
Current separation techniques cannot, for example, produce the
desired yield, or process volumes of cellular materials in a timely
fashion. Furthermore, existing microfluidic devices do not
effectively focus or orient the sperm cells.
[0004] Hence, there is need for a microfluidic device and
separation process utilizing said device that is continuous, has
high throughput, provides time saving, and causes negligible or
minimal damage to the various components of the separation. In
addition, such a device and method can have further applicability
to biological and medical areas, not just in sperm sorting, but in
the separation of blood and other cellular materials, including
viral, cell organelle, globular structures, colloidal suspensions,
and other biological materials.
BRIEF SUMMARY OF THE INVENTION
[0005] It is an objective of the present invention to provide
microfluidic devices and methods that allow for focusing and
orienting particles or cellular materials, as specified in the
independent claims. Embodiments of the invention are given in the
dependent claims. Embodiments of the present invention can be
freely combined with each other if they are not mutually
exclusive.
[0006] In some aspects, the present invention features microfluidic
devices for use in sperm cell sexing and trait enrichment. The
microfluidic device may comprise at least one flow focusing channel
where the components are focused or re-oriented by the geometry of
the channel. From an upstream end of the flow focusing channel to a
downstream end of the flow focusing channel, at least a portion of
the flow focusing channel has a reduction in height and at least a
portion of the flow focusing channel narrows in width, thereby
geometrically constricting the flow focusing channel.
[0007] One of the unique and inventive technical features of the
present invention is the physical restriction of the channel
geometry at the flow focusing region. Without wishing to limit the
invention to any theory or mechanism, it is believed that the
technical feature of the present invention advantageously
eliminates the second sheath flow structure from the microfluidic
device such that the use of a secondary sheath fluid to
focus/orient sperm cells becomes unnecessary, thus reducing the
volume of sheath fluid used as compared to existing devices that
have two focusing regions using sheath fluids for stream
compression. This provides an additional benefit of reducing
operational costs for equipment and supplies, and further
simplifying system complexity. None of the presently known prior
references or work has the unique inventive technical feature of
the present invention.
[0008] The inventive technical feature of the present invention
surprisingly resulted in equivalent purity, better performance, and
improved functionality for Y-skewed sperm cells as compared to the
prior devices having two focusing regions using sheath fluids. For
instance, the microfluidic device of the present invention
unexpectedly improved the orientation of the sperm cells, which is
believed to have increased the eligibility, i.e. higher number of
cells detected, sorted, and ablated. In addition, the device of the
present invention was able to enhance resolution between the
Y-chromosome bearing sperm cells and the X-chromosome bearing sperm
cells, which resulted in effective discrimination of
Y-chromosome-bearing sperm cells.
[0009] Further still, the prior references teach away from the
present invention. For example, contrary to the present invention,
U.S. Pat. No. 7,311,476 teaches the use of sheath fluids to focus a
fluid stream in its disclosure of microfluidic chips that have a
first focusing region and a second focusing region downstream of
the first, where each region introduces sheath fluids to focus the
sheath fluid around particles, and that the second (downstream)
focusing region requires the introduction of additional sheath
fluid to achieve the necessary focusing.
[0010] In some embodiments, the microfluidic chip includes a
plurality of layers in which are disposed a plurality of channels
including: a sample input channel into which a sample fluid mixture
of components to be isolated is inputted, and two focusing regions
comprising a first focusing region that focuses particles in the
sample fluid and a second focusing region that focuses particles in
the sample fluid, where one of the focusing regions includes
introduction of a sheath fluid via one or more sheath fluid
channels, and the other focusing region includes geometric
compression without introducing additional sheath fluid. Geometric
compression refers to physical restriction due to a narrowing in
size of the sample channel in both the vertical and horizontal axes
(i.e. from above and below and from both the left and right sides,
relative to the direction of travel along the sample channel). In
some aspects, the first focusing region may combine geometric with
the sheath fluid introduction; however, the second focusing region
does not utilize additional sheath fluid for stream focusing or
particle orienting. In other aspects, the microfluidic chip can be
loaded on a microfluidic chip cassette which is mounted on a
microfluidic chip holder.
[0011] In some embodiments, the sample input channel and the one or
more sheath channels are disposed in one or more planes of the
microfluidic chip. For instance, a sheath channel may be disposed
in a different plane than a plane in which the sample input channel
is disposed. In other embodiments, the sample input channel and the
sheath channels are disposed in one or more structural layers, or
in-between structural layers of the microfluidic chip. As an
example, the one or more sheath channels may be disposed in a
different structural layer than a structural layer in which the
sample input channel is disposed.
[0012] In one embodiment, the sample input channel may taper at an
entry point into the intersection region with the sheath channel.
In another embodiment, the sheath channel may taper at entry points
into the intersection region with the sample input channel. In some
embodiments, the microfluidic device may include one or more output
channels fluidly coupled to the sample channel. The one or more
output channels may each have an output disposed at its end. In
other embodiments, the microfluidic chip may further include one or
more notches disposed at a bottom edge of the microfluidic chip to
isolate the outputs of the output channels.
[0013] In some embodiments, the microfluidic chip system includes
an interrogation apparatus which interrogates and identifies the
components of the sample fluid mixture in the sample input channel,
in an interrogation chamber disposed downstream from the flow
focusing region. In one embodiment, the interrogation apparatus
includes a radiation source configured to emit a beam to illuminate
and excite the components in said sample fluid mixture. The emitted
light induced by the beam is received by an objective lens. In
another embodiment, the interrogation apparatus may comprise a
detector such as a photomultiplier tube (PMT), an avalanche
photodiode (APD), or a silicon photomultiplier (SiPM).
[0014] In some embodiments, the microfluidic chip includes a
sorting mechanism which sorts said components in said sample fluid
mixture downstream from said interrogation chamber, by selectively
acting on individual components in said sample fluid mixture. In
one embodiment, the sorting mechanism may comprise a laser
kill/ablation. Other examples of sorting mechanisms that may be
used in accordance with the present invention include, but are not
limited to, particle deflection/electrostatic manipulation, droplet
sorting/deflection, mechanical sorting, fluid switching,
piezoelectric actuation, optical manipulation (optical trapping,
holographic steering, and photonic/radiation pressure), surface
acoustic wave (SAW) deflection, electrophoresis/electrical
disruption, micro-cavitation (laser induced, electrically induced).
