U.S. patent application number 10/375373 was filed with the patent office on 2003-09-04 for process for sorting motile particles from lesser-motile particles and apparatus suitable therefor.
Invention is credited to Cho, Brenda S., Schuster, Timothy G., Smith, Gary D., Takayama, Shuichi.
Application Number | 20030165812 10/375373 |
Document ID | / |
Family ID | 27766144 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165812 |
Kind Code |
A1 |
Takayama, Shuichi ; et
al. |
September 4, 2003 |
Process for sorting motile particles from lesser-motile particles
and apparatus suitable therefor
Abstract
Motile particles are sorted from non-motile particles in a
microfluidic sorting device wherein a stream of sort fluid
containing motile and non-motile particles is caused to flow
adjacent a media stream in non-turbulent fashion through a sort
channel, during which flow motile particles cross the interface
between the adjacent flow streams, entering the media stream, and
forming a motile particle-depleted sort stream. The sorting devices
are easily and inexpensively fabricated and have numerous uses, in
particular sorting of motile from non-motile sperm.
Inventors: |
Takayama, Shuichi; (Ann
Arbor, MI) ; Smith, Gary D.; (Ann Arbor, MI) ;
Schuster, Timothy G.; (Ann Arbor, MI) ; Cho, Brenda
S.; (Ann Arbor, MI) |
Correspondence
Address: |
BROOKS & KUSHMAN
1000 TOWN CENTER 22ND FL
SOUTHFIELD
MI
48075
|
Family ID: |
27766144 |
Appl. No.: |
10/375373 |
Filed: |
February 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60359844 |
Feb 27, 2002 |
|
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Current U.S.
Class: |
435/4 |
Current CPC
Class: |
B01L 3/502761 20130101;
B01L 2400/0457 20130101; B01L 2200/0636 20130101; B01L 3/502753
20130101; B01L 2300/0864 20130101; G01N 15/1404 20130101; B01L
2200/0647 20130101; B01L 2300/0867 20130101; B01L 2400/0406
20130101; B01L 2200/0652 20130101; C12N 5/0612 20130101; B01L
3/502776 20130101 |
Class at
Publication: |
435/4 |
International
Class: |
C12Q 001/00 |
Claims
What is claimed is:
1. A method for sorting motile particles from lesser motile and/or
non-motile particles, comprising: a) providing a sort fluid
comprising motile particles and at least one of lesser motile
particles or non-motile particles; b) providing a media fluid to be
enriched with motile particles; c) contacting a stream of said sort
fluid and a stream of said media fluid within a sort channel of a
sorting device, said sort fluid stream and said media fluid stream
having therebetween a non-turbulent interface within said sort
channel, whereby motile particles leave the sort fluid stream and
enter the media fluid stream, forming a motile particle-depleted
sort fluid stream and a motile particle-enriched media fluid
stream, and d) separating a motile particle-depleted sort fluid
outlet stream from a motile particle-enriched media fluid outlet
stream.
2. The process of claim 1, wherein said motile particles comprise
motile sperm and said lesser motile and non-motile particles
comprise sperm of lesser motility than said motility sperm or sperm
of no motility.
3. The process of claim 1, wherein said sort fluid is provided in a
sort fluid reservoir and said media fluid is provided in a media
fluid reservoir.
4. The process of claim 1, wherein said media fluid and said sort
fluid flow in the same direction and at substantially the same flow
rate
5. The process of claim 1, wherein said sort fluid stream and said
media fluid stream exhibit laminar flow within said sort
channel.
6. The process of claim 1, wherein the transverse volume of said
media fluid stream is greater than the transverse volume of said
sort fluid stream in said sort channel.
7. The process of claim 1, wherein said motile particle-depleted
sort fluid outlet stream comprises a sort fluid inlet stream to a
second device having a sort channel, and steps a) through d) are
repeated, whereby further motile particles enter a media stream of
said second device to further deplete said sort fluid of motile
particles.
8. The process of claim 1, wherein said motile, lesser motile, and
non-motile particles are sperm particles, and the purity of motile
particles in said motile particle-enriched media outlet stream is
higher than the purity of motile sperm in the sort fluid and is at
least 80%, said percent based on the number of motile sperm in said
motile particle-enriched media outlet stream compared to total
sperm in said motile particle-enriched outlet stream.
9. The process of claim 1, wherein said motile particles comprise
motile microorganisms, and said non-motile particles comprise
non-motile microorganisms.
10. A motile particle sorting device suitable for use in the method
of claim 1, comprising: a) a sort channel having first and second
ends; b) a sort fluid inlet in fluid communication with said first
end of said sort channel; c) a media fluid inlet in communication
with said first end of said sort channel; d) a motile
particle-depleted sort fluid outlet in fluid communication with
said second end of said sort channel; and e) a motile
particle-enriched media fluid outlet in fluid communication with
said second end of said sort channel.
11. The sorting device of claim 10, wherein said first end of said
sort channel is formed by a confluence of a sort stream inlet and a
media stream inlet, and said second end of said sort channel
terminates at a divergence of a motile particle-depleted sort
stream outlet and a motile particle-enriched media stream
outlet.
12. The sorting device of claim 10, wherein said sort fluid inlet
comprises a sort fluid inlet channel, said media fluid inlet
comprises a media fluid inlet channel, said motile
particle-depleted sort fluid outlet comprises a motile
particle-depleted sort fluid outlet channel, and said motile
particle-enriched outlet comprises a motile particle-enriched
outlet channel.
13. The sorting device of claim 12, wherein each of said inlet
channels and said outlet channels are respectively in communication
with a fluid reservoir internal to said device.
14. A motile particle sorting device, comprising a device of claim
10, wherein said motile particle-depleted sort stream outlet serves
as a sort stream inlet to a second sorting device of claim 10, or
wherein said motile particle-enriched media stream outlet serves as
a sort stream inlet to a second sorting device of claim 10.
15. A plurality of sorting devices of claim 10 fabricated in a
single integral structure.
16. The plurality of sorting devices of claim 15, wherein at least
two of said sorting devices are series connected.
