U.S. patent application number 14/451015 was filed with the patent office on 2015-01-22 for system, apparatus and method for material preparation and/or handling.
The applicant listed for this patent is Keck Graduate Institute. Invention is credited to Robert Doebler, Barbara Erwin, Anna Hickerson, Bruce Irvine, Ali Nadim, James D. Sterling, Ryan P. Talbot, Denice Woyski.
Application Number | 20150024480 14/451015 |
Document ID | / |
Family ID | 40853778 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150024480 |
Kind Code |
A1 |
Doebler; Robert ; et
al. |
January 22, 2015 |
SYSTEM, APPARATUS AND METHOD FOR MATERIAL PREPARATION AND/OR
HANDLING
Abstract
Oscillating angularly rotating a container containing a material
may cause the material to be separate. Denser or heavier material
may unexpectedly tend to collected relatively close to the axis of
rotation, while less dense or light material may tend to collect
relatively away from the axis of rotation. Oscillation along an
arcuate path provides high lysing efficiency. Alternatively, a
micromotor may drive an impeller removably received in a container.
Lysing may be implemented in batch mode, flow-through stop or
semi-batch mode, or flow-through continuous mode. Lysing
particulate material may exceed material to be lysed or lysed
material and/or air may be essentially eliminated from a chamber to
increase lysing efficiency.
Inventors: |
Doebler; Robert; (Upland,
CA) ; Nadim; Ali; (San Marino, CA) ; Sterling;
James D.; (Upland, CA) ; Hickerson; Anna;
(Altadena, CA) ; Erwin; Barbara; (Ontario, CA)
; Woyski; Denice; (Anaheim, CA) ; Talbot; Ryan
P.; (South Pasadena, CA) ; Irvine; Bruce;
(Glendora, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Keck Graduate Institute |
Claremont |
CA |
US |
|
|
Family ID: |
40853778 |
Appl. No.: |
14/451015 |
Filed: |
August 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12732070 |
Mar 25, 2010 |
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14451015 |
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PCT/US2009/030622 |
Jan 9, 2009 |
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12732070 |
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61020072 |
Jan 9, 2008 |
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61117012 |
Nov 21, 2008 |
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Current U.S.
Class: |
435/306.1 |
Current CPC
Class: |
C12M 23/28 20130101;
C12M 47/06 20130101; C12M 27/00 20130101; G01N 2001/4088 20130101;
G01N 1/286 20130101; G01N 1/4077 20130101; G01N 2001/4083 20130101;
G01N 2001/4094 20130101; C12N 1/066 20130101 |
Class at
Publication: |
435/306.1 |
International
Class: |
C12M 1/00 20060101
C12M001/00; G01N 1/40 20060101 G01N001/40; G01N 1/28 20060101
G01N001/28; C12N 1/06 20060101 C12N001/06 |
Claims
1-110. (canceled)
111. An article to perform flow-through lysis by oscillation on
material to be lysed, the article comprising: at least one wall
forming at least one chamber having a first opening and at least a
second opening spaced from the first opening, the first and the
second openings to provide fluid communication into the chamber
from an exterior thereof; a particulate lysing material received in
the chamber, the particulate material including a plurality of
particles sized to lyse a material to be lysed and having a
combined particulate volume; a first filter received in the chamber
between the first opening and the particulate material, the first
filter having a plurality of apertures sized to substantially pass
the material to be lysed and to retain the particulate material; a
second filter received in the chamber between the second opening
and the particulate material, the second filter having a plurality
of apertures sized to pass the material to be lysed and to retain
the particulate material, wherein the first filter and the second
filter form a particulate retainment area therebetween having a
volume sufficiently greater than said combined particulate volume
to permit the plurality of particles to move relative to one
another to provide shear forces when in use to perform lysis by
oscillation on material to be lysed; a first tube coupled to
provide fluid communication to the first opening for material to be
lysed; and a second tube coupled to provide fluid communication
from the second opening for material that has been lysed; wherein
the first tube and the second tube do not restrict the oscillation
of the chamber and do not resonate in response to the
oscillation.
112. The article of claim 111, further comprising: an attachment
structure proximate the first opening.
113. The article of claim 111, further comprising: a first
attachment structure to attach the first tube to the first opening;
and a second attachment structure to attach the second tube to the
second opening.
114. The article of claim 111, further comprising: a nipple about
the first opening.
115. The article of claim 111, further comprising: a first nipple
to attach the first tube about the first opening; and a second
nipple to attach the second tube about the second opening.
116. The article of claim 111 wherein the at least one wall is
elongated and has a first end and a second end opposed to the first
end.
117. The article of claim 116 wherein the first opening is at the
first end and the second opening is at the second end.
118. The article of claim 116 wherein the at least one wall is
cylindrically tubular.
119. The article of claim 111 wherein the particulate material is a
plurality of beads.
120. The article of claim 119 wherein the plurality of beads
includes at least one of ceramic beads, glass beads, zirconium
beads, metal beads, plastic beads, and sand.
121. The article of claim 119 wherein the plurality of beads have
diameters in the range of approximately 100 microns.
122. The article of claim 119 wherein the plurality of beads have
diameters in the range of 50 microns to 150 microns.
123. The article of claim 111 wherein when in use a volume of the
particulate material is greater than a volume of material to be
lysed.
124. The article of claim 123 wherein when in use there is
essentially no air in the chamber.
125. The article of claim 123 wherein the chamber has a volume that
holds less than 60 .mu.l of the material to be lysed.
126. The article of claim 123 wherein the chamber has a volume that
holds approximately 10 .mu.l to approximately 40 .mu.l of the
material to be lysed.
127. The article of claim 111 wherein the first and the second
filters are fixed to the wall.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to the separation of matter,
for example particles or other material in a suspension. The
present disclosure also relates to lysing and in particular to
systems, apparatus and methods to perform lysing of a material to
be lysed using a lysing particulate material.
BACKGROUND
[0002] There are numerous applications that require the separation
of material, for example particulate or other matter in suspension.
One common approach is to employ a centrifuge to separate
relatively heavier material from relatively light material.
Centrifuges typical include a container to hold the material, a
drive system including a motor and transmission or linkage coupled
to rotate the container about a fixed axis of rotation. The
material in the container separates based on density under
centripetal acceleration, with denser or heavier material tending
to collect at a perimeter relatively away from the axis of rotation
and with the less dense or lighter material tending to collect
relatively closer to the axis of rotation.
[0003] Centrifuges may be used on a large variety of material from
particulates, to fluids, to gases, and combinations of the same.
Centrifuges are often used to separate biological material, for
example in preparing samples for analysis of the composition of
specific biological materials, such as proteins, lipids, and
nucleic acids either individually or as complexes. A centrifuge may
be used to isolate certain organelles-nuclei, mitochondria,
lysosomes, chloroplasts, and/or endoplasmic reticulum.
[0004] Lysis of biological material, for example cell lysis, is
used to analyze the composition of specific biological materials,
for example proteins, lipids, and nucleic acids either individually
or as complexes. If a cell membrane is lysed then certain
organelles-nuclei, mitochondria, lysosmes, chloroplasts, and/or
endoplasmic reticulum may be isolated. Such may be analyzed using
PCR, electron microscopy, Western blotting or other analysis
techniques.
[0005] There are numerous approaches to performing lysis. For
example, enzymatic approaches may be employed to remove cell walls
using appropriate enzymes, in preparation to cell disruption or to
prepare protoplasts. Another approach employs detergents to
physically disrupt cell membranes. These chemical approaches may
adversely affect the resulting product, for example degrading the
bio-products being released. Consequently, chemical approaches may,
in some instances, not be practical.
[0006] Yet another approach employs ultrasound to produce
cavitation and impaction for disrupting the cells. Such an approach
may not achieve as high a lysis efficiency as may be required or
desired for many applications.
[0007] Yet still another approach employs beads (e.g., glass or
ceramic) which are agitated, for example, via a vortex mixer. Such
an approach successfully addresses the issues raised by chemical
lysis approaches, yet improvements in such an approach are
desirable.
BRIEF SUMMARY
[0008] There is a need for other approaches to separating
materials. Such approaches may provide quicker separation, more
thorough separation, or may separate materials in a different
manner than previous approaches.
[0009] There is also a need for bead-based lysing apparatus and
methods that are more efficient than current lysing apparatus. Such
may reduce the amount of time required to process a sample (i.e.,
material to be lysed) and/or increase throughput. Such may also
increase the level or thoroughness of lysing, producing greater
amounts of lysed material from a given sample size. There is also a
need for lysing apparatus and methods that operate on sample sizes
that are relatively small (e.g., 10 micro-liters) compared to
conventional lysing apparatus. Such may enable lysing to be
performed where a relatively small amount of a sample is available
and/or reduce costs. Such may also reduce the amount of lysing
particulate material that is required, also providing cost
reductions. Such may also allow higher frequency oscillation,
thereby increasing efficiency, while maintaining reasonable
lifetime or fatigue characteristics. There is also a need to
efficiently and reliably lyse typically difficult to lyse material,
for example spores. There is a further need for the ability to
perform flow-through lysing. Such may allow large quantities of
small samples to be processed over time, for example processing
small samples taken every minute over a long period of time (e.g.,
day, week, month, and/or years). There is also a need for lysing
equipment that is small and hence portable, and that is relatively
inexpensive yet sufficiently robust to withstand travel or harsh
operating environments.
[0010] A system to perform lysis on material to be lysed may be
summarized as including an arm having an attachment location to at
least temporarily attach a container that at least temporarily
holds a material to be lysed and a particulate lysing material; a
motor operable to provide a drive force; and a drive mechanism
coupled to transfer the drive force of the motor into oscillation
of the attachment location of the arm along an arcuate path. The
arm may be a rigid arm that does not flex under a load in response
to the oscillation of the attachment location of the arm along the
arcuate path. The arm may be a flexible arm that flexes under a
load in response to the oscillation of the attachment location of
the arm along the arcuate path.
[0011] The system to perform lysis may further include a holder at
the attachment location, the holder configured to removably hold
the container. The system may include the container and the
particulate lysing material. In some embodiments the container is
non-removably fixed to the arm at least proximate the attachment
location. The container may have a first opening and at least a
second opening spaced from the first opening, the first and the
second openings to provide fluid communication into the chamber
from an exterior thereof. The container may include a first filter
positioned in the chamber and a second filter positioned in the
chamber spaced from the first filter to form a particulate
retainment area therebetween, the particulate retainment area
positioned between the first and the second openings, the first and
the second filters each having a plurality of apertures sized to
substantially pass the material to be lysed and to block the
particulate material. The first filter, the second filter and the
particulate material may form a cartridge that is selectively
replaceable in the chamber. The plurality of beads may include at
least one of ceramic beads, glass beads, zirconium beads, metal
beads, plastic beads, and sand and wherein the plurality of beads
have diameters in the range of approximately 10 microns to
approximately 600 microns. When in use a volume of the particulate
matter may be greater than a volume of material to be lysed. When
in use there may essentially be no air in the chamber.
[0012] The system may further include a pump operable to pump the
material to be lysed through the chamber. The pump may be
configured to intermittently pump the material to be lysed through
the chamber. The material to be lysed may have a residence time in
the chamber that may be sufficient to achieve a defined level of
lysing. The pump may continuously pump the material to be lysed
through the chamber. Given a length of the chamber, a flow rate of
the pump may be such that the material to be lysed spends
sufficient time (i.e., desired or defined residence time) in
traversing the chamber from the first opening to the second opening
to achieve a defined level of lysing.
[0013] The system may further include a first tube coupled to
provide fluid communication to the first opening for the material
to be lysed to the first opening; and a second tube coupled to
provide fluid communication from the second opening for a material
that has been lysed. Ends of at least one of the first and the
second tubes may be reinforced. Ends of at least one of the first
and the second tubes may be reinforced with additional tubes that
are concentric about the ends of the tube. A length of at least one
of the first and the second tubes may be such that the length does
not restrict the oscillation of the attachment location. The length
of at least one of the first and the second tubes may be such that
the at least one of the first and the second tubes does not
resonate in response to the oscillation of the attachment location
of the arm along an arcuate path. A respective length of each of
the first and the second tubes may be sufficiently long so as to
not restrict the oscillation of the attachment location and are
sufficiently short such that the first and the second tubes do not
resonate during use.
