U.S. patent application number 12/816183 was filed with the patent office on 2010-09-30 for methods and devices for charged molecule manipulation.
Invention is credited to Quentin T. Aten, Sandra Burnett, Larry L. Howell, Brian D. Jensen.
Application Number | 20100248343 12/816183 |
Document ID | / |
Family ID | 40229464 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100248343 |
Kind Code |
A1 |
Aten; Quentin T. ; et
al. |
September 30, 2010 |
Methods and Devices for Charged Molecule Manipulation
Abstract
Devices and methods for constraining or holding a cell are
provided. In one aspect, for example, a cellular constraint device
is provided. Such a device can include a support surface and at
least one constraining arm movably coupled to the support surface.
The constraining arm has a first position in which the constraining
arm is substantially parallel and substantially adjacent to the
support surface. Additionally, at least a portion of the
constraining arm is movable away from the support surface to a
second position where the constraining arm is operable to constrain
a single cell.
Inventors: |
Aten; Quentin T.; (Orem,
UT) ; Howell; Larry L.; (Orem, UT) ; Jensen;
Brian D.; (Orem, UT) ; Burnett; Sandra;
(Saratoga Springs, UT) |
Correspondence
Address: |
THORPE NORTH & WESTERN, LLP.
P.O. Box 1219
SANDY
UT
84091-1219
US
|
Family ID: |
40229464 |
Appl. No.: |
12/816183 |
Filed: |
June 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12668369 |
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PCT/US08/69550 |
Jul 9, 2008 |
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12816183 |
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60958624 |
Jul 9, 2007 |
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Current U.S.
Class: |
435/285.3 ;
435/283.1 |
Current CPC
Class: |
C12N 15/89 20130101;
C12M 35/02 20130101 |
Class at
Publication: |
435/285.3 ;
435/283.1 |
International
Class: |
C12M 1/00 20060101
C12M001/00 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under
National Science Foundation Grant No. 0428532. The United States
government has certain rights to this invention.
Claims
1. A cellular constraint device, comprising: a support surface; at
least one constraining arm movably coupled to the support surface;
the constraining arm having a first position in which the
constraining arm is substantially parallel and substantially
adjacent to the support surface; and at least a portion of the
constraining arm being movable away from the support surface to a
second position where the constraining arm is operable to constrain
a single cell.
2. The device of claim 1, wherein the constraining arm is operable
to constrain the single cell in a position to engage or be engaged
by a needle structure.
3. The device of claim 1, further comprising at least two
constraining arms positioned to constrain the single cell.
4. The device of claim 1, wherein the constraining arm is a lamina
emergent constraining arm, a change point constraining arm, or a
combination thereof.
5. The device of claim 1, wherein the constraining arm is a MEMs
device.
6. The device of claim 1, wherein the constraining arm is moveably
coupled to the support surface by a slider movable relative to the
support surface.
7. The device of claim 6, wherein at least a portion of the
constraining arm is operable to increase in vertical elevation
relative to the support surface as the slider is moved parallel to
the support surface.
8. The device of claim 6, further comprising a motor functionally
coupled to the slider and operable to move the slider relative to
the support surface.
9. The device of claim 6, wherein the slider is a manual
slider.
10. The device of claim 1, wherein the constraining arm when in the
second position is positioned to contact the single cell above a
midline of the single cell.
11. The device of claim 1, further comprising a needle structure
operable to carry molecular material, the needle structure being
positionable to deliver molecular material into the single
cell.
12. The device of claim 11, further comprising a charging system
electrically coupled to the needle structure, the charging system
being operable to charge and discharge the needle structure.
13. A cellular constraint device, comprising: a support structure
having an initial position and an extended position; the support
structure being moveable from the initial position to the extended
position; and wherein the support structure is operable to
constrain a cell when in the extended position.
14. The device of claim 13, wherein the support structure when in
the initial position is operable to receive the cell.
15. The device of claim 13, wherein the support structure is
operable to constrain the cell by contacting the cell above a
midline of the cell.
16. The device of claim 13, wherein the support structure is
operable constrain the cell and to prevent lateral displacement of
the cell.
17. The device of claim 13, wherein movement of the support
structure along a first axial direction causes a portion of the
support structure to move along a second axial direction that is
different from the first axial direction, wherein the portion of
the support structure is operable to contact the cell.
18. The device of claim 17, wherein the support structure is
movably coupled to a support surface, and wherein the first axial
direction is substantially parallel to the support surface.
19. A cellular constraint device, comprising: a moveable support
structure operable to constrain a cell, the movable structure being
positionable to receive the cell prior to moving to constrain the
cell.
20. The device of claim 19, wherein movement of the moveable
support structure along a first axial direction causes a portion of
the moveable support structure to move along a second axial
direction that is different from the first axial direction, wherein
the portion of the moveable support structure is operable to
contact the cell.