17. The sorting device of claim 10, which is cast of polymer
18. The sorting device of claim 13, wherein said device comprises a
first pumping system comprising a sort fluid reservoir, a sort
fluid inlet channel, a sort channel, a motile particle-depleted
sort fluid outlet, and a motile particle-depleted sort fluid
reservoir, whereby sort fluid flows from said sort fluid reservoir
to said motile particle-depleted sort fluid reservoir due to at
least one of gravity or capillary forces.
19. The sorting device of claim 13, wherein said device comprises a
second pumping system comprising a media fluid reservoir, a media
fluid inlet channel, a media channel, a motile particle-enriched
media fluid outlet, and a motile particle-enriched media fluid
reservoir, whereby media fluid flows from said media fluid
reservoir to said motile particle-enriched media fluid reservoir
due to at least one of gravity or capillary forces.
20. The sorting device of claim 19, wherein fluid flows both due to
gravity and due to capillary forces.
21. The device of claim 10, wherein said sort channel is
substantially rectangular, has a height of about 60-150 .mu.m and a
width of from about 100 .mu.m to 800 .mu.m, and a length of about
500 .mu.m to about 10,000 .mu.m, wherein said sort inlet comprises
a sort inlet channel of substantially the same height as said sort
channel and a smaller width in the range of 50 .mu.m to 500 .mu.m,
and wherein said media inlet comprises one or more media inlet
channels of substantially the same height as said sort channel, and
a total width greater than the width of said sort inlet channel and
less than the width of said sort channel, wherein total width is
the sum of the widths of each media inlet channel when there is
more than one inlet channel.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
application Serial No. 60/359,844, filed Feb. 27, 2002.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention pertains to a process for sorting
motile from lesser-motile particles, particularly motile and
non-motile particles of biological origin, and to apparatus
suitable therefor.
[0004] 2. Background Art
[0005] The separation, or "sorting" of motile from lesser-motile
and/or non-motile particles has numerous applications, but
heretofore has been technologically difficult. For example, in
analysis of water supplies, it may be desirable to separate motile
bacteria and other microorganisms, including fungi, algae, etc.,
from those which are non-motile. Identification and enumeration of
the various species may thus be facilitated. Examples of motile
organisms include flagellated and ciliated bacteria such as C.
elegans, and other microorganisms, such as paramecia and motile
plankton. Either the motile species enriched or motile
species-depleted samples, or both, may be independently analyzed,
cultured, etc.
[0006] An especially significant application is the sorting of
sperm cells. For example, in the case of in vitro fertilization, if
the donor's sperm count is low, and especially if contaminated with
non-motile sperm, deformed sperm of lesser motility than the
desired viable sperm, and other cells and seminal debris, the
success rate is raised considerably when the motile sperm are used
substantially for fertilization attempts. For example, avoidance of
anueploid sperm or DNA fragmented sperm is particularly desirable.
In many endeavors, it is desirable to be able to direct the gender
of the offspring, for example when the birthing of milk cows is
desired. In such cases, it would be advantageous to be able to sort
the X-and Y-chromosome containing sperm based on their known
motility differences.
[0007] Sperm cells from donors with oligozoospermia (low sperm
count) have previously been concentrated and to some degree
separated from cells and debris having different sizes and/or
densities by centrifugation. However, this technique allows
incorporation of non-gametes into the enriched sperm sample. These
non-gametes, however few there are, release oxygen radicals which
are detrimental to continued sperm viability. Moreover,
centrifugation is a brute force technique which damages significant
numbers of sperm, particularly at the mid-piece and tail
regions.
[0008] So-called "swim up" techniques are also known, but isolation
of the most viable sperm is challenging. S. Smith et al., FERTIL.
STERIL. 1995, 63, 591-97. Thus, doctors frequently resort to hand
sorting through dead sperm and debris to find sperm which are
motile and of distinct morphology, a very time-consuming
process.
[0009] Applications in biogenetics (biotechnology) also frequently
require separation of particles based on their motility. In
non-biological application, separation of working microrobots
(which are motile) from non-working microrobots is a possible
application.
SUMMARY OF THE INVENTION
[0010] Sorting of motile and non-motile or lesser-motile particles
is accomplished by establishing a non-turbulent and preferably
laminar flow stream ("sort stream") containing motile and
non-motile or lesser-motile particles to be sorted, and contacting
this sort stream with a second non-turbulent and preferably
co-laminar media flow stream ("media stream"), providing an exit
stream for at least a portion of a motile particle-enriched media
flow stream, and an exit stream for a motile particle-depleted sort
stream. The mobility of the motile particles allow them to enter
the media stream along the interface between the media and sort
streams, while non-motile or lesser-motile particles remain
substantially within the sort stream. Apparatus suitable for use in
the process provide for at least one sort stream inlet, at least
one media stream inlet, at least one sort channel, at least one
motile particle-depleted sort stream outlet, and at least one
motile particle-enriched media stream outlet. The apparatus are
preferably relatively small devices prepared by micromachining or
polymer casting techniques, and preferably contain all necessary
functionality integrated onto a single "chip."
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 illustrates a simple embodiment of a motile particle
sorting device of the present invention;
[0012] FIG. 2 depicts sorting of motile from non-motile particles
in a sort channel of a device of FIG. 1;
[0013] FIG. 3 illustrates one embodiment of series connected
sorting devices;
[0014] FIG. 4 illustrates a further embodiment of a sorting device
with multiple media inlets;
[0015] FIG. 5 illustrates in schematic form a further embodiment of
a sorting device in accordance with the present invention;
[0016] FIG. 6 illustrates in schematic form a further embodiment of
a sorting device in accordance with the present invention;
[0017] FIG. 7 illustrates a device similar to that of FIG. 1, in
perspective; and
[0018] FIG. 8 illustrates sorting efficiency of a device similar to
that of FIG. 1 in sorting sperm.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The description of the invention may be facilitated by
reference to FIG. 1 which represents one relatively simple
embodiment of an apparatus which may be used for practicing the
subject invention, and by reference to FIG. 2 which illustrates
sorting of motile from non-motile particles. Following these
descriptions, additional details of the functioning of the
apparatus, its geometry, the nature of fluids and fluid flow, etc.,
will be explained in greater specificity.