In some embodiments, the isolated components are moved into one of
the output channels, and unselected components flow out through
another output channel.
[0015] In further embodiments, the microfluidic chip may be
operatively coupled to a computer which controls the pumping of one
of the sample fluid mixture or the sheath fluid into the
microfluidic chip. In another embodiment, the computer can display
the components in a field of view acquired by a CCD camera disposed
over the interrogation window in the microfluidic chip.
[0016] In some embodiments, the cells to be isolated may include at
least one of viable and motile sperm from non-viable or non-motile
sperm; sperm isolated by gender and other sex sorting variations;
stem cells isolated from cells in a population; one or more labeled
cells isolated from unlabeled cells including sperm cells; cells,
including sperm cells, distinguished by desirable or undesirable
traits; genes isolated in nuclear DNA according to a specified
characteristic; cells isolated based on surface markers; cells
isolated based on membrane integrity or viability; cells isolated
based on potential or predicted reproductive status; cells isolated
based on an ability to survive freezing; cells isolated from
contaminants or debris; healthy cells isolated from damaged cells;
red blood cells isolated from white blood cells and platelets in a
plasma mixture; or any cells isolated from any other cellular
components into corresponding fractions.
[0017] Any feature or combination of features described herein are
included within the scope of the present invention provided that
the features included in any such combination are not mutually
inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art. Additional advantages and aspects of the present invention are
apparent in the following detailed description and claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0018] The features and advantages of the present invention will
become apparent from a consideration of the following detailed
description presented in connection with the accompanying drawings
in which:
[0019] FIG. 1A shows a bottom view of a top layer of a microfluidic
device according to one embodiment of the present invention.
[0020] FIG. 1B shows a close-up view and a cross-sectional side
view of an intersection region in the top layer shown in FIG.
1A.
[0021] FIG. 1C shows a close-up view and a cross-sectional side
view of a flow focusing region in the top layer shown in FIG.
1A.
[0022] FIG. 2A shows a top view of a bottom layer of the
microfluidic device.
[0023] FIG. 2B shows a close-up view and a cross-sectional side
view of the intersection region in the bottom layer shown in FIG.
2A.
[0024] FIG. 2C shows a close-up view and a cross-sectional side
view of the flow focusing region in the bottom layer shown in FIG.
2A.
[0025] FIG. 2D shows a close-up view and a cross-sectional side
view of an output channel region in the bottom layer shown in FIG.
2A.
[0026] FIG. 3A shows a top view of the microfluidic device with the
top later stacked atop the bottom layer.
[0027] FIG. 3B shows a close-up view and a cross-sectional side
view of the intersection region in the stacked layers shown in FIG.
3A.
[0028] FIG. 3C shows a close-up view and a cross-sectional side
view of the flow focusing region in the stacked layers shown in
FIG. 3A.
[0029] FIG. 3D is a side view of the stacked layers of the
microfluidic device in FIG. 3A.
[0030] FIGS. 4A-4F show multiple embodiments of the flow focusing
region in the microfluidic device of the present invention, wherein
each figure includes a top view, a side view, and various
cross-sectional views of the flow focusing region. The multiple
embodiments demonstrate geometric compression of the micro-channel
by sequential raising of the bottom surface, lowering of the top
surface, and side tapering in varying combinations.
[0031] FIG. 5 shows an embodiment of the flow focusing region in
the microfluidic device, including a top view, a side view, and
various cross-sectional views of the flow focusing region. This
embodiment demonstrates geometric compression of the micro-channel
by simultaneously raising of the bottom surface, lowering of the
top surface, and side tapering.
[0032] FIGS. 6A-6C show multiple embodiments of the flow focusing
region in the microfluidic device of the present invention, wherein
each figure includes a top view, a side view, and various
cross-sectional views of the flow focusing region. The multiple
embodiments demonstrate geometric compression of the micro-channel
by simultaneous raising of the bottom surface and lowering of the
top surface with an overlap of side tapering.
[0033] FIGS. 7A-7B show non-limiting embodiments of the flow
focusing region in the microfluidic device, wherein each figure
includes a top view, a side view, and various cross-sectional views
of the flow focusing region. The embodiments demonstrate geometric
compression of the micro-channel by simultaneous raising of the
bottom surface and lowering of the top surface with sequential side
tapering.
[0034] FIGS. 8A-8B show other non-limiting embodiments of the flow
focusing region, wherein each figure includes a top view, a side
view, and various cross-sectional views of the flow focusing
region. The embodiments demonstrate geometric compression of the
micro-channel by simultaneously compression of all four sides and
an overlapping ramp on the bottom surface or top surface.
[0035] FIGS. 9A-9B show other non-limiting embodiments of the flow
focusing region, wherein each figure includes a top view, a side
view, and various cross-sectional views of the flow focusing
region. The embodiments demonstrate geometric compression of the
micro-channel by simultaneously compression of three sides and a
sequential ramp on the bottom surface or top surface.
[0036] FIGS. 10A-10B show other non-limiting embodiments of the
flow focusing region, wherein each figure includes a top view and a
side view. The embodiments demonstrate geometric compression of the
micro-channel by overlapping compression of four sides.
[0037] FIG. 11 shows a non-limiting embodiment of a top view and a
side view of the flow focusing region. This embodiment demonstrates
geometric compression of the micro-channel by sequential
compression of only three sides.
[0038] FIG. 12 is a non-limiting example of a flow diagram for a
method of gender-skewing a semen fluid sample.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Before turning to the figures, which illustrate the
illustrative embodiments in detail, it should be understood that
the present disclosure is not limited to the details or methodology
set forth in the description or illustrated in the figures. It
should also be understood that the terminology is for the purpose
of description only and should not be regarded as limiting. An
effort has been made to use the same or like reference numbers
throughout the drawings to refer to the same or like parts.