[0014] The drive mechanism may consist of a four-bar linkage
including a first bar rotationally driven by a motor, the bar
connected by a hinge to a second bar that serves as a connecting
rod. A third and a fourth bars both pivot about a central fixed
axis with a fixed angle between them. The end of the second bar
that serves as the connecting rod is connected by a hinge to the
third bar whose length determines the angle of rotation of the
third and the fourth bars. The length of the fourth bar is the
radius of curvature of the arcuate motion of the lysis chamber,
which is coupled or connected to the fourth bar.
[0015] A method of lysing a material to be lysed may be summarized
as including receiving a material to be lysed in a chamber that
contains a particulate lysing material; oscillating the chamber
containing the material to be lysed and a particulate lysing
material along an arcuate path to produce a lysed material; and
removing the lysed material from the chamber.
[0016] The method of lysing a material to be lysed may further
include pumping the material to be lysed into the chamber.
[0017] The method may further include intermittently pumping the
material to be lysed into the chamber while oscillating the
chamber. Intermittently pumping the material to be lysed into the
chamber while oscillating the chamber may include intermittently
pumping the material to be lysed into the chamber such that the
material to be lysed spends sufficient time in the chamber to
achieve a desired level of lysing. Intermittently pumping the
material to be lysed into the chamber while oscillating the chamber
may include intermittently pumping the material to be lysed into
the chamber such that the chamber is completely evacuated of the
lysed material during each cycle of the intermittent pumping. The
chamber may be completely evacuated of the lysed material during
each cycle of the intermittent pumping by the pumping into the
chamber of more material to be lysed.
[0018] The method may further include continuously pumping the
material to be lysed into the chamber while oscillating the
chamber. The method may further include adjusting a flow rate of
the pumping of the material to be lysed into the chamber based on a
length of the chamber, a flow rate of the pump is such that the
material to be lysed spends sufficient time in the chamber (i.e.,
residence time) to achieve a desired level of lysing.
[0019] The method may further include directing the lysed material
removed from the chamber to at least one analysis device. The
method may further include evacuating the chamber with an inert
fluid.
[0020] A method of lysing a material to be lysed may be summarized
as including receiving a first cartridge having a chamber that
contains a particulate lysing material and a material to be lysed;
and oscillating the first cartridge having the chamber that
contains the material to be lysed and the particulate lysing
material along an arcuate path to produce a lysed material.
[0021] The method may further include receiving a second cartridge
in place of the first cartridge, the second cartridge having a
chamber that contains a particulate lysing material and a material
to be lysed; and oscillating the second cartridge having the
chamber that contains the material to be lysed and the particulate
lysing material along an arcuate path to produce a lysed material.
Receiving a first cartridge may include receiving the first
cartridge in a mounting bracket at an attachment point of an arm.
Oscillating the first cartridge may include oscillating a rigid arm
on which the first cartridge is mounted. Oscillating the first
cartridge may include oscillating a flexible arm on which the first
cartridge is mounted.
[0022] An article to perform flow-through lysis on material to be
lysed may be summarized as including at least one wall forming at
least one chamber having a first opening and at least a second
opening spaced from the first opening, the first and the second
openings to provide fluid communication into the chamber from an
exterior thereof; a particulate lysing material received in the
chamber, the particulate material including a plurality of
particles sized to lyse a material to be lysed; a first filter
received in the chamber between the first opening and the
particulate material, the first filter having a plurality of
apertures sized to substantially pass the material to be lysed and
to retain the particulate material; and a second filter received in
the chamber between the second opening and the particulate
material, the second filter having a plurality of apertures sized
to pass the material to be lysed and to retain the particulate
material, wherein the first filter and the second filter form a
particulate retainment area therebetween.
[0023] The article may further include an attachment structure
proximate the first opening. The article may further include a
first attachment structure to attach a first tube to the first
opening; and a second attachment structure to attach a second tube
to the second opening.
[0024] The article may further include a first nipple to attach a
first tube about the first opening; and a second nipple to attach a
second tube about the second opening. The at least one wall may be
elongated and have a first end and a second end opposed to the
first end. The first opening may be at the first end and the second
opening may be at the second end. At least one wall may be
cylindrically tubular.
[0025] The particulate material may be a plurality of beads. The
plurality of beads may include at least one of ceramic beads, glass
beads, zirconium beads, metal beads, plastic beads, and sand. The
plurality of beads may have diameters in the range of approximately
100 microns. The plurality of beads may have diameters in the range
of 50 microns to 150 microns. When in use, a volume of the
particulate matter may be greater than a volume of material to be
lysed. When in use there may be essentially no air in the chamber.
The chamber may have a volume that holds less than 60 .mu.l of the
material to be lysed. The chamber may have a volume that holds
approximately 10 .mu.l to approximately 40 .mu.l of the material to
be lysed. The first and the second filters may be fixed to the
wall.
[0026] A system to perform lysis may be summarized as including a
container having at least one chamber to hold a material to be
lysed and a lysing particulate material, the chamber having a first
opening and at least a second opening to provide fluid
communication into the chamber from an exterior thereof; an
impeller having a number of blades received in the chamber of the
container; and a micromotor coupled to turn the impeller. The first
opening may provide an entrance for material to be lysed and the
second opening may provide an exit for material that has been
lysed.
[0027] The chamber may have a third opening, at least a portion of
the micromotor may be received by the third opening and may seal
the third opening. The micromotor may be removably received in the
first third opening. The micromotor may be disposable.
[0028] The container may further include at least a first filter
positioned before the exit in a flow path, the first filter having
a plurality of apertures sized to substantially pass material that
has been lysed and to substantially block lysing material. The
container may further include at least a second filter positioned
following the entrance in the flow path, the second filter having a
plurality of apertures sized to substantially pass material to be
lysed and to substantially block lysing material.
[0029] The micromotor may pulsate. The micromotor may drive the
impeller at a rate of greater than 10,000 RPM in the presence of
liquid and beads. The micromotor may drive the impeller at a rate
of approximately 50,000 RPM, when not in the presence of liquid and
beads.
[0030] A method of system to perform lysis, may be summarized as
including receiving a material to be lysed via an entrance in at
least one chamber of a container that holds a lysing particulate
material; driving an impeller having a number of blades received in
the chamber of the container via a micromotor; and expelling a
material that has been lysed via an exit from the chamber of the
container.
[0031] Expelling a material that has been lysed via an exit may
include expelling the material that has been lysed via a first
filter positioned before the exit in a flow path, the first filter
having a plurality of apertures sized to substantially pass the
material that has been lysed and to substantially block the lysing
particulate material. Receiving a material to be lysed via an
entrance may include receiving the material to be lysed via a
second filter positioned following the entrance in the flow path,
the second filter having a plurality of apertures sized to
substantially pass the material to be lysed and to substantially
block lysing particulate material.
[0032] The method of system to perform lysis may further include
intermittently pumping the material to be lysed into the at least
one chamber via the entrance. The method may further include
continuously pumping the material to be lysed into the at least one
chamber via the entrance.
[0033] Driving an impeller may include pulsating the impeller.
Driving an impeller may include driving the impeller at a rate of
greater than 10,000 RPM in the presence of liquid and beads. The
method may further include replacing the micromotor with a new
micromotor. The method may further include disposing the
micromotor.
[0034] A system to perform lysis, may be summarized as including a
first container having at least one chamber to hold a material to
be lysed and a lysing particulate material, the chamber having a
single opening to provide fluid communication into the chamber from
an exterior thereof; an impeller having a number of blades received
in the chamber of the first container; and a micromotor coupled to
turn the impeller, at least a portion of the micromotor removably
received in the single opening of the first container to seal the
single opening in use. The micromotor may be disposable. The
micromotor may be removably received by a single opening of a
second container after removal from the single opening of the first
container. The micromotor may pulsate. The micromotor may drive the
impeller at a rate of greater than 10,000 RPM in the presence of
liquid and beads.
[0035] A method of operating a system to perform lysis may be
summarized as including receiving a material to be lysed via an
entrance in at least one chamber of a first container that holds a
lysing particulate material; locating an impeller in the chamber of
the first container via the entrance; closing the entrance of the
first container with a micromotor that is coupled to drive the
impeller; and driving the impeller to circulate the material to be
lysed and the lysing particulate material in the chamber of the
first container.
[0036] The method of system to perform lysis may further include
removing the micromotor from the entrance of the first container
and removing a material that has been lysed via the entrance of the
first container. Removing a material that has been lysed via the
entrance of the first container may include withdrawing the
material that has been lysed using a pipette. Driving the impeller
may include pulsating the impeller. The method may further include
reusing the micromotor with a second container. The method may
further include disposing of the micromotor.
[0037] A system to separate materials may be summarized as
including: a base; an actuator coupled to the base and selectively
operable to provide a drive force; and a drive mechanism coupled to
the base and coupled to transfer the drive force of the motor into
a high frequency oscillatory angular rotation of a container about
an axis of rotation. The actuator may be an electric motor.
[0038] The system may further include a holder coupled to the drive
mechanism for movement thereby, the holder configured to removably
hold the container.
[0039] The system may further include the container, wherein the
container has an interior to hold the materials to be
separated.
[0040] The system may further include the container, wherein the
container has an interior to hold the materials to be separated and
the container is non-removably fixed to the drive mechanism.
[0041] The system may further include the container, wherein the
container has an interior to hold the materials to be separated and
at least one inner port to provide fluid communication between the
interior of the container and an exterior thereof, the at least one
inner port positioned relatively proximate the axis of rotation
with respect to an arc defined by an oscillatory movement of an
outer most portion of the container from the axis of rotation.
[0042] The system may further include the container, wherein the
container has an interior to hold the materials to be separated and
at least one inner port to provide fluid communication between the
interior of the container and an exterior thereof, the at least one
inner port positioned at an inner periphery of the container.
[0043] The system may further include the container, wherein the
container has an interior to hold the materials to be separated and
at least one outer port to provide fluid communication between the
interior of the container and an exterior thereof, the at least one
outer port positioned relatively distal from the axis of
rotation.
[0044] The system may further include the container, wherein the
container has an interior to hold the materials to be separated and
at least one outer port to provide fluid communication between the
interior of the container and an exterior thereof, the at least one
outer port positioned at an outer periphery of the container.
[0045] The system may further include the container, wherein the
container has an interior to hold the materials to be separated, at
least one inner port to provide fluid communication between the
interior of the container and an exterior thereof and at least one
outer port to provide fluid communication between the interior of
the container and the exterior thereof, the at least one inner port
spaced relatively closer to the axis of rotation with respect to
the at least one outer port. The container may include at least one
filter proximate one of the inner or the outer ports. The at least
one filter may be selectively replaceable in the container.
[0046] The system may further include a pump to pump the material
to be separated through the container. The pump may be configured
to intermittently pump the material through the container. The axis
of rotation may pass through the container. The container may be
spaced from the axis of rotation. The drive mechanism may include a
four-bar linkage that may include a first member, a second member,
a third member and a fourth member, the second member coupled to
the first member, the first member rotationally driven by a motor
to eccentrically drive a first end of the second member in a
circular motion, the third bar member pivotally coupled to a second
end of the second member, the third member connected to the fourth
member at a pivot point; where an amplitude of motion of the second
member and a length of the third member define a angle of motion of
the third and the fourth members and a length of the fourth member
defines a distance of arcuate motion. The system may include a
controller coupled to control a frequency of the oscillatory
angular rotation of the container and selectively operable to set
the frequency to a sufficiently low frequency as to cause the
relatively denser material to collect relatively farther from the
axis of rotation than the relatively less dense material.
[0047] A method to separate materials may be summarized as
including: receiving a material to be separated in a container;
oscillating angularly rotating the container at a high frequency;
and removing at least some of the separated material from the
container.
[0048] The method may further include pumping the material to be
separated into the container. The method may further include
intermittently pumping the material to be separated into the
container while oscillating the container. The method may further
include directing at least some of the separated material removed
from the container to at least one analysis device. The method may
further include evacuating the container with an inert fluid. The
method may further include varying a speed of the oscillating
angular rotating to change a direction in which particles in the
material move during separation.
[0049] Such apparatus and methods may produce unexpected results.
For example, in contrast to standard centrifuges, such apparatus
and methods may cause denser or heavier materials to collected
relatively close to an axis of rotation while less dense or lighter
materials collect relatively away from the axis of rotation.