[0020] The device of FIG. 1 is shown in schematic form in plan,
i.e. as viewed from above. The device 1 embodiment consists of a
motile particle sort stream inlet 2 (or motile particle supply
reservoir serving as an inlet), a media stream inlet 3 (or media
reservoir), a motile particle-depleted sort stream outlet (or
reservoir) 4, and a motile particle-enriched stream outlet (or
reservoir) 5. Between the inlets 2 and 3 and the outlets 4 and 5 is
located a sort channel 6. Connecting the sort channel 6 to the
respective inlets and outlets are sort stream inlet channel 7,
media stream inlet channel 8, motile particle-depleted sort stream
outlet channel 9, and motile particle-enriched media stream outlet
channel 10. The width of the sort stream channel must be large
enough to allow the particles of interest to pass through
effectively without blockage, as is also true of both outlet
streams. In general, the inlet streams and outlet streams will have
a cross-sectional area less than the sort channel. The relative
cross-sections will be dependent on the flow volume and flow rates
of the various streams. The linear flow rates are preferably
similar, although the relative flow rates are only limited by the
occurrence of mixing between the sort stream and the media stream.
Depending upon numerous factors such as the viscosities of the
media and sort streams, the motility of the particles, and the
presence or absence of particles or debris of different size than
the particles desired to be sorted, the volume of the media stream
may be less than, substantially the same as, or greater than the
volume of the sort stream over any section of the sort channel.
[0021] The bulk of the description which follows is described in
relation to sorting of sperm, although the same principles apply to
other motile and non-motile particle sources.
[0022] In operation, a supply of sperm is introduced into sort
stream inlet 2 and caused to flow toward motile particle-depleted
sort stream outlet 9, initially through channel 7, then through
sort channel 6, and next to outlet channel 9. A media supply stream
compatible (i.e. not destructive) of the sperm to be sorted is
introduced into media stream inlet 3 and caused to flow through
channel 8 into sort channel 6, through channel 10, and into motile
particle-enriched media outlet 5. At the confluence of channels 7
and 8, a non-mixing, and preferably laminar flow is created, such
that the sort stream and media stream flow in parallel through the
sort channel. Non-motile (or lesser-motile) particles tend to
remain in the sort stream, while motile particles move about
randomly and enter the media stream. As a result of this random
movement, the sort stream becomes depleted of motile sperm, while
the media stream becomes increasingly enriched.
[0023] The invention may further be described broadly with
reference to FIGS. 2a, 2b, and 2c, which illustrate pictorially the
separation of motile from non-motile sperm and other non-motile
particles in the sort channel of the device of FIG. 1. In FIG. 2a,
the sort channel 6 is shown, beginning at the point of confluence
of the sort stream 11 and the media stream 12. The sort stream 11
contains motile 13 and non-motile sperm 14 as well as other
non-motile particles, here designated as "round cells" 15. Note
that the size of the media stream in plan, and hence its volume, is
considerably greater than the sort stream. Since sperm (and
similarly, other motile particles) assume an essentially random
distribution in the total liquid within a short period, a larger
media stream volume will necessarily contain a larger fraction of
total motile sperm 13.
[0024] In FIG. 2b, the randomization of motile sperm between the
two streams has begun, and continues until the desired degree of
randomization has been achieved. This degree of randomization is
preferably such that the concentration of motile sperm in the media
phase per unit of volume is the same or greater than the
concentration per unit volume in the sort stream. Note that the
sort stream and media stream are maintained as separate streams,
each preferably exhibiting laminar flow, and having a common
boundary, or interface, 16. Greater concentration of motile
particles in the media stream over the amount dictated by pure
randomization may be achievable by employing a media stream in
which the motile particles have increased mobility, i.e. by
selecting a media stream less viscous than the sort stream, or by
including additives which increase mobility of motile sperm
relative to non-motile or poorly motile sperm.
[0025] In FIG. 2c, the motile sperm-enriched media stream is
harvested by diverting it to flow into the motile particle-enriched
channel 10, while the now motile particle-depleted sort stream
continues through channel 9 into outlet 4.
[0026] The diverting juncture 17 which separates the motile
particle-depleted sort stream from the motile particle-enriched
stream may be of any geometry which avoids substantial mixing of
the streams at this point. The juncture 17 may be positioned, for
example, to provide for substantially the same outlet channel
configuration (i.e. height, width) of the sort stream inlet, at
this point. To minimize contamination of the media stream by
non-motile sperm, the juncture 17 may also be configured such that
a small portion of the media stream is also directed to the motile
particle-depleted sort stream outlet 9. In this case, a modest loss
of motile sperm will occur, however, the probability that
non-motile sperm may enter the motile sperm-enriched media stream
will be lessened as a result.
[0027] The nature of the sort stream is not critical. The sort
stream may be a biologically derived stream such as semen, or may
be washed, diluted, may be treated with additives, stains or
fluorescing dyes, viscosity modifiers, may be buffered, etc., so
long as the treatment does not impair the viability of the desired
exit stream (motile particle-depleted or motile particle-enriched).
If separation but not viability is the aim, for example with
bacterial samples, the number of potential modifications of the
sort stream are enlarged. The sort stream may also be a previously
motile particle-depleted sort stream or motile particle-enriched
media stream. For microrobotic devices, the sort stream liquid may
be any which does not impair the functioning of the device, for
example water, alcohols, ketones, glycols, esters, hydrocarbons,
etc. With biological samples, water-based sort streams are
ordinarily used.
[0028] The media stream may be selected with the same
considerations in mind which are applied to selection or
modification of the sort stream. In some cases, the sort stream may
be water, but for biological systems, it is typical to employ
streams which maintain or enhance biological activity, such as
physiological saline, buffered saline, nutrient broths, and the
like. In the case of human sperm, the preferred media is HEPES
buffered human tubal fluid.