Description
PRIORITY DATA
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/668,369, filed on Jan. 8, 2010, which is a
U.S. nationalization of PCT Application No. PCT/U.S.08/69550 filed
on Jul. 9, 2008, which claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/958,624, filed on Jul. 9, 2007, all
of which are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to the micromanipulation of
charged molecules. Accordingly, this invention involves the fields
of biotechnology, chemistry, and micromanipulation.
BACKGROUND OF THE INVENTION
[0004] Microinjection of foreign materials is often problematic,
particularly if such microinjection is being performed on a
biological structure such as a living cell. Various transfection
techniques include the microinjection of foreign genetic material
such as DNA into the nucleus of a cell to facilitate the expression
of foreign DNA. For example, when a fertilized oocyte (egg) is
transfected, cells arising from that oocyte will carry the foreign
genetic material. Thus in one application organisms can be produced
that exhibit additional, enhanced, or repressed genetic traits. As
one example, researchers have used microinjections to create
strains of mice that carry a foreign genetic construct causing
macrophages to auto-fluoresce and undergo cell death when exposed
to a certain drugs. Such transgenic mice have since played roles in
investigations of macrophage activity during immune responses and
macrophage activity during tumor growth.
[0005] Prior art microinjectors function in a similar manner to
macro-scale syringes: a pressure differential forces a liquid
through a needle and into the cell. In some cases a glass needle
that has been fire drawn from a capillary tube can be used to
pierce the cellular and nuclear membranes of an oocyte. Precise
pumps then cause the expulsion of minute amounts of genetic
material from the needle and into the nucleus.
[0006] Recently, researchers have produced fine microinjection
needles from silicon nitride and silica glass that are smaller than
fire drawn capillaries. These finer needles, however, still employ
macro-scale pumps similar to those used in traditional
microinjectors.
SUMMARY OF THE INVENTION
[0007] Accordingly, the present invention provides devices and
associated methods for constraining or holding a cell. In one
aspect, for example, a cellular constraint device is provided. Such
a device can include a support surface and at least one
constraining arm movably coupled to the support surface. The
constraining arm has a first position in which the constraining arm
is substantially parallel and substantially adjacent to the support
surface. Additionally, at least a portion of the constraining arm
is movable away from the support surface to a second position where
the constraining arm is operable to constrain a single cell. In
another aspect, the constraining arm is operable to constrain the
single cell in a position to engage or be engaged by a needle
structure. In yet another aspect the device includes at least two
constraining arms positioned to constrain the single cell.
[0008] Various constraining arm designs are contemplated. In one
aspect, for example, the constraining arm can be a lamina emergent
constraining arm, a change point constraining arm, or the like, or
a combination thereof. In another aspect, the constraining arm is a
MEMs device.
[0009] In one aspect of the present invention, the constraining arm
is moveably coupled to the support surface by a slider movable
relative to the support surface. In another aspect, at least a
portion of the constraining arm is operable to increase in vertical
elevation relative to the support surface as the slider is moved
parallel to the support surface. In yet another aspect, a motor is
functionally coupled to the slider and is operable to move the
slider relative to the support surface. In a further aspect, the
slider is a manual slider. In yet a further aspect, the
constraining arm when in the second position is positioned to
contact the single cell above a midline of the single cell.
[0010] In another aspect of the present invention, a cellular
constraint device can include a support structure having an initial
position and an extended position. The support structure is
moveable from the initial position to the extended position, and
when in the extended position, the support structure is operable to
constrain a cell. In yet another aspect, the support structure when
in the initial position is operable to receive the cell. In one
specific aspect, the support structure is operable to constrain the
cell by contacting the cell above a midline of the cell. In another
specific aspect, the support structure is operable constrain the
cell and to prevent lateral displacement of the cell.
[0011] Various motions of movement are contemplated for the support
structure. In one aspect, for example, movement of the support
structure along a first axial direction causes a portion of the
support structure to move along a second axial direction that is
different from the first axial direction, wherein the portion of
the support structure is operable to contact the cell. In another
aspect, the support structure is movably coupled to a support
surface, and the first axial direction is substantially parallel to
the support surface.
[0012] In yet another aspect of the present invention, a cellular
constraint device can include a moveable support structure operable
to constrain a cell, where the movable support structure is
positionable to receive the cell prior to moving to constrain the
cell. In another aspect, movement of the moveable support structure
along a first axial direction causes a portion of the moveable
support structure to move along a second axial direction that is
different from the first axial direction, wherein the portion of
the moveable support structure is operable to contact the cell.
DEFINITIONS OF TERMS
[0013] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set forth below.
[0014] The singular forms "a," "an," and, "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a support" includes reference to one or more
of such supports, and reference to "an oocyte" includes reference
to one or more of such oocytes.
[0015] As used herein, the term "charged molecular material" may be
used to refer to any molecular material that is capable of being
attracted to or associated with an electrically charged structure.
Accordingly, charged molecular material may be used to refer to
those molecules having a net charge, as well as those molecules
that have a net neutral charge but possess a charge distribution
that allows attraction to the structure.