Additionally or alternatively, such apparatus and method may allow
a direction (inward or outward with respect to the axis of
rotation) of material accumulation to be selected by varying a
speed of the apparatus. Such apparatus and methods may even be used
to combine separate materials.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0050] In the drawings, identical reference numbers identify
similar elements or acts. The sizes and relative positions of
elements in the drawings are not necessarily drawn to scale. For
example, the shapes of various elements and angles are not drawn to
scale, and some of these elements are arbitrarily enlarged and
positioned to improve drawing legibility. Further, the particular
shapes of the elements as drawn are not intended to convey any
information regarding the actual shape of the particular elements,
and have been solely selected for ease of recognition in the
drawings.
[0051] FIG. 1A is a front elevational view of an apparatus to
perform material separation and/or lysis, according to one
illustrated embodiment.
[0052] FIG. 1B is a front, right side, top isometric view of the
apparatus of FIG. 1A.
[0053] FIG. 1C is a front, left side, bottom isometric view of the
apparatus of FIG. 1A.
[0054] FIG. 2A is a front elevational view of the apparatus of FIG.
1A with a front cover removed, according to one illustrated
embodiment.
[0055] FIG. 2B is a front, right side, top isometric view of the
apparatus of FIG. 2A.
[0056] FIG. 2C is a front, right side, bottom isometric view of the
apparatus of FIG. 2A.
[0057] FIG. 3 is a front, right side isometric view of a motor and
drive mechanism of the apparatus of FIGS. 1A-2C.
[0058] FIG. 4 is a schematic view of a system to perform
flow-through processing, including an apparatus to perform material
separation and/or lysis, an upstream subsystem to provide material
to be separated and/or lysed, a downstream subsystem to analyze
material that has been separated and/or lysed, and a control
subsystem, according to one illustrated embodiment.
[0059] FIG. 5 is a cross-sectional view of a container having a
chamber that houses material to be lysed, particulate lysing
material, and material that has been lysed, according to one
illustrated embodiment particularly useful in flow-through
lysing.
[0060] FIG. 6 is a flow diagram of a method of operating an
apparatus, such as the apparatus of FIGS. 1A-4, to perform
lysing.
[0061] FIG. 7 is a flow diagram of a method of pumping material to
be lysed in a flow-through lysing system such as that of FIG. 4
according to one embodiment.
[0062] FIG. 8 is a flow diagram of a method of pumping material to
be lysed in a flow-through lysing system such as that of FIG. 4,
according to another illustrated embodiment.
[0063] FIG. 9 is a flow diagram of a method of pumping material to
be lysed in a flow through lysing system such as that of FIG. 4,
according to yet another illustrated embodiment.
[0064] FIG. 10 is a flow diagram of a method of pumping material to
be lysed in a flow-through lysing system such as that of FIG. 4,
according to still another illustrated embodiment.
[0065] FIG. 11 is a flow diagram of a method of evacuating lysed
material in a flow-through lysing system such as that of FIG. 4,
according to one illustrated embodiment.
[0066] FIG. 12 is a flow diagram of a method of evacuating lysed
material in a flow-through lysing system such as that of FIG. 4,
according to another illustrated embodiment.
[0067] FIG. 13 is a method of pumping material to be lysed in a
flow-through lysing system such as that of FIG. 4, according to a
further illustrated embodiment.
[0068] FIG. 14 is a flow diagram of a method of pumping material to
be lysed in a flow-through lysing system such as that of FIG. 4,
according to still a further illustrated embodiment.
[0069] FIG. 15 is a method of operating a flow-through lysing
system such as that of FIG. 4 to analyze lysed material, according
to one illustrated embodiment.
[0070] FIG. 16 is an exploded isometric view of a lysing apparatus
according to another illustrated embodiment.
[0071] FIG. 17 is a schematic diagram of a lysing system including
a lysing apparatus, an upstream subsystem to provide material to be
lysed, a downstream subsystem to analyze material that has been
lysed, and a control subsystem, according to another illustrated
embodiment.
[0072] FIG. 18 is a front elevation view of a lysing apparatus and
pipette according to one illustrated embodiment.
[0073] FIG. 19 shows a flow diagram of a method of operating a
lysing apparatus such as that of FIGS. 16 and 17, according to one
illustrated embodiment.
[0074] FIG. 20 is a flow diagram of a method of evacuating material
that has been lysed from a chamber in operating a lysing apparatus
such as that of FIGS. 16 and 17, according to another illustrated
embodiment.
[0075] FIG. 21 is a flow diagram of a method of receiving material
to be lysed in a chamber in operating a lysing apparatus such as
that of FIGS. 16 and 17, according to one illustrated
embodiment.
[0076] FIG. 22 is a flow diagram of a method of pumping material to
be lysed into a chamber in operating a lysing apparatus such as
that of FIGS. 16 and 17, according to one illustrated
embodiment.
[0077] FIG. 23 is a flow diagram of a method of pumping material to
be lysed into a chamber in operating a lysing apparatus such as
that of FIGS. 16 and 17, according to another illustrated
embodiment.
[0078] FIG. 24 is a flow diagram of a method of operating an
impeller of a lysing system such as that of FIG. 16, 17 or 18,
according to one illustrated embodiment.
[0079] FIG. 25 is a flow diagram of a method of operating an
impeller of a lysing system such as that of FIG. 16, 17 or 18,
according to one illustrated embodiment.
[0080] FIG. 26 is a flow diagram of a method of replacing a
micromotor of a lysing system such as that of FIG. 16, 17 or 18,
according to one illustrated embodiment.
[0081] FIG. 27 is a flow diagram of a method of operating a lysing
apparatus such as that of FIG. 18, according to one illustrated
embodiment.
[0082] FIG. 28 is a flow diagram of a method of operating a lysing
apparatus such as that of FIG. 18, according to one illustrated
embodiment.
[0083] FIG. 29 is a flow diagram of a method withdrawing lysed
material from a chamber of a lysing apparatus such as that of FIG.
18, according to one illustrated embodiment.
[0084] FIG. 30 is a flow diagram of a method of reusing a
micromotor of a lysing apparatus such as that of FIG. 18, according
to another illustrated embodiment.
[0085] FIG. 31 is a graph showing data representing an efficiency
of lysis as a function of lysing duration using an apparatus
similar to that of FIG. 4.
[0086] FIG. 32 is a graph showing a dependency of lysis efficiency
on frequency of oscillation.
[0087] FIG. 33 is a graph showing spore lysis as a function of
lysis duration for an apparatus similar to that of the embodiment
of FIG. 16.
[0088] FIG. 34 is an isometric view of a material separation
apparatus according to another illustrated embodiment.
[0089] FIG. 35A is a top plan view of a container to hold material
to be separated, according to one illustrated embodiment.
[0090] FIG. 35B is a side-elevational view of the container of FIG.
6A.
[0091] FIG. 36A is a top plan view of a container to hold material
to be separated, according to another illustrated embodiment.
[0092] FIG. 36B is a side-elevational view of the container of FIG.
7A.
[0093] FIG. 37A is a top plan view of a container to hold material
to be separated, according to another illustrated embodiment.
[0094] FIG. 37B is a side-elevational view of the container of FIG.
8A.
[0095] FIG. 38A is a top plan view of a container to hold material
to be separated, according to another illustrated embodiment.
[0096] FIG. 38B is a side-elevational view of the container of FIG.
9A.
[0097] FIG. 39A is a top plan view of a container to hold material
to be separated, according to another illustrated embodiment.
[0098] FIG. 39B is a side-elevational view of the container of FIG.
10A.
[0099] FIG. 40A is a top plan view of a container to hold material
to be separated, according to another illustrated embodiment.
[0100] FIG. 40B is a side-elevational view of the container of FIG.
11A.
[0101] FIG. 41 is a flow diagram of a method of operating a system
to separate materials, according to one illustrated embodiment.
[0102] FIG. 42 is a flow diagram of a method of operating a system
to separate materials, according to another illustrated
embodiment.
[0103] FIG. 43 is a flow diagram of a method of operating a system
to separate materials, according to another illustrated
embodiment.
[0104] FIG. 44 is a flow diagram of a method of operating a system
to separate materials, according to another illustrated
embodiment.
[0105] FIG. 45 is a flow diagram of a method of operating a system
to separate materials, according to another illustrated
embodiment.
[0106] FIG. 46 is a graph showing bead trajectory, linear
oscillations.
[0107] FIG. 47 is a graph showing constant distance b/w neighboring
beads.
[0108] FIG. 48 is a graph showing how particles that are denser or
heavier that the fluid may move toward the rotational axis rather
than moving away as would have been expected.
[0109] FIG. 49 is a graph showing an effect of a larger Stokes
number, hence smaller drag.
[0110] FIG. 50 is a graph showing a convergence of neighboring
beads.
[0111] FIG. 51A is a plan view of a lysing apparatus having
Luer-Lock couplers, according to one illustrated embodiment, and
two syringes coupleable to the lysing apparatus via the
couplers.
[0112] FIG. 51B is an isometric view of the lysing apparatus of
FIG. 51A.
[0113] FIG. 52 is a plan view of a plurality of lysing apparatus
coupled sequentially to one another, according to one illustrated
embodiment.
[0114] FIG. 53A is an isometric view of a manifold or array of
lysing apparatus, according to one illustrated embodiment.
[0115] FIG. 53B is an isometric view of the manifold or array of
lysing apparatus carried by a frame, according to one illustrated
embodiment, the lysing apparatus positioned to deposit lysed
material into respective wells of a plate.
[0116] FIG. 54A is an isometric view of a cartridge style container
for use in flow through lysing showing an end cap removed from a
body of the cartridge style container, according to one illustrated
embodiment.
[0117] FIG. 54B is an isometric view of the cartridge style
container of FIG. 54A with the end cap secured to a body of the
cartridge style container.
[0118] FIG. 55A is a side elevational view of a stopcock style
lysing device, according to one illustrated embodiment, showing an
inner portion rotated or configured to provide a first flow path
via two selected ports.
[0119] FIG. 55B is a side elevational view of the stopcock style
lysing device of FIG. 55A, showing the inner portion rotated or
configured to provide a second flow path via two selected
ports.
[0120] FIG. 56A is an exploded isometric view of a stopcock style
lysing device, according to one illustrated embodiment, showing an
inner vessel having an open bottom portion, the inner vessel in a
first orientation with respect to an outer vessel.
[0121] FIG. 56B is an isometric view of a stopcock style lysing
device of FIG. 56A, showing an inner vessel received in an outer
vessel, and an drive device including a motor and impeller received
in the inner vessel.
[0122] FIG. 56C is an isometric view of the inner vessel of FIG.
56A, showing the inner vessel in a second orientation, different
from the orientation illustrated in FIG. 56A.
[0123] FIG. 57A is an exploded side elevational view of a stopcock
style lysing device, according to another illustrated embodiment,
showing an inner vessel with a closed bottom portion.
[0124] FIG. 57B is an bottom plan view of a stopcock style lysing
device of FIG. 57A.
DETAILED DESCRIPTION
[0125] In the following description, certain specific details are
set forth in order to provide a thorough understanding of various
disclosed embodiments. However, one skilled in the relevant art
will recognize that embodiments may be practiced without one or
more of these specific details, or with other methods, components,
materials, etc. In other instances, well-known structures
associated with micromotors, controllers including motor
controllers, and control systems such as programmed general purpose
computing systems and the like have not been shown or described in
detail to avoid unnecessarily obscuring descriptions of the
embodiments.
[0126] Unless the context requires otherwise, throughout the
specification and claims which follow, the word "comprise" and
variations thereof, such as, "comprises" and "comprising" are to be
construed in an open, inclusive sense, that is as "including, but
not limited to."
[0127] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment. Thus, the appearances of the
phrases "in one embodiment" or "in an embodiment" in various places
throughout this specification are not necessarily all referring to
the same embodiment. Furthermore, the particular features,
structures, or characteristics may be combined in any suitable
manner in one or more embodiments.
[0128] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the content clearly dictates otherwise. It should also be noted
that the term "or" is generally employed in its sense including
"and/or" unless the content clearly dictates otherwise.
[0129] The headings and Abstract of the Disclosure provided herein
are for convenience only and do not interpret the scope or meaning
of the embodiments.