[0029] The nature of the media fluid and the sort fluid may be
selected, if possible, to avoid interfacial mixing due to osmotic
effects. This is the case, for example, when the base fluid (e.g.
water) of both the sort and media streams have substantially the
same amounts of soluble ingredients such as salts, acids, bases,
buffers, dissolved organic material, and the like. The fluids may
also be selected, when possible, to avoid interfacial mixing by
diffusion. However, complete absence of any diffusion is an
unlikely goal in this respect.
[0030] The relative fluid volumes may be selected with respect to
the desired degree of incorporation of the motile particles within
the media phase. For the highest degree of incorporation, the media
volume should be large with respect to the sort fluid volume.
However, proportionately smaller media volumes may also be used,
particularly when sequential (serial) sorting is performed. Ratios
of media fluid to sort fluid of from 1:1000 to 1000:1 are
preferably used, more preferably 1:100 to 100:1, and most
preferably within the range of 1:10 to 10:1. For typical
applications, the ratio of media volume to sort fluid volume is
within the range of 1:1 to 3:1. The media fluid volume is most
preferably higher than the sort fluid volume.
[0031] The volumes referred to here are the volumes at a given
cross-section of the sort channel. For example, a sort channel
which is rectangular in shape having dimensions of 100
.mu.m.times.200 .mu.m will have a "transverse volume" of
2.times.10.sup.4 .mu.m.sup.2. This "transverse volume," actually a
cross-sectional area, can be converted into true volume by
multiplying by channel length or an increment thereof. Thus, the
same rectangular channel previously described and having a
transverse volume of 2.times.10.sup.4 .mu.m.sup.2 will have an
actual volume over a 100 .mu.m length of 2.times.10.sup.6
.mu.m.sup.3.
[0032] The linear flow rates of the sort fluid and media fluid are
preferably substantially the same, i.e. within a range of flow
rates of 1.5:1 to 1:1.5. If the linear flow rate of the media fluid
is greater than that of the sort fluid, correspondingly less
transverse volume of media fluid can be used for the same degree of
motile particle incorporation. Flow is preferably concurrent,
although counterconcurrent flow is also possible provided that
interfacial mixing is not exacerbated beyond that which facilitates
the desired degree of depletion/enrichment of the sort and media
fluids.
[0033] The interface between the sort and media fluids is
preferably a substantially non-mixing interface. By "non-mixing" is
meant an absence of mixing which occurs due to excessive turbulence
between the two fluids. For example, it is most desired that
parallel, concurrent, laminar flow take place such that a
substantially "static" appearing interface is obtained, as opposed
to an interface which exhibits waves, currents, eddys, and the
like. Turbulent flow generally results in partial to full mixing of
the streams, rendering depletion/enrichment of motile particles
less efficient or even completely impossible. The theoretically
best resolution of motile particles occurs when a static-appearing
interface or "streamline" is created where interfacial mixing
occurs only due to diffusional and osmotic effects. However, it
would not depart from the spirit of the invention to allow some
turbulence along the interface. The turbulence is excessive when
the desired degree of resolution cannot be obtained, even with
multiple stages of devices. The turbulence, expressed as a Reynolds
number, should in any case be less than 2000, more preferably less
than 100, yet more preferably less than 10, and most preferably 1
or less. High performance devices such as those illustrated by
example herein, exhibit a Reynolds number of approximately 0.1.
[0034] The nature of the interface, i.e. its degree of turbulence,
may be assessed by the degree of resolution. However, the
turbulence may also be assessed in numerous additional ways. For
example, in PDMS devices as described hereinafter, the optically
transparent nature of the device allows the interface itself to be
observed microscopically, for example by coloring one or both of
the fluids and observing the interface by the change of color at
the interface. By conventional optical techniques, the interface
between media of differing refractive index are also easily
observed. The degree of mixing of the sort and media streams may
also be monitored by introducing a taggant, i.e. a radioactive
soluble compound or non-motile particle, a visual or fluorescent
dye, etc., into one stream but not the other. Appearance of the
taggant in the outlet stream of the stream initially containing no
taggant provides evidence of interfacial mixing, either of a
turbulent kind, or by diffusion or osmosis. Some incorporation due
to the latter two effects is expected, but is also expected to be
quite minimal. An incorporation of 50% of the taggant into the
non-tagged stream essentially constitutes complete mixing. Mixing
of less than 20% of the taggant into the non-tagged stream,
preferably less than 10%, more preferably less than 5%, and most
preferably less than 1% is desired. So long as the Reynolds number
is kept reasonably low, the degree of turbulence will be
satisfactory. A flow which satisfies the above criteria is termed a
"substantially non-turbulent flow" herein. It should be noted that
concurrent flow streams exhibit much less turbulence, and hence
interfacial mixing, than countercurrent flow streams.
[0035] Provided the fluid flow rate meets the non-turbulent
requirements just described, the rate itself may vary widely. The
walls of the sorting device also create the possibility for
turbulence, since they are static with respect to the fluid flow.
The effect of the walls will be most important when narrow channels
are employed, and particularly at the walls which abut the narrower
of the sort or media streams. Since the devices of interest are
rather small and have rather small channels, linear flow rates of
less than 10 cm/s, preferably less than 10 mm/s are preferred. Flow
rates of between 0.10 mm/s to 10 mm/s are particularly preferred.
The low end of linear flow rate is determined by the mixing of
non-motile particles from the sort stream into the media stream by
Brownian motion. For example, at a flow rate of zero, with
identical base fluid compositions (e.g. buffered saline),
distribution of non-motile particles into the media phase would
eventually be complete over time such that their concentrations
become identical. The higher the flow rate, the less Brownian
redistribution of non-motile particles will occur. The upper limit
of the flow rate is reached when the interfacial flow becomes
turbulent, as evidenced by a high degree of mixing.
[0036] Determining the relative flow volumes, relative flow rates,
and absolute flow rates of any given stream can be routinely
accomplished by one skilled in the art by simple calculations
and/or measurements of resolution, for example by varying the
respective rates and volumes and determining the relative
enrichment and depletion of particles between the sort and media
streams.