[0040] Following is a list of elements corresponding to a
particular element referred to herein: [0041] 100 microfluidic chip
[0042] 110 first/sample micro-channel [0043] 112 micro-channel
inlet [0044] 115 intersection region [0045] 120 flow focusing
channel/region [0046] 121 bottom surface [0047] 122 top surface
[0048] 123 bottom ramp [0049] 124 top ramp [0050] 125 first
sidewall [0051] 126 second sidewall [0052] 127 upstream end of the
flow focusing channel [0053] 128 downstream end of the flow
focusing channel [0054] 129 cross-sectional area [0055] 130 sheath
fluid micro-channel [0056] 140 output micro-channel [0057] 150
output notches
[0058] In one aspect, the present disclosure relates to a
microfluidic chip design and methods that can isolate particles or
cellular materials, such as sperm and other particles or cells,
into various components and fractions. For example, the various
embodiments of the present invention provide for isolating
components in a mixture, such as isolating viable and motile sperm
from non-viable or non-motile sperm; isolating sperm by gender, and
other sex sorting variations; isolating stems cells from cells in a
population; isolating one or more labeled cells from un-labeled
cells distinguishing desirable/undesirable traits; isolating genes
in nuclear DNA according to a specified characteristic; isolating
cells based on surface markers; isolating cells based on membrane
integrity (viability), potential or predicted reproductive status
(fertility), ability to survive freezing, etc.; isolating cells
from contaminants or debris; isolating healthy cells from damaged
cells (i.e., cancerous cells) (as in bone marrow extractions); red
blood cells from white blood cells and platelets in a plasma
mixture; and isolating any cells from any other cellular
components, into corresponding fractions.
[0059] In other aspects, the various embodiments of the present
invention provide systems and methods particularly suited for
sorting sperm cells to produce a sexed semen product in which the
live, progressively motile sperm cells are predominantly
Y-chromosome bearing sperm cells. In some embodiments, the systems
and methods of the present invention can produce a sex-sorted or
gender skewed semen product comprising at least 55% of Y-chromosome
bearing sperm cells. In other embodiments, the systems and methods
can produce a sexed semen product comprising about 55% to about 90%
of Y-chromosome bearing sperm cells. In yet other embodiments, the
systems and methods can produce a sexed semen product comprising at
least 90%, or at least 95%, or at least 99% of Y-chromosome bearing
sperm cells.
[0060] While the description below focuses on the separation of
sperm into viable and motile sperm from non-viable or non-motile
sperm, or isolating sperm by gender and other sex sorting
variations, or isolating one or more labeled cells from unlabeled
cells distinguishing desirable/undesirable traits, etc., the
present invention may be extended to other types of particulate,
biological or cellular matter, which are capable of being
interrogated by fluorescence techniques within a fluid flow, or
which are capable of being manipulated between different fluid
flows into different outputs.
[0061] The various embodiments of the microfluidics chip utilize
one or more flow channels having substantially laminar flow, and a
flow focusing region for focusing and/or orienting one or more
components in the fluid, allowing the one or more components to be
interrogated for identification and to be isolated into flows that
exit into one or more outputs. In addition, the various components
in the mixture may be subjected to one or more sorting processes
on-chip using various sorting techniques, such as, for example,
particle deflection/electrostatic manipulation; droplet
sorting/deflection; mechanical sorting; fluid switching;
piezoelectric actuation; optical manipulation (optical trapping,
holographic steering, and photonic/radiation pressure); laser
kill/ablation; surface acoustic wave (SAW) deflection;
electrophoresis/electrical disruption; micro-cavitation (laser
induced, electrically induced); or by magnetics (i.e., using
magnetic beads). The various embodiments of the present invention
thereby provide focusing and separation of components on a
continuous basis without the potential damage and contamination of
prior art methods, particularly as provided in sperm separation.
The continuous process of the invention also provides significant
time savings in isolating the fluid components.
[0062] Microfluidic Chip Assembly
[0063] FIGS. 1-3 show an illustrative embodiment of a microfluidic
chip (100) of the present invention. The microfluidic chip may be
manufactured of glass (e.g. soda lime, quartz, borosilicate, etc.)
or a suitable thermoplastic (e.g., low auto-fluorescing polymer
etc.). Glass can be manufactured through etching and bonding
processes, known to one of ordinary skill in the art.
Thermoplastics can be manufactured through an embossing process or
injection molding process, as well known to one of ordinary skill
in the art, and is of suitable size. In certain embodiments,
microfluidic chips can be manufactured from a combination of glass
and thermoplastic.
[0064] Referring now to FIGS. 1-11, the present invention features
microfluidic chip (100) comprising at least one flow focusing
channel (120) in which from an upstream end (127) to a downstream
end (128) thereof, at least a portion of the flow focusing channel
(120) has a reduction in height and at least a portion of the flow
focusing channel (120) narrows in width, thereby geometrically
constricting the flow focusing channel (120). The geometrical
constriction of the flow focusing channel (120) is configured to
focus a material in a fluid that is flowing through the flow
focusing channel (120).
[0065] In some embodiments, a ramp, a step, or constriction is
disposed on a bottom surface (121) of the flow focusing channel
(120), a top surface (122) of the flow focusing channel (120), or
both, which reduces the height reduction of the portion of the flow
focusing channel (120). The ramp, step, or constriction narrows the
channel in the vertical or longitudinal direction. In other
embodiments, the flow focusing channel (120) has a first sidewall
(125) and a second sidewall (126) opposite the first sidewall
(125). The sidewalls (125, 126) taper the flow focusing channel
(120) to form the portion of the flow focusing channel (120) that
narrows in width. The sidewalls (125, 126) are angled so that the
width of the channel narrows in a horizontal or lateral direction.
In some other embodiments, the narrowing effect is created by at
least a portion of the flow focusing channel (120) having a shape
of a cone with a larger diameter of the cone biased towards the
upstream end (127) and a smaller diameter of the cone biased
towards the downstream end (128).
[0066] As shown in FIGS. 4A-4F and 11, in one embodiment, the
portion of the flow focusing channel (120) with the height
reduction and the portion of the flow focusing channel (120)
narrowing in width are sequential. In another embodiment, the
portion of the flow focusing channel (120) with the height
reduction and the portion of the flow focusing channel (120)
narrowing in width are simultaneous, as shown in FIGS. 5-7B and
9A-9B, or overlapping, as shown in FIGS. 8A-8B and 10A-10B.