[0130] A number of embodiments of apparatus and systems to separate
materials are described herein. The material separation apparatus
and systems perform separation on a material to be separated, for
example a particulate material in suspension, to produce separated
material or material that has been separated. The material to be
separated may take the form of biological materials, for example
cells, spores, tissue, yeast, fungi, plants, bacteria, etc.,
typically suspended in a liquid medium. For instance the material
may take the forms of organelles-nuclei, mitochondria, lysosomes,
chloroplasts, endoplasmic reticulum, etc. The material may include
lysing particulate material, for instance beads.
[0131] A number of embodiments of lysis apparatus and systems are
described herein. The lysis apparatus and systems perform lysis on
a material to be lysed using lysing particulate material, to
produce lysed material or material that has been lysed. The
material to be lysed may take the form of biological materials, for
example cells, spores, tissue, yeast, fungi, plants, bacteria,
etc., typically suspended in a liquid medium. The lysing
particulate material may take a variety of forms. While generally
referred to herein as beads, the term bead is not meant to be
limiting with respect to size or shape. The beads may, for example,
take the form of ceramic beads, glass beads, zirconium beads,
zirconium/silica beads, metal beads, plastic beads, and/or sand.
The lysed material may likewise take a variety of forms, for
example organelles-nuclei, mitochondria, lysosomes, chloroplasts,
endoplasmic reticulum, etc.
[0132] Various embodiments of the material separation and/or lysis
apparatus and systems may, for example, operate in: 1) a batch
mode, 2) flow-through stop or semi-batch mode, or 3) continuous
flow-through mode. In batch mode, a container having a chamber
holding a sample of material to be separated or lysed is located in
a holder and oscillated. The container is removed after sufficient
oscillation and the separated and/or lysed material recovered. In
the flow-through stop or semi-batch mode, a sample of material to
be separated or lysed flows into to fill the chamber. The container
is then oscillated until sufficiently separated and/or lysed. The
chamber is evacuated of the separated and/or lysed material. In the
flow-through mode, a sample of material to be separated and/or
lysed flows through the chamber of the container during oscillation
at a desired flow rate, providing a desired or defined residence
time within the chamber. In the flow-through stop or semi-batch
mode, the sample may abutted by an immiscible liquid or gas and the
chamber may be evacuated by a blast of a fluid, for example a
liquid or a gas.
[0133] At least some of the embodiments take advantage of the
understanding that the forces responsible for mechanical rupture of
biological samples scale with the oscillation frequency squared,
and that by employing relatively small sample sizes, the various
embodiments described herein can achieve relatively higher
frequencies than commercially available apparatus, resulting in
rapid and efficient lysis. Various specific embodiments will now be
discussed.
[0134] At least some of the embodiments take advantage of a
recently identified property of material to undergo an
"anti-centrifugal" force when oscillated at a sufficiently high
frequency, which frequency is a function of various characteristics
of the particles. Such may be advantageously employed to change a
direction of motion of particles or to achieve a direction of
separation not previously thought to be achievable. Such may be
employed with a variety of materials and is not limited or
restricted to lysing.
[0135] FIGS. 1A-1C and 2A-2C show an apparatus 10 operable to
perform separation and/or lysing on a material to be separated
and/or lysed contained in a container 12, according to one
illustrated embodiment. In some embodiments, off-the-shelf vials
and tubes may be employed as the container 12 to hold specimens of
material to be separated and/or lysed and the lysing particulate
material or other material, for example PCR or Eppendorf tubes.
While illustrated in FIGS. 1A-1C and 2A-2C in a batch mode, the
separation and/or lysis apparatus 10 may be used in a flow-through
stop or semi-batch mode or in a continuous mode as illustrated in
FIG. 4.
[0136] The container 12 may be removably coupled to an arm 14 via a
holder 16. The holder 16 may take a variety of forms. For example,
the holder 16 may take the form of a U-shaped clamp or other
member. The holder 16 may include a fastener (e.g., screw, bolt,
etc.) 16a operable to secure the holder 16 in a container securing
configuration. Alternatively, the holder 16 may be resilient and
biased into the container securing configuration.
[0137] The arm 14 may be coupled to pivot about an axle 18 such
that the container 12 oscillates along an arcuate path 20.
Oscillation along an arcuate path 20 achieves confined periodic
flow fields with angular accelerations that provide strong
particulate flow fields and large shear rates between beads in a
liquid solution or slurry. Experiments by the applicants have
demonstrated that miniaturized geometries can provide superior
lysis through the application of high frequencies (e.g., greater
than approximately 100 Hz). Since the relative forces on
non-neutral density beads in a liquid scale according to
.omega..sup.2r, where .omega. represents angular velocity and r is
the distance of a bead from the center of rotation, a small
increase in angular speed can allow for a substantial decrease in
size to attain similar performance. Linear oscillatory motions,
even at high frequencies result in little lysis of biological
samples, while those with an arc motion may achieve lysis that is
superior to commercially available bead-based lysis apparatus.
High-speed movies clearly show that linear motions result in
periodic concentration of beads followed by expansion of beads away
from one another, but relatively little relative motion of beads
that is not along the axis of motion. In contrast, where a
container oscillates in an arc, the beads are seen to compress to
higher density just as a strong swirl is induced, resulting in very
effective lysing. Collisions and shearing provided by the relative
motion of the suspended beads contribute to the high efficiency of
the lysing.
[0138] The arm 14 may be a rigid arm, i.e., an arm that does not
appreciably bend during oscillation with a load having a mass at
least roughly equivalent to an expected load of a container
containing a material to be lysed and a lysing particulate
material. Alternatively, the arm 14 may be a flexible arm, i.e., an
arm that does appreciably bend during oscillation with a load
having a mass at least roughly equivalent to an expected load of a
container containing a material to be separated and/or lysed and
optionally a lysing particulate material.
[0139] As best illustrated in FIGS. 2A-2C and 3 in which a cover
plate 24 is removed, the arm 14 may be driven via a motor 22 and a
drive mechanism 26, which may take the form of a four-bar linkage.
In particular, a shaft 28 of the motor 22 drives a first member
such as a bar, here in the form of eccentric cam 30. The eccentric
cam 30 is received in a bore 32 of a second member or connecting
arm 34. The connecting arm 34 is drivingly coupled to the holder 16
by the axle 18 of a rocker arm 36. The drive mechanism 26 provides
a low cost, reliable mechanism to realize relatively high frequency
oscillatory motion along the arcuate path 20. While such
frequencies may not be considered high for other types of devices,
of instance rotating devices or ultra-sonic devices, such
frequencies are considered high oscillating type devices.
[0140] FIG. 4 shows a flow-through separation and/or lysis system
400 according to one illustrated embodiment. As described in more
detail herein, the flow-through separation and/or lysis system 400
may be operated in a flow-through stop or semi-batch mode, or in a
continuous flow mode.
[0141] The flow-through system 400 includes a separation and/or
lysing apparatus 410 and a container 412, which may be similar to
those described in previous embodiments. For example, the
separation and/or lysing apparatus 410 may include an arm 414 and
holder 416 to hold the container 412 as the container pivotally
oscillates about an axle 418.
[0142] The flow-through separation and/or lysis system 400 may
include an upstream subsystem 438 to deliver material to be
separated and/or lysed. For example, the upstream subsystem 438 may
include a pump 440 operable to pump or otherwise deliver material
to be separated and/or lysed to the container 412. The upstream
subsystem 438 may also include a reservoir 442 that holds the
material to separated and/or lysed.
[0143] The upstream subsystem 438 may additionally or alternatively
include a mechanism to collect material to be separated and/or
lysed, for example a sampling apparatus 439. The sampling apparatus
439 may be manually operated or may be automatic. The sampling
apparatus 439 may, for example, sample the ambient environment, for
example the air or atmosphere, water or fluids, soil or other
solids. The sampling apparatus 439 may include a vacuum or
mechanism to create a negative pressure to extract a sample. The
sampling apparatus 439 may include an actuator, for example an arm
with a shovel or broom to retrieve samples. The sampling apparatus
may include an actuator, for example a needle and syringe to
example samples.
[0144] The material to be separated and/or lysed may be delivered
via one or more conduits, for example, a tube 444a to an entrance
446a of the container 412. The tube 444a may be reinforced at one
or both ends, for example, being reinforced with multiple layers of
concentrically arranged tubes 448a. The tube 444a may have a length
L.sub.1 that is sufficiently long to allow the container 412 and
arm 414 to oscillate, while being sufficiently short as to prevent
resonance in the tube. The length L.sub.1 would be a function of
the density, the rigidity, or the attachment method of the tube
444a as well as the density, mass and/or rigidity of any material
to be separated and/or lysed carried therein.
[0145] The flow-through separation and/or lysis system 400 may
further include a downstream analysis subsystem 449. The downstream
analysis subsystem 449 may include one or more downstream analysis
apparatus 450. The downstream analysis apparatus 450 may take any
of a variety of forms. For example, the downstream analysis
apparatus 450 may include a nucleic acid amplification instrument,
electron-microscope, western blotting apparatus, mass spectrometer,
gas chromatograph, etc.
[0146] The downstream analysis subsystem 449 may further include
one or more computing systems 452 communicatively coupled to the
downstream analysis apparatus 450. The computing system 452 may be
coupled to one or more networks 453, for example a local area
network (LAN), a wide area network (WAN) such as the Internet,
and/or a wireless wide area network (WWAN). The computer system 452
may provide information about the results of an analysis performed
on separated and/or lysed material via the network 453. For
example, the computing system 452 may automatically provide an
alert or other message to suitable system based on the results of
the analysis. Such may, for example, be used to provide an alert
when a toxic or dangerous substance or condition is detected.
[0147] The downstream analysis apparatus 450 may be fluidly
communicatively coupled to an exit 446b of the container 412 via
one or more conduits, for example, tube 444b. The tube 444b may be
reinforced at one or both ends, for example, by one or more
concentrically arranged lengths of tube 448b. The tube 444b may
have a length L.sub.2 that is sufficiently long as to allow the
container 412 and arm 414 to oscillate freely while being
sufficiently short as to prevent resonance of the tube 444b. The
length L.sub.2 may be based on the density, the rigidity, or the
attachment method of the tube 444b as well as a density, mass
and/or rigidity of any material carried therein.
[0148] The flow-through separation and/or lysis system 400 may
further include one or more control systems 454. The control system
454 may take the form of one or more motor controllers and/or
computing systems. The control system 454 may be configured to
operate the flow-through system 400 in a flow-through stop or
semi-batch mode and/or in a flow-through continuous flow mode. The
control systems 454 may, for example, be communicatively coupled to
control the separation and/or lysing apparatus 410 and/or pump
440.
[0149] The flow-through system 400 provides a number of advantages
over batch based apparatus. For example, some types of beads may
have an affinity for certain bio-products that are released on
lysis, so some of the cell contents may be "lost" due to adsorption
on the bead surfaces. The flow-through design may advantageously
automatically elute the adsorbed biomolecules. It also avoids
difficult or additional acts that may be required in batch mode
configurations to evacuate the chamber. For example, the
flow-through embodiments may eliminate any possible need to blast
the chamber with a fluid such as air to clear the chamber of the
separated and/or lysed material.
[0150] FIG. 5 shows a container 512 according to one illustrated
embodiment.
[0151] The container 512 may have an entrance 546a to provide fluid
communication from an exterior 560 of the container to a chamber
562 of the container 512. The container 512 may include an exit
546b providing fluid communication between the exterior 560 and the
chamber 562 of the container 512. A first tube 544a may be coupled
to the container 512 to provide material to be lysed 564 to the
chamber 562 via the entrance 546a. As noted previously, the tube
544a may be reinforced, for example, with one or more layers of
concentrically arranged tubing 548a. A second tube 544b may be
coupled to the container 512 via the exit 546b to remove lysed
material 566 via the exit 546b. In some embodiments, the container
512 may include attachment structures to attach or otherwise couple
or secure the tubes 544a, 544b. For example, the container 512 may
include a ribbed nipple 568a at the entrance 546a and/or a ribbed
nipple 568b at or proximate the exit 546b.
[0152] The container includes lysing material 570. The lysing
material 570 may take a variety of forms, for example, a plurality
of beads. The beads may take a variety of forms including one or
more of ceramic beads, glass beads, zirconium beads,
zirconium/silica beads, metal beads, plastic beads, and/or sand.
The beads may have a variety of diameters, for example, between
approximately 10 microns and approximately 600 microns.