[0037] The geometry of the devices can vary. Sort channel length,
for example, is generally a function of the rapidity at which
motile particles randomize themselves between the two phases, and
the flow rates. For example, at a given flow rate, motile particles
which have limited motility will require a longer sort channel,
while at a given sort channel length, less motile particles will
require a slower rate of flow. Interfacial surface area also
effects the geometry of the device. For example, flat rectangular
sort channels with one fluid located parallel to and abutting a
channel face of greater dimension, and with the other fluid
adjacent, will exhibit faster randomization and thus require less
sort channel length than the same channel when the first fluid is
located parallel to and abutting a channel face of lesser
dimension. In the latter case, the interfacial area is much reduced
as compared to the former.
[0038] While it is theoretically possible to construct devices of
macroscopic size, even of greater than 10 cm in length, for most
purposes, the sort channel will be quite short, in almost all cases
less than 2-5 cm, and for most devices, in the range of 100 .mu.m
to 1 cm. For sperm sorting, for example, a sort channel length of
5000 .mu.m (5 mm) has proven quite satisfactory. In staged devices,
shorter sort channel lengths may be desirable.
[0039] The cross-sectional geometry of the sort channel is not
critical. Square, rectangular, ellipsoidal, circular, trapezoidal,
triangular or other cross-sections may be used. For ease of
manufacturing, non-undercut channels such as square, rectangular,
triangular, trapezoidal, and half-round or half-elliptical sections
are preferred. These shapes are preferred, for example, when neat
casting or solution casting methods of construction are employed.
In the case of construction by stereolithography techniques
("SLA"), more complex shapes can easily be fabricated. Complex
shapes with undercut channels can also be provided by casting
techniques when the device is cast in successive layers which are
then attached together, for example by bonding. However, the
channel width must be such that both the media stream and sort
stream can both incorporate particles. For human sperm sorting, for
example, a substantially rectangular channel with a height of 50
.mu.m and a width of 500 .mu.m has proven satisfactory. For a point
of reference, human sperm have a head of about 2.5 .mu.m in
diameter and about 5 .mu.m long, and are about 60 .mu.m in overall
length.
[0040] The cross-sectional areas and hence size of the supply
channels and outlet channels are generally smaller than those of
the sort channel. The minimum size of the sort stream inlet channel
is dictated by the size of the particles which are present in the
sort stream. Preferably, the sort stream channel provides a free
channel from 3 to 10 times the size of the particles expected to be
contained therein. The same considerations apply to the size of the
media stream outlet channel, but not necessarily to the media
stream inlet channel. Preferably, the sort stream inlet and outlet
channels will have comparable sizes, although in some instances, as
described earlier, it may be desirable that the outlet channel is
larger than the inlet, thus incorporating a portion of the media
stream into the sort stream. For sperm sorting, a rectangular sort
stream inlet channel of 50 .mu.m height, 100 .mu.m width, and 5000
.mu.m length has proven satisfactory.
[0041] The length of the various inlet and outlet channels is not
critical. It is preferred that at least the inlet channels have
some substantial length, to encourage formation of a laminar flow
stream prior to the confluence of the sort and media stream
channels. In general, more viscous fluids will not require as long
a channel length as less viscous fluids. In some cases, the inlet
channels may be completely dispensed with, i.e. the sort stream
inlet (or reservoir) and/or media stream inlet (or reservoir) may
feed directly into the sort channel. For most purposes, however,
and to facilitate construction of sorting devices, it is preferable
that inlet channels be employed. For the sperm sorting device
described later, for example, inlet channel lengths of about 3 mm
have proven satisfactory.
[0042] The junction 18 (FIG. 1) of confluence of the sort and media
streams is preferably configured to encourage a smooth joining of
the fluid streams without excessive mixing. In general, therefore,
the junction will be a relatively acute angle. The included angles
between the sort stream inlet channel and the sort channel and
between the media stream inlet channel and sort channel may be the
same or different, i.e. the devices are not necessarily
symmetrical. The same considerations apply to the junction 17 where
the sort stream and media stream are separated, or "diverted" from
each other. However, it is preferred that the sort stream inlet
channel, sort channel, and sort stream outlet channel be
substantially linear to provide as little disturbance of the
non-motile particles in the sort stream as possible.
[0043] The material of construction of the sorting devices may be
any suitable material, and the fabrication of the device may
involve any fabrication process. For example, devices may be
micromachined chemically by etching of glass, silica, silicon,
metals, or by solution etching of polymers, etc. The devices may
also be individually fabricated by known stereolithography
techniques. The devices may be injection molded of moldable
polymers, for example silicone rubber, thermoplastic polyurethane,
polyethylene, polypropylene, polytetrafluoroethylene, polyvinyl
chloride, polyvinylidene chloride, polyamide, polyester, and the
like.
[0044] It is at present preferable to cast the sorting devices by
supplying a negative "master" and casting a castable material over
the master. Preferred castable materials are polymers, including
epoxy resins, curable polyurethane elastomers, polymer solutions,
i.e. solutions of acrylate polymers in methylene chloride or other
solvents, and preferably, curable polyorganosiloxanes, most
preferably for cost reasons, polyorganosiloxanes which
predominately bear methyl groups, such as polydimethylsiloxanes
("PDMS"). Curable PDMS polymers are well known and available from
many sources. Both addition curable and condensation-curable
systems are available, as also are peroxide-cured systems. All
these PDMS polymers have a small proportion of reactive groups
which react to form crosslinks and/or cause chain extension during
cure. Both one part (RTV-1) and two part (RTV-2) systems are
available. Addition curable systems are preferred when biological
particle viability is essential.
[0045] In many instances, transparent devices are desirable. Such
devices may be made of glass or transparent polymers. PDMS polymers
are well suited for transparent devices. A benefit of employing a
polymer which is slightly elastomeric is the case of removal from
the mold and the potential for providing undercut channels, which
is generally not possible with hard, rigid materials. Methods of
fabrication of microfluidic devices by casting of silicone polymers
is well known. See, e.g. D. C. Duffy et al., "Rapid Prototyping of
Microfluidic Systems in Poly(dirnethylsiloxane)," ANALYTICAL
CHEMISTRY 70, 4974-4984 (1998). See also, J. R. Anderson et al.,
ANALYTICAL CHEMISTRY 72, 3158-64 (2000); and M. A. Unger et al.,
SCIENCE 288, 113-16 (2000).