[0016] As used herein, the term "peptide" may be used to refer to a
natural or synthetic molecule comprising two or more amino acids
linked by the carboxyl group of one amino acid to the alpha amino
group of another. A peptide of the present invention is not limited
by length, and thus "peptide" can include polypeptides and
proteins.
[0017] As used herein, the term "uncharged" when used in reference
to a needle structure may be used to refer to the relative level of
charge in the needle structure as compared to a charged molecular
material. In other words, a needle structure may be considered to
be "uncharged" as long as the amount of charge on the needle
structure is insufficient to attract a useable portion of the
charged molecular material. Naturally what is a useable portion may
vary depending on the intended use of the molecular material, and
it should be understood that one of ordinary skill in the art would
be aware of what a useable portion is given such an intended use.
Additionally it should be noted that a needle structure with no
measurable charge would be considered "uncharged" according to the
present definition.
[0018] As used herein, the term "sample" when used in reference to
a sample of a molecular material may be used to refer to a portion
of molecular material that has been purposefully attracted to or
associated with the needle structure. For example, a sample of a
molecular material such as DNA that is described as being
associated with a needle structure would include DNA that has been
purposefully attracted thereto, but would not include DNA that is
attracted thereto through the mere exposure of the needle structure
to the environment. One example of DNA that would not be considered
to be a "sample" includes airborne DNA fragments that may associate
with the needle structure following exposure to the air.
[0019] As used herein, "adjacent" refers to near or close
sufficient to achieve a desired effect.
[0020] As used herein, "associate" is used to describe molecular
material that is in electrostatic contact with a structure due to
attraction of opposite charges. For example, DNA that has been
attracted to a structure by a positive charge is said to be
associated with the structure.
[0021] As used herein, the term "substantially" refers to the
complete or nearly complete extent or degree of an action,
characteristic, property, state, structure, item, or result. For
example, an object that is "substantially" enclosed would mean that
the object is either completely enclosed or nearly completely
enclosed. The exact allowable degree of deviation from absolute
completeness may in some cases depend on the specific context.
However, generally speaking the nearness of completion will be so
as to have the same overall result as if absolute and total
completion were obtained. The use of "substantially" is equally
applicable when used in a negative connotation to refer to the
complete or near complete lack of an action, characteristic,
property, state, structure, item, or result. For example, a
composition that is "substantially free of" particles would either
completely lack particles, or so nearly completely lack particles
that the effect would be the same as if it completely lacked
particles. In other words, a composition that is "substantially
free of" an ingredient or element may still actually contain such
item as long as there is no measurable effect thereof.
[0022] As used herein, the term "about" is used to provide
flexibility to a numerical range endpoint by providing that a given
value may be "a little above" or "a little below" the endpoint
without affecting the desired result.
[0023] As used herein, a plurality of items, structural elements,
compositional elements, and/or materials may be presented in a
common list for convenience. However, these lists should be
construed as though each member of the list is individually
identified as a separate and unique member. Thus, no individual
member of such list should be construed as a de facto equivalent of
any other member of the same list solely based on their
presentation in a common group without indications to the
contrary.
[0024] Concentrations, amounts, and other numerical data may be
expressed or presented herein in a range format. It is to be
understood that such a range format is used merely for convenience
and brevity and thus should be interpreted flexibly to include not
only the numerical values explicitly recited as the limits of the
range, but also to include all the individual numerical values or
sub-ranges encompassed within that range as if each numerical value
and sub-range is explicitly recited. As an illustration, a
numerical range of "about 1 to about 5" should be interpreted to
include not only the explicitly recited values of about 1 to about
5, but also include individual values and sub-ranges within the
indicated range. Thus, included in this numerical range are
individual values such as 2, 3, and 4 and sub-ranges such as from
1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5,
individually.
[0025] This same principle applies to ranges reciting only one
numerical value as a minimum or a maximum. Furthermore, such an
interpretation should apply regardless of the breadth of the range
or the characteristics being described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a view of a molecular material manipulation system
in accordance with one embodiment of the present invention.
[0027] FIG. 2 is a view of a molecular material manipulation system
in accordance with another embodiment of the present invention.
[0028] FIG. 3 is a view of a molecular material manipulation system
in accordance with yet another embodiment of the present
invention.
[0029] FIG. 4 is a view of a molecular material manipulation system
in accordance with a further embodiment of the present
invention.
[0030] FIG. 5 is graphical plot of DNA concentration to pixel
intensity in accordance with yet a further embodiment of the
present invention.
DETAILED DESCRIPTION
[0031] Aspects of the present invention provide methods and systems
for manipulating molecular material. Such methods and systems can
include the actual manipulation of the molecular material as well
as the movement and positioning of a device or devices used for the
manipulation.