[0153] In the flow through embodiments, the container 512 may
include a first filter 572a positioned relatively proximate the
entrance 546a and a second filter 572b positioned relatively
proximate the exit 546b. The first and second filters 572a, 572b
form a particulate retainment area 574 in which the lysing
particulate material 570 is retained. In particular, the filters
572a, 572b may have a plurality of openings sized to substantially
pass the material to be lysed 564 and the lysed material 566,
respectively, while blocking the particulate lysing material 570.
The container 512 may include one or more structures, for example,
tabs or annular ridges 576a, 576b to retain the first and second
filters 572a, 572b in place. Filters may, for example take the form
of nylon or stainless steel mesh filter.
[0154] The embodiments of FIGS. 1A-5 may advantageously allow
extremely high packing densities. In these embodiments, the volume
of particulate material may advantageously exceed the volume of
material to be lysed or may exceed the volume of material that has
been lysed. Additionally or alternatively, these embodiments may
advantageously have essentially no air in the chamber. As used
herein, essentially no air means that the chamber is free of air
other than small bubbles which may be unintentionally entrapped in
the chamber. Such may increase lysing efficiency and prevent
undesirable heating of the system from friction associated with
liquid-air contact line motions.
[0155] FIG. 6 shows a method 600 of operating an apparatus such as
that illustrated in FIGS. 1A-4 to lyse material, according to one
illustrated embodiment.
[0156] At 602, material to be lysed is received in the chamber of
the container. The chamber may already hold lysing particulate
material. At 604, the container is oscillated along an arcuate
path. The oscillation produces large variations in movement between
respective ones of the lysing particulate material. Such variations
are more pronounced than in translational or rotational movements.
At 606, the lysed material is removed from the chamber of the
container.
[0157] FIG. 7 shows a method 700 of pumping material to be lysed in
a flow-through lysing system such as the one of FIG. 4, according
to one illustrated embodiment.
[0158] At 702, the material to be lysed is pumped into the chamber
of the container.
[0159] FIG. 8 shows a method 800 of pumping material to be lysed in
a flow-through lysing system such as that of FIG. 4, according to
one illustrated embodiment.
[0160] At 802, the material to be lysed is intermittently pumped
into the chamber of the container while the container is
oscillated. Such is suitable for the flow-through stop or
semi-batch mode.
[0161] FIG. 9 shows a method 900 of pumping material to be lysed in
a flow-through lysing system such as that of FIG. 4, according to
another illustrated embodiment.
[0162] At 902, the material to be lysed is intermittently pumped
into the chamber such that the material to be lysed spends a
sufficient time in the chamber to achieve a desired level of
lysing. Thus, if is determined that 30 seconds of oscillation
achieves a desired level of lysing, the pump may be intermittently
operated to load the chamber with material to be lysed
approximately every 30 seconds. Oscillation times of few seconds or
tenths of seconds may be suitable. Such operation is suitable for
the flow-through stop or semi-batch mode.
[0163] FIG. 10 shows a method 1000 of pumping material to be lysed
in a flow-through lysing system such as that of FIG. 4, according
to another illustrated embodiment.
[0164] At 1002, the material to be lysed is intermittently pumped
into the chamber such that the chamber is completely evacuated of
the lysed material during each cycle of the intermittent pumping.
Such is suitable for the flow-through stop or semi-batch mode.
[0165] FIG. 11 shows a method 1100 of evacuating lysed material in
a flow-through lysing system such as that of FIG. 4, according to
another illustrated embodiment.
[0166] At 1102, the chamber is evacuated of the lysed material
during each cycle of the intermittent pumping by pumping into the
chamber more material to be lysed. Such is suitable for the
flow-through stop or semi-batch mode.
[0167] FIG. 12 shows a method 1200 of operating a lysing apparatus
such as that of FIG. 4, according to another illustrated
embodiment.
[0168] At 1202, the chamber is evacuated of the lysed material each
cycle of the intermittent pumping by pumping an inert fluid into
the chamber. The inert fluid may take the form of a liquid or gas,
and may be immiscible with the lysed material or material to be
lysed. Such is suitable for the flow-through stop or semi-batch
mode.
[0169] FIG. 13 shows a method 1300 of operating a continuous lysing
apparatus, according to one illustrated embodiment.
[0170] At 1302, the material to be lysed is continuously pumped
into the chamber of the container while the container is
oscillated. Such is suitable for the flow-through continuous
mode.
[0171] FIG. 14 shows a method 1400 of operating a flow-through
lysing apparatus, according to another illustrated embodiment.
[0172] At 1402, a flow rate of the pumping of the material to be
lysed is adjusted based at least in part on the length and free
volume of the chamber such that the material to be lysed spends
sufficient time in the chamber (i.e., desired or defined residence
time) to achieve a desired level of lysing. Such is suitable for
the flow-through continuous mode.
[0173] FIG. 15 shows a method 1500 of operating a flow-through
lysing apparatus, such as that of FIG. 4, according to another
illustrated embodiment.
[0174] At 1502, the lysed material removed from the chamber of the
container is directed to at least one analysis device. At 1504, the
lysed material is analyzed. Analysis may take a variety of forms,
for example analysis with electron-microscope, western blotting,
mass spectrometry, gas chromatography, etc. Such is suitable for
any of the modes, and particularly suited to the flow-through
modes.
[0175] FIG. 16 shows a flow-through lysing apparatus 1600 according
to another illustrated embodiment. As described in more detail
herein, the flow through lysis system 1600 may be operated in a
flow-through stop or semi-batch mode, or in a continuous flow
mode.
[0176] The flow-through lysing apparatus 1600 includes a container
1602 having a chamber 1604, and a micromotor 1606 coupled to drive
an impeller 1608.
[0177] As illustrated, the chamber 1604 may have a first opening
1604a that serves as an entrance providing fluid communication from
an exterior 1610 of the container 1602 to the chamber 1604. Also as
illustrated, the chamber 1604 may have a second opening 1604b that
serves as an exit, providing fluid communication from the chamber
1604 to the exterior 1610. The container 1602 may further have a
third opening 1604c sized to receive the impeller 1608 and to
sealingly engage an outer portion of the micromotor 1606. Some
embodiments may include a bushing or O-ring to form or enhance the
sealing between the micromotor 1606 and third opening 1604c.
[0178] A first coupler 1610a may include a stem 1612a sized to be
sealingly received in the opening 1604a to provide fluid
communication into the chamber 1604. The stem 1612a may be threaded
with the hole 1604a having a complementary thread. The first
coupler 1610a may include an attachment structure, for example, a
ribbed nipple 1614a to secure a tube 1616a and provide a flow of
material to be lysed to the chamber 1604. An O-ring 1618a, or other
similar structure, may enhance a seal between a flange of the first
coupler 1610a and the container 1602.
[0179] A second coupler 1610b may include a stem 1612b sized to be
sealingly received in the opening 1604b to provide fluid
communication into the chamber 1604. The stem 1612b may be threaded
with the hole 1604b having a complementary thread. The second
coupler 1610b may include an attachment structure, for example, a
ribbed nipple 1614b to secure a tube 1616b and provide a flow of
material to be lysed to the chamber 1604. An O-ring 1618b, or other
similar structure, may enhance a seal between a flange of the
second coupler 1610b and the container 1602.
[0180] Filters 1619a, 1619b may be positioned in the chamber to
retain lysing particulate material therebetween. The filters 1619a,
1619b may, for example, take the form of nylon mesh filters with 50
micron openings mounted to suitable fittings.
[0181] The micromotor 1606 may, for example, take the form of a
micromotor having a 4 mm diameter, and may be capable of driving
the impeller at high speed, for example approximately 50,000 RPM,
when not in the presence of liquid and beads. The impeller 1608 may
be a nylon or acrylic impeller having a number of vanes. The vanes
may be straight, without curvature or angle of attachment, such
that movement of material is primarily circumferential. Should
axial/horizontal movement of the material through the chamber be
desirable, for example in a flow-through mode (e.g., FIGS. 16 and
17), such axial or flow movement comes from pumping and not from
rotation of the impeller. This allows more precise control over
amount of time that the material remains in the chamber and hence
is subject to lysis. The vanes may, for example, produce a periodic
flow at a frequency nearly 5 times as high as the embodiments of
FIGS. 1A-4, however with a smaller amplitude of motion.
[0182] The lysing apparatus 1600 may also include a controller 1620
coupled to control the micromotor 1606. The controller 1620 may,
for example include a motor controller and/or a programmed general
purpose computing system, a special purpose computer, an
application specific integrated circuit (ASIC) and/or field
programmable gate array (FPGA). The controller 1620 may for
example, be programmed or configured to cause the motor to pulsate.
Pulsating may increase the effectiveness of the lysing.
[0183] FIG. 17 shows a flow-through lysing system 1700 according to
one illustrated embodiment. As described in more detail herein, the
flow-through lysis system 1700 may be operated in a flow-through
stop or semi-batch mode, or in a continuous flow mode.
[0184] The flow-through lysing system 1700 includes a container
1702 having a chamber (not illustrated in FIG. 17), openings 1704a,
1704c (only two illustrated), and a micromotor 1706 coupled to an
impeller (not shown in FIG. 17). The opening or entrance 1704 may
be fluidly communicatively coupled to a pump 1720 that delivers
material to be lysed from a reservoir 1722 via a first conduit or
tube 1716a. A second opening or exit may deliver lysed material to
one or more downstream analysis apparatus 1724 via one or more
conduits such as tubes 1716b. As previously noted, downstream
analysis may take a variety of forms, for instance nucleic acid
amplification, electrophoresis, western blotting, mass
spectrometry, gas chromatography, etc. The downstream analysis
apparatus 1724 may be communicatively coupled to one or more
computing systems 1726. The flow-through lysing system 1700 may
also include one or more control systems 1728 which may control the
micromotor 1706 and/or pump 1720. The control system 1728 may for
example synchronize the pumping and oscillation, for example to
implement a flow-through stop or semi-batch mode. The control
system 1728 may for example control the pumping to attain a desired
or defined residence time of the material in the chamber to achieve
a desired or defined level of lysing, for example to implement a
flow-through continuous mode.
[0185] The embodiments of FIGS. 16 and 17 may advantageously allow
extremely high packing densities. In these embodiments, the volume
of particulate material may advantageously exceed the volume of
material to be lysed or may exceed the volume of material that has
been lysed. Additionally or alternatively, these embodiments may
advantageously have essentially no air in the chamber. As used
herein, essentially no air means that the chamber is free of air
other than small bubbles which may be unintentionally entrapped in
the chamber. Such may increase lysing efficiency and prevent
undesirable heating of the system from friction associated with
liquid-air contact line motions.
[0186] FIG. 18 shows a lysing system 1800 according to another
illustrated embodiment. The lysing system 1800 is particularly
suitable for batch mode lysing operations.
[0187] The lysing system 1800 includes a container 1802 having a
chamber 1804 that has a single opening 1804a to provide fluid
communication with an exterior of the container 1802. The apparatus
1800 includes a micromotor 1806 coupled to drive an impeller 1808
that is received in the chamber 1804. A portion of the micromotor
1806 is sized to form a sealing engagement with the container 1802
to seal the opening 1804a. Some embodiments may include one or more
bushings or O-rings (not shown) to ensure the seal.
[0188] Initially, the chamber 1804 is packed with material to be
lysed 1810 and lysing particulate material 1812. After rotation of
the impeller 1808, for a sufficient length of time, the chamber
1804 contains material that has been lysed and the lysing
particulate material 1812. The micromotor 1806 and impeller 1808
may then be removed and the lysed material may be extracted, for
example using a pipette 1814. The chamber 1804 of the batch mode
embodiments may not be as densely packed as in flow-through
embodiments since room may be required for the apparatus to
withdraw the lysed material.
[0189] In some embodiments, off-the-shelf vials and tubes may be
employed as the container 1802 to hold specimens of material to be
lysed and the lysing particulate material, for example PCR or
Eppendorf tubes.
[0190] The embodiment of FIG. 18 may advantageously allow extremely
high packing densities. In these embodiments, the volume of
particulate material may advantageously exceed the volume of
material to be lysed or may exceed the volume of material that has
been lysed. This embodiment is less likely to ensure that there is
essentially no air in the chamber since room may be required for
receiving the withdrawal apparatus (e.g., pipette). However, where
possible, elimination of air in the chamber may increase lysing
efficiency and prevent undesirable heating of the system from
friction associated with liquid-air contact line motions.