[0046] The nature of the channel and reservoir walls of the devices
may be selected in view of the application of the device and the
fluids contemplated for use therein. For example, the walls may be
hydrophobic or hydrophilic, or some portions of the device may be
hydrophobic while other portions are hydrophilic. In addition, the
walls may be treated or derivitized to modify their surfaces with
biologically compatible or bioactive coatings, or to provide
chemical functionality. For sperm sorting, coating the channels
with bovine serum albumin (BSA) has proven useful in improving
liquid flow within the channels and to minimize non-specific
adsorption of cells to channel walls.
[0047] Fluids may be supplied to the inlets or inlet channels of
the device by any suitable method. Fluids may, for example, be
supplied from syringes, from microtubing attached to or bonded to
the inlet channels, etc. In preferred devices, the sort stream
inlet and media stream inlet are in the form of "on-chip"
reservoirs capable of holding and supplying the requisite amounts
of liquids. These reservoirs may be filled by syringe, pipet,
etc.
[0048] Fluid flow may be established by any suitable method. For
example, external micropumps suitable for pumping small quantities
of liquids are available. Micropumps may also be provided in the
device itself, driven by thermal gradients, magnetic and/or
electric fields, applied pressure, etc. All these devices are known
to the skilled artisan. Integration of passively-driven pumping
systems and microfluidic channels has been proposed by B. H. Weigl
et al., PROCEEDINGS OF MICROTAS 2000, Enshede, Netherlands, pp.
299-302 (2000).
[0049] Preferably, however, fluid flow is established by a gravity
flow pump, by capillary action, or by combinations of these
methods. A simple gravity flow pump consists of a fluid reservoir
either external or internal to the device, which contains fluid at
a higher level (with respect to gravity) than the respective device
outlet. Such gravity pumps have the deficiency that the hydrostatic
head, and hence the flow rate, varies as the height of liquid in
the reservoir drops. For many devices, a relatively constant and
non-pulsing flow is desired.
[0050] To obtain constant flow, a gravity-driven pump as disclosed
in published PCT application No. WO 03/008102 A1 (Jan. 18, 2002),
herein incorporated by reference, may be used. In such devices, a
horizontal reservoir is used in which the fluid moves horizontally,
being prevented from collapsing vertically in the reservoir by
surface tension and capillary forces between the liquid and
reservoir walls. Since the height of liquid remains constant, there
is no variation in the hydrostatic head.
[0051] Flow may also be induced by capillary action. In such a
case, fluid in the respective outlet channel or reservoir will
exhibit greater capillary forces with respect to its channel or
reservoir walls as compared to the capillary forces in the
associated inlet channel or reservoir. This difference in capillary
force may be brought about by several methods. For example, the
walls of the outlet and inlet channels or reservoirs may have
differing hydrophobicity or hydrophilicity. Alternatively, and
preferably, the cross-sectional area of the outlet channel or
reservoir is made smaller, thus exhibiting greater capillary
force.
[0052] Most preferably, integrated, on-board reservoirs which serve
as constant flow rate gravity-driven pumps and which also exhibit a
difference in capillary forces between inlet and outlet are used.
Flow in such devices may begin as soon as the devices are filled
with liquid or when blocking valves or plugs are opened, or may be
initially assisted by a pressure differential between the inlet and
outlet, for example by applying suction briefly to the outlet.
[0053] Multiple devices may be connected in many ways to effect
complex separations, to provide enhanced yield, to provide
increased resolution (sorting efficiency) or any combination of
these. In addition, multiple sort and/or media streams may be
employed. When multiple sort or media streams are used, the sort
streams may be the same or different, as may be the media
streams.
[0054] For enhanced efficiency, for example, the motile
particle-depleted sort stream outlet of a device such as that
depicted in FIG. 1 may be connected to the sort inlet of a second
device, this second device also having a media supply. As a result
of this sequential contact with two media streams, the sort stream
will be further depleted of motile particles. The motile
particle-enriched streams from both devices may be combined. Use of
several sequential stages in this manner allows for virtually 100%
recovery of motile particles. Preferably, when multiple devices are
employed, they are fabricated on the same structure with integral
connecting channels. One such device is shown schematically in FIG.
3.
[0055] In FIG. 3, the series configured two-stage motile particle
sorter consists of a single sort fluid reservoir 20, connected to
first sort channel 22 by sort stream channel 21. The first stage
also consists of first media supply reservoir 23, media stream
channel 24, motile particle-enriched first media channel 25, and
motile particle-enriched first media reservoir 26. The motile
particle-depleted sort outlet stream from the first stage flows
through connecting passage 27 to serve as the sort stream inlet to
the second stage sort channel 28. Second media reservoir 29
supplies media to the second stage sort channel through media inlet
channel 30. Sort fluid further depleted of motile particles exits
the device through channel 31 into motile particle-depleted sort
stream reservoir 32, while a second stream of motile
particle-enriched media fluid exits the sort channel through media
outlet channel 33 and into reservoir 34. The two motile
particle-enriched media reservoirs 26, 34 can be connected to a
common exit channel or reservoir, optionally through valved
passages, or may be emptied manually, e.g. using a syringe or
pipet, and their contents combined, if desired.
[0056] Additional devices are shown in FIGS. 4, 5, and 6. In the
device of FIG. 4, two media supply reservoirs 41 supply media fluid
to the device 40, motile particle-enriched media being collected in
the two media outlet reservoirs 42. A single sort fluid reservoir
supplies fluid containing motile and non-motile particles from sort
fluid reservoir 43, and the motile particle-depleted sort fluid is
collected in sort fluid outlet reservoir 45 after passing through
sort channel 44. In this case, a central sort stream 46 is flanked
on each side by media streams 47.