[0032] It has thus now been discovered that molecular material may
be manipulated through the use of a needle structure without the
use of a pump mechanism. Such a "pump-less" needle structure
utilizes electrical charge to associate and release charged
molecular material therefrom. For example, in one aspect a method
for manipulating molecular material can include positioning an
uncharged needle structure in electrical proximity with a charged
molecular material at a first locus in a liquid environment,
charging the needle structure such that at least a portion of the
charged molecular material becomes associated with the needle
structure, moving the needle structure and the first locus relative
to one another, and discharging the needle structure to
disassociate the charged molecular material at a second locus. Such
a method utilizes the charge of a molecular material to facilitate
the transfer from the first locus to the second locus. In the case
of DNA, for example, such a transfer is made feasible by the
unequal charge distributions within DNA molecules. More
specifically, the phosphate backbone of DNA has a net charge of one
electron per phosphate, giving a total of two electrons per base
pair. This net negative charge on the outer backbone of the DNA
molecule makes it possible to move DNA from the first locus to the
second locus using a charged needle structure.
[0033] As further description, an electrical charge is introduced
into the needle structure to attract the charged molecular material
onto its outer surface. The needle structure can then be moved from
the first locus to the second locus along with the associated
molecular material. It should be noted that such movement may
include moving the needle structure to the second locus, moving the
second locus to the needle structure, or a combination of both.
Moving to the second locus may also include moving from the outside
of a cell to the inside of the cell, moving from one portion of a
liquid to another portion of the same liquid, moving from one
liquid to another liquid, and the like. Following arrival at the
second locus, the molecular material can then be released from the
surface of the needle structure. In one aspect, such release may be
accomplished by releasing the charge of the needle structure and
allowing the molecular material to diffuse away from the structure.
In another aspect, the polarity of the charge of the needle may be
reversed to electrostatically repel the molecular material from the
needle's outer surface at the second locus.
[0034] Numerous types of charged molecular material are
contemplated for use according to aspects of the present invention,
all of which would be considered to be within the present scope.
Non-limiting examples include DNA, RNA, peptides, polymers, organic
molecules, inorganic molecules, ions, and combinations thereof. In
one specific aspect, DNA may be any form of natural or synthetic
DNA, including genomic DNA, cDNA, plasmid DNA, and the like. In
another specific aspect, RNA may be any form of RNA, including
RNAi, siRNA, shRNA, mRNA, tRNA, rRNA, microRNA, and hybrid
sequences or synthetic or semi-synthetic sequences thereof.
[0035] The manipulation of molecular material according to aspects
of the present invention may be useful in a variety of situations
and environments. For example, the transfer of such material may be
utilized to transfer molecular material into a single cell.
Although any single cell would be considered to be within the
present scope, in one specific aspect the single cell may be an
oocyte. Other non-limiting examples of single cells include
neuronal cells, fibroblasts, cancer cells, and the like.
Additionally, it is also contemplated that molecular material may
be transferred to particular regions or biological structures
within a single cell. In one specific example, molecular material
may be transferred into the nucleus of a single cell.
[0036] A variety of motions are contemplated to move the needle
structure and the associated molecular material from the first
locus to the second locus. In one aspect, for example, the needle
structure can be moved along a linear or substantially linear path
from the first locus to the second locus. One example of such a
motion would include situations where the first locus and the
second locus are substantially aligned with an elongate axis of the
needle structure, with the first locus at or near the tip of the
needle structure. By moving the needle structure forward along the
elongate axis, the tip of the needle will move along a linear path
from the first locus to the second locus. Alternatively, in other
aspects the needle structure may also be moved in an additional
direction that is out of the linear path of the needle structure.
In such cases, however, it may be beneficial to maintain the
orientation of the needle structure, particularly in those aspects
where molecular material is being transferred into a cell. If such
an orientation is not maintained, there may be a risk of damage to
the cell being injected. More specifically, if a loci axis is
defined between the first locus and the second locus, the angular
relationship between the elongate axis of the needle structure and
the loci axis should remain constant as the needle structure and
the first locus are moved relative to one another. As such, in
situations where the needle structure is moved in a direction away
from the loci axis, the orientation of the needle structure would
still be maintained parallel to the elongate axis established prior
to movement. In another aspect, the angular relationship between
the loci axis and the elongate axis need not remain constant,
provided the needle structure be positioned so as the second loci
is approached in a direction that is along the elongate axis. In
other words, the needle structure may be moved to an intermediate
locus, and movement of the needle structure from the intermediate
locus to the second locus can be in a direction along the elongate
axis of the needle structure at the intermediate locus.
[0037] Upon arrival of the needle structure at the second locus,
the molecular material can be released. This can be accomplished in
a variety of ways, and any method that releases the molecular
material should be considered to be within the scope of the present
invention. In one aspect, the charge of the needle structure can
simply be released, thus allowing the molecular material to diffuse
away at the second locus. It should be noted that the needle
structure may not necessarily be completely discharged, but in some
cases could be discharged to a degree sufficient to substantially
release the charged molecular material associated with the needle
structure. In another aspect, discharging the needle structure can
include reversing the polarity of the needle structure charge. By
utilizing this method, the molecular material may be actively
driven from the needle structure, thus minimizing the amount of
time the needle structure is present at the second locus.