[0067] According to other embodiments, the present invention
features a microfluidic chip (100) comprising a first micro-channel
(110) and at least one flow focusing region (120) fluidly coupled
to the first micro-channel (110). The first micro-channel (110) may
include an inlet (112) through which a sample fluid enters the
first micro-channel (110). The flow focusing region (120) is formed
by a bottom surface (121), a top surface (122), a first sidewall
(125) and a second sidewall (126) opposite the first sidewall
(125). From an upstream end (127) of the flow focusing region to a
downstream end (128) of the flow focusing region, at least a
portion of the bottom surface (121) is raised, at least a portion
of the top surface (122) is lowered, and at least a portion of the
first and second sidewalls (125, 126) taper the flow focusing
region (120), thereby reducing a cross-sectional area (129) of the
flow focusing region at the downstream end (128) relative to the
upstream end (127). The cross-sectional views in FIGS. 4A-9B
illustrate this reduction in the cross-sectional area (129).
[0068] In some embodiments, the microfluidic chip may further
comprise one or more sheath fluid micro-channels (130) intersecting
the first micro-channel (110), which forms an intersection region
(115). The one or more sheath fluid micro-channels (130) are
configured to flow a sheath fluid into the intersection region
(115) and into the first micro-channel (110) to cause laminar flow.
It is to be understood that the one or more sheath fluid
micro-channels (130) are upstream of the flow focusing region
(120). In one embodiment, the device may have one sheath fluid
micro-channel (130) intersecting the first micro-channel (110) at
the intersection region (115) to cause laminar flow. In another
embodiment, the device may have at least two sheath fluid
micro-channels (130) intersecting the first micro-channel (110) at
the intersection region (115) to cause laminar flow. In other
embodiments, the microfluidic chip may further comprise one or more
output micro-channels (140) fluidly coupled to the first
micro-channel (110) downstream of the flow focusing region (120).
The one or more output micro-channels (140) are configured to
output fluids, which may have components such as particles or
cellular material, from the first micro-channel (110).
[0069] In one embodiment, the bottom surface (121) includes a ramp
(123) having a positive slope or a step that raises the portion of
the bottom surface. For example, the entire bottom surface (121)
may be a ramp (123) that gradually increases a height of the bottom
surface from the upstream end (127) to the downstream end (128). In
one embodiment, the top surface (122) includes a ramp (124) having
a negative slope or a step that lowers the portion of the top
surface. The ramp (124) may form the entire top surface (122) such
that a height of the top surface gradually decreases from the
upstream end (127) to the downstream end (128). In yet another
embodiment, the entire first and second sidewalls (125, 126) are
angled so as to taper the flow focusing region (120) from the
upstream end (127) to the downstream end (128).
[0070] In some embodiments, at least two of the raised portion of
the bottom surface (121), the lowered of the portion of the top
surface (122), and the sidewall tapering occur simultaneously as
shown in FIGS. 5, 7A-7B, and 9A-9B. In other embodiments, at least
two of the raised portion of the bottom surface (121), the lowered
portion of the top surface (122), and the sidewall tapering are
overlapping as shown in FIGS. 8A-8B and 10A-10B. In yet other
embodiments, the raised portion of the bottom surface (121), the
lowered portion of the top surface (122), and the sidewall tapering
occur in a pre-determined sequence as illustrated in FIGS.
4A-4F.
[0071] In preferred embodiments, the microfluidic chip of the
present invention utilizes physical features, such as ramps, steps,
constrictions, and the like, to narrow the main micro-channel (110)
from a minimum of three directions, or more preferably, from all
four directions. In some embodiments, the narrowing can occur
simultaneous from all four directions, or the narrowing can be
sequential or overlapping so that narrowing at any instantaneous
point along the channel can be from a single direction or from more
than one direction. In other embodiments, the narrowing at an
instantaneous point upstream or downstream can be from a different
direction or include a different direction in the more than one
direction. Without wishing to limit the invention to a particular
theory or mechanism, the focusing effect that results from a
particular physical feature (e.g., a vertical ramp) is not
restricted only to the immediate location in which the physical
feature is present; preferably, the effect may extend up- or
downstream. Accordingly, a focusing region may comprise a series of
physical features that appear to be separate features, but together
create a single focusing region through the overlap of the focusing
effects of each feature.
[0072] In some embodiments, the amount of change in the physical
features (e.g. the height of a vertical ramp or step, or the amount
of tapering) can differ amongst the individual physical features in
a given focusing region. For example, a bottom ramp can raise the
bottom surface of the channel by 30 .mu.m, while the ramp in the
top surface of the channel lowers the top of the channel by 15
.mu.m. Thus, the total reduction in the channel height is 45 .mu.m,
but the individual features account for different proportions of
the total change.
[0073] As shown in FIG. 3D, the microfluidic chip may include two
or more structural layers in which are disposed micro-channels that
can serve as a sample input channel, a sheath or buffer fluid
channel, or an output channel, etc. However, one of ordinary skill
in the art would know that less or additional layers may be used,
and the channels may be disposed in any of the layers as long as
the object of the present invention is achieved. The micro-channels
are of suitable size to accommodate a particulate laminar flow, and
may be disposed in any of the layers of the chip in the appropriate
length, as long as the object of the present invention is realized.
The desired flow rate through the microfluidic chip may be
controlled by a predetermined introduction flow rate into the chip,
maintaining the appropriate micro-channel dimensions within the
chip, by pumping mechanisms, providing narrowing of the
micro-channels at various locations, and/or by providing obstacles
or dividers within the micro-channels.
[0074] A plurality of inputs is provided into the microfluidic
chip, which provides access to the micro-channels. In one
embodiment, a sample input is used for introducing components in a
sample fluid mixture into a sample input channel from a reservoir
source. The microfluidic chip also includes a sheath/buffer input
for the introduction of a sheath or buffer fluid. Sheath or buffer
fluids are well known in the art of microfluidics, and in one
embodiment, may contain nutrients well known in the art to maintain
the viability of the components (i.e., sperm cells) in the fluid
mixture.
[0075] In one embodiment, one or more of output channels (140)
fluidly coupled to the main micro-channel (110) are provided for
removal of fluid which has flowed through the microfluidic chip,
including isolated fluid components and/or sheath or buffer fluids.