[0191] FIG. 19 shows a method 1900 of operating a flow-through
lysing apparatus and/or system according to one illustrated
embodiment. Such may be useful in a flow-through stop or semi-batch
mode or in a flow-through continuous mode.
[0192] At 1902, material to be lysed is received in the chamber of
a container via an entrance. The chamber may already hold lysing
particulate material. At 1904, the micromotor drives the impeller
to cause the lysing particulate material to lyse the material to be
lysed. At 1906, material that has been lysed is expelled from the
chamber of the container via an exit.
[0193] FIG. 20 shows a method 2000 of evacuating material that has
been lysed from a chamber, according to one illustrated
embodiment.
[0194] At 2002, the material that has been lysed may be expelled
via a first filter position before the exit in a flow path of
material through the apparatus or system.
[0195] FIG. 21 shows a method 2100 of receiving material to be
lysed in a chamber, according to another illustrated
embodiment.
[0196] At 2102, the material to be lysed is received in the chamber
via a second filter positioned following the entrance of the
chamber in the flow path through the apparatus or system.
[0197] FIG. 22 shows a method 2200 of pumping material to be lysed
into a chamber, according to another illustrated embodiment.
[0198] At 2202, the material to be lysed is intermittently pumped
into the chamber via the entrance. Such may be particularly
suitable for flow-through stop or semi-batch mode operation.
[0199] FIG. 23 shows a method 2300 of pumping material to be lysed
into a chamber, according to one illustrated embodiment.
[0200] At 2302, the material to be lysed is continuously pumped
into the chamber of the container via the entrance, at a flow rate
that provides for a resident time of the material to be lysed in
the chamber that is sufficiently long to achieve a desired or
defined level of lysing. The micromotor may continuously drive the
impeller to lyse the material. Such may be particularly suitable
for flow-through continuous mode operation.
[0201] FIG. 24 shows a method 2400 of operating an impeller of a
lysing system, according to one illustrated embodiment.
[0202] At 2402, the micromotor pulsatingly drives the impeller.
Pulsations may be achieved by varying a voltage or current
delivered to the micromotor. Pulsating may achieve a higher
efficiency of lysing, thereby increasing throughput or decreasing
time required to achieve a desired or defined level of lysing.
[0203] FIG. 25 shows a method 2500 of operating an impeller of a
lysing system according to one illustrated embodiment.
[0204] At 2502, the micromotor drives the impeller at greater than
10,000 RPM in the presence of liquid and beads. Driving the
impeller at a relatively high speed achieves a desired or defined
level of lysing.
[0205] FIG. 26 shows a method 2600 of replacing a micromotor of a
lysing system according to one illustrated embodiment.
[0206] At 2602, the micromotor may be replaced with a new
micromotor. At 2604, the old micromotor may be disposed or
recycled. This may be particularly useful since it is difficult to
seal the internal elements (e.g., rotor, stator) of the high speed
micromotor from exposure to the ambient environment, thus the
micromotors may fail more frequently than in other embodiments or
environments.
[0207] FIG. 27 shows a method 2700 of operating a batch based
lysing apparatus according to one illustrated embodiment. The
method 2700 may be particularly useful for use with the embodiment
of FIG. 18.
[0208] At 2702, material to be lysed is received in a chamber of a
first container via an entrance. The chamber may already hold a
lysing particulate material or the lysing material may be provided
into the chamber with or after the material to be lysed.
[0209] At 2704, an impeller is located in the chamber of the first
container. At 2706, the entrance to the first container is closed
or sealed with a micromotor. At 2708, the micromotor drives the
impeller to circulate the material to be lysed and the lysing
particulate material. The micromotor may drive the impeller for a
sufficient length of time at a sufficient speed until a desired or
defined level of lysing has occurred.
[0210] FIG. 28 shows a method 2800 of operating a lysing apparatus
according to one illustrated embodiment. The method 2800 may be
particularly useful for use with the embodiment of FIG. 18.
[0211] At 2802, the micromotor may be removed from the entrance of
the first container. At 2804, the material that has been lysed is
removed from the chamber of the first container via the
entrance.
[0212] FIG. 29 shows a method 2900 of removing material that has
been lysed according to one illustrated embodiment.
[0213] At 2902, the material that has been lysed may be withdrawn
using a pipette.
[0214] FIG. 30 shows a method 3000 of operating a lysing apparatus
according to another illustrated embodiment.
[0215] At 3002, the micromotor may be reused with one or more
additional containers. It is noted that the micromotor,
particularly when operated at high speed, may not be particularly
well protected from the material to be lysed, lysing particulate
material, or lysed material. Consequently, the micromotor may wear
out. In many applications the micromotor may be employed to lyse
multiple samples before failing.
[0216] FIG. 31 shows data on efficiency of lysis using an apparatus
similar to that of FIG. 4.
[0217] A first curve 3102 represents measured fluorescence versus
time of oscillation using an embodiment similar to that illustrated
in FIG. 4. Fluorescence is proportional to the amount of nucleic
acid released from cells. A second curve 3105 represents measured
fluorescence versus time of oscillation using a commercially
available "MINI-BEADBEATER-1 product from Biospec Products, Inc. of
Bartlesville, Okla. As seen by comparison of the first curve 3102
and second curve 3105, the embodiment of FIG. 4 causes the release
of cell contents more efficiently than the commercially available
apparatus.
[0218] FIG. 32 illustrates a dependency of lysis efficiency on the
frequency.
[0219] A curve 3202 appears to indicate a nearly quadratic
dependence of the degree of lysis on frequency as controlled by
changes to the applied voltage for a fixed amount of time.
[0220] FIG. 33 shows data representing spore lysis as a function of
time for an embodiment similar to that illustrated in FIGS. 16 and
17.
[0221] The curves 3302, 3304 illustrate that the time to saturation
is comparable to that of the embodiments of FIG. 4, but with peak
efficiency of only 80%. The power required for this efficiency was
only 400 mW, which is lower than the power used for various other
embodiments.
[0222] FIG. 34 shows a material separation apparatus 3410 according
to one illustrated embodiment.
[0223] The material separation apparatus 3410 has a base 3412. The
material separation apparatus 3410 includes an actuator in the form
of an electric motor 3414 and a transmission or drive mechanism
3416 coupled to the base 3412. The electric motor 3414 is
selectively operable to drive the drive mechanism 3416 to
oscillatingly angularly rotate (i.e., oscillating pivot) a
container 3418, about an axis of rotation 3420 as indicated by
double headed arrow 3422. Notable in this embodiment, the axis of
rotation 3420 passes through a portion of the container 3418. The
container 3418 has an interior 3424 that holds material 3426. The
material 3426, is material to be separated at a first time, and is
separated material at a second time.
[0224] The drive mechanism 3416 may include a first drive member
3430 that is rotated by a drive shaft 3432 of the motor 3414. A
second drive member 3434 may be coupled to the first drive member
3430 may a connecting rod or member 3436 such that the second drive
member eccentrically rotates the container 3418. Other drive
members may be employed, for example eccentric gears or cams. The
second drive member 3434 is coupled to a holder 3436 to which the
container 3418 is removably attached or permanently fixed.
[0225] FIGS. 35A and 35B show a container 3500 according to one
illustrated embodiment.
[0226] As illustrated, the container 3500 may have an oval or
circular outer periphery. The container 3500 may be mounted
concentrically with respect to an axis of rotation 3502, for
oscillating angular rotation thereabout as indicated by double
headed arrow 3504. Thus, the axis of rotation 3502 passes through a
portion of the container 3500.
[0227] The container 3500 may include at least one port 3506 to
transfer material between an interior 3508 of the container 3500
and an exterior 3510 thereof. The container 3500 may include one or
more filters (now shown), which may, for example take the form of
nylon or stainless steel mesh filter. One or more of the ports,
collectively 3506, may include a valve and/or filter.
[0228] FIGS. 36A and 36B show a container 3600 according to one
illustrated embodiment.
[0229] As illustrated, the container 3600 may have a rectangular or
square outer periphery. The container 3600 may be mounted
concentrically with respect to an axis of rotation 3602, for
oscillating angular rotation thereabout as indicated by double
headed arrow 3604. Thus, the axis of rotation 3602 passes through a
portion of the container 3600.
[0230] The container 3600 may include at least one port 3606 to
transfer material between an interior 3608 of the container 3600
and an exterior 3610 thereof. The container 3600 may include one or
more filters (now shown), which may, for example take the form of
nylon or stainless steel mesh filter. One or more of the ports,
collectively 3606, may include a valve and/or filter.
[0231] FIGS. 37A and 37B show a container 3700 according to one
illustrated embodiment.
[0232] As illustrated, the container 3700 may have an annular
cross-section with an oval or circular outer periphery 3700a and an
oval or circular inner periphery 3700b. The container 3700 may be
mounted concentrically with respect to an axis of rotation 3702,
for oscillating angular rotation thereabout as indicated by double
headed arrow 3704. Thus, the axis of rotation 3702 passes through a
portion of the container 3700.
[0233] The container 3700 may include a number of outer ports
3706a, 3706b to transfer material between an interior 3708 of the
container 3700 and an exterior 3710 thereof. In particular, the
outer ports 3706a, 3706b may be formed in the outer periphery 3700a
of the container 3700. The container 3700 may include a number of
inner ports 3706c, 3706d to transfer material between the interior
3708 of the container 3700 and the exterior 3710 thereof. In
particular, the inner ports 3706c, 3706d may be formed in the inner
periphery 3700b of the container 3700. The container 3700 may
include one or more filters (now shown), which may, for example
take the form of nylon or stainless steel mesh filter. One or more
of the ports, collectively 3706, may include a valve and/or
filter.
[0234] FIGS. 38A and 38B show a container 3800 according to one
illustrated embodiment.
[0235] As illustrated, the container 3800 may have an annular
cross-section with an oval or circular outer periphery 3800a and an
oval or circular inner periphery 3800b. The container 3800 may be
mounted concentrically with respect to an axis of rotation 3802,
for oscillating angular rotation thereabout as indicated by double
headed arrow 3804. Thus, the axis of rotation 3802 passes through a
portion of the container 3800.
[0236] The container 3800 may include a number of outer ports
3806a, 3806b to transfer material between an interior 3808 of the
container 3800 and an exterior 3810 thereof. In particular, the
outer ports 3806a, 3806b may be formed in the outer periphery 3800a
of the container 3800. The container 3800 may include a number of
inner ports 3806c, 3806d to transfer material between the interior
3808 of the container 3800 and the exterior 3810 thereof. In
particular, the inner ports 3806c, 3806d may be formed in the inner
periphery 3800b of the container 3800. The container 3800 may
include one or more filters (now shown), which may, for example
take the form of nylon or stainless steel mesh filter. One or more
of the ports, collectively 3806, may include a valve and/or
filter.
[0237] FIGS. 39A and 39B show a container 3900 according to one
illustrated embodiment.
[0238] As illustrated, the container 3900 may have an oval or
circular cross section with an oval or circular outer periphery
3900a and an oval or circular inner periphery 3900b. The container
3900 may be mounted for oscillating angular rotation about an axis
of rotation 3902 as indicated by double headed arrow 3904. Thus,
the axis of rotation 3902 does not pass through any portion of the
container 3900.
[0239] The container 3900 may include a number of outer ports 3906a
to transfer material between an interior 3908 of the container 3900
and an exterior 3910 thereof. The container 3900 may include a
number of inner ports 3906b to transfer material between the
interior 3908 of the container 3900 and the exterior 3910 thereof.
In particular, the outer port 3906a may spaced relatively farther
from the axis of rotation 3902 than the inner port 3906b. The
container 3900 may include one or more filters (now shown), which
may, for example take the form of nylon or stainless steel mesh
filter. One or more of the ports, collectively 3906, may include a
valve and/or filter.
[0240] FIGS. 40A and 40B show a container 4000 according to one
illustrated embodiment.
[0241] As illustrated, the container 4000 may have an oval or
circular cross section with an oval or circular outer periphery
4000a and an oval or circular inner periphery 4000b. The container
4000 may be mounted for oscillating angular rotation about an axis
of rotation 4002 as indicated by double headed arrow 4004. Thus,
the axis of rotation 4002 dos not pass through any portion of the
container 4000.