[0057] FIGS. 5 and 6 are schematics of multiple stage devices which
rely on alternative connections of various flow paths to improve
one or more aspects of the sorting process. In both Figures, double
lines represent sort channels. The device of FIG. 5 is capable of
not only sorting motile from non-motile particles, but also into
fractions of different motilities, and has three sort channels. The
device of FIG. 6 splits the outlet of a single sort channel into
fractions, the furthest away from the sort stream containing
proportionately more of the particles with highest motility. As can
be seen, the present devices can be configured simply or with great
complexity. Devices may also operate in parallel, series parallel,
or other modes. Parallel processing may be desired for sorting
larger samples, or to measure sorting efficiency, etc., while
comparing different media fluids. Such comparisons are more
statistically accurate when measurements are made in a single
device.
[0058] While much of the description herein refers to separation of
motile from non-motile particles, the subject invention processes
and devices are also suitable for separating motile particles of
differing motility. The most motile particles will enter the media
stream at a higher rate than particles of lesser motility. The
residence time in the sort channel is preferably selected such that
the most motile particles will assume a random or near random
distribution in the total fluid. In contrast to separation of
motile from completely non-motile particles, however, where
additional sort channel length can be tolerated, and distribution
of non-motile particles into the media stream is due substantially
only to Brownian motion and to turbulence and like effects, when
motile and lesser motile particles are separated, the lesser motile
particles will also assume a random distribution given sufficient
time. The sort channel length must be adjusted downward such that
this cannot occur. The media stream will be enriched with both
motile and lesser motile particles, but will be correspondingly
more greatly enriched by the particles of greater motility.
Multiple sequential processing of a first media stream (serving as
the sort stream to a further stage) will cause higher resolution
between the differently motile particles. Second and further
sorting of the sort streams and their subsequent treatment in like
fashion will increase the yield.
EXAMPLE 1
[0059] A microfluidic sperm sorting device was prepared from Dow
Corning SYLGARD.RTM. 184 curable silicone resin, using the soft
lithography technique described by D. C. Duffy et al., cited
previously. The curable PDMS was cast onto a master having the
desired reservoir and channel features as protuberances. The cast
PDMS sorting devices were plasma oxidized to seal the open channel
side of the casting to a glass cover slide. Channels and reservoirs
were coated with 1% bovine serum albumin fraction V from Sigma,
dissolved in phosphate buffered saline (PBS) from Invitrogen
Corporation. The entire device was approximately 6 mm thick,
exclusive of the cover slide, and somewhat larger than a U.S. penny
coin. A perspective view of the device is shown in FIG. 7.
[0060] In FIG. 7, the PDMS casting is transparent, and only the
reservoirs and channels are depicted. The cover slide would be
bonded to the bottom plane of the device. The numerals are the same
as those of FIG. 1. The channels are rectangular in cross-section,
with a channel height of 50 .mu.m, while the reservoirs are roughly
semi-circular. Both inlet reservoirs 2 and 3 are approximately 3 mm
in height, while the outlet reservoirs are approximately 2 mm in
height. The inlet and outlet channels 7, 8, 9, 10 are about 5000
.mu.m long. The sperm inlet channel 7 and the motile depleted sperm
outlet channel 9 have a width of 100 .mu.m, while the media inlet
channel 8 and outlet channel 10 have a width of about 300 .mu.. The
sort channel 6 has a width of 500 .mu.m and a length of 5000
.mu.m.
[0061] Semen samples were obtained with institution Review Board
approval from men undergoing infertility evaluation. Sorting tests
were performed using washed semen samples. In the order listed, 60
.mu.L of processing media was added to the media inlet reservoir,
50 .mu.L of a washed semen sample to the sample inlet reservoir,
and 2 .mu.L of media to each of the outlet reservoirs. Sperm
sorting yields were calculated taking these dilution factors into
account. The numbers of motile sperm were determined by a Makler
Counting Chamber (Sefi-Medical Instruments, Haifi, Israel). For
visualization of membrane-compromised sperm, which generally
corresponds to non-motile sperm, 3 .mu.L of propidium iodide
(Molecular Probes, www.probes.com, 60 mM dissolved in processing
media) was added to sperm samples prior to sorting. A Texas Red
filter set (577 nm excitation, 620 nm emission) was used to view
red fluorescence from stained cells. An inverted microscope (NIKON
TE 300, www.nikon-usa.com) with a CCD camera (Hamamatsu ORCA-100,
www.hamamatsu.com) was used to capture images and record
movies.
[0062] The sorting device uses a sorting system where non-motile
sperm flow along their initial streamlines and exit one outlet
whereas motile sperm can deviate from their initial streamlines and
exit through a different outlet. This sorting mechanism is related
to the "filtering" mechanism used in an "H-filter" where rapidly
diffusing small molecules exit through a different outlet from
larger molecules and particles that diffuse more slowly. The
difference between the two devices is that the sorting device of
the present invention takes advantage of active movement of cells
whereas an H-filter takes advantage of passive diffusion of
particles. This type of sorting is possible because in small
channels, multiple laminar streams can flow parallel to each other
with no turbulent mixing at the interface between the streams.
Typical Reynolds Numbers for the flow of sperm sample and media
inside the sorting device were on the order of 0.1. Non-motile
human sperm, approximately 60 .mu.m in length, and non-motile
particles on the same order of magnitude in size diffuse slowly
(D=1.5.times.10.sup.-13 m.sup.2/sec; 690 sec to diffuse 10 .mu.m)
and remained within their initial streamlines. In contrast, motile
human sperm swim at velocities greater than 20 .mu.m/sec at
20.degree. C. This rapid mobility allows motile sperm, but not the
non-motile sperm, to distribute themselves randomly within the
width of a 500 .mu.m channel within seconds. The sorting device was
designed specifically to give sperm a residence time of 20 seconds
in the main separation channel. A bifurcation placed at the end of
this separation channel allows efficient collection of only the
motile sperm that deviated from its initial inlet stream.