As shown in FIG. 3, one embodiment of the device may comprise three
output channels (140), which include two side output channels and a
center output channel disposed between said side channels. Each
channel (140) may have its own output; however, the number of
outputs may be less or more depending on the number of components
to be isolated from the fluid mixture. In some embodiments, instead
of a straight edge, where necessary, one or more notches or
recesses (150) are disposed at an edge of the microfluidic chip to
separate the outputs and for attachment of external tubing etc. For
example, the chip may have notches (150) that create a "W" shaped
edge at the output.
[0076] In one embodiment, the sample fluid mixture including the
components is introduced into the sample input (112), and the fluid
mixture flows through the main channel (110). The sheath or buffer
fluids are introduced into the sheath/buffer channels (130), and
flow into the main channel (110) to join with the fluid mixture at
the intersection region (115). The sheath or buffer fluids and the
fluid mixture undergo laminar flow towards the flow focusing region
(120) before flowing out through the one or more output channels
(140). In some embodiments, the micro-channels of the microfluidic
chip may be dimensioned so as to achieve a desired flow rate(s)
that meets the objective of the present invention. In one
embodiment, the micro-channels may have substantially the same
dimensions, however, one of ordinary skill in the art would know
that the size of any or all of the channels in the microfluidic
chip may vary in dimension (i.e., between 50 and 500 microns), as
long as the desired flow rate(s) is achieved.
[0077] In another embodiment, as shown in FIG. 2B, the main channel
(110) may taper at the entry point into the intersection region
(115) to control and speed up the flow into the intersection, and
allow the sheath or buffer fluids from the sheath micro-channels
(130) to compress the sample fluid mixture in a first direction
(i.e., horizontally) on at least two sides. For example, the sheath
or buffer fluids from the sheath micro-channels (130) intersect the
main micro-channel (110) at a substantially perpendicular angle or
less, such as an angle of 45.degree., thereby compressing the fluid
mixture flow such that the components in the fluid mixture are
compressed or flattened, and oriented in the selected or desired
direction. Thus, the sample fluid mixture becomes a relatively
smaller, narrower stream, bounded or surrounded by sheath or buffer
fluids, while maintaining laminar flow in the main micro-channel
(110). However, one of ordinary skill in the art would appreciate
that the depicted configurations, angles, and structural
arrangements of the microfluidic chip layers and channels may be
different as long as they achieve the desired features of the
present invention.
[0078] In some embodiments, downstream from the flow focusing
region (120), the components in the fluid mixture flow through the
main micro-channel (110) into an interrogation apparatus where the
components are interrogated and identified. In one embodiment, the
interrogation apparatus includes a chamber with an opening or
window cut into the microfluidic chip. The opening or window can
receive a covering to enclose the interrogation chamber. The
covering may be made of any material with the desired transmission
requirements, such as plastic, glass, or may even be a lens. In one
embodiment, the window and covering allow the components of the
fluid mixture flowing in the main micro-channel (110) through the
interrogation chamber to be viewed, and acted upon by a suitable
radiation source configured to emit a high intensity beam with any
wavelength that matches the excitation of the components.
[0079] Although a laser may be used, it is understood that any
suitable other radiation sources may be used, such as a light
emitting diode (LED), arc lamp, etc. to emit a beam which excites
the components. In another embodiment, the light beam can be
delivered to the components by an optical fiber that is embedded in
the microfluidic chip at the opening.
[0080] In some embodiments, a high intensity laser beam from a
suitable laser of a preselected wavelength--such as a 355 nm
continuous wave (CW) (or quasi-CW) laser--is required to excite the
components in the fluid mixture (i.e., sperm cells). The laser
emits a laser beam through the window so as to illuminate the
components flowing through the main micro-channel (110) in the
interrogation region of the chip. Since the laser beam can vary in
intensity widthwise along the main micro-channel, with the highest
intensity generally at the center of the main micro-channel (e.g.,
midsection of the main channel width) and decreasing therefrom, it
is imperative that the flow focusing region focuses the sperm cells
at or near the center of the fluid stream where optimal
illumination occurs at or near the center of the illumination laser
spot. Without wishing to be bound to a particular belief, this can
improve accuracy of the interrogation and identification
process
[0081] In some embodiments, the high intensity beam interacts with
the components such that the emitted light, which is induced by the
beam, is received by an objective lens. The objective lens may be
disposed in any suitable position with respect to the microfluidic
chip. In one embodiment, the emitted light received by the
objective lens is converted into an electronic signal by an optical
sensor, such as a photomultiplier tube (PMT) or photodiode, etc.
The electronic signal can be digitized by an analog-to-digital
converter (ADC) and sent to a digital signal processor (DSP) based
controller. The DSP based controller monitors the electronic signal
and may then trigger a sorting mechanism.
[0082] In other embodiments, the interrogation apparatus may
comprise a detector such as a photomultiplier tube (PMT), an
avalanche photodiode (APD), or a silicon photomultiplier (SiPM).
For example, the optical sensor of the interrogation apparatus may
be APD, which is a photodiode with substantial internal signal
amplification through an avalanche process.
[0083] In some embodiments, a piezoelectric actuator assembly may
be used to sort the desired components in the fluid mixture in main
micro-channel (110) as the components leave the interrogation area
after interrogation. A trigger signal sent to the piezoelectric
actuator is determined by the sensor raw signal to activate a
particular piezoelectric actuator assembly when the selected
component is detected. In some embodiments, a flexible diaphragm
made from a suitable material, such as one of stainless steel,
brass, titanium, nickel alloy, polymer, or other suitable material
with desired elastic response, is used in conjunction with an
actuator to push target components in the main micro-channel into
an output channel (140) to isolate the target components from the
fluid mixture. The actuator may be a piezoelectric, magnetic,
electrostatic, hydraulic, or pneumatic type actuator.
[0084] In alternative embodiments, a piezoelectric actuator
assembly or a suitable pumping system may be used to pump the
sample fluid into the main micro-channel (110) toward the
intersection region (115). The sample piezoelectric actuator
assembly would be disposed at sample input (112). By pumping the
sample fluid mixture into the main micro-channel, a measure of
control can be made over the spacing of the components therein,
such that a more controlled relationship may be made between the
components as they enter the main micro-channel (110).
[0085] Other embodiments of sorting or separating mechanisms that
may be used in accordance with the present invention include, but
are not limited to, droplet sorters, mechanical separation, fluid
switching, acoustic focusing, holographic trapping/steering, and
photonic pressure/steering. In a preferred embodiment, the sorting
mechanism for sex-sorting of sperm cells comprises laser
kill/ablation of selected X-chromosome-bearing sperm cells.