[0242] The container 4000 may include a number of outer ports 4006a
to transfer material between an interior 4008 of the container 4000
and an exterior 4010 thereof. The container 4000 may include a
number of inner ports 4006b to transfer material between the
interior 4008 of the container 4000 and the exterior 4010 thereof.
In particular, the outer port 4006a may spaced relatively farther
from the axis of rotation 4002 than the inner port 4006b. The
container 4000 may include one or more filters (now shown), which
may, for example take the form of nylon or stainless steel mesh
filter. One or more of the ports, collectively 4006, may include a
valve and/or filter.
[0243] FIG. 41 shows a method 4100 of operating an apparatus to
separate materials, according to one illustrated embodiment.
[0244] At 4102, a material to be separated is received in a
container. The material may, for example, include a particulate
material in a suspension.
[0245] At 4105, the container is oscillating angularly rotated at a
high frequency. Such may be implemented by supplying power to a
motor to drive a drive mechanism coupled to the container.
[0246] At 4106, at least some of the separated material is removed
from the container. For example, the relatively dense or heavier
material may be removed. The relatively dense or heavier material
may collect at a portion of the interior of the container that is
relatively closer to an axis of rotation than other portions of the
interior of the container. Thus, such dense or heavier material may
be removed, for instance, via an inner port of the container. Also
for example, the relatively less dense or lighter material may be
removed. The relatively less dense or lighter material may collect
at a portion of the interior of the container that is relatively
farther from an axis of rotation than other portions of the
interior of the container. Thus, such less dense or lighter
material may be removed, for instance, via an outer port of the
container. The separated material being removed may pass through
one or more filters to further separate materials.
[0247] FIG. 42 shows a method 4200 of operating an apparatus to
separate materials, according to one illustrated embodiment.
[0248] At 4202, the material to be separated is pumped into the
container.
[0249] FIG. 43 shows a method 4300 of operating an apparatus to
separate materials, according to one illustrated embodiment.
[0250] At 4302, the material to be separated is intermittently
pumped into the container while oscillating the container.
[0251] FIG. 44 shows a method 4400 of operating an apparatus to
separate materials, according to one illustrated embodiment.
[0252] At 4402, at least some of the separated material removed
from the container is directed to at least one analysis device.
Such may be accomplished using gravity flow, pumps, valves,
etc.
[0253] FIG. 45 shows a method 4500 of operating an apparatus to
separate materials, according to one illustrated embodiment.
[0254] At 4502, the container is evacuated of the separated
materials using an inert fluid. For example the container may be
flushed with an inert gas or liquid. Such may prepare the container
for a next specimen, sample or batch of material to be
separated.
[0255] To summarize, apparatus and methods cause separation of
particles (e.g., cells, bio-molecules, etc.) in a fluid suspension
by imparting angular oscillations to the fluid container, which
essentially undergoes oscillatory rigid-body rotation. Particles
whose density is different from the fluid can be separated radially
similar to centrifugation. However, the direction of particle
motion and accumulation can unexpectedly be opposite to ordinary
centrifugation. One can thus collect the relatively heavy or denser
particles near the rotation axis while the relatively light or less
dense particles are thrown away from the axis of rotation. In
contrast, in ordinary centrifugation, particles denser than the
fluid move away from the rotation axis.
[0256] As taught here, it is shown that if instead of rotating
steadily such as in an ordinary centrifuge, the container undergoes
high-frequency, purely oscillatory, angular rotation, dense or
relatively heavy particles can be made to move toward the rotation
axis while light or relatively less dense particles can be moved
away from the axis of rotation.
[0257] Thus, such provides an approach to separating particles
based on their density difference (but also dependent upon their
size) in a manner similar to a centrifuge. However, the direction
of particle migration can be manipulated (for instance by changing
the frequency of oscillations) to be opposite to what one expects
in an ordinary centrifuge. Such can potentially be applied to
separation of red and white blood cells or other bio-particles or
bio-molecules. In addition to particle separation and
concentration, one can envision using such for re-suspension of
particles that have already been separated in an ordinary
centrifuge. For instance, heavy particles are centrifuged out, but
are then re-suspended by putting the container in an oscillatory
angular rotation mode, rather than in its original steady
rotation.
[0258] In practice, a particle suspension is introduced into and
completely fills a container (for instance a chamber having a
square cross-section, thin side walls, and a top cover) and the
container is made to undergo oscillatory angular rotations about an
axis perpendicular to the centerline of the container (e.g. center
of the square cross-section). The frequency and amplitude of
oscillations can be varied. Particles migrate radially and collect
near the rotation axis or near the side walls, depending on their
density and size.
[0259] The above approach is based on a theoretical analysis of
particle motion, set out below. The theoretical analysis neglects
some effects that are assumed to be of minor importance (e.g.
Basset history-integral forces and lift forces on the particles as
well as hydrodynamic interactions among the particles and between
the particles and the walls). These effects may end up being
significant and may modify the current predictions. Experimental
verification is planned.
[0260] Applicants have observed that linear sliding motion is not
as effective at lysing spores as the "wagging" or oscillatory
motion described herein and in U.S. provisional patent application
Ser. No. 61/020,072 filed Jan. 9, 2008, which is incorporated by
reference herein in its entirety.
[0261] The equations of motion for a bead include:
m p V t = m f Du Dt - 1 2 m f ( V t - Du Dt ) - 6 .pi..mu. a ( V -
u ) + ( m p - m f ) g Equation 1 ##EQU00001##
[0262] Where the first term after the equal sign represents
pressure stress, the second term represents added mass, the third
term viscous drag and the forth term represents gravity, but can be
ignored or neglected.
[0263] Where cartridge displacement is represented by:
.DELTA. sin(.omega.t)i Equation 2
[0264] And fluid acceleration is represented by:
Du/Dt=-.omega..sup.2.DELTA. sin(.omega.t)i Equation 3
The equation of motion for the bead becomes:
(m.sub.P+1/2m.sub.f){umlaut over
(x)}=-3/2m.sub.f.omega..sup.2.DELTA. sin
(.omega.t)-6.pi..mu..alpha.[{dot over (x)}-.omega..DELTA.
cos(.omega.t)] Equation 4
with initial conditions:
x(0)=0{dot over (x)}(0)=.omega..DELTA., Equation 5
[0265] In moving frame and dimensionless, the equation is
represented as:
{umlaut over (X)}=(1=.alpha.)sin(t)-.beta.{dot over (X)} Equation
6
[0266] where
.alpha. = 3 m f 2 m p + m f .beta. = 6 .pi..mu. a .omega. ( m p - m
f / 2 ) = St - 1 Equation 8 ##EQU00002##
[0267] and with initial conditions:
X(0)=0{dot over (X)}(0)=0 Equation 9
[0268] The solution is given by:
X ( t ) = ( 1 - .alpha. ) { 1 .beta. - - .beta. t .beta. ( 1 +
.beta. 2 ) - 1 1 + .beta. 2 [ sin ( t ) + .beta. cos ( t ) ] }
Equation 10 ##EQU00003##
[0269] FIG. 46 shows bead trajectory, linear oscillations.
[0270] FIG. 47 shows constant distance b/w neighboring beads.
[0271] Oscillatory rotational motion is represented by:
.phi.(t)=.DELTA. sin(.omega.t)
.OMEGA.(t)={dot over (.phi.)}=.omega..DELTA. cos(.omega.t)
{dot over (.OMEGA.)}(t)=-.omega..sup.2.DELTA. sin(.omega.t)
Equations 11
[0272] And fluid acceleration by:
Du Dt = .OMEGA. . r e ^ .theta. - .OMEGA. 2 r e ^ r Equations 12
##EQU00004##
[0273] The equations of motion are represented as:
{umlaut over (r)}=r({dot over
(.theta.)}).sup.2=-.alpha.r.OMEGA..sup.2-.omega..beta.{dot over
(r)}
r{umlaut over (.theta.)}-2{dot over (r)}{dot over
(.theta.)}=.alpha.{dot over (.OMEGA.)}r-.omega..beta.r({dot over
(.theta.)}-.OMEGA.)
r(0)=r.sub.o.theta.(0)=0{dot over (r)}(0)=0{dot over
(.theta.)}(0)=.omega..DELTA. Equations 13
[0274] In rotating frame and dimensionless, the equations of motion
are become:
{umlaut over (r)}=-.beta.{dot over
(r)}+.DELTA..sup.2r[(1-.alpha.)cos.sup.2(t)+2 cos(t)+2 cos(t){dot
over (.delta.)}+{dot over (.delta.)}.sup.2]
{umlaut over (.delta.)}=(1=.alpha.)sin(t)-.beta.{dot over
(.delta.)}-2({dot over (r)}/r)[{dot over (.delta.)}+cos(t)]
r(0)=1.delta.(0)=0{dot over (r)}=0{dot over (.delta.)}(0)=0
Equations 14
[0275] with parameters:
( 1 - .alpha. ) = m p - m f m p + m f / 2 .beta. = 6 .pi. .omega. _
a .omega. ( m p + m f / 2 ) = St - 1 Equations 15 ##EQU00005##
[0276] FIG. 48 represents how particles that are denser or heavier
that the fluid may move toward the rotational axis rather than
moving away as would have been expected.
[0277] FIG. 49 represents the effect of a larger Stokes number,
hence smaller drag.
[0278] FIG. 50 represents convergence of neighboring beads.
[0279] An approximate may be made via a method of averaging.
Where
.beta.< {square root over (.alpha.)}
[0280] particles move radially inward, while where
.beta.< {square root over (.alpha.)}
[0281] particles move radially outward.
[0282] FIGS. 51A and 51B show a lysing apparatus 5100, according to
another illustrated embodiment.
[0283] The lysing apparatus 5100 includes a body 5102 that forms a
chamber 5104. The body 5102 may have an opening 5106 sized and
dimensioned to receive an impeller 5108 therethrough such that the
impeller resides in the chamber 5104. The opening 5106 may
optionally receive part or all of a drive motor, for instance a
micro electric motor 5110. The electric motor 5110 is coupled to
drive the impeller 5108. The electric motor 5110 is selectively
operable in response to power supplied thereto. The electric motor
5110 may be secured in the opening 5106 via a press type fitting or
interference fit. In particular, an inner wall forming the opening
5106 and/or chamber 5104 may be slightly tapered to sealing engage
a side wall of the electric motor 5110 as the electric motor is
advanced through the opening 5106 and into the chamber 5104.
Alternatively, or additionally, a side wall of the electric motor
5110 may be slightly tapered to sealing engage a side wall of the
opening 5106 and/or the chamber 5104 as the electric motor 5110 is
advanced through the opening 5106 and into the chamber 5104.
Alternatively, the electric motor 5110 and the opening 5106 and/or
chamber 5104 may include coupler structures. For instance, the
electric motor 5110 and the opening 5106 and/or chamber 5104 may
include threads (not shown) which sealing mate together as the
electric motor 5110 is advanced through the opening 5106 and into
the chamber 5104. Alternatively, a bayonet (not shown) or lug type
(not shown) coupler structure may be employed. Other sealing
structures may be employed. For example, one or more gaskets,
washers or O-rings (not shown) may be employed, with or without a
seat or peripheral ring to seat the gasket, washers or O-rings. The
seal may be a fluid tight seal and/or a gas tight seal.
[0284] The lysing apparatus includes a first port 5112a and a
second port 5112b (collectively 5112). The first and second ports
5112 include passages 5114a, 5114b, respectively, (collectively
5114) to provide fluid communication with the chamber from an
exterior thereof. The ports 5112 may be used to as input ports to
supply material to the chamber 5104 and/or as output ports to
remove material from the chamber 5104.
[0285] Each port 5112 may have a coupler 5116a, 5116b (collectively
5116) that allows selective coupling to the respective port 5112a,
5112b. For example, each of the ports 5112 may include a respective
Luer-Lock.RTM. fitting or Luer-Slip.RTM. fitting, male or female.
The Luer-Lock.RTM. or Luer-Taper.RTM. fittings allow the coupling
of syringes 5118a, 5118b (FIG. 51A, collectively 5118) to the
lysing apparatus 5100. For example, a first syringe 5118a may be
coupled to the first port 5112a to allow sample or specimen
injection, while a second syringe 5118b may be coupled to the
second port 5112b to allow removal of a sample or specimen after
lysing (i.e., lysed material). Such may allow the passage of a
sample or specimen back and forth through the chamber 5104, for
instance to enhance performance of the lysing or of DNA capture.