[0038] The present invention additionally provides systems for
manipulating molecular material. In one aspect for example, a
system for manipulating molecular material can include a needle
structure, a charging system electrically coupled to the needle
structure, the charging system being operable to charge and
discharge the needle structure, and a movement system operable to
move the needle structure from a first locus to a second locus. In
one specific aspect, the system may further include a charged
molecular material sample associated with the needle structure. As
has previously been described, such molecular material may include
any molecular material sample that is purposefully attracted to or
associated with the needle structure. Non-limiting examples of such
samples include DNA, RNA, peptides, polymers, organic molecules,
inorganic molecules, ions, and combinations thereof. A more
specific list of non-limiting examples may include DNA, RNA,
peptides, and combinations thereof.
[0039] In another specific aspect, the system can further include a
single cell positioned to receive the needle structure upon
operation of the movement system. Although a variety of single
cells are contemplated, in one aspect the single cell is an
oocyte.
[0040] Numerous needle structure configurations and materials are
contemplated, and it should be noted that any material and
configuration that allows the manipulation of molecular material
through electrical charge and discharge should be considered to be
within the present scope. In one aspect, for example, the material
of the needle structure may include a metal or metal alloy, a
conductive glass, a polymeric material, a semiconductor material,
and combinations thereof. Non-limiting examples of metals for use
in the needle structure include indium, gold, platinum, silver,
copper, associated alloys, palladium, tungsten, aluminum, titanium,
and combinations thereof. Polymeric materials that can be used to
construct the needle structure can include any conductive polymer,
non-limiting examples of which include polypyrrole doped with
dodecyl benzene sulfonate ions, SU-8 polymer with embedded metallic
particles, and combinations thereof. Non-limiting examples of
useful semiconductor materials can include monocrystalline silicon,
polycrystalline silicon, germanium, gallium arsenide, indium-tin
oxide, and combinations thereof. It should additionally be noted
that in some aspects the conductive material may be a conductive
layer that is coated on a second material, where the second
material provides the physical structure of the needle structure.
Additionally, the needle structure may be comprised of a hollow,
non-conductive material, such as a glass, where the hollow material
is filled with a conductive material such as a conductive liquid.
The needle structure may be of a variety of sizes depending on the
intended use of the device. In one aspect, however, the tip of the
needle structure may be less than about 5 microns across. In
another aspect, the tip of the needle structure may be less than
about 1 microns across. In yet another aspect, the tip of the
needle structure may be less than about 100 nanometers across.
[0041] The charging system operatively coupled to the needle
structure may include any system capable of electrically charging,
maintaining the charge, and subsequently discharging the device.
Non-limiting examples can include batteries, DC power supplies,
photovoltaic cells, static electricity generators, capacitors, and
the like. The charging system can include a switch for activation
and deactivation, and in some aspects can also include a polarity
switch to reverse polarity of the charge on the needle structure.
In one aspect the system may additionally include multiple charging
systems, one system for charging the needle structure with a
charge, and another charging system for charging the needle
structure with an opposite polarity charge. In one example
scenario, an initially uncharged needle structure is brought into
contact with a sample of a charged molecular material. The
molecular material may be in water, saline, or any other liquid
useful for molecular material manipulation. A charge opposite in
polarity to the charge of the charged molecular material is applied
to the needle structure, thus attracting a portion of the sample of
molecular material thereto. The needle structure is then moved from
the first locus to the second locus while the molecular material is
held in place by the charge. Once at the second locus, the polarity
of the charge at the needle structure can be reversed, thus
releasing the molecular material from the needle structure, and in
some cases actively driving the molecular material into the region
surrounding the second locus. The needle structure can subsequently
be withdrawn from the second locus.
[0042] A variety of movement systems are contemplated to move the
needle structure from the first locus to the second locus. Any
technique capable of moving the needle structure with sufficient
precision to allow the manipulation of molecular material is
considered to be within the present scope. Non-limiting examples of
movement systems include, mechanical systems, magnetic systems,
piezoelectric systems, electrostatic systems, thermo-mechanical
systems, pneumatic systems, hydraulic systems, and the like. The
needle structure may also be moved manually by a user. For example,
a user may push the needle structure along a track from the first
locus to the second locus. Additional movement systems are
described more fully below.
[0043] In another aspect of the present invention, a system for
manipulating molecular material is provided including a moveable
support frame, a needle structure associated with the moveable
support frame, the needle structure operable to carry molecular
material therewith, where the moveable support frame is operable to
move the needle structure from an initial position to an extended
position, and wherein an elongate axis of the needle structure is
maintained in a substantially constant orientation as the moveable
support frame moves the needle structure from the initial position
to the extended position. In a more specific aspect, a vertical
elevation of the needle structure in the extended position is
different from a vertical elevation of the needle structure in the
initial position. In such a case, the elongate axis of the needle
structure in the extended position is parallel to or substantially
parallel to the elongate axis of the needle structure in the
initial position. Furthermore, the system can include a charging
system as described above. It should be noted that the term
"vertical" as has been used herein refers to movement relative to
the elongate axis of the needle structure in the initial
position.