[0086] In laser ablation, the laser is activated when an
X-chromosome-bearing sperm cell is detected during interrogation.
The laser emits a high intensity beam directed at the
X-chromosome-bearing sperm cell centered within the fluid stream.
The high intensity beam is configured to cause DNA and/or membrane
damage to the cell, thereby causing infertility or killing the
X-chromosome-bearing sperm cell. As a result, the final product is
comprised predominantly of Y-chromosome-bearing sperm cells. In
preferred embodiments, the reduction in the cross-sectional area
(129) of the flow focusing region geometrically compresses the
fluid that carries sperm cells. The geometric compression of the
fluid centralizes the sperm cells within the fluid such that the
sperm cells are focused at or near a center of the first
micro-channel (110). Since the laser beam varies in intensity
widthwise along the main micro-channel, with the highest intensity
generally at the center of main micro-channel and decreasing
therefrom, it is imperative that the flow focusing region focuses
the sperm cells at or near the center of the fluid stream where the
laser beam has the highest intensity to impart maximum damage to
the selected sperm cells.
[0087] Chip Operation
[0088] In one embodiment, as previously stated, the components that
are to be isolated include, for example: isolating viable and
motile sperm from non-viable or non-motile sperm; isolating sperm
by gender, and other sex sorting variations; isolating stems cells
from cells in a population; isolating one or more labeled cells
from un-labeled cells distinguishing desirable/undesirable traits;
sperm cells with different desirable characteristics; isolating
genes in nuclear DNA according to a specified characteristic;
isolating cells based on surface markers; isolating cells based on
membrane integrity (viability), potential or predicted reproductive
status (fertility), ability to survive freezing, etc.; isolating
cells from contaminants or debris; isolating healthy cells from
damaged cells (i.e., cancerous cells) (as in bone marrow
extractions); red blood cells from white blood cells and platelets
in a plasma mixture; and isolating any cells from any other
cellular components, into corresponding fractions; damaged cells,
or contaminants or debris, or any other biological materials that
are desired to isolated. The components 160 may be cells or beads
treated or coated with, linker molecules, or embedded with a
fluorescent or luminescent label molecule(s). The components may
have a variety of physical or chemical attributes, such as size,
shape, materials, texture, etc.
[0089] In one embodiment, a heterogeneous population of components
may be measured simultaneously, with each component being examined
for different quantities or regimes in similar quantities (e.g.,
multiplexed measurements), or the components may be examined and
distinguished based on a label (e.g., fluorescent), image (due to
size, shape, different absorption, scattering, fluorescence,
luminescence characteristics, fluorescence or luminescence emission
profiles, fluorescent or luminescent decay lifetime), and/or
particle position etc.
[0090] In one embodiment, a two-step focusing method may be used in
order to position the components in the main micro-channel (110)
for interrogation in the interrogation chamber. The first focusing
step of the present invention is accomplished by inputting a fluid
sample containing components, such as sperm cells etc., through
sample input (112), and inputting sheath or buffer fluids through
the sheath or buffer micro-channels (130). In some embodiments, the
components are pre-stained with dye (e.g., Hoechst dye), in order
to allow fluorescence, and for imaging to be detected. Initially,
the components in the sample fluid mixture flow through
micro-channel (110) and have random orientation and position. At
the intersection region (115), the sample mixture flowing in the
micro-channel (110) is compressed by the sheath or buffer fluids
flowing from the sheath or buffer micro-channels (130), in a first
direction (e.g., at least horizontally on at least both sides of
the flow, if not all sides depending on where the main
micro-channel (110) enters the intersection region (115)), when the
sheath or buffer fluids meet with the sample mixture. As a result,
the components are focused and compressed into a thin stream and
the components (e.g., sperm cells) move toward a center of the
channel width.
[0091] In preferred embodiments, the present invention includes a
second focusing step where the sample mixture containing the
components is further compressed in the flow focusing region (120)
using physical or geometric compression, instead of a second
intersection of sheath fluids. Thus, with the second focusing step
of the present invention, the sample stream is focused at the
center of the channel, and the components flow along the center of
the channel in approximately a single file formation. Without
wishing to be bound to a particular theory or mechanism, the
physical/geometric compression has the advantage of reducing the
volume of sheath fluid since the second intersection of sheath
fluids is eliminated.
[0092] Accordingly, the microfluidic devices described herein may
be used in the two-step focusing method described above. In one
embodiment, the present invention provides a method of focusing
particles in a fluid flow. The method may comprise providing any
one of the microfluidic devices described herein, flowing a fluid
mixture comprising the particles into the sample micro-channel
(110), flowing a sheath fluid through one or more sheath fluid
channels (130) into a first focusing region such that the sheath
fluid compresses the fluid mixture from at least one side while
maintaining laminar flow in the sample micro-channel (110), and
flowing the fluid mixture and sheath fluid through a second
focusing region (120) comprising physical structures. The second
focusing region (120) does not include the introduction of
additional sheath fluid. Compression of the fluid mixture, by the
introduction of sheath fluid and/or the physical structures at the
first focusing region constricts the particles of the fluid mixture
into a relatively smaller, narrower stream bounded by the sheath
fluids. For example, sheath fluid introduced into the sample
micro-channel (110) by two sheath fluid channels (130) can compress
the fluid mixture stream from two sides into a relatively smaller,
narrower stream while maintaining laminar flow. Flow of the fluid
mixture and sheath fluids in the second focusing region causes
further constriction of the fluid mixture stream and re-orienting
of the particles within the stream, which is caused by the physical
structures such as the raised portion of the bottom surface (121),
the lowered portion of the top surface (122), and the tapering
portions of the first and second sidewall (125, 126), thus focusing
the particles.
[0093] In some embodiments, the components of the sample are sperm
cells, and because of their pancake-type or flattened teardrop
shaped head, the sperm cells can re-orient themselves in a
predetermined direction as they undergo the focusing step--i.e.,
with their flat surfaces perpendicular to the direction of a light
beam. Thus, the sperm cells develop a preference on their body
orientation while passing through the two-step focusing process.
Specifically, the sperm cells tend to be more stable with their
flat bodies perpendicular to the direction of the compression.