Use of syringes 5118 may occur at either port 5112a, 5112b or at
both ports 5112. The advantages of using a syringe 5118 as a sample
or specimen delivery system include the fact that syringes 5118 are
inexpensive, disposable, and employ positive displacement of fluid
for a high degree of reliability in rapidly dispensing volumes. The
Luer-Lock.RTM. design exemplifies a universal attachment that seals
reliably and mates with many devices that also have complimentary
Luer-Lock.RTM. fittings.
[0286] As illustrated in FIG. 52, selectively fastenable fittings,
such as the Luer-Lock.RTM. fittings, may allow multiple lysing
apparatus 5100a-5100b (collectively, 5100, only three illustrated)
to be connected in succession. Such may advantageously be used to
sequentially process a sample or specimen through multiple stages.
Additionally, or alternatively, lysing particulate (e.g., beads) in
the different sequential lysing apparatus 5100 may each have a
respective receptivity for different molecules. For instance, the
particulate in successive ones of the sequential lysing apparatus
may be conferred with receptors (e.g., binding sites) to capture
different respective molecules from the same sample or specimen.
Each lysing apparatus 5100 with a different captured molecule, may
then be easily separated from one another, and processed
individually using different types of elution acts or steps.
[0287] FIGS. 53A and 53B show a lysing manifold or array 5300,
according to one illustrated embodiment. The lysing manifold or
array 5300 includes a block or frame 5302 that has a plurality of
positions 5304a, 5304h (collectively 5304, only two called out in
FIG. 53A) to hold respective ones of one or more individual lysing
apparatus 5306a-5306h (collectively 5306, six illustrated). The
individual lysing apparatus 5306 may, for example, take the form of
distinct lysing apparatus which employ a chamber that receives an
impeller and electric motor, for instance, the individual lysing
apparatus 53006 may be identical or similar to the lysing apparatus
5100 (FIG. 51). Each individual lysing apparatus 5306 may include a
respective disposable electric motor coupled to drive the impeller.
Each individual lysing apparatus 5306 may include a first port
5308a and a second port 5308b (collectively 5308, only two called
out in FIG. 53A). The ports 5308 may function as inlet and/or
outlets to a chamber (not called out in FIG. 53A or 53B).
[0288] As illustrated in FIG. 53B, the lysing manifold or array
5300 may include a support structure 5310 to support one or more
blocks or frames 5302 and associated individual lysing apparatus
5306. In particular, the support structure 5310 may include rails
5310a, 5310b to hold the block or frame 5302 and associated
individual lysing apparatus 5306 positioned relative to a structure
that receives the lysed material, for example a plate such as a
micro-titer plate 5312. For instance, the support structure 5310
may hold the block or frame 5302 such that the associated
individual lysing apparatus 5306 are positioned above respective
ones of a plurality of wells 5312a (only one called out in FIG.
53B) of the micro-titer plate 5312. FIG. 53B shows only a single
lysing manifold or array 5300 carrying a single row of individual
lysing apparatus 5306, constituting a one-dimensional array of
lying apparatus 5306. Alternatively, the support structure 5310 may
carry additional lysing manifolds or arrays, each carrying a
respective single row of individual lysing apparatus 5306. The
individual lysing apparatus 5306 carried by the plurality of lysing
manifolds or arrays 5300 can constitute a two-dimension array. As a
further alternative, a single lysing manifold or array 5300 may
carry individual lysing apparatus 5306 arranged in a
two-dimensional array. As an even further alternatively, a motor
and drive mechanism may be coupled to move a single lysing manifold
or array 5300 carrying the individual lysing apparatus 5306 along
the rails 5310a, 5310b of the support structure 5310. Thus, the
one-dimensional array of lysing apparatus 5306 may be moved to
address a two-dimensional array of positions. Movement may be
controlled manually or automatically, for example via one or more
computer processors.
[0289] As described immediately above, individual lysing apparatus
5306 can be bundled together into a lysing manifold or array 5300
(e.g., one- or two dimensions) to facilitate multiplex processing.
The distance between centers for these individual lysing apparatus
5306 can, for example, be 9 mm or a multiple of 9 mm to match a
standard format of a micro-titer plate 5312 (e.g., with 9 mm
spacing, 96 well plate or greater). Similarly, the use of electric
motors with diameters below 4.5 mm allows the manifold or array of
lysing apparatus 5300 to be used for micro-titer plate formats with
4.5 mm spacing (e.g., 384 well plate). Bundling the individual
lysing apparatus 5306 in strips or rows of 4, 8, 6 or 12 may
facilitate use for automated or semi-automated processing of
samples in a micro-titer format. Additionally, if intake ports
5308a of the individual lysing apparatus 5306 are designed to
receive sample or specimen from pipette tips, then the individual
lysing apparatus 5306 may be addressed by multichannel pipettors
for either manual or robotic operation. The block or frame 5302 may
be fabricated monolithically from a single block of material that
has been molded or cut-extruded with multiple sites for the
individual lysing apparatus 5306.
[0290] FIG. 54A and 54B show a cartridge style container 5400
configured to perform flow through lysis processing, according to
one illustrated embodiment. In particular, FIG. 54A shows the
container 5400 with one end cap 5400a removed to provide access to
a chamber (not called out in FIG. 54A or 54B) formed by a body
5400b of the container 5400, and the other end cap 5400c fixed to
the body 5400b. FIG. 54B shows the container 5400 with both end
caps 5400a, 5400c fastened to the body 5400b of the container 5400.
The cartridge style container 5404 may, for example, be employed
with the oscillating arcuate motion based apparatus (FIGS. 1-5),
sometimes referred to herein as a bead beater.
[0291] The body 5400b of the container 5400 may have openings 5400d
(only one illustrated, in FIG. 54A) at opposed ends 5400e, 5400f
thereof, providing access to the chamber formed by the body 5400b
of the container 5400. These openings 5400d may be relatively large
to accommodate samples or specimens in various states. As noted
above, at least one end cap 5400a, 5400b of the container 5400 is
selectively removable from the body 5400b of the container 5400.
Such provides access to the interior of the chamber for relatively
large samples or specimens. In some embodiments, both end caps
5400a, 5400c are selectively removable and fastenable to the body
5400b of the container 5400, while in other embodiments only one
end cap 5400a, 5400c may be removable. In such embodiments, the
other end cap 5400a, 5400c may be a monolithic portion of the body
5400b, or may be permanently secured thereto, for example via an
adhesive, heat sealing or radio frequency (RF) welding.
[0292] Each of the end caps 5400a, 5400c may include a respective
port 5402a, 5402b (collectively 5402) that provides fluid
communication to the interior chamber of the body 5400b from an
exterior thereof. Such may accommodate flow through operation.
[0293] The cartridge style container 5400 and flow through lysing
operation may be used on virtually any cell type, for example
plants, bacteria, spores, yeast, invertebrates and vertebrates.
Additionally, the re-closable end caps 5400a, 5400c advantageously
allows the placement of a piece of sample tissue in the chamber,
while maintaining flow through capability after the end cap 5400a,
5400c is fastened to close the chamber. This may allow the lysing
apparatus to function as a homogenizer of tissues, for instance
biopsy samples, mouse tail slices, leaf punches, seeds, etc. Such
may eliminate the need to precede cell lysis with a separate tissue
homogenization act or step, which would otherwise typically require
a separate piece of equipment.
[0294] A small disposable electric motor, such as that used to mix
the beads in the embodiment employing an impeller received in a
chamber of bead blender (e.g., FIG. 16), may also be used for other
integrated functions related to analytical biochemistry. For
example, such small disposable electric motors may be used as part
of, but not limited to, a pump, a reversible pump, a valve, a mixer
of reagents, a micro centrifuge, etc. Such disposable electric
motors may even perform these functions in combination with
performing other functions, such as, but not limited to, lysing
cells in the presence of lysing particulate or beads, while pumping
fluid (e.g., material to be lysed, lysed material, cleansers) at
the same time. For example, a pump may comprises an assay device
with impeller and a disposable electric motor, such as illustrated
in FIG. 16, with a check valve on either or both ports. The check
valve(s) direct flow in one direction, while the fluid is driven by
motion the motor imparts to the fluid via the impeller.
[0295] In both configurations (i.e., lysing apparatus with rapidly
oscillating arcuate motion sometimes referred to herein as bead
beating or lysing apparatus with high angular velocity impeller
sometimes referred to herein as bead blender), high energy is
imparted to the fluid, in turn causing high velocities of lysing
particulate or beads relative to each other, which in turn causes
high shear forces between lysing particulate or beads as they pass
by relative to each other. Not to be limited by theory, these shear
forces are a possible explanation for surprising ultra rapid lysis
of the cells. Other configurations that impart similar shear forces
between the lysing particulate or beads may also provide rapid cell
lysing.
[0296] The flow through nature of some embodiments may allow for
reuse of the system for processing additional samples or specimens.
For example, the flow through nature may facilitate performance of
one or more wash acts or steps to sterilize or otherwise sanitize
or cleanse the system. Containers may be reused by cleaning and/or
sterilizing the container between uses. This may be coordinated
with downstream processing of one sample or specimen such that the
container may be made ready for another sample or specimen during
the downstream processing. One or more acts may be employed to
clean and/or sterilize the container, for example using a high pH
or low pH solution, bleach, detergent or combinations thereof.
Adjusting pH may advantageously reduce the number of wash acts or
steps, since the pH can be easily neutralized. An alternative
approach may be the use of di-ethyl-pyrocarbonate (DEPC). DEPC
compound can destroy proteins and nucleic acid. This treatment may
be followed by a single wash and then a flow of hot air. Because
DEPC is so volatile, it may be removed by degradation and
evaporation during the act of passing heated air over any surfaces
treated with the DEPC.
[0297] Lysis efficiency or cell disruption appears to be affected
by the ratio of particulate or bead volume to chamber volume.
Higher efficiency appears to occur when the volume of lysing
particulate or beads is greater than 50% of the volume of the
chamber, with an upper limit. Not to be limited by theory, the
assumption is that a denser population of lysing particulate or
beads leads to a higher rate of collisions and/or a higher rate of
proximal passes between lysing particulate or beads with high shear
force, thereby increasing the efficiency of lysis. Clearly, this
advantage diminishes when the lysing particulate or beads are
packed too densely to move or are too dense to permit the electric
motor to function (e.g., over packed in bead blender apparatus). In
theory, the ratio of chamber volume to lysing particulate or bead
volume for both the oscillating arcuate motion based apparatus
(i.e., bead beater) and the rotational impeller based apparatus
(i.e., bead blender) can be any number, but higher efficiencies
will occur when the ratio is greater than 1 to 1.
[0298] Lysis efficiency appears to be affected by a ratio of the
volume of the lysis chamber to the volume of fluid in the lysis
chamber. The high energy methods of lysis of cells by mechanical
means with lysis particulate or beads such as by rapid oscillation
of the chamber or fast rotation of a vane (i.e., impeller) are
primarily designed to fill the lysis chamber entirely with fluid.
It is possible to include a gap of air in the chamber during lysis,
however doing so will disadvantageously reduce lysis efficiency as
the air gap is increased. This approach of allowing an air gap
tends to generate heat. However, the heat may advantageously be
used to further denature components of the sample matrix or assist
in elution of captured analyte. For example, in the case of capture
of DNA by sequence specific capture probes, the heat generated by
lysing in the presence of an air gap or pocket may be used to
enhance the release and elution of DNA from the capture probes.
[0299] The various embodiments described above can be combined to
provide further embodiments. U.S. provisional patent application
Ser. No. 61/020,072 filed Jan. 9, 2008; International Patent
Application Serial No. PCT/US2009/030622 filed Jan. 9, 2009 and
published as WO 2009/089466; U.S. provisional patent application
Ser. No. 61/117,012 filed Nov. 21, 2008; U.S. provisional patent
application Ser. No. 61/220,984 filed Jun. 26, 2009; U.S.
provisional patent application Ser. No. 61/317,604, filed Mar. 25,
2010; and U.S. non-provisional application Ser. No. 12/732,070,
filed Mar. 25, 2010 are incorporated herein by reference, in their
entirety. Aspects of the embodiments can be modified, if necessary
to employ concepts of the various patents, applications and
publications to provide yet further embodiments.
[0300] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
* * * * *