[0044] Additional components of the system are contemplated,
depending on the nature of the molecular material manipulation. For
example, a system that is utilized to transfer a molecular material
such as DNA to the nucleus of a single cell can include a support
structure positioned adjacent to the moveable support frame, where
the support structure is operable to secure a single cell in a
position to receive the needle structure when in the extended
position. One example of a system having such a support structure
for holding an oocyte is shown in FIG. 1. A pair of oocyte supports
12 are used to hold the oocyte 18 in a position to receive the
needle structure 14 when moved into the extended position by the
moveable support frame 16. In this aspect the center of the oocyte
18 is located such that the nucleus of the oocyte is aligned with
the needle structure when it is brought to the extended position.
In another aspect, an oocyte may be placed in a recess in the
substrate holding the moveable support frame such that the
approximate center of the oocyte is aligned with the elongate axis
of the needle structure when in the initial position (not shown).
The extended position in this configuration can thus be achieved by
moving the needle structure along the elongate axis toward the
oocyte. Additional configurations for support structures would be
readily apparent to one of ordinary skill in the art once in
possession of the present disclosure, and such configurations are
considered to be within the present scope.
[0045] In yet another aspect of the present invention, a system for
manipulating molecular material can include a moveable support
frame, a needle structure associated with the moveable support
frame, the needle structure being chargeable between a first
charged state and a second charged state, the moveable support
frame being operable to move the needle structure from an initial
position to an extended position, wherein an elongate axis of the
needle structure is maintained in a substantially constant
orientation as the moveable support frame moves the needle
structure from the initial position to the extended position.
Furthermore, in one specific aspect a vertical elevation of the
needle structure in the extended position is different from a
vertical elevation of the needle structure in the initial
position.
[0046] As has been described, a variety of configurations are
contemplated for manipulating molecular material according to
aspects of the present invention. In one specific example, a
pumpless microelectromechanical system (MEMS) device is provided
for the introduction of foreign molecular material into a single
cell. Due to the small size of such devices, it may be beneficial
to create a system that is at least substantially self-contained.
The use of the molecular material manipulation techniques of the
present invention eliminates the need for precise injection pumps,
thus facilitating such a self-contained system.
[0047] In order to inject foreign molecular material into a single
cell such as an oocyte, the system should effectively constrain the
cell and introduce foreign molecular material into the cell's
nucleus. As was shown in FIG. 1, the system may utilize three
polysilicon arms arranged around the oocyte. Two of these arms
(support structures 12) are arranged and configured to constrain
the cell, while the third (moveable support frame 16) includes a
needle structure for introducing molecular material into the cell.
In one aspect, the three arms will cause the oocyte to align in a
proper position as they come into contact. As has also been
described, any of the three arms may be actuated manually by the
user, or they may be actuated by motors or other movement
systems.
[0048] To hold an oocyte in place, the two support structure arms
rise up out of plane and move toward the cell. The arms are
lamina-emergent, change point, parallel-guiding, six-bar
mechanisms, kinematically grounded to the chip substrate, and
receive a linear input from a slider. The constraining arms are
designed to contact the cell wall above the cell's midline to
prevent both lateral and vertical displacements during transfer of
molecular material. For oocytes such as mouse cells (80-100 .mu.m
in diameter), the constraining arms are designed to have a total
vertical displacement of 68.2 .mu.m and a total horizontal
displacement of 84.6 .mu.m from its fabricated position. Given such
dimensions, one of ordinary skill in the art could readily modify
the present system to be adaptable to other cell sizes. The
constraining arm's large horizontal displacement thus allows for a
larger area into which the cell can be initially placed. The needle
arm, similar to the constraining arms, can be made of a
lamina-emergent, change-point, parallel-guiding, six-bar mechanism
receiving a linear input from a slider. However, the needle
structure mechanism is attached to a moveable support frame. The
moveable support frame is a fully compliant, parallel guiding
mechanism with, for mouse oocytes, 50 .mu.m of in-plane travel. In
one aspect, the moveable support frame is sufficiently stiff so
that as the slider moves forward, the needle rises up out of the
plane with minimal horizontal displacement in the translating
stage. Further forward movement causes the moveable support frame
to deflect and move the needle structure horizontally in its
extended position toward the second locus, thus maintaining a
parallel relationship between the elongate axis of the needle
structure in the initial position with the elongate axis in the
extended position. The previously described system is shown more
fully in FIGS. 2 and 3. FIG. 2 shows the system in the initial
position, including the moveable support frame 22, the needle
structure, 24, and a compliant folded beam suspension 26 to
facilitate the horizontal movement of the needle structure 24 once
in the extended position. The aspect shown in FIG. 2 additionally
shows a manual slider 28 to allow a user to manually actuate the
device from the initial position to the extended position. The
system can additionally includes electrical contacts to charge the
needle structure (not shown).
[0049] FIG. 3 shows the system in the extended position. Note that
as the moveable support frame 32 is moved from the initial position
shown in FIG. 2, the needle structure 34 is maintained in an
orientation that is parallel to the needle structure in the initial
position.