Hence, with the control of the sheath or buffer fluids, the sperm
cells which start with random orientation, now achieve uniform
orientation. Thus, the sperm cells not only make a single file
formation at the center of the channel, but they also achieve a
uniform orientation. Thus, the components introduced into sample
input, which may be other types of cells or other materials as
previously described, undergo the two-step focusing steps, which
allow the components to move through the main micro-channel (110)
in a single file formation, and in a more uniform orientation
(depending on the type of components), which allows for easier
interrogation of the components.
[0094] In conjunction with the preceding embodiment, the present
invention also provides a method of producing a fluid with
gender-skewed sperm cells implementing the two-step focusing
procedure. Referring to FIG. 12, the method may comprise providing
any one of the microfluidic devices described herein, flowing a
semen fluid comprising sperm cells into the sample micro-channel
(110), flowing a sheath fluid through the one or more sheath fluid
channels (130) such that the sheath fluid compresses the semen
fluid from at least one side while maintaining laminar flow in the
sample micro-channel (110), thereby constricting the semen fluid
into a relatively smaller, narrower stream, flowing the semen fluid
and sheath fluids into the focusing region (120) to further
constrict the semen fluid and centralize the sperm cells within the
stream, without that addition of additional sheath fluid, such that
the sperm cells are focused at or near a center of the sample
micro-channel (110), determining a chromosome type of the sperm
cells in the focused stream, and sorting Y-chromosome-bearing sperm
cells from X-chromosome-bearing sperm cells, thereby producing the
fluid comprising gender-skewed sperm cells that are predominantly
Y-chromosome-bearing sperm cells.
[0095] In some embodiments, the chromosome type of the sperm cells
may be determined using any one of the interrogation apparatus
described herein. In one embodiment, the interrogation apparatus is
disposed downstream from the focusing region (120). The
interrogation apparatus may comprise a radiation source that
illuminates and excites the sperm cells, and a response of the
sperm cell is indicative of the chromosome type in the sperm cell.
In preferred embodiment, the Y-chromosome-bearing sperm cells are
sorted from the X-chromosome-bearing sperm cells by laser ablation,
which exposes the cells to a high intensity laser source that
damages or kills cells that are determined to bear an X-chromosome.
In one embodiment, the gender-skewed sperm cells are comprised of
at least 55% of Y-chromosome-bearing sperm cells. In another
embodiment, the gender-skewed sperm cells are comprised of about
55%-99% of Y-chromosome-bearing sperm cells. In yet another
embodiment, the gender-skewed sperm cells are comprised of at least
99% of Y-chromosome-bearing sperm cells.
[0096] In one embodiment, further downstream in the micro-channel
(110), the components are detected in the interrogation chamber
using a radiation source. The radiation source emits a light beam
(which may be via an optical fiber) which is focused at the center
of the channel widthwise. In one embodiment, the components, such
as sperm cells, are oriented by the focusing region such that the
flat surfaces of the components are facing toward the beam. In
addition, all components are preferably aligned in a single file
formation by focusing as they pass under a radiation source. As the
components pass under the radiation source and are acted upon by a
light beam, the components emit the fluorescence which indicates
the desired components. For example, with respect to sperm cells, X
chromosome cells fluoresce at a different intensity from Y
chromosome cells; or cells carrying one trait may fluoresce in a
different intensity or wavelength from cells carrying a different
set of traits. In addition, the components can be viewed for shape,
size, or any other distinguishing indicators.
[0097] In one embodiment, interrogation of the sample containing
components (i.e., biological material), is accomplished by other
methods. Overall, methods for interrogation may include direct
visual imaging, such as with a camera, and may utilize direct
bright-light imaging or fluorescent imaging; or, more sophisticated
techniques may be used such as spectroscopy, transmission
spectroscopy, spectral imaging, or scattering such as dynamic light
scattering or diffusive wave spectroscopy. In some cases, the
optical interrogation region may be used in conjunction with
additives, such as chemicals which bind to or affect components of
the sample mixture or beads which are functionalized to bind and/or
fluoresce in the presence of certain materials or diseases. These
techniques may be used to measure cell concentrations, to detect
disease, or to detect other parameters which characterize the
components.
[0098] However, in another embodiment, if fluorescence is not used,
then polarized light back scattering methods may also be used.
Using spectroscopic methods, the components are interrogated and
the spectrum of those components which had positive results and
fluoresced (i.e., those components which reacted with a label) are
identified for separation. In some embodiments, the components may
be identified based on the reaction or binding of the components
with additives or sheath or buffer fluids, or by using the natural
fluorescence of the components, or the fluorescence of a substance
associated with the component, as an identity tag or background
tag, or met a selected size, dimension, or surface feature, etc.,
are selected for separation. In one embodiment, upon completion of
an assay, selection may be made, via computer and/or operator, of
which components to discard and which to collect.
[0099] Continuing with the embodiment of beam-induced fluorescence,
the emitted light beam is then collected by the objective lens, and
subsequently converted to an electronic signal by the optical
sensor. The electronic signal is then digitized by an
analog-digital converter (ADC) and sent to an electronic controller
for signal processing. The electronic controller can be any
electronic processor with adequate processing power, such as a DSP,
a Micro Controller Unit (MCU), a Field Programmable Gate Array
(FPGA), or even a Central Processing Unit (CPU). In one embodiment,
the DSP-based controller monitors the electronic signal and may
then trigger a sorting mechanism when a desired component is
detected. In another embodiment, the FPGA-based controller monitors
the electronic signal and then either communicates with the DSP
controller or acts independently to trigger a sorting mechanism
when a desired component is detected. In some other embodiments,
the optical sensor may be a photomultiplier tube (PMT), an
avalanche photodiode (APD), or a silicon photomultiplier (SiPM). In
a preferred embodiment, the optical sensor may be an APD that
detects the response of the sperm cell to interrogation.
[0100] In one embodiment of the sorting mechanism, the selected or
desired components in the main micro-channel (110) in the
interrogation chamber are isolated into a desired output channel
using a piezoelectric actuator. In an exemplary embodiment, the
electronic signal activates the driver to trigger the actuator at
the moment when the target or selected component arrives at a
cross-section point of jet channels and the main micro-channel
(110). This causes the actuator to contact a diaphragm and push it,
compressing a jet chamber, and squeezing a strong jet of buffer or
sheath fluids into the main micro-channel (110), which pushes the
selected or desired component into a desired output channel.