[0063] The sorting device described integrates all functions
necessary for sperm sorting, for example, inlet/outlet ports, fluid
reservoirs, pumps, power source, sort channel, etc., onto a simple
chip design that is practical to manufacture and use. A key design
feature of this embodiment is the set of four horizontally-oriented
fluid reservoirs that also function as sample inlet/outlet ports
and a fluid pumping system. The orientation, geometry, and size of
these reservoirs are designed to balance gravitational forces and
surface tension forces, and provide a pumping system that generates
a steady flow rate over extended periods of time regardless of the
volume of fluid in the reservoirs. This contrasts with conventional
gravity-driven pumping systems whose flow rates decrease over time
as the volume of fluid in the inlet reservoir decreases. The
diameters of the reservoirs were selected to be small enough that
surface tension prevents liquid from spilling out of the
horizontally-oriented reservoirs, but large enough to hold
sufficient amounts of sample (tens to hundreds of microliters) and
allow convenient sample introduction and recovery. This balance of
forces allows the reservoirs to be arranged horizontally without
the liquid inside spilling out. The horizontal reservoir
arrangement, in turn, holds the height difference between the fluid
in the inlet and outlet reservoirs the same (1.0 mm height
difference hetween inlet and outlet reservoir ceilings) regardless
of the volume of fluid present in the reservoirs and maintains a
constant hydraulic pressure even as the amount of fluid in the
reservoirs changes.
[0064] The passively-driven pumping system described here is unique
in that it uses horizontally-oriented reservoirs to overcome the
problem of traditional gravity-driven pumping, where the pressure
decreases as the amount of liquid in the reservoir decreases.
Furthermore, the structure of the pump is greatly simplified
compared to other mechanical or non-mechanical pumping systems
allowing easy manufacture and integration of the pump into a small,
integrated device. Finally, the use of gravity and surface tension
as the driving-force contributes to the overall small size of the
sorting device by eliminating the need for power supplies such as
batteries. Taking gravity, surface tension, and channel resistance
into consideration, the sorting device was designed to give a
steady flow rate of sperm with a residence time of approximately 20
seconds inside the main sort channel. More specifically, the device
is designed so that the flow resistance of the fluid reservoirs is
more than 10 times less than that of the microfluidic channels, and
therefore negligible. Thus, the resistance of the channels,
calculated to be 2.8.times.10.sup.12 kg/(sec/m.sup.4), approximates
the total resistance of the system. Since a bulk flow rate of 0.008
.mu.L/sec is required to achieve the desired residence time of 20
seconds and the total resistance is 2.8.times.10.sup.12
kg/(sec/m.sup.4), the net pressure drop required to drive the fluid
is 23 N/m.sup.2. To achieve this desired pressure drop, we designed
the dimensions of the reservoirs such that capillary forces (3.0 mm
diameter inlet reservoir vs. 2.0 mm diameter outlet reservoir)
would be 13 N/m.sup.2 and the pressure drop across the microfluidic
channel of the sorting device due to hydrostatic forces (1.0 mm
height difference) would be 9.8 N/m.sup.2. For calculation of the
capillary force, the contact angle was assumed to be 0.degree. (the
contact angle of water on BSA coated PDMS is very small), the
surface tension of the washed semen sample assumed to be
approximately 0.040 N/m (less than that of water due to
"impurities" such as proteins), and the viscosity of the washed
semen sample to be similar to that of water. The observed bulk flow
rate of 0.008 .mu.L/sec for a dilute particle suspension in 1% BSA
solution was approximately equal to that of the calculated flow
rate. Actual sperm samples sometimes had lower flow rates due to
larger apparent viscosity. Smaller flow rates for the sperm sample
stream would result in slightly lower yields but does not affect
the purity of the sperm recovered at the sorted sperm outlet.
[0065] Sperm sorting efficiencies of the sorting device were
evaluated by three methods: (i) tracking the movement of motile
sperm in the channel by phase contrast microscopy, (ii) tracking
movement of propidium iodide (PI) stained cells in the channel by
fluorescence microscopy, (iii) using a Makler Counting Chamber, a
grid-based sperm counting device, to determine numbers of motile
sperm and non-motile sperm in the inlet and outlet reservoirs (FIG.
8). The sperm tracking experiments shows the process of how motile
sperm can swim out of its initial streamline. PI stains membrane
compromised cells such as dead cells, and thus allows the
non-motile sperm to be highlighted and visualized with red
fluorescence while the motile sperm remain unstained. The bar
graphs in FIG. 8 compare percentage of sperm that are motile before
and after sorting. The unshaded bars represent the initial sperm
sample, while the solid bars represent the motile particle-enriched
media stream. The purity of motile sperm after sorting was nearly
100% regardless of motile sperm purity before sorting. The yields
(39%, 42%, 43%), defined as the ratio of the number of motile sperm
in the motile sperm outlet reservoir to the total number of motile
sperm in the sperm sample inlet reservoir, were comparable to or
greater than the recovery rates (0.8% to 50%) of sperm processed
using conventional sorting methods such as direct swim-up, swim-up
from a pellet of centrifuged sperm, or density gradient separation.
It was also observed that sperm morphology, another important trait
that correlates with successful pregnancies, also improved after
sorting with the device (Strict Sperm Morphology: 9.5.+-.1.1%
normal before sorting to 22.4.+-.3.3% normal after sorting). Kruger
Strict sperm morphology is a set of criteria or standards whereby
sperm must fit within specific measurements (head width and length,
tail length, acrosome making up a certain percentage of the sperm
head) and lack abnormalities (e.g. pin head, round head, crimped
tail).
[0066] As can be seen from the above, the motile particle sorting
devices are small, easily manufactured, simple in operation, and
highly efficient. In the claims which follow, the terms "a" and
"an" mean "one or more than one" unless indicated otherwise.
[0067] While embodiments of the invention have been illustrated and
described, it is not intended that these embodiments illustrate and
describe all possible forms of the invention. Rather, the words
used in the specification are words of description rather than
limitation, and it is understood that various changes may be made
without departing from the spirit and scope of the invention.
* * * * *
References