[0050] The following description relates to DNA, however the
present scope should not be limited to such. Rather, DNA is used to
describe various embodiments for convenience. Though it has a net
charge of zero, DNA has an unequal distribution of internal charges
resulting in a negative character equal to two electrons for every
base pair. Exploiting the electrical characteristics of DNA, the
needle structure may be designed to attract, associate, and release
molecular material using static electric charges. Thus, the needle
requires no pumps, no capillaries, and consists only of an easily
fabricated, solid, pointed body. The needle and the bottommost
monosilicon layer of a MEMS chip may form a capacitor. Voltage is
applied to the needle structure using a compliant beam attached to
either side the moveable support frame. The compliant beam may be
highly folded and attach to the needle structure about halfway
between the two vertical legs to prevent the generation of moments
that might cause the needle structure to rotate about a horizontal
axis. The compliant beams are electrically coupled to fixed
electrical bonding pads near the device through which charge can be
applied. In this case, a positive charge would be applied to
attract the negatively charged DNA molecules. The negative terminal
of the voltage supply can be attached to a bonding pad connected to
the underlying monosilicon substrate.
[0051] The MEMS construction can be achieved by a variety of
methods. The MEMS methods themselves are well known, and will not
be discussed in detail. One exemplary process, however, is
polyMUMPs (Polysilicon MEMS Multi-User Processes), a fabrication
process for surface micromachined polysilicon structures (MEMSCAP
USA).
[0052] Accordingly, the MEMS system described can be operated as
follows: DNA is introduced into the solution containing the needle
structure. A positive charge is applied to the needle structure to
attract the DNA sample. A single cell such as an oocyte is
positioned at a point to receive the needle structure and the
associated DNA. As is shown in FIG. 4, constraining structures 42
are raised to align and constrain the oocyte. In FIG. 4, the oocyte
would be located on top of the pad shown at 44. The moveable
support frame 46 is raised to the extended position, and the needle
structure 48 is extended horizontally forward into the oocyte until
the nucleus has been punctured by the needle. The polarity of the
electrical charge can then be reversed to release the DNA into the
oocyte nucleus, and the needle structure can be retracted from the
cell.
[0053] In one aspect, to be useful a practical, self-contained
pumpless MEMS injection device should satisfy three principle
constraints. First, to be practically useful, the MEMS device
should must concentrate measurable amounts of DNA on its surface in
a reasonably short amount of time. Second, to increase the
likelihood of cell survival, the MEMS needle structure should
remain inside the cell for as little time as possible; e.g. the
MEMS needle structure should be capable of repelling DNA
concentrated on its surface in a matter of seconds. Third, to
prevent damage to the device or the cell, and to prevent unwanted
bubble formation, the MEMS needle structure should not cause
electrolysis of the surrounding water. In some cases, not causing
electrolysis of the water surrounding the MEMS needle structure,
may be particularly limiting. In initial feasibility testing,
electrolysis occurred at approximately 1.8 V on gold bond pads
submerged in an aqueous solution of 0.9% NaCl (i.e. saline
solution). In initial testing, when electrolysis was allowed to
occur, delamination of gold-on-polysilicon bond pads, and near
complete removal of gold from gold on polysilicon bond pads were
observed. Thus, it appears that for some applications the operating
voltage for the MEMS needle structure should be less than 1.8
V.
EXAMPLES
[0054] The following examples are provided to promote a more clear
understanding of certain embodiments of the present invention, and
are in no way meant as a limitation thereon.
Example 1
DNA Visualization and Imaging Methods
[0055] 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) is used
to visualize the DNA in the following example. DAPI exhibits low
toxicity and its strong fluorescence under ultra-violet
illumination. When dissolved in water and not bound to DNA, DAPI
has an excitation maximum of 355 nm (ultra-violet light) and an
emission maximum of 453 nm (blue light). When DAPI is bound to DNA
its excitation maximum changes to 388 nm and its emission maximum
shifts to 454 nm, and the intensity of the emitted light increases
roughly twenty-fold compared to free DAPI. The increase in emission
intensity when DAPI binds with DNA makes it possible to distinguish
between unbound DAPI and DAPI-stained DNA.
[0056] DAPI-stained DNA is visualized using a Zeiss Axioskop
Fluorescence Microscope with UV illumination and a purpose-built
blue light filter for imaging DAPI stained samples. Because the
DAPI-DNA complex fluoresces in the blue portion of the visible
spectrum, the blue color channel is isolated from raw RGB images to
simplify image analysis. To provide quantitative estimates of the
concentration of DNA on or near the needle structure, a regression
model is made of blue pixel intensity (I) as a function of DNA
concentration (C). DAPI stained DNA samples of known and uniform
concentration are imaged using the aforementioned parameters, and
the mean blue channel pixel intensities of the images are
calculated using a MATLAB script. The relationship between
concentration in ng/.mu.L and blue pixel intensity is linear as
shown in FIG. 5. A linear fit to the data gives the relationship
shown in Equation I
C=(I-83.07)/36:788 I
The blue channel intensity measurements are shown with 95% error
bounds, and the linear model of the data given in Equation I is
represented by the solid line. Because of the long exposure used
(six seconds), the intercept value (blue pixel intensity at 0
ng/.mu.L) is highly susceptible to changes in ambient lighting
conditions. In cases where the ambient lighting conditions cause
the intercept to deviate from the value of 83.07 shown in Equation
I, a reference image of a MEMS die submerged in the appropriate
media (either distilled water or 0.9% NaCl) with no DNA present can
be used to calculate the value of the intercept under those
lighting conditions.