[0101] In some embodiments, the isolated components are collected
from their respective output channel (140) for storing, further
separation, or processing, such as cryopreservation. In some
embodiments, the outputted components may be characterized
electronically, to detect concentrations of components, pH
measuring, cell counts, electrolyte concentration, etc.
[0102] Chip Cassette and Holder
[0103] In some embodiments, the microfluidic chip may be loaded on
a chip cassette, which is mounted on chip holder. The chip holder
is mounted to a translation stage to allow fine positioning of the
holder. For instance, the microfluidic chip holder is configured to
hold the microfluidic chip in a pre-determined position such that
the interrogating light beam intercepts the fluid components. In
one embodiment, the microfluidic chip holder is made of a suitable
material, such as aluminum alloy, or other suitable
metallic/polymer material. A main body of the holder may be any
suitable shape, but its configuration depends on the layout of the
chip. In further embodiments, the main body of the holder is
configured to receive and engage with external tubing for
communicating fluids/samples to the microfluidic chip. A gasket of
any desired shape, or O-rings, may be provided to maintain a tight
seal between the microfluidic chip and the microfluidic chip
holder. The gasket may be a single sheet or a plurality of
components, in any configuration, or material (i.e., rubber,
silicone, etc.) as desired. In one embodiment, the gasket
interfaces, or is bonded (using an epoxy) with a layer of the
microfluidic chip. The gasket is configured to assist in sealing,
as well as stabilizing or balancing the microfluidic chip in the
microfluidic chip holder. The details of the cassette and holder
and the mechanisms for attachment of the chip to the cassette and
holder, are not described in any detail, as one of ordinary skill
in the art would know that these devices are well-known and may be
of any configuration to accommodate the microfluidic chip, as long
as the objectives of the present invention are met.
[0104] In some embodiments, a pumping mechanism includes a system
having a pressurized gas which provides pressure for pumping sample
fluid mixture from reservoir (i.e., sample tube) into sample input
of the chip. In other embodiments, a collapsible container having
sheath or buffer fluid therein, is disposed in a pressurized
vessel, and the pressurized gas pushes fluid such that fluid is
delivered via tubing to the sheath or buffer input of the chip.
[0105] In one embodiment, a pressure regulator regulates the
pressure of gas within the reservoir, and another pressure
regulator regulates the pressure of gas within the vessel. A mass
flow regulator controls the fluid pumped via tubing, respectively,
into the sheath or buffer input. Thus, tubing is used in the
initial loading of the fluids into the chip, and may be used
throughout the chip to load a sample fluid into sample input.
[0106] In accordance with the present invention, any of the
operations, steps, control options, etc. may be implemented by
instructions that are stored on a computer-readable medium such as
a memory, database, etc. Upon execution of the instructions stored
on the computer-readable medium, for example, by a computing device
or processor, the instructions can cause the computing device or
processor to perform any of the operations, steps, control options,
etc. described herein. In some embodiments the operations described
in this specification may be implemented as operations performed by
a data processing apparatus or processing circuit on data stored on
one or more computer-readable storage devices or received from
other sources. A computer program (also known as a program,
software, software application, script, or code) can be written in
any form of programming language, including compiled or interpreted
languages, declarative or procedural languages, and it can be
deployed in any form, including as a stand-alone program or as a
module, component, subroutine, object, or other unit suitable for
use in a computing environment. A program can be stored in a
portion of a file that holds other programs or data, in a single
file dedicated to the program in question, or in multiple
coordinated files. A program can be deployed to be executed on one
computer or on multiple computers interconnected by a communication
network. Processing circuits suitable for the execution of a
computer program include, by way of example, both general and
special purpose microprocessors, and any one or more processors of
any kind of digital computer.
[0107] In one embodiment, a user interface of the computer system
includes a computer screen which displays the components in a field
of view acquired by a CCD camera over the microfluidic chip. In
another embodiment, the computer controls any external devices such
as pumps, if used, to pump any sample fluids, sheath or buffer
fluids into the microfluidic chip, and also controls any heating
devices which set the temperature of the fluids being inputted into
the microfluidic chip.
[0108] It should be noted that the orientation of various elements
may differ according to other illustrative embodiments, and that
such variations are intended to be encompassed by the present
disclosure. The construction and arrangements of the microfluidic
chip, as shown in the various illustrative embodiments, are
illustrative only. Although only a few embodiments have been
described in detail in this disclosure, many modifications are
possible (e.g., variations in sizes, dimensions, structures, shapes
and proportions of the various elements, values of parameters,
mounting arrangements, use of materials, colors, orientations,
etc.) without materially departing from the novel teachings and
advantages of the subject matter described herein. Some elements
shown as integrally formed may be constructed of multiple parts or
elements, the position of elements may be reversed or otherwise
varied, and the nature or number of discrete elements or positions
may be altered or varied. The order or sequence of any process,
logical algorithm, or method steps may be varied or re-sequenced
according to alternative embodiments. Other substitutions,
modifications, changes and omissions may also be made in the
design, operating conditions and arrangement of the various
illustrative embodiments without departing from the scope of the
present disclosure.
[0109] As used herein, the term "about" refers to plus or minus 10%
of the referenced number.
[0110] Although there has been shown and described the preferred
embodiment of the present invention, it will be readily apparent to
those skilled in the art that modifications may be made thereto
which do not exceed the scope of the appended claims. Therefore,
the scope of the invention is only to be limited by the following
claims. In some embodiments, the figures presented in this patent
application are drawn to scale, including the angles, ratios of
dimensions, etc. In some embodiments, the figures are
representative only and the claims are not limited by the
dimensions of the figures. In some embodiments, descriptions of the
inventions described herein using the phrase "comprising" includes
embodiments that could be described as "consisting essentially of"
or "consisting of", and as such the written description requirement
for claiming one or more embodiments of the present invention using
the phrase "consisting essentially of" or "consisting of" is
met.
[0111] The reference numbers recited in the below claims are solely
for ease of examination of this patent application, and are
exemplary, and are not intended in any way to limit the scope of
the claims to the particular features having the corresponding
reference numbers in the drawings.
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