Example 2
DNA Attraction Experiment
[0057] The DNA attraction and repulsion experiments are performed
both in distilled water and in 0.9% saline solution. In both cases,
the experiments follow identical protocols, with the exception of
the media into which the device is submerged. A MEMS device as has
been described herein is covered in approximately 2 mm of either
distilled water or 0.9% saline solution. A 1-2 .mu.L drop of 306
ng/.mu.L DAPI stained DNA is placed in the solution near the device
using a calibrated pipette. The needle structure of the MEMS device
is connected to the positive terminal of a voltage source providing
1.5 V DC. The substrate of the MEMS device is connected to the
negative terminal of the voltage source. Images can be taken to
verify the DAPI-stained DNA on the surface of the needle structure.
Approximate concentrations of the DNA can be calculated using the
linear model of Equation I.
Example 3
DNA Repulsion Experiment
[0058] DNA is attracted to the tip of a MEMS needle structure as is
described in Example 2, by connecting the needle structure to the
positive terminal of a 1.5 V DC voltage source and connecting the
MEMS device substrate to the negative terminal of the voltage
source. Following attraction of DNA, the polarity of the electrical
charge is then reversed so that the positive terminal is connected
to the MEMS device substrate and the negative terminal is connected
to the needle structure. Images can be taken from the time the
polarity is reversed to verify DNA release and repulsion.
Additionally, the time between connecting the MEMS needle structure
to the negative terminal and when DNA is clearly repelled from the
needle structure can be calculated, and approximate concentrations
can be calculated using the linear model given in Equation I.
Example 4
Distilled Water Experiment
[0059] A MEMS device as has been described herein is covered in
approximately 2 mm of distilled water. A 1-2 .mu.L drop of 306
ng/.mu.L DAPI stained DNA is placed in the distilled water near the
device using a calibrated pipette. The needle structure of the MEMS
device is connected to the positive terminal of a voltage source
providing 1.5 V DC. The substrate of the MEMS device is connected
to the negative terminal of the voltage source. Images can be taken
to verify the DAPI-stained DNA on the surface of the needle
structure. After 1 hour 17 minutes of incubation, approximately
2.4-2.5 ng/.mu.L is attracted to the tip of the charged needle
structure. The approximations are derived from the linear model of
Equation I.
[0060] Following attraction of DNA, the polarity of the electrical
charge is then reversed so that the positive terminal is connected
to the MEMS device substrate and the negative terminal is connected
to the needle structure. The negatively charged needle structure
repels measurable amounts of DNA from its tip within six seconds of
the polarity change.
Example 5
0.9% Saline Solution Experiment
[0061] A MEMS device as has been described herein is covered in
approximately 2 mm of 0.9% saline solution. A 1-2 .mu.L drop of 306
ng/.mu.L DAPI stained DNA is placed in the saline solution near the
device using a calibrated pipette. The needle structure of the MEMS
device is connected to the positive terminal of a voltage source
providing 1.5 V DC. The substrate of the MEMS device is connected
to the negative terminal of the voltage source. When connected in
this manner and submerged in 0.9% NaCl solution, the MEMS needle
structure has a capacitance of approximately 230 pf. Images can be
taken to verify the DAPI-stained DNA on the surface of the needle
structure. After 5 minutes 46 seconds of incubation, approximately
2.2-2.4 ng/.mu.L is attracted to the tip of the charged needle
structure. The approximations are derived from the linear model of
Equation I.
[0062] Following attraction of DNA, the polarity of the electrical
charge is then reversed so that the positive terminal is connected
to the MEMS device substrate and the negative terminal is connected
to the needle structure. The negatively charged needle structure
repels measurable amounts of DNA from its tip within six seconds of
the polarity change.
[0063] It is to be understood that the above-described compositions
and modes of application are only illustrative of preferred
embodiments of the present invention. Numerous modifications and
alternative arrangements may be devised by those skilled in the art
without departing from the spirit and scope of the present
invention and the appended claims are intended to cover such
modifications and arrangements. Thus, while the present invention
has been described above with particularity and detail in
connection with what is presently deemed to be the most practical
and preferred embodiments of the invention, it will be apparent to
those of ordinary skill in the art that numerous modifications,
including, but not limited to, variations in size, materials,
shape, form, function and manner of operation, assembly and use may
be made without departing from the principles and concepts set
forth herein.
* * * * *